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10562319 | Mice homozygously transgenic (tg) 1 for the DO11.10 α/β-TCR (OVA-TCR tg/tg 22 ) on BALB/c background (gift from Dennis Y. Loh, Washington University School of Medicine, St. Louis, MO) and BALB/c mice were bred under specific pathogen-free conditions in our animal facility. All animal experiments were performed in accordance with institutional, state, and federal guidelines. Magnetic isolation of naive CD62L + CD4 + T cells was performed using two-parameter high-gradient magnetic cell separation (MACS ). Splenic cells from OVA-TCR tg/tg mice were stained with FITC-conjugated anti-CD4 mAb (GK1.5 23 ) and MultiSort anti-FITC microbeads (Miltenyi Biotec). CD4 + cells were isolated by positive selection on VS + columns using the MidiMACS system (Miltenyi Biotec 24 ). After release of MultiSort microbeads, the CD4 + cells were stained with anti-CD62L MACS microbeads (Miltenyi Biotec). CD62L + CD4 + cells were positively selected on VS + columns to a purity of >99%, as determined by cytometric analysis. Labeling of naive CD62L + CD4 + cells with 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes) was performed as described 25 . In brief, cells were washed and resuspended at a concentration of 10 7 /ml in PBS. CFSE was added at a final concentration of 5 μM and incubated for 5 min at room temperature. The reaction was stopped by washing the cells with RPMI 1640 (Life Technologies) containing 10% FCS (Sigma Chemical Co.). Cell cultures were set up with 2 × 10 6 cells/ml in complete RPMI 1640, containing 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, and 10 μM 2-ME. The antigenic peptide OVA 323–339 (Neosystem) was used at 0.5 μM. Irradiated spleen cells (3,000 rad) from BALB/c mice were used as APCs for OVA-TCR tg/tg T cells at a 5:1 ratio. Th cell cultures were split on day 2 or day 3. Recombinant murine IL-12 (gift from Maurice Gately, Hoffmann-La Roche, Nutley, NJ) was used at 1 ng/ml and neutralizing anti–IL-4 mAb (11B11 26 ) at 6 μg/ml. IL-4 was added at 30 ng/ml (culture supernatant of murine myeloma cell line P3-X63 Ag.8.653 transfected with murine IL-4 cDNA 27 ), anti–IFN-γ mAb (AN18.17.24 28 ) at 5 μg/ml, and anti–IL-12 mAb (C17.8.6 29 ) at 6 μg/ml. Recombinant human IL-2 (Hoffmann-La Roche) was used at 50 U/ml where indicated. Progression of the cell cycle was inhibited by supplement of the following drugs: 300 μM l -mimosine (ICN 30 ), 2 μg/ml aphidicolin (Sigma Chemical Co. 31 ), 1 μg/ml nocodazole (Sigma Chemical Co. 32 ), or 200 nM paclitaxel (ICN 33 ). In the presence of the inhibitors, 86–93% of the Th cells were viable on day 2 and 62–83% on day 3, with 83% for l -mimosine. Without inhibitors, 95% viable Th cells were detectable on day 2 and 90% on day 3 according to staining with propidium iodide, in the experiment shown in Fig. 4 . Calculating back from the numbers of Th cells in the various generations to the number of naive Th cells that had proliferated in culture, an estimated >50% of originally seeded cells were represented even after 4 d of culture. Also in the cultures with cell cycle inhibitors, 40–60% of the starting Th cells were still alive on day 2, and at least 20% on day 4, in the presence of l -mimosine. In all experiments using cell cycle inhibitors, they were also added to washing buffers and during the restimulation with PMA/ionomycin. Activation of Th cells was controlled by staining with anti-CD25 (PharMingen), anti-CD44 (IM7.8.1 34 ), anti-CD62L (MEL14 35 ), and anti-CD69 (PharMingen). In the experiment described in Fig. 7 , activation of naive Th cells immediately after isolation resulted in 76% CD25 + Th cells 36 h after onset of activation. Without TCR triggering, 17–30% and 8–25% of the originally seeded naive Th cells survived for 72 and 96 h, respectively. Cells treated with IL-4 showed the higher and cells cultivated without IL-4 the lower survival rate. Of the remaining Th cells, 50–62.5% and 41.9–59%, cultivated for 72 and 96 h respectively, reacted to TCR stimulation with upregulation of CD25, analyzed 36 h after activation by antigen and APCs. Cells (2 × 10 6 /ml) were restimulated with 10 ng/ml PMA and 1 μg/ml ionomycin (both Sigma Chemical Co.) for 5 h. Brefeldin A (Sigma Chemical Co.) was added at 5 μg/ml for the last 3 h of stimulation. Cells were then fixed with 2% formaldehyde 6 . For intracellular cytokine staining, cells were permeabilized with 0.5% saponin (Sigma Chemical Co.) in PBS/BSA/azide and incubated with the following cytokine-specific mAbs: PE-coupled anti–IL-4 (11B11), anti–IL-5 (TRFK5), and anti–IL-10 (JES5-16E3) at 3 μg/ml (PharMingen); digoxigenin (Dig)-conjugated anti–IL-2 (S4B6 36 ) at 3 μg/ml, anti–IL-4 (11B11) at 1 μg/ml, anti–IL-10 (JES5-2A5 ) at 1.5 μg/ml, anti–IFN-γ (AN18.17.24) at 1 μg/ml, and anti–TNF-α (MP6-XT22; PharMingen) at 1 μg/ml. Digoxigenized primary mAbs were detected with anti-Dig Fab fragments (Boehringer Mannheim) conjugated to Cy5. Anti-Dig–Cy5 and PE-labeled isotype control mAbs (3 μg/ml; PharMingen) were used as controls. In parallel, the specificity of the staining for intracellular IL-4 and IL-10 was controlled by blocking the staining of digoxigenized anticytokine mAbs by incubating the cells 15 min before and during the staining of IL-4 and IL-10, with an excess of the respective unconjugated anticytokine mAbs. OVA-TCR tg/tg cells were identified according to staining with the clonotypic mAb KJ1-26.1 39 . Four-color cytometric analysis of intracellular cytokines and cell proliferation was performed by gating on KJ1-26.1 + lymphocytes and on individual cell generations, identified by CFSE staining. In cells of these subpopulations, coexpression of two cytokines was analyzed as indicated. The population of undivided Th cells was identified according to CFSE staining intensity of nonactivated, KJ1-26.1 − Th cells in the same cultures. Cytometric analysis was performed on a FACSCalibur™ using CELLQuest™ research software (Becton Dickinson). Dead cells were excluded according to forward and side scatter and staining with propidium iodide (0.4 μg/ml). The observed (obs.) frequencies of cytokine-coexpressing cells were compared with the expected (exp.) values calculated for random coincidence of two independent variables using the test for phi (φ) correlation coefficients (values ranging from −1 to 1) 40 . A φ value of −1 indicates that two analyzed cytokines are never coexpressed, a value of 0 identifies random coexpression, and a φ coefficient of 1 marks a pair of cytokines that is always coexpressed in individual cells. We observed only φ values ≥ 0 41 . Here we analyze whether cellular proliferation is involved in the regulation of cytokine expression during Th cell differentiation from naive precursors into cytokine-producing cells. Naive CD62L + CD4 + cells were purified to >99% from the spleens of OVA-TCR tg/tg BALB/c mice by two-parameter MACS. The isolated cells were labeled with CFSE, allowing for the cytometric discrimination of individual generations of proliferating cells. CFSE-labeled, naive, OVA-specific Th cells were stimulated with the antigenic peptide OVA 323–339 and congenic APCs, i.e., irradiated BALB/c splenocytes, either in the presence of IL-12 and anti–IL-4, to analyze expression of IFN-γ, IL-2, and TNF-α, or in the presence of IL-4, anti–IL-12, and anti–IFN-γ, to analyze expression of IL-4, IL-5, and IL-10. Expression of the cytokines was evaluated by intracellular immunofluorescence, after recall stimulation of the cells with PMA/ionomycin, at various time points after primary activation. The frequencies of cytokine-expressing Th cells were determined for each T cell population that had undergone a defined number of cell divisions, according to CFSE staining, as shown in Fig. 1 A. In Fig. 1 B, the frequencies of cells capable of expressing the various cytokines at different time points of stimulation are compared for cells of different generations. Cells that had not divided could be analyzed up to day 2 in IL-4–supplemented cultures and up to day 3 in IL-12–supplemented cultures. In those cells, expression of IFN-γ, IL-2, TNF-α, IL-4, and IL-10 was clearly detectable, revealing that the expression of these cytokines in Th cells does not require cell division. For IL-5, the low frequency of expressing cells did not allow us to make a reliable statement. In cells that had divided from one up to seven times, we observed expression of all cytokines analyzed, generally at higher frequencies in populations that had divided more often. For example, IL-4 was expressed on day 5 by 10.8% of the cells that had divided three times but by 30.7% of the cells that had divided seven times. IL-10 was expressed on day 5 by 7.4% of cells divided three times and by 23.1 and 21.8% of cells divided six and seven times. Within a given generation, the frequencies of cytokine-producing cells were not constant but varied over time of stimulation. For cells divided five times, the frequencies of IL-4–expressing cells rose from 9.7% on day 3 to 20.9 and 20.4% on days 4 and 5 and then dropped to 10.1% on day 6. In summary, the ability of primarily activated Th cells to produce IL-4, IL-10, and IFN-γ upon restimulation is correlated with but not dependent on cell proliferation. The degree of coexpression of the cytokines IL-2, IL-4, and IL-10 is not correlated with the proliferative activity of Th cells, showing that the Th cells are not sequentially instructed to express the various cytokine genes upon restimulation. φ correlation coefficients for the simultaneous expression of two cytokines in individual Th cells were calculated for the total population and separately for cells of each generation at various time points after initial activation and polarization with IL-4 ( Table ). In the total cell population, the cytokines IL-2 and IL-4 were expressed independently of each other until day 4, with φ values ranging from 0.03 to 0.04. On day 5 and day 6, expression was correlated with φ = 0.13 and φ = 0.23. φ correlation coefficients for IL-4 and IL-10 rose from 0.11 on day 3 to 0.34 on day 5. φ values for IL-2 and IL-10 coexpression ranged from 0.02 on day 4 to 0.06 on days 5 and 6. The correlation of cytokine coexpression in cells of different generations at each time point analyzed was similar to that of the total populations at that time point. As shown in Fig. 2 for cells of the seventh generation, analyzed on days 4 and 6 after priming, cells with a given record of cell divisions showed an increased coexpression of IL-2 and IL-4, whereas coefficients of IL-4 and IL-10 coexpression and IL-2 and IL-10 coexpression were similar at both time points. Thus, a certain number of cell cycles is not linked to a certain degree of cytokine coexpression nor does higher division number necessarily imply a higher probability of cytokine coexpression, arguing against the idea of a cell cycle counting mechanism being involved in the instruction of Th cells to express cytokines upon restimulation, with different cell cycle numbers required for each cytokine 21 . We have analyzed the time point when Th cells become first instructed to express IL-4 upon later restimulation. CFSE-labeled, naive CD62L + CD4 + OVA-TCR tg/tg cells were activated with antigen, APCs, IL-4, anti–IL-12, and anti–IFN-γ, and analyzed for expression of IL-4 and IL-2 after recall stimulation with PMA/ionomycin . Although IL-2 production was inducible in naive Th cells and those activated for 17 and 42 h, IL-4 was expressed neither by naive Th cells nor by cells stimulated for 17 h. 42 h after onset of primary activation, IL-4 expression was readily detectable after restimulation both in cells that had divided once or twice, and in cells that had not divided, at frequencies ranging from 2.5 to 6.2%. Since instruction of Th cells to express IL-4 upon restimulation is not dependent on completion of the first cell division after initial activation, as shown above, we analyzed next whether entry of the cells into the first cell cycle may be required. Using inhibitors to arrest activated Th cells in different phases of the first cell cycle, we determined the frequencies of Th cells that were induced to express IL-4, IL-10, and IL-2 after PMA/ionomycin restimulation . The analysis was performed 3 d after onset of primary activation, when a high frequency of cells was expected to express IL-4 and/or IL-10 upon restimulation . At that time point, nonarrested cells had divided up to four times with 17.7–32.6% of them expressing IL-4, 6.9–17.9% IL-10, and 3.7–14.4% IL-2. In contrast, cells arrested with l -mimosine in the late G1 phase of the first cell cycle expressed neither IL-4 nor IL-10. IL-2 expression was clearly detectable in 6.2% of these cells. For cells that had been allowed to progress into the early S phase and had been arrested by aphidicolin, blocking the elongation process during DNA replication, 2.6% expressed IL-4 and 0.4% IL-10. When cells were arrested in the G2/M phase of the first cell cycle by nocodazole, inhibiting the formation of metaphase microtubules, i.e., the initiation of metaphase, 14.9% produced IL-4 and 1.1% IL-10. When arrested with paclitaxel, which prevents depolymerization of microtubules in metaphase, i.e., metaphase/anaphase transition, 37% of the cells expressed IL-4 and 14.7% IL-10. To exclude that the observed differences in the abilities of Th cells to express the various cytokines upon restimulation were caused by decreased overall Th cell activation in cultures with cell cycle inhibitors, expression of the markers of Th cell activation CD25, CD44, CD62L, and CD69 was analyzed in comparison with expression of IL-2 . None of the cell cycle inhibitors could block expression of any of the activation markers CD25, CD44, and CD69, or prevent downregulation of expression of CD62L, which occurs after Th cell activation, or block expression of IL-2. The above results show that processes of the early S phase of the first cell cycle after initial activation are involved in instructing Th lymphocytes for IL-4 expression upon restimulation. We now analyzed whether Th cells can be primed to become instructed for IL-4 expression, independently of entry into cell cycling. As illustrated in Fig. 6 A, we activated CFSE-labeled, naive CD62L + CD4 + OVA-TCR tg/tg Th cells in the presence of l -mimosine for 2 d, to prevent the S phase–dependent instruction of the cells for IL-4 expression. IL-4 and antigen were removed from the culture after 1 d, and anti–IL-4 was added. After 2 d, l -mimosine was also removed, and the cells were further cultivated for 2 d in the absence of antigen and IL-4. At that time, some cells had undergone one or two cell divisions, and all cells were examined for their ability to express IL-4, IL-10, and IL-2 upon recall stimulation with PMA/ionomycin . Compared with the previous experiments, the overall frequencies of IL-4–expressing cells were reduced and IL-2 levels were enhanced in the control cell population, which had not been blocked by l -mimosine. This was probably because IL-4 had only been added for the first day of stimulation, and had been blocked throughout the last 3 d. However, IL-4–expressing cells were clearly detectable in cells of all generations, at frequencies ranging from 0.9 to 7.0%. As expected, control cultures with permanent inhibition of DNA synthesis by l -mimosine showed no IL-4–expressing cells. Stimulating naive Th cells with antigen and IL-4, while inhibiting instruction of the Th cells for IL-4 expression with l -mimosine, led to priming of the cells to become instructed later, when relieved from l -mimosine, and allowed to enter S phase. After 2 d of culture in the absence of l -mimosine, 6.7, 8.5, and 4.1% of IL-4–expressing cells could be readily detected among cells that had divided once or twice or not at all. This priming of Th cells to become instructed for expression of cytokines upon restimulation, as induced by IL-4, was restricted to IL-4. Expression of IL-10 was not detectable on day 4 at significant levels in any of the cell populations initially activated in the presence of antigen and IL-4 for only the first day. Inhibiting the entry of an activated T cell into the S phase of the first cell cycle by l -mimosine had shown that Th cells can maintain the signal(s) for instruction of IL-4 expression for at least 1 d. Is this priming due to signaling via the IL-4R alone, or is signaling of the TCR also required? To analyze this, naive Th cells were first stimulated with IL-4 for 36 h, then with antigen at various time points after removal of IL-4, before they were analyzed for expression of IL-4 upon restimulation. CFSE-labeled, naive CD62L + CD4 + OVA-TCR tg/tg Th cells were stimulated with IL-4, but not with antigen, in the presence of anti–IL-12 and anti–IFN-γ. After 36 h, IL-4 was removed and anti–IL-4 was added to the cultures. After 72 or 96 h, the Th cells were stimulated with antigen and APCs. Induction of IL-4 expression was compared in these cells with Th cells that had been activated as described above, but either in the presence or absence of IL-4 for the entire culture period, and with Th cells that had been stimulated directly after isolation with or without IL-4, antigen, and APCs for 36 h. In all cultures, the antigen was withdrawn 36 h after it was added. On day 4 after onset of stimulation with antigen and APCs, the cells were restimulated with PMA/ionomycin and analyzed for expression of IL-4, IL-10, and IL-2 by intracellular immunofluorescence. 2.6% of the Th cells that had been activated simultaneously and directly after isolation with antigen, APCs, and IL-4 for 36 h expressed IL-4, compared with 0.2% of Th cells activated alike but without IL-4 . Of the Th cells that had been activated 72 or 96 h after isolation with antigen and APCs for 36 h, 2.8 and 4.3% expressed IL-4, if they had been exposed to IL-4 for the entire culture period, and only 0.5 and 0.8% if not. Cells that had been stimulated with IL-4 for 36 h, but IL-4 then having been withdrawn for another 36 or 60 h before TCR stimulation, did not express IL-4 at higher frequencies than those cells that had never been exposed to IL-4 in vitro (0.3 and 0.6% vs. 0.5 and 0.8%). Thus, the initial stimulation with IL-4 for 36 h in the absence of TCR triggering did not result in priming for IL-4 instruction. According to CFSE staining, the various cell populations showed similar proliferation activities under all culture conditions after antigen stimulation (data not shown). IL-10 was hardly detectable in cell populations that had been activated with IL-4 for only 36 h. However, in those cultures containing IL-4 for the entire culture period, IL-10 was expressed by 15.5 and 23.5% of the cells. IL-2 was expressed in all Th cell populations at high frequencies, except those that had been incubated with IL-4 all the time and that expressed IL-10 at high frequencies. IL-2 and IL-10 expression and the analysis of proliferation using CFSE staining indicate that the Th cells were still able to respond to TCR stimulation, even 72 and 96 h after isolation, and that their failure to react with expression of IL-4 is due to a lack of priming for IL-4 expression. From this experiment, it is clear that simultaneous IL-4R and TCR stimulation is required to prime a Th cell to become instructed for IL-4 recall expression. Cytokine expression in Th lymphocytes is transient, but can be memorized by the cells upon neutral or even adverse restimulation 6 7 8 9 10 11 12 13 14 . Here we show that in primarily activated Th lymphocytes, entry into the first cell cycle is required and sufficient to instruct the Th cells to produce IL-4 and IL-10 upon restimulation, whereas IL-2 expression is independent of cell cycling. Moreover, independently of DNA synthesis, Th cells are primed to become instructed for IL-4 recall expression, by simultaneous signaling via IL-4R and TCR but not IL-4R alone. This coordinate signaling can be maintained for at least 1 d. Two groups have recently used a similar technical approach to analyze the dependency of cytokine expression on proliferation in activated T cells 19 21 . Gett and Hodgkin 21 claim that cell cycling may act as an intrinsic clock, allowing expression of distinct cytokines time-independently only after a certain number of cell divisions. We find a similar correlation between proliferative activity of the cells and their capacity to express cytokines. However, already in activated but nondivided Th cell populations, cells expressing IL-2, IL-4, IL-10, IFN-γ, and TNF-α are clearly detectable. Our results rule out the possibility that even completion of the first cell cycle after activation is required to allow expression of any of these cytokines upon restimulation. Moreover, if cell division controlled the successive expression of various cytokine genes, the frequencies of cells coexpressing cytokines, which are switched on early and later, would increase in correlation to proliferation rather than time. We find here that coexpression of any two of the cytokines IL-2, IL-4, and IL-10 is not correlated with the number of cell divisions a cell has performed after activation. However, in cells that had divided a given number of times, the capacity for cytokine expression was correlated to the time of stimulation. We did not observe differentiation of Th cells for IL-4 expression as early as 17 h after primary stimulation. After 42 h, IL-4–producing cells were evident upon restimulation, also in the nondivided population. The time required to become instructed for IL-4 expression after recall stimulation could reflect the time needed by a naive Th cell to progress into the S phase of the first cell cycle after activation. The correlative increase in frequencies of cells expressing cytokines with increasing number of cell cycles performed may rather be explained by assuming that the cells get instructed for cytokine expression not only in the first but also in later cell generations, increasing the frequencies of cytokine-expressing cells among those cell populations that have divided most often. Bird et al. 19 report that instruction of an activated Th cell to express IFN-γ requires entry of the cells into the S phase of the first cell cycle, whereas cells would have to complete three cell cycles to become instructed for IL-4 expression. In contrast, we find Th cells instructed for expression of IL-4 already in the nondivided population, but only among those cells that had entered the S phase of the first cell cycle. We can exclude that the IL-4–expressing Th cells we observe are preactivated Th cells, since none of the purified naive Th cells or Th cells stimulated for 17 h with antigen expressed IL-4 upon restimulation. The reason for the discrepancy between the present data and those of Bird et al. 19 could be the overall lower frequency of IL-4–expressing cells in the cultures of Bird et al., making it difficult to identify them in the nondivided cell population. Gett and Hodgkin 21 , analyzing the capacity for IL-4 expression in Th cells once divided, apparently detected a low frequency of IL-4–expressing cells but did not check expression of IL-4 in nondivided cells. Taking the work of Bird et al. 19 and our results together, instruction of Th cells for IFN-γ, IL-4, or IL-10 expression requires the cells to enter the S phase of the first cell cycle after activation, pointing to a common molecular mechanism for instruction of the cells to express these “effector” cytokines upon restimulation. For IL-10 instruction, the cells apparently have to complete the S phase. IL-2 expression in naive cells does not seem to require entry into the cell cycle. It is likely that those cells have already been instructed for IL-2 expression in the thymus 20 42 . The molecular basis for instruction of Th cells to express particular cytokines after restimulation is less clear, as is its stability and plasticity. Apart from cytokine-dependent modulations of the signaling and transcription repertoire of Th cells expressing particular cytokines, restricting their potential to respond to signals inducing expression of other cytokines 43 44 45 46 47 48 49 , modifications of the cytokine gene loci themselves have been demonstrated. Demethylation has been described for the DNA comprising the IL-3, IL-4, IL-5, or IFN-γ genes of Th cell lines, clones, and ex vivo–activated T cell populations containing cells expressing the respective cytokines upon restimulation 15 16 17 19 . Furthermore, it has been shown for such T cell populations that transcription of the IL-4, IL-13, and IFN-γ genes parallels the appearance of particular DNase I hypersensitive sites within the expressed cytokine gene loci 18 20 . These results suggest that the instruction for expression of particular cytokines might be maintained in Th cells by epigenetic somatic imprinting of the respective cytokine gene loci. The initiation of the somatic imprinting of cytokine genes remains enigmatic. This is even more so in light of the monoallelic expression of cytokine genes in many Th cells, suggesting that the instruction of a Th cell to express a particular cytokine is a stochastic event for each allele in a given population of Th cells 50 51 52 53 . We describe here that instruction of a Th cell for IL-4 and IL-10 recall expression is dependent on progression of the activated Th cell through the S phase of the first cell cycle, while Bird et al. 19 have shown the same for IFN-γ. The most obvious characteristic of the S phase of the first cell cycle is that the first DNA synthesis after activation of the naive T cell occurs. One might speculate that an epigenetic modification of the DNA, most likely demethylation, may be the molecular correlate of cytokine gene instruction. How would demethylation be targeted to the cytokine gene locus? The presence of sequence- or site-specific demethylating enzymes has not been demonstrated to date 54 . “Unspecific” demethylation machineries would have to be directed to a particular area of DNA, most likely by flagging of the DNA through sequence-specific proteins, which may or may not belong to the transcription machinery. We here provide indirect evidence for such a flagging of cytokine genes for molecular modification. Th cells that had been activated via TCR and IL-4R, but inhibited to enter the cell cycle, are primed to become instructed for IL-4 expression later, when allowed to progress through S phase, even in the absence of the inductive stimuli. For the priming for IL-4 instruction, signaling by IL-4 alone or sequential stimulation by IL-4 and antigen is not sufficient. The IL-4R and TCR have to be stimulated simultaneously, although it cannot be excluded at present that activation by antigen, followed by IL-4 stimulation, would not work as well. This is in accordance with the results of Agarwal and Rao 20 , who showed that chromatin remodeling of the IL-4 gene is detectable only after stimulation of naive Th cells with IL-4 and anti-CD3 and not with IL-4 alone. Default induction of IL-4 expression is dependent on IL-4R/signal transducer and activator of transcription (STAT)6 signaling 55 56 57 . However, IL-4R– or STAT6-independent IL-4 induction has also been observed. An IL-4–independent pathway for the induction of IL-4 has been demonstrated in IL-4R–deficient NK T cells 58 . IL-4 expression is also inducible in CD3 + T cells deficient for STAT6 and BCL-6 59 , suggesting a role for BCL-6 as an inhibitor of IL-4 expression, probably by competing with STAT6 for DNA binding motifs 60 . According to the present results, in wild-type cells components of both the IL-4R and TCR signaling pathways are required to prime Th cells for IL-4 instruction. Evidence has been obtained that STAT6 is crucial for chromatin remodeling and expression of IL-4, using STAT6-deficient cells 19 . In those cells, expression of IL-4 could be induced by trichostatin A, an inhibitor of histone deacetylation (i.e., an activator of nucleosome displacement), and azacytidine, preventing DNA methylation. For STAT6, as well as for nuclear factor of activated T cells (NFAT)1 and activating protein 1 (AP-1), involved in TCR signaling, binding to and activation by the coactivators p300/cAMP response element–binding protein (CREB)–binding protein (CBP), which exhibit histone acetyltransferase activity, have been described 61 62 63 64 . In line with this, BCL-6, the inhibitor of STAT6, binds to silencing mediator of retinoid and thyroid receptor (SMRT), a protein able to recruit histone deacetylase activity 65 66 . The speculative sequence of events for induction of molecular memory of cytokine expression appears to be IL-4R– and TCR-mediated chromatin rearrangement by specific acetylation of histones, perhaps involving STAT6 and displacement of BCL-6, followed by binding of specific, yet unidentified flagging factors to the DNA of the cytokine gene locus of the primed cell, in the G0/G1 phase preceding the first cell cycle of the activated cell. Finally, imprinting of the cytokine gene locus by demethylation during the S phase of the first cell cycle after activation would provide the molecular basis for cytokine memory. | Study | biomedical | en | 0.999996 |
10562320 | Generation of STAT1 −/− 36 , IFN-α receptor (IFNAR)1 −/− 37 , and IFN-γ receptor (IFNGR)1 −/− mice 38 has been described. IFNAR and IFNGR double-knockout mice (AR+GR) −/− were derived by interbreeding IFNAR −/− and IFNGR −/− mice and screening for compound homozygous mutant offspring. Strain backgrounds were either 129 or C57BL/6 (eighth backcross generation) and were compared with wild-type mice of the same strain. Mice were housed under specific pathogen–free conditions, and all work with animals conformed to guidelines approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. Lymphocytes of thymi, spleens, and lymph nodes were prepared from 6–8-wk-old mice. Triple staining for surface markers was performed by incubating ∼10 6 cells first with M1/42.3 (TIB-126; American Type Culture Collection), a rat mAb that recognizes a framework epitope common to mouse MHC class I, followed by staining cells with FITC-conjugated anti–rat IgG (Caltag Labs.). Cells were subsequently incubated with anti–CD4-TC (tricolor) and anti–CD8-PE or anti–B220-PE antibodies (Caltag Labs.) in the presence of normal rat serum to block cross-reactions. Stained cells were washed two times with cold staining buffer (0.2% BSA and 0.1% sodium azide in PBS) and fixed with 1% paraformaldehyde in staining buffer, followed by FACS™ analysis. Additional surface markers were analyzed using anti–H-2K b –PE (PharMingen) and anti–I-A b –biotin, anti–IgM-biotin, and streptavidin–allophycocyanin (Caltag Labs.). For comparing MHC class I induction in response to IFN-γ and IL-7, selected lymphocyte populations were incubated in the presence or absence of cytokine for 48 h, followed by FACS™ analysis. Mean channel shift was the average increase in mean fluorescence intensity 39 and varied <10% for three separate experiments. 20 μg of total RNA prepared from splenocytes or thymocytes using Trizol reagent (Life Technologies) was resolved on 1.5% denaturing agarose gels and transferred to nitrocellulose membranes. cDNA probes were derived from splenic RNA by reverse transcriptase–PCR. The primer sets for amplifying different genes were as follows: LMP2, forward primer, 5′-ATGCTGCGGGCAGGAGCACCTACCGC-3′ and reverse primer, 5′-TCACTCATCGTAGAATTTTGGCAGCTC-3′; LMP7, forward primer, 5′-ATGGCGTTACTGGATCTGTGCGGTGC-3′ and reverse primer, 5′-TCACAGAGCGGCCTCTCCGTACTTGTA-3′; TAP1, forward primer, 5′-AGTGTCTCGGGAATGCTGCTGAAGGTG-3′ and reverse primer, 5′-AGT-GTGCAGTCCAGAGGCCTTGTCGTCTG-3′; β-actin, forward primer, 5′-GTGGGGCGCCCCAGGCACCA-3′ and reverse primer, 5′-CTCCTTATTGTCACGCACGATTTC-3′; MHC I, forward primer, 5′-GCACAGATTCCCCAAAGG-3′ and reverse primer, 5′-ATCTCAGGGTGAGGGGCTCA-3′; β2m, forward primer, 5′-GTGACCCTAGTCTTTCTGGTG-3′ and reverse primer, 5′-TGAATCTTCAGAGCATCATG-3′; IRF-1, forward primer, 5′-TCCATGGAAGCACGCTGCTA-3′ and reverse primer, 5′-AGACTGCTGCTGACGACACA-3′. The amplified DNA fragments were purified and labeled using RadPrime DNA labeling kits (Life Technologies). Hybridization, washing, and stripping of the nitrocellulose membrane followed standard procedures 40 . To quantify specific signals, membranes were exposed to PhosphorImager screens (Molecular Dynamics) and analyzed according to the manufacturer's instructions. Quantitative measurements were normalized to values for actin. Production of retrovirus was as described 41 . A murine stem cell virus vector expressing humanized green fluorescent protein (GFP; a gift of Dr. W. Pear, University of Pennsylvania, Philadelphia, PA) was transfected into the Phoenix packaging cell line (a gift of Dr. G.P. Nolan, Stanford, CA), and culture supernatant was used as viral stock. Transduction of bone marrow cells with retrovirus was as described 42 . In brief, C57BL/6 wild-type or STAT1 −/− mice were treated with 150 mg/kg 5-fluorouracil (Sigma Chemical Co.) 6 d before harvest. Bone marrow cells were harvested and incubated on Retronectin TM (Takara-Shuzo) coated dishes containing bone marrow transfer medium and 1 ml of recombinant virus for 48 h. Cultures were supplemented with 1 ml of fresh virus after the first 24 h. 1.6 million bone marrow cells were injected intravenously into lethally irradiated (900 rads) wild-type or STAT1 −/− recipients. Mononuclear cells recovered from peripheral blood of recipient mice were analyzed by double staining with anti–H-2K b –PE and anti–TCR-allophycocyanin or IgM-allophycocyanin after gating for GFP fluorescence to score donor-derived lymphocytes. Freshly isolated splenocytes were incubated with rat anti-B220 at 0.2 μg/10 6 cells for 30 min at 4°C. Antibody-bound cells were washed twice with 1× PBS and incubated with anti–rat antibody–coated M450 Dynabeads (Dynal) for 30 min at 4°C on a mixer. B cells were collected using a magnet after three washes with 1× PBS containing 0.2% BSA. Nuclear extracts prepared from positively selected B cells and negatively selected T cells were resolved in 7% SDS-PAGE, followed by Western blot analysis as described 43 . The antibodies used for immunoblots were anti–phosphotyrosyl-STAT1 (Zymed Labs.) and anti–STAT1 COOH terminus (STAT1C; a gift from Dr. C. Schindler, Columbia University, New York, NY). Purity of selected cell populations was verified by flow cytometry. B cell fractions were typically >95% pure; T cell fractions were >80% pure. Nuclear extracts prepared from Dynabead-selected T or B cells, which were left untreated or treated with human recombinant IL-7 (10 ng/ml; StemCell) or IFN-γ (100 U/ml; Boehringer Mannheim) for 30 min, were analyzed using a 32 P-labeled probe containing a high-affinity GAS sequence 44 in the presence or absence of anti-STAT1C antibody as described 43 . Type I and type II IFNs are known modulators of MHC antigen expression during inflammation and viral infection 45 . Because STAT1 is a signal transducer shared by both types of IFNs, we examined the expression of MHC antigens on freshly isolated lymphocytes from wild-type or STAT1 −/− mice with antibody to either MHC class I (M1/42.3, an mAb recognizing a framework epitope of MHC class I) or class II (I-A b ). Cells from STAT1 −/− mice housed under pathogen-free conditions showed a two- to threefold reduction of surface MHC class I compared with lymphocytes from wild-type mice . The expression of MHC class II, however, was comparable on lymphocytes of both genotypes , even though both class I and class II expression can be induced by IFN, and both show an absolute requirement for STAT1 for induction 21 . Reduced expression and absence of induction of MHC class I was not due to a global defect, however, because class I expression was induced in response to mitogenic stimulation of both B and T splenocytes devoid of STAT1 (data not shown), reflecting an intact NF-κB pathway 46 47 48 . To explore the mechanisms underlying the reduction of cell surface expression of MHC class I in the absence of STAT1, we examined the levels of MHC class I heavy chain and light chain (β2m) mRNA by Northern hybridization. To assess the potential role of constitutive IFN in this regulation, we included lymphocytes lacking receptors for either IFN-α or IFN-γ derived from IFNAR −/− or IFNGR −/− mice. As shown in Fig. 2 A, MHC class I heavy chain and β2m mRNA levels were reduced significantly in both thymi and spleens of STAT1 −/− mice. Quantitation of the mRNA levels of MHC class I heavy and light chains after normalization with the values for actin showed ∼50–70% reduction compared with wild-type levels. The mRNA levels of MHC class I heavy chain and, to a lesser degree, light chain were also decreased in the thymi of IFNAR −/− and IFNGR −/− mice, showing that at least part of the regulation of basal class I expression requires an intact IFN signaling pathway. Similar reductions were also observed in splenocytes. To study the possible role of other transcription factors in reduced MHC class I expression, we examined the level of IRF-1 mRNA, which is a STAT1 target and has been shown to modulate the levels of both MHC class I heavy and light chain in response to IFN-γ 49 50 . IRF-1 mRNA levels were decreased substantially (30–50%) in spleens and thymi of STAT1 −/− mice and to a lesser extent in lymphocytes from IFNAR −/− and IFNGR −/− mice. These results suggest that IFNs, operating through STAT1 and possibly IRF-1, are required to maintain full basal mRNA levels of MHC class I heavy and light chains. Interestingly, class I heavy and light chain mRNA expression was much higher in spleen than thymus . Similarly increased cell surface class I protein levels were observed in peripheral relative to immature lymphocytes, suggesting a developmental upregulation of class I molecules during maturation. As antigen processing and presentation molecules are also critical for surface expression of the MHC class I complex, we measured the mRNA levels of these accessory molecules. As shown in Fig. 2 B, TAP1, LMP2, and LMP7 expression was reduced to ∼35% of wild-type levels in splenocytes and thymocytes from STAT1 −/− mice. Smaller reductions of TAP1, LMP2, and LMP7 mRNA levels were seen in thymus and spleen of IFNAR −/− and IFNGR −/− mice. These results showed that all of the MHC class I complex and related genes examined were regulated by STAT1 and that the regulation was at least partially due to constitutive IFN signaling. In all cases, loss of STAT1 produced a more severe reduction in MHC class I and accessory molecules than did loss of a single IFN receptor, suggesting that the phenotype of STAT1 −/− mice was due to either the additive effect of loss of both type I and type II IFN receptors or to an IFN-independent mechanism. A nuclear protein recently implicated in the control of MHC class I gene expression and antigen processing is the promyelocytic leukemia (PML) protooncogene 51 . PML mRNA expression was reduced approximately twofold in spleen cells from STAT1 −/− mice . Because this protein may act as a master regulator of MHC class I cell surface expression that functions downstream of STAT1, its reduction in STAT1 −/− cells may contribute to reduced MHC class I cell surface levels. Interestingly, like class I heavy and light chain genes, PML was also expressed at higher levels in spleen relative to thymus, indicating its potential involvement in the developmental switch in class I expression in lymphocytes. Reduced expression of MHC class I molecules observed in the absence of STAT1 could be due to either a cell-intrinsic role for STAT1 in lymphocytes, e.g., downstream of a cytokine receptor, or to a more global role elsewhere in the animal, e.g., regulating production of a secreted cytokine that ultimately targets lymphocytes. To determine if the reduction of MHC class I was a primary, cell-autonomous defect, we performed bone marrow transplantation (BMT). Bone marrow cells from either wild-type or STAT1 −/− mice were used to reconstitute either STAT1 +/+ or STAT −/− animals that had been lethally irradiated. To distinguish donor-derived lymphocytes from residual endogenous cells of the recipient mice, donor cells were infected with a retrovirus expressing GFP. The levels of MHC class I on mature peripheral blood lymphocytes were monitored 6 wk after BMT by staining with antibodies against MHC class I plus TCR for mature T cells or plus IgM for mature B cells and gating on GFP-positive lymphocytes. Four types of transplants were performed: STAT1 +/+ cells transferred into STAT1 +/+ or STAT1 −/− mice and STAT1 −/− cells transferred into STAT1 +/+ or STAT1 −/− mice. Wild-type and STAT1 −/− bone marrow was capable of reconstituting both mature T and B lymphocytes in mice of both genotypes . STAT1 −/− bone marrow was somewhat less efficient at reconstituting mature T lymphocytes than wild-type donors, although the cause of this deficiency is unclear. Nonetheless, on both subsets of lymphocytes, MHC class I expression levels reflected the genotype of the donor rather than that of the recipient. STAT1 +/+ cells transferred into STAT1 −/− hosts acquired an MHC class I phenotype similar to that of wild-type cells , whereas STAT1 −/− cells transferred to STAT1 +/+ hosts maintained their reduced levels of MHC class I . Autologous transplants also maintained the level of MHC class I appropriate for their genotype . These results demonstrated that basal expression of MHC class I on T and B lymphocytes is regulated by a cell-intrinsic process that depends on the STAT1 status of the cell itself, independent of the environment in which it matures. The greater reduction of class I heavy and light chain mRNA in STAT1 −/− cells relative to IFN receptor mutants suggested that STAT1 may be important even in the absence of IFN. To further examine the role of constitutive IFN signaling in MHC expression patterns on different subsets of lymphocytes, we compared MHC class I antigen levels on lymphocytes from mice devoid of IFN signaling by combined loss of receptors for both IFN-α and IFN-γ . As noted above, higher levels of MHC class I expression were detected on lymphocytes from peripheral organs than on thymocytes. Different subsets of T lymphocytes also displayed different levels of class I. CD4 + CD8 + DP thymocytes expressed very limited amounts of surface MHC class I compared with CD4 − CD8 − DN or CD4 − CD8 + and CD4 + CD8 − SP thymocytes ( Table ). In peripheral organs, the levels of MHC class I on both CD4 and CD8 T lymphocytes were further elevated compared with those of SP thymocytes. In spite of the differential expression of MHC class I on different lymphocyte populations, when the levels of MHC class I were compared between STAT1 −/− and wild-type animals, all subsets of lymphocytes, whether from thymus, spleen, or lymph nodes, showed reduced expression of MHC class I in the absence of STAT1 . Whereas DP and DN thymocytes and B lymphocytes from spleen and lymph nodes showed 40–50% reduction of MHC class I, SP thymocytes (CD4 − CD8 + and CD4 + CD8 − ) and peripheral T lymphocytes (both CD4 and CD8) showed a more pronounced reduction of MHC class I expression (only 16–26% of wild-type levels). MHC class I expression was also reduced on other cells types, including monocytes, macrophages, NK cells, and mouse embryonic fibroblasts (data not shown), suggesting that STAT1 is generally required for maintaining constitutive levels of MHC I expression. RNA expression studies suggested that class I levels regulated by STAT1 were at least partially independent of the presence of either IFN receptor. This observation left open the possibility that the more severe defect observed in the absence of STAT1 could be due to the combined loss of IFN-α and IFN-γ signaling. We therefore tested class I protein levels in the absence of both IFN-α and IFN-γ receptors using cells from (AR+GR) −/− mice. To exclude the possibility that any variation in MHC class I expression was due to differences in genetic background, wild-type, STAT1 −/− , IFNAR −/− , and (AR+GR) −/− mice were maintained on the 129 (H-2 b ) strain background. A similar reduced level of MHC class I was observed on B cells of both spleens and lymph nodes from (AR+GR) −/− and STAT1 −/− mice . Therefore, the STAT1 requirement in B cells is likely to be entirely downstream of IFN signaling. Surprisingly, however, SP thymocytes and CD4 and CD8 T lymphocytes from STAT1 −/− mice displayed a more pronounced reduction of MHC class I (∼50%) than those from (AR+GR) −/− mice. Therefore, STAT1 was required for the expression of MHC class I antigens on maturing and peripheral T lymphocytes even in the absence of any IFN signaling. The preceding results showed that endogenous IFNs were present and functional in normal animals but also indicated that STAT1-dependent gene expression could be independent of IFN signaling. These results prompted us to test the tyrosine phosphorylation and nuclear accumulation of STAT1, an indication of its activation by cytokines. Nuclear extracts prepared from freshly isolated splenocytes of wild-type, STAT1 −/− , and (AR+GR) −/− mice were subjected to immunoblot with antibody specific for tyrosine-phosphorylated STAT1. As shown in Fig. 5 A, top panel, phospho-STAT1α and phospho-STAT1β were indeed detected in nuclear extracts of wild-type resting splenocytes, presumably indicative of the action of IFN. Strikingly, a lower but still significant level of phospho-STAT1 was also detected in the (AR+GR) −/− splenocytes. Whereas the decreased activation of STAT1 in (AR+GR) −/− lymphocytes relative to wild-type cells was presumably due to the loss of IFN responsiveness, the residual phospho-STAT1 in (AR+GR) −/− lymphocytes must result from an IFN-independent mechanism. These results demonstrate that both IFN-dependent and -independent pathways contributed to the basal activation of STAT1 in lymphocytes. MHC class I expression was differentially regulated in T and B cells; specifically, STAT1-mediated expression in B cells was entirely IFN dependent, yet both IFN-dependent and -independent pathways were involved in STAT1-mediated expression in T cells . Therefore, we tested whether the regulation of basal activation of STAT1 also varied in these two subsets of lymphocytes. Nuclear extracts from isolated splenic T and B lymphocytes of wild-type and (AR+GR) −/− mice were analyzed for phospho-STAT1. As shown in Fig. 5 B, activated STAT1α and STAT1β were detected in both T and B lymphocytes of wild-type mice. In contrast to wild-type lymphocytes, phospho-STAT1 levels were undetectable in B cells from (AR+GR) −/− mice but could still be detected in T cells. Therefore, STAT1 activity in B cells was entirely dependent on IFN signaling, whereas both IFN-dependent and -independent pathways mediated STAT1 activation selectively in T cells. This dual mode of STAT1 activation was not limited to splenic T lymphocytes; basal activation of STAT1 was also present in (AR+GR) −/− thymocytes . This pattern of STAT1 activation mirrors the regulation we found for MHC class I gene expression. The previous results defined an IFN-independent activation of STAT1 in T but not B cells. We reasoned that factors involved in selective activation of STAT1 might be cytokines preferentially acting on thymic and splenic T cells but not on mature B cells. One such candidate is IL-7, whose receptor is expressed on both T and B lineage progenitors 52 53 54 but not on DP thymocytes or mature peripheral B lymphocytes 55 . STAT1 phosphorylation in freshly isolated splenocytes was blocked by the kinase inhibitor staurosporine (data not shown), suggestive of the action of a cytokine signaling pathway. Therefore, we first examined whether IL-7 could activate STAT1 in lymphocytes. STAT1 activation was significantly enhanced after either IL-7 or IFN-γ treatment of splenic T cells from wild-type mice . A comparable level of activated STAT1 was present in T cells of (AR+GR) −/− mice after IL-7 treatment but not after IFN-γ treatment, due to the absence of IFN-γ receptors. STAT1 was also activated in thymocytes of wild-type and (AR+GR) −/− mice in response to IL-7 (data not shown). In marked contrast to T cells, no activation of STAT1 was observed in IL-7–treated B lymphocytes, although IFN-γ was still capable of activating STAT1 in B cells of wild-type though not (AR+GR) −/− mice . To further confirm the selective response to IL-7 in T versus B cells, electrophoretic mobility gel shift (EMSA) was performed using nuclear extracts from purified lymphocyte populations. Whereas IL-7 treatment induced the formation of two complexes in T cells from wild-type mice, IFN-γ only activated a complex with mobility corresponding to the lower band of the IL-7–activated complexes . Antibodies to STAT1 and STAT5 (not shown) identified these complexes as STAT1 and STAT5 . However, whereas a STAT1 complex was activated by IFN-γ in B cells from wild-type mice, no complexes were activated in response to IL-7 in these cells . IL-2 and IL-4, other cytokines capable of activating STAT5, failed to activate STAT1 (data not shown). Therefore, IL-7 selectively activated STAT1 in T but not B cells and functioned independently from IFN, consistent with the pattern previously observed for MHC class I expression. As STAT1 is essential for MHC class I expression, the differential activation of STAT1 in T and B cells by IL-7 prompted us to examine IL-7–dependent modulation of class I expression. Splenocytes and thymocytes of wild-type, (AR+GR) −/− , and STAT1 −/− mice were treated with IL-7 or IFN-γ for 48 h, and induction of class I protein expression was followed by FACS™ analysis ( Table ). Whereas IFN-γ induced MHC class I expression on both T and B cells of wild-type mice, IL-7 selectively induced class I expression only on T cells ( Table , top). This IL-7–dependent induction of MHC class I was not secondary to IFN signaling, as a comparable induction of MHC class I expression occurred on T cells from (AR+GR) −/− mice. IL-7 also induced class I on DN and SP thymocytes from wild-type mice but not on DP thymocytes that lack IL-7Rα ( Table , bottom). In contrast, neither IL-7 nor IFN-γ caused significant class I induction on cells deficient in STAT1, similar to the lack of effect of IL-7 on receptor-negative B cells. Analogous results were obtained with IL-7R–positive pre-B cell lines derived from bone marrow; MHC class I was induced in response to IL-7 on wild-type but not STAT1 −/− cells (data not shown). Taken together, these results demonstrate that STAT1 is capable of mediating MHC class I induction in response to IL-7, and we conclude that STAT1 and IL-7 contribute to the basal level of MHC class I on mature T lymphocytes. We have demonstrated a role for constitutive IFN signaling in the absence of inflammation for homeostatic gene regulation in normal lymphocytes. This genetic and biochemical analysis has also revealed underlying differences in the regulation of MHC class I expression in T and B cells. Moreover, these data uncovered a novel IFN-independent but IL-7–dependent STAT1 activity that operates selectively in T lymphocytes. A model for these different modes of regulation is illustrated in Fig. 7 . In both T and B cells, constitutive IFN signaling activates STAT1 to maintain MHC class I gene expression either directly in combination with STAT2 and IRF-9 or possibly indirectly through its downstream target, IRF-1. In addition, both cell types have IFN-independent mechanisms for regulation of class I levels. In T cells, an IL-7–dependent but IFN-independent mechanism activates STAT1 phosphorylation, and this STAT1-dependent signaling is necessary for full class I expression. In B cells, the IFN-independent mechanism is also STAT1 independent, possibly acting through the B cell–specific protein CIITA that has been shown to modulate MHC class I gene expression 12 13 . The role of IL-7 has been mainly characterized during lymphoid development, where it is required to prevent apoptosis in lymphoid precursors 34 and to serve as a cofactor for V(D)J recombination in both T and B lymphocytes 53 54 . These actions of IL-7 depend on JAK1, 56 JAK3 57 , and STAT5, though the requirement for STAT1 signaling is less clear 58 59 . Here we provide evidence that IL-7 is also important in mature T cells for maintenance of constitutive expression of MHC class I through activated STAT1. However, the role of STAT1 activation in the upregulation of MHC class I in response to IL-7 is likely indirect, possibly through IRF-1, a downstream target of STAT1. STAT1 can be activated in response to cytokines and growth factors other than IFN, at least in cell culture, but initial evidence from in vivo studies largely failed to support these alternative activators, suggesting instead that STAT1 was dedicated to IFN signaling 21 36 60 . Recent evidence has suggested that fibroblast growth factor also functions through STAT1 in developing chondrocytes, at least in organ culture 28 . IL-7–mediated STAT1 activation demonstrates a novel role for STAT1 outside the scope of IFN. For B cells, no phospho-STAT1 accumulated in the absence of IFN signaling, and MHC class I levels were equivalent between STAT1 −/− and (AR+GR) −/− mice. A STAT1-independent mechanism was operative for maintaining basal expression , possibly through B cell–specific proteins such as CIITA, first characterized as regulators of MHC class II 12 13 . Indeed, overexpression of CIITA in human G3A cells, which are defective in CIITA, enhances the expression of MHC class I. It is likely that the action of CIITA compensates for the loss of IFN and STAT1 signaling in B cells derived from (AR+GR) −/− and STAT1 −/− mice, allowing them to retain modest expression of MHC class I. The unchanged patterns of surface MHC class II and equal levels of CIITA mRNA in all mouse strains tested (data not shown) suggest that CIITA-dependent pathways are intact in the absence of STAT1, although their responsiveness to IFN is lost. Interestingly, expression of MHC class I on peripheral macrophages from STAT1 −/− and (AR+GR) −/− mice were similar (data not shown). Macrophages also express CIITA and display high constitutive levels of MHC class II, consistent with a class I phenotype similar to that of B cells. CIITA is unable to perform this basal function in most tissues, as its expression outside the antigen presentation system is dependent on IFN induction. The important role of B lymphocytes and macrophages as APCs may explain why they have evolved independent mechanisms to maintain MHC expression. We have demonstrated that basal activation of STAT1 in resting lymphocytes relies on the action of at least two different cytokines, namely IFN and probably IL-7. Although both IFN and IL-7 are involved in activating STAT1 in T lymphocytes, this action is entirely IFN dependent in B lymphocytes. Similarly, STAT1 basal phosphorylation is maintained by both IFN and IL-7 in SP and DN thymocytes but not in DP thymocytes that lack IL-7Rα and display very low levels of MHC class I. One of the functions of the constitutively activated STAT1 is to maintain MHC class I expression, although it is also likely that other targets exist as well. Interestingly, it has recently been shown that constitutively phosphorylated STAT1 accumulates in lung epithelium of asthmatic patients in the absence of IFN 61 , suggesting that the tight regulation of STAT1 phosphorylation is important for normal physiology. Although it has been known that IFN is responsible for increased class I expression during immune responses, we provide evidence for the continuous presence of IFNs in vivo in the absence of overt infection in the maintenance of basal levels of expression. The presence of endogenous IFNs in vivo has been suggested from low levels of IFN mRNA detected in various human organs 62 , but the presence or significance of IFN protein in the absence of an inflammatory response has been less clear. Here we provide genetic and biochemical evidence for IFNs exerting a constitutive biological response. The source of basal IFN has not been determined, and we cannot exclude a role for subclinical infection or possibly normal intestinal flora. However, we suggest that basal IFN production, whether spontaneously produced or induced by common environmental stimuli, contributes to normal immune homeostasis. Consistent with this notion, gene-targeted loss of the inhibitor SOCS1 produced severe pathology 63 that appeared to be entirely due to unopposed IFN signaling 64a , again suggesting the presence of biologically active IFN in the absence of overt infection. Likewise, the transcriptional repressor IRF-2 appears to be essential to inhibit basal IFN responses, and ablation of IRF-2 leads to IFN-dependent pathology 64 . These findings suggest a novel function for IFN as a homeostatic cytokine in addition to its role as an inflammatory mediator. Constitutive IFN signaling is required for beneficial responses, such as maintenance of MHC class I expression and perhaps priming a state of readiness for combating viral infection 65 , whereas the less benign effects required only during inflammation are held in check by negative regulators, such as IRF-2 and SOCS1. It seems likely that other cytokines would have similar dual roles in both homeostatic and stress situations. The mechanisms of developmental control of MHC class I expression are still largely uncharacterized. We show that expression of MHC class I is downregulated during the transition from DN to DP thymocytes, followed by reexpression on SP thymocytes. It was further enhanced when SP thymocytes matured to peripheral CD4 and CD8 T lymphocytes. Relative changes of MHC class I levels from DN to DP and from DP to SP thymocytes or SP thymocytes to peripheral T lymphocytes were comparable in wild-type and IFN receptor and STAT1 mutant mice ( Table ), suggesting that neither IFN nor STAT1 is responsible for this developmental control. Interestingly, PML expression, a possible regulator of MHC class I downstream of STAT1 51 , was also higher in spleen than thymus; therefore, it may be one of the factors underlying developmental control. IRF-1 has also been shown to affect MHC class I expression, inducing mRNA levels for heavy chain, LMP2, and TAP1 66 67 . IRF-1 is a STAT1 target and was substantially reduced in STAT1 −/− mice and, therefore, is likely one of the downstream mediators of STAT1 in this process. However, IRF-1 is not essential for regulation of MHC class I expression, at least in response to IFN-α/β, as MHC class I expression was still induced in IRF-1 −/− cells, presumably through the action of the ISGF3 complex 24 . Nonetheless, STAT1 −/− and IRF-1 −/− mice do not display identical phenotypes. Despite the decreased expression of MHC class I on STAT1 −/− cells, we have not observed any abnormalities in CD8 development (data not shown), suggesting that the levels of MHC class I on thymic epithelium were still sufficient to rescue CD8 + SP cells during positive selection. In contrast, IRF-1 −/− mice show an increased ratio of CD4/CD8 cells due to an intrinsic requirement of IRF-1 for CD8 cell development 66 . One of the main functions of MHC class I is to facilitate CD8 T lymphocyte–mediated cell cytotoxicity during intracellular infection by viruses or bacteria. However, these pathogens have evolved many strategies to escape from immune responses by targeting the class I complex 68 . For example, cytomegalovirus and adenovirus are able to retain MHC class I molecules in the endoplasmic reticulum or Golgi complex through a retrieval signal in the cytoplasmic tails of their viral proteins 69 70 71 . HIV Nef protein modifies the endocytic machinery to downmodulate surface expression of MHC class I 72 . Recently, JAK–STAT signaling was also shown to be disrupted during viral infection, resulting in suppression of MHC induction by IFN-γ 73 74 . The multiple modes of regulation of class I genes in B cells and other APCs may provide a backup system for the host during viral infection. When IFN signaling is targeted by viruses, the professional APCs would be less sensitive to the loss of IFN responsiveness in MHC class I regulation by relying on STAT1-independent pathways. Indeed, STAT1 −/− mice displayed comparable virus clearance compared with wild-type mice after influenza virus infection 75 . In vitro CTL activity against autologous targets after infection with influenza virus was also indistinguishable in STAT1 −/− and wild-type mice (data not shown). | Study | biomedical | en | 0.999996 |
10562321 | Specific pathogen-free female Dunkin-Hartley guinea pigs (180–300 g; Hilltop Lab Animals) were used. All animals were shipped in filtered crates and kept in high-efficiency particulate-filtered air. Guinea pigs were fed a normal diet (Prolab; Agway) and were handled in accordance with the standards established by the United States Animal Welfare Acts set forth in National Institutes of Health guidelines and the “Policy and Procedures Manual” published by the Johns Hopkins University School of Hygiene and Public Health Animal Care and Use Committee. Animals were sensitized to OVA by intraperitoneal injection on days 1, 3, and 5 (see sensitization below). 3 wk later, animals were exposed to either parainfluenza virus or control medium via nasal instillation (see viral infection below). Some animals were treated with AbMBPs on the day of infection and 2 d later. Another group of animals was treated with antibody to IL-5 (AbIL5), 5 and 3 d before infection. All studies of airway function, inflammatory responses, and lung viral content were done 4 d after infection. Pathogen-free guinea pigs were sensitized to OVA. Animals were injected intraperitoneally with 10 mg·kg −1 OVA (0.3 ml) every other day for a total of three injections. Nonsensitized, pathogen-free guinea pigs were used as controls. Sensitization to OVA was confirmed 21 d later by intravenously injecting 1.0 ml of 2.5% OVA randomly into a few animals from each group. This dose caused acute, lethal anaphylaxis in all groups of sensitized guinea pigs, but had no effect in nonsensitized guinea pigs. Parainfluenza type 1 (Sendai virus, VR-105; American Type Culture Collection) was grown in rhesus monkey kidney cell monolayers in L-15 medium for 1 wk at 34°C. Cells and medium were frozen and thawed, cleared by low-speed centrifugation, and stored in aliquots at −70°C. Animals were anesthetized intramuscularly with ketamine (30 mg·kg −1 ) and xylazine (5 mg·kg −1 ), and were inoculated intranasally with 0.5 ml of virus stock diluted in Hank's PBS to produce a solution containing a 10 5 tissue culture ID 50 (TCID 50 ) ml −1 (10 5 times the concentration required to produce infection in 50% of rhesus monkey kidney monolayers). Infected and noninfected animals were housed in separate laminar flow rooms. Some animals from both the sensitized virus-infected and nonsensitized virus-infected groups were given 240 μl·kg −1 of AbIL5 intraperitoneally 3 and 5 d before infection. Some of the sensitized virus-infected guinea pigs were pretreated intraperitoneally with AbMBP 13 1.5 ml immediately before infection, and again 2 d after infection. Experiments were conducted 4 d after viral infection. The guinea pigs were anesthetized intraperitoneally with urethane (1.5 g·kg −1 ). This dose produces a deep anesthesia lasting 8–10 h, although none of these experiments lasted longer than 4 h. Both jugular veins were cannulated for the administration of drugs. One internal carotid artery was cannulated for measurement of blood pressure using a DTX™ pressure transducer (Viggo-Spectramed), and the heart rate was derived from the blood pressure tracing using a tachograph. The trachea was cannulated, and the animals were ventilated with a positive pressure, constant volume rodent respirator (Harvard Apparatus, Inc.) at a tidal volume of 10 ml·kg −1 and a respiratory rate of 100 breaths·min −1 . The animals were paralyzed by intravenously infusing succinylcholine (10 μg·kg −1 ·min −1 ). All animals were pretreated intraperitoneally with guanethidine (10 mg·kg −1 ) to deplete norepinephrine. Pulmonary inflation pressure ( P pi ) was measured at the trachea using a DTX™ pressure transducer. All signals were recorded on a polygraph (Grass Instrument Co.). Bronchoconstriction was measured as the increase in P pi above the basal inflation pressure produced by the ventilator. The sensitivity of the method was increased by taking the output P pi signal from the driver of one channel to the input of the preamplifier of a different channel on the polygraph. Thus, baseline P pi was recorded on one channel and increases in P pi above the baseline were recorded on a separate channel. With this method, increases in P pi as small as 2–3 mm H 2 O can be accurately recorded. Both vagus nerves were cut, and the distal ends were placed on shielded electrodes immersed in liquid paraffin. Electrical stimulation of both vagus nerves produced bronchoconstriction and bradycardia. The vagus nerves were stimulated at frequencies ranging 2.0–25.0 Hz for 5 s at 120-s intervals, keeping both pulse duration (0.1 ms) and voltage (10.0 V) constant between groups. At the end of each experiment, heparin (2,000 U) was given intravenously to both sensitized virus-infected and nonsensitized virus-infected animals. After 15–20 min, the response to vagal stimulation was remeasured as above. After the completion of each experiment, atropine (1 mg·kg −1 ) was administered intravenously to confirm that bronchoconstrictions were due to cholinergic nerve stimulation. Changes in P pi were recorded on a Grass polygraph as described above. 30 min after administering guanethidine, baseline responses to electrical stimulation of the vagus nerves were obtained. Both vagus nerves were simultaneously stimulated at 1-min intervals (2 Hz, 0.2 ms, 2.5–30 V, 44 pulses per train). The function of autoreceptors is frequency dependent. Stimulation of M 2 Rs by endogenous acetylcholine is greatest at high frequencies of stimulation. Therefore, the ability of exogenous agonists to inhibit vagally induced bronchoconstriction via stimulation of M 2 Rs is more readily apparent at low frequencies of stimulation 5 . Thus, the studies with the agonist pilocarpine were carried out at 2 Hz. The voltage was chosen at the beginning of each experiment (within a range of 2.5–30.0 V; mean 9.11 ± 0.92 V) to give an increase in P pi of ∼20 mmH 2 O (21.4 ± 0.75 mmH 2 O). Cumulative doses of pilocarpine (1–100 μg·kg −1 ) were administered intravenously, and the effect on vagally induced bronchoconstriction was measured. 30–100 μg·kg −1 of pilocarpine produced a small, transient bronchoconstriction. Therefore, the effect of these doses of pilocarpine on vagally induced bronchoconstriction was measured after the P pi had returned to baseline. At the end of each experiment, heparin (2,000 U) was given intravenously in both the sensitized virus-infected and nonsensitized virus-infected guinea pigs. After 15–20 min, the response to vagal stimulation was remeasured as above. After the completion of each experiment, atropine (1 mg·kg −1 ) was given intravenously to confirm that bronchoconstrictions were due to vagal stimulation. The results are expressed as a ratio of bronchoconstriction in the presence of pilocarpine to the bronchoconstriction in the absence of pilocarpine. Thus, a ratio <1 would indicate that pilocarpine was inhibiting vagally induced bronchoconstriction. The function of muscarinic receptors on airway smooth muscle was tested in vagotomized guinea pigs by measuring bronchoconstriction in response to increasing intravenous doses of acetylcholine (1–10 μg·kg −1 ). At the end of the experiment, bronchoalveolar lavage was performed in situ via the tracheal cannula. The lungs were lavaged with five aliquots of 10.0 ml PBS. The recovered lavage fluid (40–45 ml) was centrifuged (350 g for 7 min). The cells were resuspended in 10 ml of deionized water to remove any erythrocytes before an additional 40 ml of PBS was added. Cells were centrifuged again, the supernatant was poured off, and the cells were resuspended in 10 ml of PBS. Cells were counted using a Neubauer Hemocytometer (Hausser Scientific Co.). Aliquots of the cell suspension were cytospun onto glass slides, stained with Diff-Quik ® (Baxter Healthcare Corp.), and counted to obtain differential cell counts. Viral infection was confirmed in all guinea pigs that were exposed to parainfluenza virus by infection of rhesus monkey kidney cells with aliquots of lung homogenate from each animal . After physiological studies were completed, the guinea pig lungs were removed and stored at −70°C. Frozen samples were thawed, weighed, and homogenized in 2 ml PBS (Polytron™; Brinkmann). Virus was eluted from the tissue homogenate by incubating at 34°C for 1 h. The suspensions were centrifuged at 450 g for 30 min, and the supernatants were inoculated in serial 10-fold dilutions into fresh rhesus monkey kidney cell monolayers. After 1 wk of incubation at 34°C, the monolayers were washed and the medium replaced with a 0.5% suspension of guinea pig erythrocytes in Hank's PBS. After 1 h, the erythrocytes were washed off and the monolayers were examined under an inverted phase–contrast microscope (Olympus Corp.) for evidence of hemadsorption (sticking of erythrocytes to the surface of cells because of expression of viral hemagglutinin on these surfaces). Viral content was determined as the amount of lung homogenate required to produce infection in 50% of rhesus monkey kidney monolayers (the TCID 50 ), and is expressed as TCID 50 /g lung wet wt. Only data from virus-exposed guinea pigs with confirmed parainfluenza infection are reported. Acetylcholine, atropine, guanethidine, heparin, OVA, pilocarpine, succinylcholine, and urethane were purchased from Sigma Chemical Co. Purified rabbit AbMBP was produced as described previously 13 . Purified rat anti–mouse/human AbIL5 (TRFK-5) was purchased from PharMingen. All drugs were dissolved and diluted in 0.9% NaCl or PBS. All data are expressed as mean ± SEM. Acetylcholine, frequency, and pilocarpine responses were analyzed using two-way analyses of variance for repeated measures. Baseline heart rates, blood pressures, P pi , and changes in P pi (before pilocarpine administration), histological measurements, and bronchoalveolar lavage were analyzed using analysis of variance (Statview 4.5; Abacus Concepts, Inc.). A P value of 0.05 was considered significant. There were no significant differences among the groups for baseline heart rate ([beats per min] control, 261 ± 3.7; virus-infected, 255 ± 6.8; sensitized, 261 ± 5.7; sensitized virus-infected, 260 ± 4.2; virus-infected with AbIL5, 252.5 ± 11.0; sensitized virus-infected with AbIL5, 268 ± 6.5; and sensitized virus- infected with AbMBP, 270 ± 6.6), systolic blood pressure ([mmHg) control, 51.2 ± 1.7; virus-infected, 47.9 ± 2.3; sensitized, 51.3 ± 2.2; sensitized virus-infected, 49.0 ± 2.3; virus-infected with AbIL5, 42.5 ± 2.5; sensitized virus-infected with AbIL5, 45.6 ± 2.1; and sensitized virus-infected with AbMBP, 47.1 ± 1.5), diastolic blood pressure ([mmHg] control, 29.4 ± 1.5; virus-infected, 28.6 ± 2.1; sensitized, 30.8 ± 1.0; sensitized virus-infected, 29.0 ± 1.3; virus-infected with AbIL5, 28.8 ± 3.1; sensitized virus-infected with AbIL5, 27.8 ± 2.2; and sensitized virus-infected with AbMBP, 33.6 ± 1.8), or animal weight ([kg] control, 0.449 ± 0.02; virus-infected, 0.516 ± 0.03; sensitized, 0.476 ± 0.03; sensitized virus-infected, 0.434 ± 0.02; virus-infected with AbIL5, 0.424 ± 0.02; sensitized virus-infected with AbIL5, 0.461 ± 0.04; and sensitized virus-infected with AbMBP, 0.406 ± 0.02). A positive pressure of 70–220 mmH 2 O (mean 128 ± 4.43 mmH 2 O) was needed to ventilate the animals. Sensitization of pathogen-free guinea pigs did not alter baseline P pi (90.7 ± 4.1, n = 14) compared with nonsensitized controls (92.4 ± 3.6, n = 17). Regardless of sensitization, viral infection increased baseline P pi . The increased baseline was not prevented by pretreatment with AbIL5 (sensitized 143.3 ± 9.6, n = 9; nonsensitized 143.3 ± 3.3, n = 3), but was attenuated by treatment with AbMBP in the sensitized virus-infected guinea pigs . Simultaneous electrical stimulation of both cut vagus nerves at 2 Hz produced bronchoconstriction that was rapidly reversible upon cessation of stimulation. There were no significant differences among the groups for the voltages used (control, 5.8 ± 1.4; virus-infected, 6.8 ± 1.6; sensitized, 10.7 ± 3.4; sensitized virus-infected, 8.9 ± 1.6; virus-infected with AbIL5, 8.9 ± 2.0; sensitized virus-infected with AbIL5, 5.8 ± 1.4; and sensitized virus-infected with AbMBP, 11.6 ± 1.7) or the degree of bronchoconstriction produced ([mmH 2 O] control, 20.9 ± 0.5; virus-infected, 19.8 ± 1.1; sensitized, 25.0 ± 2.0; sensitized virus-infected, 20.6 ± 1.4; virus-infected with AbIL5, 19.2 ± 2.9; sensitized virus-infected with AbIL5, 23.2 ± 2.0; and sensitized virus-infected with AbMBP, 19.2 ± 2.6). Pilocarpine inhibited vagally induced bronchoconstriction in a dose-dependent manner in pathogen-free guinea pigs, demonstrating that there are functional M 2 Rs on the parasympathetic nerves . Sensitization to OVA did not affect the ability of pilocarpine to inhibit vagally induced bronchoconstriction. In virus-infected animals, irrespective of sensitization, pilocarpine no longer inhibited vagally induced bronchoconstriction . This demonstrates that the neuronal M 2 Rs were dysfunctional in virus-infected guinea pigs. Pretreatment with an AbIL5 did not prevent virus-induced M 2 R dysfunction in nonsensitized guinea pigs . In contrast, pretreatment with AbIL5 did prevent virus-induced M 2 R dysfunction in virus-infected guinea pigs sensitized to OVA . Likewise, pretreatment of sensitized guinea pigs with AbMBP before viral infection also protected the function of the neuronal M 2 Rs . Heparin was administered after the maximal dose of pilocarpine had been given. Heparin did not inhibit vagally induced bronchoconstriction in the nonsensitized virus-infected guinea pigs . However, in sensitized virus-infected guinea pigs, heparin did significantly inhibit vagally induced bronchoconstriction. Simultaneous electrical stimulation of both cut vagus nerves (1–25 Hz) caused frequency-dependent bronchoconstriction. There was no difference between vagally induced bronchoconstriction in the uninfected sensitized and nonsensitized guinea pigs . In contrast, after viral infection, vagally induced bronchoconstriction in both sensitized and nonsensitized animals was significantly greater than in their respective controls. This difference was not apparent at lower frequencies of stimulation, but became greater with increasing frequencies. Vagally induced bronchoconstriction was not significantly different between the sensitized and nonsensitized virus-infected groups. Pretreatment with AbIL5 did not prevent virus-induced vagal hyperreactivity in the nonsensitized guinea pigs . In contrast, pretreatment with AbIL5 did prevent virus-induced vagal hyperreactivity in guinea pigs sensitized to OVA . Pretreatment of the sensitized guinea pigs with AbMBP before viral infection prevented virus-induced vagal hyperreactivity in the sensitized guinea pigs . Likewise, administration of heparin 30 min before testing vagal responses reversed virus-induced hyperreactivity to stimulation of the vagus nerves in sensitized animals . In vagotomized guinea pigs, intravenous acetylcholine (2–10 μg·kg −1 ) caused dose-dependent bronchoconstriction. Acetylcholine-induced bronchoconstriction was not affected by sensitization, viral infection , or by any of the treatments (data not shown). The total number of cells recovered by lung lavage at the end of each experiment was increased by viral infection regardless of whether the animals were sensitized . This increase was comprised of macrophages and neutrophils. Lymphocyte numbers were not significantly affected by either sensitization or viral infection. Sensitization significantly increased the number of eosinophils recovered in the lavage fluid . This increase with sensitization was not further potentiated by viral infection (compare gray with hatched bars). Viral infection did not increase the number of eosinophils recovered in the lavage fluid in nonsensitized guinea pigs (compare white with black bars). Treatment with AbIL5 not did not change leukocyte numbers in the lavage fluid of nonsensitized virus-infected guinea pigs In contrast, the total number of inflammatory cells in the sensitized virus-infected animals was decreased by AbIL5 . This decrease was due to a significant decrease in eosinophils. Pretreatment with AbMBP caused a small but statistically significant decrease in the number of eosinophils recovered from the airways . Neither AbIL5 nor AbMBP significantly altered the numbers of lymphocytes recovered in the lavage fluid. Infectious virus was quantified in all guinea pigs that were exposed to parainfluenza virus . Viral content of the lungs was significantly decreased in sensitized guinea pigs compared with nonsensitized virus-infected guinea pigs. Pretreatment with AbIL5 did not alter viral content of nonsensitized virus-infected guinea pigs. In contrast, there was a significant increase in the viral content of AbIL5-treated, sensitized virus-infected animals. Sensitized virus-infected animals treated with AbMBP did not have any significant change in the viral content. It is important to note that these results were collected from all of the infected animals, but the actual titer measurements were performed on separate days over a 4-mo period. The experiments described in this paper were designed to test whether the mechanism of M 2 R dysfunction and associated airway hyperreactivity in virus-infected guinea pigs differs between sensitized and nonsensitized animals. In control guinea pigs, the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction in a dose-dependent manner, confirming that neuronal M 2 Rs were functioning to inhibit release of acetylcholine 5 . Similarly, pilocarpine also inhibited vagally induced bronchoconstriction in the guinea pigs sensitized to OVA, confirming that sensitization alone does not inhibit neuronal M 2 R function 14 . Likewise, sensitization does not induce vagal hyperreactivity, since vagally induced bronchoconstriction was also similar between sensitized and nonsensitized guinea pigs. In contrast, viral infection of both the sensitized and nonsensitized guinea pigs caused loss of M 2 R function, since pilocarpine no longer inhibited vagally induced bronchoconstriction . Viral infection also induced hyperreactivity in both the sensitized and nonsensitized guinea pigs, since vagal nerve stimulation was potentiated in both compared with their respective noninfected controls . The degree of vagal hyperreactivity was similar in the sensitized and nonsensitized virus-infected animals. This virus-induced hyperreactivity was mediated by the parasympathetic nerves and not by a change in the responsiveness of airway smooth muscle, since bronchoconstriction induced by intravenous acetylcholine was not different among the groups . Thus, viral infection causes loss of M 2 R function and vagal hyperreactivity, regardless of sensitization status. In antigen-challenged guinea pigs, hyperreactivity to electrical stimulation of the vagus nerves as well as to intravenous histamine is mediated entirely by loss of neuronal M 2 R function 15 . We have demonstrated that bronchoconstriction in response to stimulation of the vagus nerves is potentiated by viral infection in both sensitized and nonsensitized guinea pigs. Since the responsiveness of airway smooth muscle is not changed by viral infection or by sensitization, virus-induced hyperreactivity to vagal nerve stimulation results from increased release of acetylcholine due to loss of neuronal M 2 R function. Eosinophils, through release of MBP, mediate loss of M 2 R function in antigen-challenged guinea pigs 10 . In contrast, we have shown that virus-induced M 2 R dysfunction and vagal hyperreactivity in nonsensitized guinea pigs are not mediated by eosinophils. Depletion of eosinophils with AbIL5 did not prevent virus-induced M 2 R dysfunction or vagal hyperreactivity . Furthermore, heparin, which binds and neutralizes eosinophil MBP, did not reverse virus-induced M 2 R dysfunction or vagal hyperreactivity . Thus, virus-induced loss of M 2 R function and hyperreactivity in nonsensitized guinea pigs is not mediated by eosinophils. In contrast, virus-induced loss of M 2 R dysfunction and vagal hyperreactivity in sensitized guinea pigs is mediated by eosinophils, and specifically via eosinophil MBP. Depletion of eosinophils in sensitized, virus-infected guinea pigs prevented M 2 R dysfunction, and also prevented vagal hyperreactivity . Removing positively charged proteins with heparin both acutely restored M 2 R function and reversed vagal hyperreactivity in the sensitized virus-infected guinea pigs . The role of MBP was confirmed, since treatment with AbMBP prevented both M 2 R dysfunction and vagal hyperreactivity in the sensitized virus-infected guinea pigs . Thus, by sensitizing guinea pigs before viral infection, the mechanism of virus-induced M 2 R dysfunction and vagal hyperreactivity was switched to be clearly dependent on eosinophils and eosinophil MBP. Traditionally, eosinophils are not prominent in the response of nonasthmatics to viruses. Airway viral infections cause a neutrophil and mononuclear cell influx into the airways 16 . Typically, viral clearance has been attributed to T cell release of IFN-γ and TNF-α, and to direct killing by CD8 + cells and NK cells 17 . A CD4 + T lymphocyte response may also contribute to viral clearance, and here again it is production of INF-γ by T cells that appears to be beneficial 18 . Under some circumstances, viral infections do cause airway eosinophilia. Children who wheeze with respiratory syncytial virus (RSV) infection have both increased eosinophils and eosinophil cationic protein (ECP) in their airways 19 . With viral infections other than RSV, eosinophilia is seen in atopic individuals. During naturally acquired viral infection, children with asthma have large increases in eosinophil MBP regulated on activation, normal T cell expressed and secreted (RANTES), and macrophage-inhibitory protein 1α in their nasal secretions 20 . After intranasal infection with rhinovirus, biopsies of the lower airways of individuals with asthma contain increased eosinophils that persist even into convalescence 21 . In patients with asthma, the presence of eosinophils and MBP in their airways during periods of exacerbations has been well established 22 . However, the pathogenesis and role of this eosinophilia in hyperresponsiveness have been unclear, considering that many of these exacerbations are triggered by viral infection 1 23 . In mice, viral infection can induce airway eosinophilia by production of IL-5 by T lymphocytes 24 25 . It has been shown that, although under normal circumstances CD8 + T lymphocytes produce IFN-γ in response to viral infections, in an allergic milieu these CD8 + cells respond to viral infection by producing IL-5 12 . Studies by Coyle et al. 12 used a transgenic mouse model in which the CD8 + T lymphocytes expressed the receptor for a particular viral glycoprotein. When these mice inhaled this glycoprotein, there was increased IFN-γ and an influx of neutrophils and mononuclear cells into the lung lavage. However, if the mice were first sensitized to a nonviral antigen, OVA, the response to inhaled viral glycoprotein was production of IL-5 and an influx of eosinophils. These investigators demonstrated that exposure of the transgenic CD8 + T lymphocytes to IL-4 changed their in vitro response to the viral glycoprotein from IFN-γ to IL-5. Thus, it was postulated that in an “atopic” animal (i.e., one that had been sensitized), the production of IL-4 conditions the CD8 + T lymphocytes so that their response to viral infection is to produce IL-5 and to promote pulmonary eosinophilia. In vivo and in vitro experiments have investigated the specific interactions between eosinophils and viruses. Viral infection of epithelial cells causes release of the chemokines for eosinophil migration such as RANTES, monocyte chemotactic protein 1, and macrophage inhibitory protein 1α 26 27 . Eosinophils also respond directly to RSV infection 28 . After infection with rhinovirus, eosinophils have been shown to activate T cells by acting as APCs 29 . This demonstrates that eosinophils are being recruited and activated by viral infections. The possible mechanism of virus-induced eosinophil activation and granulation has been studied by Olszewska-Pazdrak et al. 30 . Incubation of eosinophils with RSV-infected epithelial cells increases expression of the adhesion molecule CD18 on the eosinophils. Upregulation of Mac-1 (CD11b/CD18) is critical to eosinophil activation 31 , allowing the eosinophils to interact with infected respiratory epithelium and to release ECP. The increased expression of CD18 also allows virus-specific T cells to bind and activate eosinophils. An unexpected finding was an IL-5– and eosinophil-dependent antiviral effect in sensitized animals. Sensitization before viral infection decreased the viral content of the lungs by >80% . AbIL5 pretreatment reversed the effect of sensitization, increasing viral titers in the lungs of sensitized virus-infected guinea pigs. In contrast, pretreatment with AbIL5 in the nonsensitized virus-infected guinea pigs had no effect on viral titers. This suggests that in addition to changing the mechanism of M 2 R dysfunction, sensitization before viral infection has also changed the guinea pig's ability to clear virus. The increased eosinophils in sensitized virus-infected animals were associated with decreased viral titer, whereas depleting eosinophils with AbIL5 was associated with increased viral titers. Although treatment with AbMBP did cause a small but significant decrease in eosinophil number, this decrease was much less than the decrease associated with AbIL5 treatment . Treatment with AbMBP did not alter the viral content in the lungs of sensitized virus-infected guinea pigs. Therefore, the mechanism of the eosinophil's apparent ability to enhance viral clearance does not involve eosinophil MBP, as AbMBP treatment did not interfere with the ability of sensitization to lower viral titers. Although the activation of eosinophils by viral infection has been shown, the possible direct antiviral role of eosinophils is less clear. ECP and eosinophil-derived neurotoxin are both RNases 32 that can inhibit replication of RNA viruses such as parainfluenza 33 . Domachowske et al. have demonstrated, in vitro, that eosinophils can directly inhibit both RSV and parainfluenza virus infectivity 34 . They found that the mechanism of eosinophil antiviral activity is through the RNase activity of eosinophil-derived neurotoxin and ECP 35 . Another possible antiviral mechanism for eosinophils may be through release of nitric oxide (NO), which is an effective inhibitor of viral replication 36 . While the virucidal effects of eosinophils through production of NO have not been investigated, eosinophils do use NO production to kill parasites 37 . NO production by eosinophils is increased in sensitized guinea pigs 38 . Exhaled NO levels are increased in patients with asthma 39 , and specifically during virus-induced asthma attacks 40 . In addition to the possible antiviral effects of eosinophils, other effectors in the Th2 pathway may also play a role in viral clearance. In mice, IL-5 plays an important role in mucosal immunity through the induction of plasma cell release of IgA 41 . IL-5 may also play a role in systemic immunity through B cell growth and differentiation 42 and induction of receptors for IL-2 43 , although studies in human B cells have yielded conflicting results 44 45 . IL-5 may also act to stimulate differentiation of cytotoxic T lymphocytes 46 . Although viral clearance is primarily dependent on cytotoxic T cell responses, antibody responses can also affect viral clearance 47 . Therefore, it is possible that the effect of AbIL5 on the viral titers recovered in our sensitized virus-infected guinea pigs could also have occurred through disruption of a noneosinophil inflammatory pathway. We have demonstrated that virus-induced loss of M 2 R function in nonsensitized virus-infected guinea pigs is not mediated by eosinophils. In contrast, by sensitizing guinea pigs to a nonviral protein before viral infection, we have switched the response to subsequent viral infection to depend on eosinophils. We have demonstrated that in sensitized animals, virus-induced hyperreactivity results from activation of eosinophils, release of MBP, and inhibition of neuronal M 2 R function. M 2 Rs are present in humans 48 , and loss of M 2 R function is associated with airway hyperreactivity in patients with asthma 49 50 . Although the neuronal M 2 Rs are functional in humans with stable asthma, their function is lost during naturally acquired viral infection 51 . Asthma attacks are often precipitated by viral infection and are characterized by an eosinophilic airway inflammatory response, yet viral infection is not traditionally associated with eosinophilia. We have shown that sensitizing guinea pigs to a nonviral protein before viral infection switches the inflammatory response to viral infection to include eosinophils. Many asthmatics are atopic and have increased eosinophils in their airways even during periods of quiescence 52 . Our data suggest that eosinophils play an important role in the responses of patients with atopic asthma to viral infection. This altered mechanism may be important in understanding the role viruses play in triggering asthma exacerbations. In addition, under these circumstances, eosinophils may play an additional role in viral clearance. | Study | biomedical | en | 0.999998 |
10562322 | C57BL/6J × 129/SvEv-Gpi1c RAG2 −/− mice (derived from the ES cell line EK.CCE 129/SvEv-Gpi1c; reference 29 ) were provided by Drs. H. Bluethmann and E. Wagner (Basel Institute for Immunology). TNF −/− C57BL/6J × 129/SvEv mice were generated using the ES cell line GS1 as previously described 28 . C57BL/6J × 129SvJ mice were obtained from Dr. H. Bluethmann and the Biotechnology and Animal Breeding Division of RCC (Füllinsdorf, Switzerland). TNF −/− mice were backcrossed with (C57BL/6J × 129/SvEv-Gpi1c) RAG2 −/− mice in our animal facility. PCR procedures were used to determine RAG2 and TNF genotypes. Three different primers were used to characterize the RAG2 genotype: Rag1, 5′-GGGAGGACACTCACTTGCCAGTA-3′; Rag2, 5′-AGTCAGGAGTCTCCATCTCACTGA-3′; and Ragneo, 5′-CGGCCGGAGAACCTGCGTG-CAA-3′, to yield a 350-bp fragment for the mutated and a 263-bp fragment for the wild-type allele. The TNF genotype was defined using the TNF10 (5′-CCTCAGCAAACCACCAAGTGGA-3′) and TNF12 (5′-TTGGGCAGATTGACCTCAGCG-3′) primers, where a 350-bp fragment corresponds to wild-type allele, and no band is detected for the mutant allele. As the RAG2 −/− , TNF −/− , and 129 × B6 mice were obtained from different sources and the number of backcrosses to C57BL/6J mice was not documented for the two gene-deficient mouse lines, several control experiments were performed before using these mouse lines in the CD4 T cell transfer model of colitis to exclude potential graft-versus-host reactions. These controls included mixed lymphocyte reactions using splenocytes from donor and recipients as responders and, upon irradiation (2,000 rads), as stimulators, as well as the careful histopathological examination of small intestine, skin, and liver from all experimental animals. Both the mixed lymphocyte reactions and the histopathological analyses revealed no indications of minor histoincompatibilities or graft-versus-host reactions in all of the different donor–recipient combinations used (data not shown). TNF-α −/− mice (C57BL/6) were obtained from Dr. M. Marino (Ludwig Institute for Cancer Research, New York, NY) for subsequent backcrossing to C57BL/6J RAG2 −/− mice (>10 backcross generations; obtained from E. Wagner, Basel Institute for Immunology). The TNF-α genotype was defined using three different primers (TNF-α1, 5′-AGATAGCAAATCGGCTGACGG-3′; TNF-α2, 5′-ATCAGTTCTATGGCCCAGACC-3′; and TNF-αneo, 5′-CCTTCTATCGCCTTCTTGACG-3′), where TNF-α1/TNF-αneo yielded a band of 1,200 bp for the mutant allele, and the TNF-α1/TNF-α2 primers yielded a band of 750 bp for the wild-type allele. Mice were kept at specific pathogen–free conditions in the central animal facility of the Medical School, University of Bern, Bern, Switzerland. PE-conjugated anti-CD4 (clone GK1.5), FITC-conjugated anti-CD45RB (clone 16A), biotin-conjugated anti–TNF-α pAb, biotin-conjugated anti–IL-4 mAb (clone BVD6-24G2), anti–TNF-α mAb (clone MP6-XT22), and anti–IL-4 mAb (BVD4-1D11) were purchased from PharMingen. Protein G–purified antibodies from the hybridoma supernatants anti-B220 (clone RA3-6B2), anti–macrophage-specific mAb (clone F4/80), anti-CD8α (clone 53-6.7), anti–IFN-γ mAb , anti–Mac-1 mAb (clone M1/70), and anti–IFN-γ mAb (clone OIE703B2) were biotinylated or FITC conjugated according to standard protocols. Lamina propria lymphocytes (LPLs) from small and large bowel were isolated as previously described 30 . In brief, small pieces of appropriate intestinal tissues were incubated for 20 min at 37°C in Ca 2+ - and Mg 2+ -free HBSS containing 2% horse serum (GIBCO-BRL Life Technologies), 1 mM dithiothreitol, and 0.5 mM EDTA. After washing pieces of intestine with HBSS containing 5% horse serum, the tissues were incubated with 100 U/ml collagenase (Sigma Chemical Co.) and 10 mg/ml DNase (Boehringer Mannheim) twice for 60 min each at 37°C. Cells were passed through 70- and then 40-μm nylon cell strainers and further fractionated on a 40–70% Percoll gradient (Pharmacia Biotech; 15 min, 800 g , room temperature). For the sorting of macrophages and CD4 cells, isolated cells from small and large bowel were stained with PE-conjugated anti-CD4 mAb, FITC-conjugated anti-F4/80, and FITC-conjugated anti-Mac-1 mAb for subsequent sorting on a FACS Vantage™ (Becton Dickinson). After osmotic lysis of erythrocytes, splenocytes were incubated with 0.5–1 μg biotinylated anti-CD8α and anti-B220 mAb per 10 6 cells for 15 min on ice. Avidin magnetic beads (Miltenyi Biotec) were used as second step reagents, and the CD8α + and B220 + splenocytes attached to the beads were removed by magnetic separation. The negative fraction, enriched for CD4 T cells, was stained using FITC-conjugated anti-CD45RB mAb and PE-conjugated anti-CD4 mAb. Subsequently, CD4 T cells were sorted according to the expression of CD45RB on a FACS Vantage™. CD4 T cells were divided into three different subpopulations (low, intermediate, and high) according to expression level of CD45RB. The CD4 cell subset with lowest expression of CD45RB was collected as CD45RB lo , and the subset with highest expression of CD45RB was termed CD45RB hi . CD4 + T cells with intermediate cell surface expression of CD45RB were discarded. Sorted CD4 + CD45RB hi and CD4 + CD45RB lo T cells were washed and resuspended at 10 6 cells/ml in sterile PBS. 2 × 10 5 cells were injected intraperitoneally into each of 8–12-wk-old recipient mice. Upon transfer, body weight of recipient mice was measured every other day. Mice were killed and analyzed either on day 10 or between 3 and 4 wk after adoptive transfer when the mice showed a weight loss between 20 and 30% of their initial weight or 6 wk after transfer in experimental groups where no sign of colitis was observed. Intestinal tissue sections from large and small bowel were fixed in 4% paraformaldehyde (in 1× PBS) for subsequent paraffin embedding or frozen in O.C.T. compound (Bayer AG) for preparation of cryostat sections. Paraffin-embedded sections were cut and stained with hematoxylin and eosin. To assess the histopathological alterations in the colon present after adoptive transfer of CD4 T cell subpopulations from different donors, a scoring system was established using the following parameters: (a) cellular infiltration in the lamina propria of the large bowel (score from 0 to 3); (b) mucin depletion (score from 0 to 2); (c) crypt abscesses (score from 0 to 2); (d) epithelial erosion (score from 0 to 2); (e) hyperemia (score from 0 to 3); and (f) thickness of colonic mucosa (score from 1 to 3). Hence, the range of histopathological scores was from 1 (no alteration) to 15 (most severe signs of colitis). A 1,108-bp cDNA fragment of the murine TNF-α gene (position 1–1,108; provided by Genentech Inc.) and a 900-bp EcoRI–HindIII cDNA fragment of the IFN-γ gene (provided by Dr. K. Arai, DNAX, Inc., Palo Alto, CA) were subcloned into pGEM-2 (Promega Corp.). After linearization of both plasmids, sense and antisense RNA probes were prepared using the appropriate RNA polymerase as previously described 31 . Serial frozen sections of intestinal tissue were hybridized in situ with an antisense RNA probe specific for the TNF-α and IFN-γ genes. In situ hybridizations were performed as previously described 31 . In brief, cryostat sections were fixed in 4% paraformaldehyde in PBS for 20 min, washed in PBS, and incubated in the presence of 1 μg/ml proteinase K (Boehringer Mannheim) at 37°C for 30 min. After postfixation and acetylation, the hybridization was performed with 2 × 10 5 cpm of 35 S-labeled RNA probe per microliter of hybridization solution for 18 h at 48°C. After digestion of nonhybridized single-stranded RNA and washing, the slides were dipped in NTB2 nuclear track emulsion (Eastman Kodak Co.). The slides were exposed for 3 wk in the dark at 4°C, developed, and counterstained with nuclear fast red (Sigma Chemical Co.) by standard techniques. To determine the number of TNF-α–, IL-4–, and IFN-γ–producing cells in the LPLs of large versus small intestine, an enzyme-linked immunospot (ELISPOT) assay was performed as previously described 32 . In brief, nitrocellulose-backed microtiter plates (96-well; Millipore Corp.) were coated at 4°C overnight with anti–TNF-α, anti–IFN-γ, and anti–IL-4 mAbs and subsequently blocked with TBS containing 5% BSA. After washing the plate with TBS containing 0.025% Tween-20, freshly isolated LPLs or sorted CD4 T cells and macrophages were incubated in IMDM containing 10% FCS in a CO 2 incubator at 37°C overnight. The plates were extensively rinsed with TBS plus 0.025% Tween-20 and incubated for 2 h at 37°C with biotinylated anti–TNF-α pAb, anti–IFN-γ mAb, and anti–IL-4 mAb, respectively. The plate was subsequently rinsed and incubated for 2 h at 37°C with avidin–alkaline phosphatase (Sigma Chemical Co.), diluted 1:1000. 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NTB) solution (Kirkegaard and Perry Labs., Inc.) was used as a substrate for the alkaline phosphatase. Positive spots were counted under a dissecting microscope. When 2 × 10 5 CD4 + CD45RB hi T cells from B6 × 129 donor animals (TNF +/+ ) were transferred into sex-matched, 8–10-wk-old syngeneic RAG2 −/− mice, the recipients rapidly started to lose weight 12–14 d after adoptive T cell transfer. This dramatic wasting disease led to an average weight loss of 25–30% 24 d after transfer and is associated with clinical signs of severe colitis, in particular, persistent diarrhea and occasionally also bloody stool and anal prolapses. RAG2 −/− recipients of 2 × 10 5 CD4 + CD45RB lo T cells did not, however, show weight loss, and clinical signs of colitis were completely absent throughout the entire observation period . To assess the contribution of TNF-α and the structurally related LT-α (TNF-β) to the course of disease in this mouse model of colitis, B6 × 129 RAG2 −/− mice were first backcrossed to TNF-deficient B6 × 129 mice (TNF −/− ) to obtain TNF −/− RAG2 −/− mice. The consequences of an adoptive transfer of CD4 + CD45RB hi T cells from TNF −/− donors into TNF −/− RAG2 −/− mice on the body weights of the recipients is depicted in Fig. 1 B. Throughout the entire observation period of 42 d, no sign of weight loss was observed. Clinical signs of colitis, such as persistent diarrhea, or more severe signs, such as anal prolapse or bloody stool, were absent in all animals throughout the entire observation period. No difference in the kinetics of body weight or clinical signs of colitis were apparent when TNF −/− RAG2 −/− recipients of TNF −/− CD4 + CD45RB hi and CD4 + CD45RB lo T cells were compared . When recipients of CD4 + CD45RB hi T cells had lost between 20 and 30% of their initial body weight or at the end of the observation period on day 42 after transfer, all mice were killed and part of each colon was paraffin embedded for subsequent detailed histopathological analyses. To quantitate the histological alterations present in the large intestines of recipient mice, a semiquantitative colitis score system ranging from 1 (no histopathological alterations) to 15 (most severe histopathological alterations) was established (see Materials and Methods). The results of these microscopical analyses of colonic tissue sections are summarized in Fig. 2 A. TNF +/+ RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells revealed massive alterations of the colon, with colitis scores between 12 and 15 (mean 13.2) for individual animals. Control TNF +/+ RAG2 −/− mice reconstituted with TNF +/+ CD4 + CD45RB lo splenocytes, however, showed only minimal signs of histopathological alterations (mean 4.5). Histological changes of the colon in TNF −/− RAG2 −/− mice after adoptive transfer of TNF −/− CD4 + CD45RB hi T cells were minimal to moderate (mean 5.8) and comparable to the colitis scores observed in TNF −/− RAG2 −/− recipients of the corresponding TNF −/− CD4 + CD45RB lo T cells . Representative examples of cross-sections of the colon from a TNF +/+ RAG2 −/− recipient of colitis-inducing TNF +/+ CD4 + CD45RB hi T cells (colitis score 14) and from a TNF −/− RAG2 −/− recipient of TNF −/− CD4 + CD45RB hi T cells (colitis score 5) are shown in Fig. 2B and Fig. C , respectively. Extensive inflammatory cell infiltrates, glandular elongation, loss of goblet cells, epithelial erosion, and crypt abscesses were observed in colonic tissue sections of mice affected with wasting disease . In contrast, minimal colonic pathology was observed in tissue sections from TNF −/− CD4 + CD45RB hi T cells→TNF −/− RAG2 −/− mice. Limited cellular infiltration, together with minimal glandular elongation and minimal loss of goblet cells, was present in the lamina propria and submucosa, whereas epithelial erosion and crypt abscesses were completely absent in these mice . These experiments clearly demonstrate that TNF is required for the induction of clinical and histopathological signs of severe colitis and epithelial erosion of the intestinal wall. Thus, we attempted to determine if TNF production by the transferred colitogenic CD4 + CD45RB hi T cells is required for the development of colitis or whether local production of TNF by resident non-T cells of the recipients is sufficient for disease induction and perpetuation. To this end, two reciprocal donor–recipient combinations were analyzed: transfer of TNF +/+ CD4 T cell subsets into TNF −/− RAG2 −/− recipients and transfer of TNF −/− CD4 T cell subsets into TNF +/+ RAG2 −/− recipients. The consequences of adoptive transfer of TNF −/− CD4 + CD45RB hi T cells into TNF +/+ RAG2 −/− mice on body weights are illustrated in Fig. 3 A. Similar to when TNF +/+ CD4 + CD45RB hi T cells were transferred into TNF +/+ RAG2 −/− mice, recipients started to lose weight 12–14 d after adoptive T cell transfer. This weight loss paralleled with clinical signs of colitis such as persistent diarrhea, anal prolapse, and bloody stool. TNF −/− RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells, however, did not show any clinical signs of colitis during the entire observation period . The results of the semiquantitative histopathological analysis of the colon in these two different donor–recipient combinations are shown in Fig. 4 A. Massive alterations of the colon with colitis scores ranging from 10 to 14 for individual mice (mean colitis score 11.7) were observed in tissue sections from TNF +/+ RAG2 −/− recipients of TNF −/− CD4 + CD45RB hi T cells. A representative cross-section of an affected colon from this donor–recipient combination is shown in Fig. 4 B (colitis score 12). Hence, CD4 + CD45RB hi T cells from TNF −/− donors induce histological alterations of the colonic mucosa comparable to those of CD4 + CD45RB hi T cells from TNF +/+ donors upon transfer into TNF +/+ RAG2 −/− mice . In contrast, only minimal colonic pathologies were observed in TNF −/− RAG2 −/− mice reconstituted with TNF +/+ CD4 + CD45RB hi splenocytes , demonstrating that TNF produced exclusively by transferred CD4 T cells is not sufficient to induce wasting disease. The absence of a wasting disease is associated with only minimal to moderate histopathological signs of colitis, with scores ranging from 2 to 8 for individual animals of this group (mean colitis score 4.7). Control animals of both groups reconstituted with the corresponding CD4 + CD45RB lo T cells showed only mild to moderate histopathological alterations of the colonic mucosa . Whereas protection from onset of severe colitis in TNF −/− RAG2 −/− mice reconstituted with TNF −/− CD4 + CD45RB hi T cells can be attributed to the complete absence of TNF production in both transferred T cells and recipient cells, the absence of clinical signs of colitis and severe histopathological alterations in the colons of TNF −/− RAG2 −/− mice reconstituted with potentially TNF-producing TNF +/+ CD4 + CD45RB hi T cells is less obvious. Thus, we defined the frequency of TNF-α–producing cells in the colon in the four different donor–recipient combinations to assess whether protection from colitis in the TNF +/+ CD4 + CD45RB hi T cells into the TNF −/− RAG2 −/− group is associated with low production of TNF-α by donor-derived T cells. To this end, inflammatory cells were isolated from the colonic mucosa for subsequent analysis in an ELISPOT assay. The results obtained clearly show that TNF-α is produced by a massive number of inflammatory cells isolated from the colons of TNF +/+ RAG2 −/− mice, transferred with either TNF +/+ or TNF −/− CD4 + CD45RB hi T cells 24 d after adoptive transfer of the respective CD4 + CD45RB hi T cell subset. In contrast, in the experimental group that did not develop colitis, i.e., in TNF −/− RAG2 −/− mice reconstituted with TNF +/+ CD4 + CD45RB hi T cells, TNF-α was produced only by very few infiltrating cells isolated from the large intestinal mucosa. This demonstrates that only a small fraction of transferred CD4 T cells produces TNF-α in the colonic mucosa of TNF −/− RAG2 −/− recipients . To further confirm that absence of colitis induction and perpetuation of disease correlate with the absence of significant numbers of TNF-α–producing cells in the colonic mucosa, we also determined the frequency of TNF-α–producing cells in the colonic mucosa of recipients of CD4 + CD45RB lo T cells, i.e., in the control groups that did not show signs of clinical or histopathological colitis. As seen in Fig. 5 , right panels, absence of colitis induction always correlates with the absence of TNF-α production by isolated colonic cells from recipients of CD4 + CD45RB lo splenocytes. To determine if the absence of TNF production by host-derived cells affects the induction of TNF production in the transferred CD4 T cells, thus resulting in the low frequency of TNF-α–producing T cells detected in TNF −/− RAG2 −/− recipients of TNF +/+ colitogenic T cells , we assessed the frequency of TNF-α–producing cells in fractionated CD4 T cells and, as a main TNF-α–producing, recipient-derived cell type, in macrophages isolated from the affected colonic mucosa of TNF +/+ RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells. 8.7‰ of the macrophages isolated from the affected colons produced TNF-α, whereas only 0.17‰ of isolated colonic CD4 T cells produced TNF-α in an ELISPOT assay. The frequency of TNF-α–secreting macrophages in the colonic mucosa in diseased animals was ∼10-fold higher than in macrophages isolated from the small intestinal mucosa of the same animals, where no signs of histopathological alterations were observed (8.7 and 0.8‰ TNF-α–producing macrophages, respectively). To assess how the presence or absence of TNF production by recipient cells and donor-derived CD4 T cells might affect the cytokine pattern produced in the colonic mucosa, ELISPOT assays were performed to determine the frequency of IL-4– and IFN-γ–secreting cells in the isolated inflammatory cells from the colonic mucosa. In all four combinations of transfer, CD4 + CD45RB hi T cells preferentially induced production of the Th1 cytokine IFN-γ in the colonic mucosa. Similar frequencies of IFN-γ–producing infiltrating CD4 T cells were detected in experimental groups of mice that developed severe colitis and in recipient mice that were protected from onset of severe disease . Hence, production of IFN-γ does not correlate with the presence of clinical signs of colitis but correlates with the transfer of CD4 + CD45RB hi T cells, as the frequency of IFN-γ–producing cells is markedly reduced in recipients of CD4 + CD45RB lo T cells from both TNF +/+ and TNF −/− donor mice. Colonic IL-4–producing cells, however, were less frequent in recipients of CD4 + CD45RB hi T cells when compared with recipients of CD4 + CD45RB lo T cells. The differences in IL-4 production by infiltrating CD4 T cells were only minimal among the four recipient groups of CD4 + CD45RB hi T cells . To assess not only the frequency but also the histological distribution of TNF-α–producing cells, in situ hybridization of cryostat sections of the affected colonic mucosa (day 24 after cell transfer) with TNF-α gene probes were performed. As shown in Fig. 6A and Fig. E , high frequencies of TNF-α mRNA–expressing cells were detected on colonic tissue sections of TNF +/+ RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells and TNF +/+ RAG2 −/− recipients of TNF −/− CD4 + CD45RB hi T cells . In both groups, TNF-α mRNA–expressing cells were not randomly distributed over the entire colonic mucosa but were preferentially clustered in the apical areas of colonic crypts proximal to the gut lumen and also at sites close to epithelial erosions of the colonic mucosa. In contrast, only a limited number of TNF-α mRNA CD4 T cells were detected in TNF −/− RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells . In these mice, rare TNF-α mRNA–expressing CD4 T cells were almost randomly distributed in the colonic lamina propria. In situ hybridization with an IFN-γ–specific probe revealed strong IFN-γ mRNA expression in the colonic mucosa of all groups of recipients of CD4 + CD45RB hi T cells . In the two donor–recipient combinations in which no severe colitis was induced, TNF −/− RAG2 −/− recipients of TNF +/+ and TNF −/− CD4 + CD45RB hi T cells , the strong IFN-γ mRNA expression observed in some mice was always associated with elevated cellular infiltration of the colonic mucosa (colitis score ≥7). IFN-γ mRNA + cells were not, however, restricted to the sites proximal to the lumen of the colon but were focally distributed throughout the entire colonic mucosa . As a negative control, affected intestinal colonic tissues were hybridized with sense probes of the TNF-α and IFN-γ genes . To further determine if the correlation between absence of clinical signs of colitis and absence of TNF-α–expressing cells is also true at early time points of disease induction where histopathological signs of a deleterious inflammation of the colon are still absent, in situ hybridization for the localization of TNF-α mRNA–expressing cells were performed on colonic tissue obtained on day 10 . Despite the absence of significant histopathological alterations of the colonic mucosa 10 d after transfer in the two groups that later developed colitis (TNF +/+ CD4 + CD45RB hi → TNF +/+ RAG2 −/− and TNF −/− CD4 + CD45RB hi →TNF +/+ RAG2 −/− mice , the frequency of TNF-α mRNA–expressing cells was comparable to that observed on day 24 after cell transfer with a preferential localization of TNF-α mRNA in cells close to, or even facing, the intestinal lumen. In TNF −/− RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells that did not develop clinical colitis, TNF-α mRNA–expressing cells were almost completely absent on day 10 . To exclude the possibility that the failure of TNF +/+ CD4 + CD45RB hi T cells to induce colitis in TNF −/− RAG2 −/− mice was due to the absence of peripheral lymph nodes and Peyer's patches or the aberrant splenic architecture found in mice deficient for LT-α 28 , we decided to back-cross C57BL/6J TNF-α −/− mice to the syngeneic RAG2 −/− background for subsequent transfer experiments using TNF-α −/− mice as donors. As depicted in Fig. 8 A, transfer of the CD4 + CD45RB hi T cells initially leads to a slight decrease in body weight around day 10 after cell transfer, but thereafter, recipients begin to gain weight to reach the body weight of the control animals transferred with the same number of CD4 + CD45RB lo T cells from the same donor animals. During the entire observation period of 35 d, the recipients of CD4 + CD45RB hi T cells did not show any signs of clinical colitis such as loose stool or even diarrhea or anal prolapse. The histopathological analyses performed 35 d after adoptive cell transfer demonstrated only mild signs of colitis, in particular focal accumulation of inflammatory cells. The histopathological scores determined for recipients of CD4 + CD45RB hi and CD4 + CD45RB lo T cells were not markedly different and even overlapped . Microphotographs of the colon from a TNF-α −/− RAG2 −/− recipient of TNF-α −/− CD4 + CD45RB hi T cells 35 d after adoptive transfer are shown in Fig. 8C and Fig. D . In recent years, several studies attempted to elucidate the role of TNF-α in IBD. Overproduction of TNF-α has been well documented in the affected intestinal mucosa in different animal models 14 16 33 and human patients with active Crohn's disease 17 19 . After administration of anti-TNF-α/LT-α (TNF) antibodies, an attenuation of disease progression has been observed in different mouse models of colitis and patients with IBD 15 16 19 . However, the beneficial effects of an anti-TNF treatment are often transient 19 . The reasons for the limited effects are not entirely understood and can possibly be attributed to a variety of factors, including the limited bioavailability of TNF-neutralizing agents at the site of mucosal lesions and redundant effects mediated by TNF at various stages of disease. Furthermore, recent results suggest that neutralizing antibodies to TNF can reverse suppressive effects mediated by TNF-α on T cells in vivo 34 , thus leading to higher functional and proliferative activity of disease-promoting T cells. Such an antiinflammatory effect of TNF-α has been demonstrated by Liu et al. in experimental allergic encephalomyelitis in mice where TNF-α can limit the extent and duration of severe central nervous system pathology 35 . Hence, the accelerated onset of weight loss and clinical signs of colitis in TNF +/+ RAG2 −/− mice after transfer of CD4 + CD45RB hi T cells from TNF −/− rather than TNF +/+ donors is thus in agreement with the notion that systemic TNF-α may attenuate T cell functions in vivo. On the other hand, our study also unambiguously demonstrates the nonredundant role of TNF in the induction of colitis in this mouse model. The nature of the TNF-producing cells (T cells versus non-T cells) relevant for the development of colitis in experimental animal models and human patients with IBD has not been directly determined so far. Several studies 22 36 found large numbers of TNF-α–secreting macrophages in the inflamed colonic mucosa. Other studies reported high frequencies of TNF-producing T cells, in particular CD4 + T cells, in the affected colonic mucosa 14 37 , suggesting that TNF produced by T cells may represent a critical factor directly affecting the extent of disease. Our results, however, clearly indicate that CD4 + T cell–produced TNF is neither sufficient nor required for induction of disease in this mouse model of colitis and that TNF production by recipient non-T cells is essential and sufficient for induction of severe histopathological alterations and onset of clinical signs of colitis. The observed preferential TNF-α production by colonic macrophages and their nonrandom distribution close to sites with epithelial erosion (data not shown) implies an important role of this cell subset in the pathogenesis of colitis. However, it remains to be determined if macrophages are the only recipient-derived cellular source of TNF-α crucial for the development of clinical signs of disease. In particular, as intestinal epithelial cells have also been demonstrated to produce TNF-α 24 , it is possible to speculate that epithelial cells are induced directly or indirectly by activated CD4 T cells to produce TNF-α. This assumption is supported by the results of in situ hybridizations of tissue specimens from the affected colon, where TNF-α mRNA–expressing cells are consistently observed in the intestinal epithelium even at early stages of disease when only minimal infiltration of the colon with inflammatory cells is observed . Although the number of cells recovered from the colonic mucosa from animals at the time of initial signs of weight loss, i.e., between days 10 and 14 after cell transfer, was too low for a quantitative assessment of the frequency of TNF-α–secreting cells, the results of the in situ hybridizations clearly indicate that TNF-α mRNA expression is detectable at levels similar to those observed on day 24, during the most active stage of disease. This indicates that elevated TNF-α expression is not the consequence of the strong inflammatory cell infiltrates but in fact precedes the severe histopathological alterations. As comparable numbers of TNF-α mRNA–expressing cells are detected in TNF +/+ RAG2 −/− mice reconstituted with either TNF +/+ or TNF −/− CD4 + CD45RB hi T cells, we conclude that recipient non-T cells are already the main TNF-producing cell type at the very early stages of disease. Similar findings have been reported in IL-2–deficient mice where, in the intestines of 10-d-old mice, 20-fold higher TNF-α mRNA expression levels were observed compared with older mice 24 . This increase, however, was found both in the small and the large intestines, whereas in our model, elevated expression of TNF-α mRNA as detected by in situ hybridization was restricted to the colon (data not shown). The precise effects mediated by locally produced TNF-α during development of colitis must remain speculative. The location of TNF-α–producing cells close to or even in the colonic epithelium concomitant with the appearance of histopathological alterations and subsequent clinical onset of severe colitis suggests that TNF-α may have a direct cytotoxic effect on epithelial cells by inducing an accelerated apoptosis of epithelial cells. Induction of TNF-α–mediated apoptosis via TNFR p55 in intestinal epithelial cells, first at the top of the villi and subsequently extending along the villus axis towards the intestinal crypts, has been recently reported by Guy-Grand et al. 38 and Piguet et al. 39 . Such an enhanced apoptosis of colonic epithelial cells may lead to the extensive erosion of the epithelium with subsequent severe inflammatory reactions, as observed during later stages of disease. Furthermore, TNF-α can induce the production of other inflammatory mediators such as free radicals, tissue degrading enzymes such as matrix metalloproteases 40 41 , and further proinflammatory cytokines, including IL-1β 42 . TNF-α can further amplify a local immune reaction through its effects on professional APCs such as dendritic cells, which are activated by TNF-α to become potent inducers of immune reactions 43 . Because LT-α, in addition to TNF-α, either as a homotrimer or heterotrimer, also binds to TNFR p55 and TNFR p75, we decided to use donor and recipient mice that were deficient for both TNF-α and LT-α (TNF −/− ). The protection of TNF −/− RAG2 −/− recipients of TNF +/+ CD4 + CD45RB hi T cells from developing severe colitis clearly demonstrates that not only CD4 T cell–derived TNF-α but also CD4 T cell–produced LT-α is not sufficient for disease induction, nor is it required for initiation and progression of disease. The observed protection of TNF-α −/− LT-α +/+ RAG2 −/− mice from onset of colitis upon transfer of TNF-α −/− CD4 + CD45RB hi T cells directly demonstrates that TNF-α plays a nonredundant role in the development of colitis, and its functions cannot be compensated for by the action of LT-α. These findings confirm the results of a previous report on the respective roles of TNF-α and LT-α in a mouse model of experimental allergic encephalomyelitis in which no redundancy in the functions of TNF-α and LT-α was observed in vivo 44 . The observed protection of TNF-α −/− recipients from colitis induction seems to be in contrast to the reported attenuation of disease when experimental mice are treated with a soluble LT-βR–human Fcγ1 fusion protein in two models of colitis, CD4 + CD45RB hi T cells→SCID mice and bone marrow–transplanted tg∈26 mice 45 . However, it cannot be excluded that binding of the soluble LT-βR–human Fcγ1 fusion protein to activated, LT-bearing transferred T cells or recipient cells may affect the subsequent functional differentiation and/or survival of these LT-β + cells. Evidence for opsonizing effects of receptor–Fcγ fusion proteins bound to cell surface ligands has recently been provided by the recombinant cell surface expression of human Fcγ in a reversed opsonization 46 . TNF-α has also been previously found to affect the trafficking of T cells into distinct organs such as the central nervous system 47 . However, when we examined the presence of CD4 T cells in the colonic mucosa at early time points (10 d) after adoptive cell transfer, in all donor–recipient combinations, CD4 T cells were found in the lamina propria of the small bowel and also, in slightly lower numbers, in the colon. 24 d after adoptive cell transfer, the frequency of colonic CD4 T cells recovered from the recipients of CD4 + CD45RB hi T cells varied considerably among individual animals. However, no clear correlation between the frequency of CD4 T cells recovered from the inflammatory infiltrates of the colon and development of or protection from colitis was found. A possible explanation for the presence of CD4 T cells in the intestinal lamina propria might be the TNF-independent expression of MAdCAM (mucosal addressin cellular adhesion molecule) 1 on endothelial cells in the small intestine, which we also observed in TNF −/− RAG2 −/− mice before adoptive cell transfer (data not shown). Although clinical signs of colitis are completely abrogated in the absence of TNF, histopathological analyses revealed in some recipients moderate alterations of the large intestinal mucosa. The enhanced cellularity of the colonic mucosa (colitis score ≥7) was always associated with enhanced expression of IFN-γ at a protein and mRNA level. This is in agreement with previous reports that indicated an involvement of IFN-γ in the induction of inflammatory reactions in the colonic mucosa 6 15 48 . The function of IFN-γ in the pathogenesis of colitis, however, seems to be nonessential, as described by the recent report by Simpson et al., who clearly demonstrated a redundant role of IFN-γ in the CD4 + CD45RB hi transfer model and the bone marrow transplantation into tg∈26 transgenic mouse model of colitis 23 . In agreement with this notion is our calculation of the absolute numbers of IFN-γ–producing cells isolated from the colons of recipients of CD4 + CD45RB hi T cells 24 d after transfer that revealed no clear correlation between the number of IFN-γ–secreting cells and presence or absence of severe colitis (data not shown). In conclusion, we provide evidence that (a) resident non-T cells are induced in situ directly or indirectly by CD4 T cells to produce TNF, which is essential for the development of clinical signs of colitis; (b) TNF produced by CD4 T cells alone is not sufficient to induce colitis; (c) activation of resident non-T cells by CD4 T cells is not TNF dependent, and hence, other mechanisms, either cell–cell contact such as CD40–CD40 ligand interactions 49 or soluble mediators are involved in the induction of TNF-α production in non-T cells of the affected colonic mucosa; and (d) the functions exerted by TNF-α in the development of colitis can not be compensated for by LT-α. | Study | biomedical | en | 0.999998 |
10562323 | p55- and p75-deficient mice, originally supplied by Immunex Corp. to J.L.M. Ferrara, and C57BL/6 WT animals (The Jackson Laboratory) were bred and maintained at the Redstone Animal Facility of the Dana-Farber Cancer Institute. Congenic Ly5.1 + C57BL/6 mice were purchased from the National Institutes of Health. All animals were kept under microisolators and provided with food and water ad libitum. To obtain peripheral blood (PB) samples, mice were first anesthetized by metaphane inhalation. For white blood cell (WBC) and RBC counts, the mice were terminally bled via a cardiac puncture. The counts were performed on a Coulter MaxM. A small blood sample was obtained by puncture of the venous sinus in the eye for analysis for the presence of Ly-5.2 + cells (see below). BM cells were obtained by flushing both tibiae and femurs of donor mice with PBS containing 2% FCS. The cell suspension was layered on a cushion of FCS and washed once in PBS, 2% FCS. Nucleated cells were counted with a hemocytometer. Assuming that the cell content of a tibia is ∼0.6 that of a femur 35 , the total cell number was divided by 3.2 to calculate the cell number per femur. To determine the presence of the different lineages in the BM, cells were incubated on ice for 35 min with the FITC-conjugated lineage (Lin) markers B220 (RA3-6B2), Mac-1 (M1/70), Gr-1 (RB6-8C5), Ly-1 (53-7.3), or PE-labeled Ter119 (PharMingen). Cells were then washed twice with PBS, 2% FBS. During the last wash, the medium contained propidium iodide (PI, 2 μg/ml; Sigma Chemical Co.) to stain the dead cells. Cells were analyzed on a single laser FACScan™ (Becton Dickinson). For purification, cells were labeled with a cocktail of mAbs, including Sca-1, derivatized with cyanine 5 succinimidyl ester (a gift from Dr. P. Lansdorp, Terry Fox Laboratory, Vancouver, British Columbia, Canada), FITC-labeled Lin markers, and (PE)-conjugated c-Kit (2B8; PharMingen). Where indicated, CD34-FITC (RAM34; PharMingen) was used to further subdivide the Sca-1 + Lin −/lo c-Kit + subpopulation into CD34 + and CD34 − cells. A preenrichment step was included in the purification protocol when cells were sorted; total BM cells were depleted of cells that expressed mature Lin markers by magnetic separation according to the manufacturer's directions (StemCell Technologies). Multicolor analysis to identify and sort Sca-1 + Lin −/lo c-Kit + progenitor cells was performed on a Coulter Epics ® Elite ESP. To calculate the absolute number of each cell type per femur (i.e., the different mature cell lineages and Sca-1 + Lin −/lo c-Kit + cells), the total number of cells per femur was multiplied by the fraction of each cell population in total BM cells as obtained by FACS ® analysis. Unseparated BM cells or highly purified progenitor cells were fixed in ice-cold 80% ethanol and kept on ice overnight. The next day, cells were spun down and washed once in PBS with 0.1% Triton X-100. Cells were resuspended in 0.5 ml PBS containing 50 μg/ml PI, incubated at 37°C for 30 min, and then kept on ice until analysis, which was within 1–2 h of finishing the staining. The sample was analyzed on a Coulter Epics ® XL analyzer. To assay for erythroid (BFU-E), granulocyte and/or macrophage (CFU-G/M), multilineage (CFU-GEMM), and pre-B clonogenic progenitors, cells were resuspended in IMDM containing 2% FCS. A portion of the cell suspension was resuspended in methylcellulose containing medium that was supplemented with the appropriate cytokines (StemCell Technologies). Pre-B cell colonies were scored in situ after 7 d of culture, the other colonies after 12–14 d of culture. The number of each clonogenic progenitor per femur was calculated by multiplying the number of nucleated cells per femur with the frequency of each progenitor, i.e., the number of respective colonies counted divided by the number of cells plated (5 × 10 4 cells for the pre-B cell assay and 1.5 × 10 4 cells for the erythroid/myeloid colony assay). The BM content of CFU-S day 12 cells was determined as originally described 36 . In brief, 7 × 10 4 unseparated BM cells were injected into C57BL/6 recipients that were irradiated with 950 cGy given in two equally split doses (5 h apart) from a 137 Cs source. The spleens were harvested 12 d later, and the macroscopic surface colonies were counted. The spleens of control animals that were irradiated but not transplanted with BM cells showed none or one to two pin-point size colonies. This size of colony was not included in the colony counts of the spleens from test animals. The number of CFU-S per femur was calculated by multiplying the number of nucleated cells per femur with the frequency of CFU-S, i.e., number of colonies counted divided by 7 × 10 4 . A slightly modified competitive repopulating units (CRU) assay was used to determine the number of HSCs in BM 37 38 . In brief, varying doses of unseparated test cells (Ly5.2 + ) were injected together with 1 × 10 5 unseparated adult BM “helper” cells (Ly5.1 + ) into lethally irradiated (as for CFU-S) recipients (Ly5.1 + ). The allelic difference at the Ly5 locus between the recipient/helper cells and the test cell population was used to follow hematopoietic reconstitution from the test cells. 4, 8, and 16 wk after the transplantation, a small blood sample was taken from each mouse and analyzed for the presence of Ly5.2 + cells. A mouse was considered to be reconstituted by CRU present in the test cell population (“positive”) when, 16 wk after the transplantation, >1% of the PB cells were Ly5.2 + and included both myeloid and lymphoid cells, as determined by their respective distinct side scatter (SSC) properties. CRU frequencies for every type of BM tested were calculated by analysis of the proportion of negative mice using Poisson statistics 39 . The number of CRU per femur was calculated by multiplying the number of nucleated cells per femur with the frequency of CRU. Some transplanted mice were used as donors for secondary BM transplantations. Marrow cells from both femurs and tibiae were isolated, and a proportion of these cells together with Ly5.1 + helper cells was injected into three to five irradiated Ly5.1 + recipients (analogous to a normal CRU assay). The Student's t test was used for the comparison of the mean of various hematological parameters from WT animals with that from p55 −/− or p75 −/− animals, unless otherwise stated. Several assays were performed to assess the various lineages and subpopulations of the hematopoietic system ( Table ). In young TNFR-deficient mice (<3 mo), the WBC and RBC counts in the PB did not differ from those of WT mice, nor did the relative contributions to the several myeloid and lymphoid lineages (data not shown). The PB indices were mirrored by similar BM cellularities in these three strains of mice. Functional analysis of marrow cell suspensions revealed a significantly higher number of myeloid clonogenic cells in p55 −/− mice compared with WT mice ( P < 0.01), but no significant differences were observed in the number of erythroid and pre-B clonogenic cells, or CRU. We hypothesized that an effect of perturbed TNFR signaling in HSCs may only be measurable with increased age. Therefore, we performed the same series of hematopoietic cell assays with BM cells from older mice (>6 mo). Neither the WT nor the p75 −/− mice showed any significant change with aging, aside from the normalization of the WBC counts in p75 −/− mice. In contrast, a profound disturbance of hematopoiesis in older p55 −/− mice could be demonstrated ( Table ). Nearly twice as many WBCs were found in the blood , and this was paralleled by the BM cell counts ( P < 0.005). Moreover, FACS ® analysis of the various mature lineages showed a tendency to increased cell numbers in all lineages except the B cell lineage. The same tendency was observed for clonogenic progenitors . When all myeloid CFCs, i.e., BFU-E, CFU-G/M, and CFU-GEMM, were combined, the total number of colonies in p55 −/− marrow was shown to be significantly higher than in WT or p75 −/− marrow ( P < 0.01): 128,000 ± 38,300 vs. 85,400 ± 25,000 and 78,300 ± 28,000, respectively. To assess the status of immature progenitors, we performed CFU-S day 12 assays and immunolabeled BM cells to identify the Sca-1 + Lin −/lo c-Kit ++ subpopulation. Similar to the mature myeloid clonogenic progenitors, the number of CFU-S day 12 in p55 −/− BM cells was higher compared with WT (not significant) and p75 −/− ( P < 0.03) BM cells: 2,700 ± 500 vs. 2,100 ± 260 and 1,700 ± 450, respectively . Fig. 2 depicts a representative FACS ® profile of BM cells showing the parameters used to calculate the absolute number of Sca-1 + Lin −/lo c-Kit ++ cells per femur. There is a considerably larger, although not statistically significant subpopulation of Sca-1 + Lin −/lo c-Kit ++ cells in p55 −/− BM compared with WT or p75 −/− BM: 11,300 ± 4,800 vs. 4,800 ± 2,200 and 5,200 ± 2,400 cells, respectively. Since this subpopulation includes long-term repopulating HSCs 40 , this phenotypic finding may indicate an increase in the number of HSCs in p55 −/− BM. However, it has been clearly shown that phenotype does not always predict function 38 41 . Therefore, we performed competitive repopulating experiments under limiting dilution conditions to determine the number of CRU per femur ( Table ). To our surprise, we found a significant decrease in the frequency of CRU among p55 −/− BM cells compared with WT ( P < 0.001). When corrected for BM cellularity, this translated into a near fourfold reduction of the absolute number of CRU in p55 −/− mice (350 vs. 1,275 in WT mice). Taken together, these data show a tendency with aging in p55-deficient mice to increased sizes of most mature and immature hematopoietic (progenitor) cell populations, which could explain the overall increase in marrow cellularity found in these animals. At the same time, the number of HSCs that give rise to this variety of cell populations is reduced. To further elucidate the extent of the stem cell defect in old p55 −/− mice, we analyzed in detail the hematopoiesis of three sets of mice transplanted with low numbers of BM cells . Groups 1 and 2 represent animals transplanted with 16,000 total BM cells (WT and p55 −/− , respectively). A comparison of their donor repopulation kinetics as measured at various time points after transplant illustrates a dramatic decline in donor reconstitution over a 4-mo time period by p55 −/− BM cells, whereas the donor reconstitution by WT cells showed a slight increase over the same time period tested . 16 wk after transplantation, the level of donor cells in recipients of p55 −/− BM cells (group 2) was significantly lower ( P < 0.05) compared with that in recipients of WT BM cells (group 1) (1.1 ± 1.0 vs. 10.2 ± 12.2, respectively). The difference in repopulating ability between WT and p55 −/− HSCs becomes even more apparent when individual mice are analyzed ( Table ). Between 4 and 16 wk after the transplantation, Ly5.2 + donor reconstitution did not increase in any of the recipients of p55 −/− (Ly5.2 + ) BM cells. In contrast, more than half of all the animals that had received WT (Ly5.2 + ) BM cells showed an increase in Ly5.2 + cells, by as much as 38% in one of the recipients. Moreover, only 22% of the group 2 animals that were multilineage reconstituted at week 4 remained so at week 16, compared with nearly 80% of the animals in group 1 ( Table ). Thus, when equal numbers of BM cells are transplanted, the repopulating ability of p55 −/− marrow cells is inferior to that of WT cells. However, when the CRU frequencies ( Table ) were used to calculate the average number of CRU received by individual transplanted animals (i.e., the total number of cells transplanted divided by the frequency of CRU), it was calculated that group 1 had received on average 1 HSC, whereas group 2 had received on average 0.3 HSC. This could potentially explain, or at least contribute to, the observed differences. Therefore, we compared group 1 WT mice with a group of mice that received on average 1 p55 −/− HSC, equivalent to 64,000 total BM cells (group 3). As expected, approximately the same number of mice remained multilineage reconstituted after 16 wk ( Table ; 78% in group 1 and 70% in group 3). However, although 4 wk after the transplantation the level of donor reconstitution in group 3 (p55 −/− transplant) recipients was significantly higher than in group 1 (WT transplant) recipients ( P < 0.01), the percentage of donor p55 −/− cells, again, gradually decreased with each subsequent analysis . 90% of all animals that received p55 −/− marrow cells showed as much as a 34% decline in donor reconstitution ( Table ). Thus, not only does p55 −/− marrow contain fewer HSCs, these data also indicate that the proliferative potential of p55 −/− HSCs is less than that of WT HSCs. To further stress the proliferative response of the Ly5.2 + transplanted cells, BM cells of some of the primary recipients were transplanted into secondary recipients. The results of these experiments are shown in Table . Eight primary recipients of WT marrow cells and three of p75 −/− marrow cells were used in these experiments. Because we had not been able to demonstrate remarkable differences between WT and p75 −/− hematopoiesis in older mice, we grouped these mice together to compare their secondary transplantation results to the results of the serially transplanted p55 −/− marrow cells. Of 11 WT/p75 −/− primary recipients, 4 animals (36%) contained Ly5.2 + HSCs that generated multilineage progeny upon transplantation into secondary recipients. This proportion is in accordance with previous published data 42 . Only 2 (18%) of the 11 primary recipients of p55 −/− BM gave secondary reconstitution upon serial passage. As observed in the primary recipients of p55 −/− cells, and in contrast to secondary recipients of WT donor cells, the kinetics of Ly-5.2 + reconstitution in the secondary recipients showed a marked decline over time (data not shown). One possible explanation for the observed differences in HSC number and proliferative potential between WT and p55 −/− mice is a higher cycling rate of p55 −/− HSCs. Purified Sca-1 + Lin −/lo c-Kit ++ cells still include large numbers of committed progenitor cells, as confirmed by our finding that between 20 and 30% of these cells (WT and knockout cells) were in the S/G2/M phase of the cell cycle (data not shown). Therefore, we further subdivided this population using an mAb against the CD34 antigen. The Sca-1 + Lin −/lo c-Kit ++ CD34 − cells, which include the long-term reconstituting HSCs 43 , comprise ∼10% of all Sca-1 + Lin −/lo c-Kit ++ cells. The sorting strategy for obtaining these cells is depicted in Fig. 4 . The results of cell cycle analyses of unseparated BM cells and highly purified cells (box 3) are shown in Table . As expected, DNA staining of unseparated BM cells did not demonstrate a difference in cycling activity between the WT and either knockout mouse. Moreover, the result obtained with the highly purified CD34 − subpopulation of Sca-1 + Lin −/lo c-Kit ++ cells isolated from p55 −/− marrow cells did not strongly suggest that cycling activity of p55 −/− HSCs was altered. Therefore, a difference in percentage of cycling HSCs does not seem to explain the observed qualitative and quantitative differences between HSCs from WT and p55-deficient mice. The p55 and p75 TNFRs are ubiquitously expressed on all hematopoietic cells, with the exception of erythroid and unstimulated T cells 2 44 . There is ample evidence from in vitro culture systems that, through one or both of these receptors, TNF-α can promote the expansion and differentiation of various hematopoietic subpopulations, as well as inhibit their proliferative response 10 11 12 13 . However, the long-term effects of this cytokine in vivo as a possible regulator of steady state hematopoiesis, especially with regard to the stem cell compartment, are not well defined. The present studies were undertaken to address this question by analyzing the hematopoietic system of mice in which TNF-α signaling is impaired. We and others 45 have shown that most hematopoietic subpopulations tested are present in young p55 −/− and p75 −/− mice at levels comparable to those of WT mice. In older mice, no significant differences in the assays tested between p75 −/− and WT mice could be demonstrated. However, when older p55 −/− mice were compared with WT mice they showed an interesting phenotype: their HSC compartment was significantly smaller than that of their WT counterparts (approximately fourfold), whereas the Sca-1 + Lin −/lo c-Kit ++ progenitor compartment had increased in size (approximately twofold). This was reflected in significant increases in total cellularity of the BM, total myeloid CFCs, and WBC and RBC counts in the PB. Thus, with aging of the mice, the lack of signaling through the p55 TNFR seems to affect the balance between generating new HSCs (self-renewal) and producing committed progenitors (differentiation). Interestingly, only the B cell lineage deviated from this pattern, in that the numbers of B220 + cells in the marrow and in the blood (data not shown) were similar to their WT counterparts, whereas the number of pre-B CFCs was significantly decreased in p55-deficient mice . Previous studies have shown that the p55 TNFR is essential for the structural organization of lymphoid organs 46 47 48 49 and the number of B cells in the Peyer's patch 48 . Our data also suggest that the generation of B cells in the marrow depends on p55 TNFR signaling. Whether this is mediated by TNF-α or lymphotoxin α remains to be studied. Exhaustion of the HSC pool can be explained by loss of HSCs due to cell death or differentiation. TNF-α has been shown to both induce and suppress apoptosis in murine Sca-1 + Lin − cells. Analogous to the effects of TNF-α on the proliferation of hematopoietic cell populations, its effect on apoptosis seems to depend on which additional cytokine was present: suppression in cultures with IL-1α present, and induction when Steel factor was supplied 50 . The apoptotic effects of TNF-α are well documented and mainly exerted through its p55 receptor 51 52 , although an apoptotic role for the p75 receptor has been demonstrated in mature CD8 + cells 9 . Increased apoptosis in HSCs will definitely decrease the HSC pool. However, if this were the case in p55 −/− mice, one would expect to find an accompanying decrease in the number of progenitor cells. The opposite was found in our p55-deficient mice. The mechanisms that control self-renewal versus commitment decisions in HSCs are not known. However, it is generally accepted that an increase in the number of HSC cell divisions is accompanied by an increased chance of HSCs to commit and differentiate among one of the lineages 53 54 55 . TNF-α has been shown to prevent WT but not p55 −/− Sca-1 + Lin − c-Kit + cells from entry into S phase 31 . Increased cycling of p55 −/− HSCs could explain our results. However, we could not detect a difference in cell cycle activity between Sca-1 + Lin − c-Kit + subpopulations (CD34 + or CD34 − ) sorted from WT or p55 −/− BM cells. The effects of TNF-α on the cell cycle of HSCs may be very subtle, which would explain why HSCs in young mice seem unaffected and why it is not detectable by the methods used in this study. One study has demonstrated the importance of TGF-β in maintaining HSCs in a quiescent, noncycling state in unperturbed steady state hematopoiesis 30 . Since TNF-α cooperates with TGF to inhibit the proliferation of primitive progenitor cells 32 , it is not inconceivable that more HSCs escape their quiescent state when one of the inhibitory signals is missing. There are two important considerations with respect to the finding that the phenotype in p55 −/− mice manifests only with aging (>6 mo of age). First, TNF-α production and TNF-α signaling cascades seem to change with aging. Aged mice produce more TNF-α in response to LPS 56 , and T cells from older humans express more p55 but less p75 TNFR subunits and are more susceptible to TNF-α–induced apoptosis 57 . It is possible that TNF-α signaling in HSCs also changes with aging, and that any deficiencies in this signaling pathway have greater consequences later in life. It may be of interest to look at mice of 20 mo or older since the number of cycling HSCs increases dramatically at this age 58 . Second, hematopoiesis during fetal and/or neonatal development, which is at that time expanding tremendously, not only contains HSCs with properties different from adult HSCs 42 59 , but is also probably regulated differently than adult, steady state hematopoiesis 60 . The regulatory mechanisms of fetal/neonatal hematopoietic cells may not depend on TNF-α signaling at all. This, in addition to the possibility that older hematopoietic cells, and HSCs in particular, may be more sensitive to TNF-α, could explain the lack of a phenotype in young p55 −/− mice. Thus, our findings suggest that, with age, a lack of signaling through the p55 TNFR increases the number of committed hematopoietic progenitors and their subsequent progeny at a cost to the stem cell pool. The scarcity of secondary recipients with multilineage p55 −/− progeny confirms the lack of self-renewal or exhaustion of p55 −/− HSCs. However, what is surprising and contrary to our expectations is the lower level of multilineage reconstitution per HSC from p55 −/− donors compared with WT. The explanation for this reduction may be that hematopoietic regeneration in myeloablated recipients is different from both adult steady state hematopoiesis and fetal/neonatal hematopoiesis. Although little is known about the cytokines and other regulatory elements in the activation and/or recruitment of transplanted HSCs, there are some important differences compared with steady state hematopoiesis 61 . One important difference is that transplanted HSCs must home to an appropriate microenvironment. It is possible that altered TNF-α signaling affects the homing properties of p55 −/− cells. In this regard, it is of interest that TNF-α can affect the expression of CXC chemokine receptor 4 62 63 64 , a chemokine receptor critical for murine BM engraftment and regeneration as well as B cell development 65 66 . Moreover, in our experimental design, the transplanted HSCs are influenced by stress-induced environmental changes associated with the myeloablation of the recipient. Stress can induce elevated levels of glucocorticoid hormones, which, in synergy with other cytokines, make BM cells more susceptible to IL-1 through upregulation of the IL-1 receptors, which favors myeloid differentiation 67 68 . A beneficial effect of IL-1 may be the suppression of apoptosis. However, for this effect to be delivered, the combination of TNF-α may be essential 50 . Therefore, it is possible that p55 −/− HSCs acquire elevated levels of IL-1 receptors after transplantation but are more likely to differentiate than their WT counterparts when they do not receive the appropriate TNF signal. Thus, the loss of HSCs and all other hematopoietic cell types after transplantation of p55 −/− marrow cells may not necessarily reflect steady state hematopoiesis, but rather the inability of these cells to adequately respond to extreme proliferative stimuli. The need for intact TNFR signaling in HSCs during hematopoietic regeneration of a myeloablated recipient may be more crucial for its survival than during steady state hematopoiesis. The G0/G1 arrest that TNF-α has been shown to induce in Sca-1 + Lin − c-Kit + cells may be imperative in restraining the proliferative response of HSCs to external stimuli and thereby preventing their extinction. In this respect, it is of interest that in TNF −/− mice it was shown that TNF is important for the generation of an inflammatory response, but its presence was far more essential in limiting the extent and duration of this response 69 . This possible regulatory role in limiting the number of cell divisions in HSCs warrants further investigation, for example in retroviral gene therapy protocols with HSCs as target cells. Future studies of the role of TNF in steady state hematopoiesis should include the analysis of mice older than 20 mo and the generation of allophenic mice, which would allow the tracking of HSC progeny over time, without perturbing the hematopoietic system 70 . | Study | biomedical | en | 0.999998 |
10562324 | The following mAbs were produced in our lab: JT3A (IgG2a, anti-CD3); BAB281 22 and KL247 (IgG1 and IgM, respectively, anti-NKp46); Z231 23 and KS38 (IgG1 and IgM, respectively, anti-NKp44); KD1 and c127 (IgG2a and IgG1, respectively, anti-CD16); c218 and GPR165 (IgG1 and IgG2a, respectively, anti-CD56); A6-136 (IgM, anti-HLA class I ); GL183 (IgG1, anti-p58.2 ); EB6 (IgG1, anti-p58.1 ); and Z199 (IgG2b, anti-NKG2A ). D1.12 (IgG2a, anti–HLA-DR) mAb was provided by Dr. R.S. Accolla (University of Insubria, Varese, Italy). HP2.6 (IgG2a, anti-CD4) mAb was provided by Dr. P. Sanchez-Madrid (Hopital de la Princessa, Madrid, Spain). The novel mAbs were derived by immunizing 5 wk–old Balb/C mice with activated (CD3 − , CD56 + , and CD16 + ) NK cells, either NK clones (EC1 and SA260 for A76 and Z25 mAbs, respectively) or a polyclonal NK cell population (for AZ20 mAb). After different cell fusions, the mAbs were selected for the ability to induce lysis in redirected killing assays against the FcγR + P815 target cells. PBLs were derived from healthy donors by Ficoll-Hypaque gradients and depletion of plastic-adherent cells. To obtain enriched NK cells, PBLs were incubated with anti-CD3 (JT3A), anti-CD4 (HP2.6), and anti–HLA-DR (D1.12) mAbs (30 min at 4°C), followed by goat anti–mouse-coated Dynabeads (Dynal; 30 min at 4°C) and immunomagnetic depletion 21 22 23 . CD3 − 4 − DR − cells were used in cytolytic assays or cultured on irradiated feeder cells in the presence of 100 U/ml rIL-2 (Proleukin; Chiron Corporation) and 1.5 ng/ml PHA (GIBCO BRL) to obtain polyclonal NK cell populations or, after limiting dilution 27 , NK cell clones. Cells were stained with the appropriate mAb followed by PE- or FITC-conjugated isotype-specific goat anti–mouse second reagent (Southern Biotechnology Associates Inc.). Samples were analyzed by one- or two-color cytofluorimetric analysis (FACScan™; Becton Dickinson), as described previously 7 . The following FcγR-negative targets were used: MEL15 ; M14 (human melanoma ); SMMC (human hepatocarcinoma ); A549 (human lung adenocarcinoma, no. CCL-185.1; American Type Culture Collection). FO-1 and 1174 mel (human melanomas) were provided by Dr. S. Ferrone (Roswell Park Cancer Center, Buffalo, NY); AUMA (human melanoma) was provided by Dr. P. Coulie (Catholic University of Louvain, Brussels, Belgium). The FcγR-positive target used was P815 (murine mastocytoma). PHA blasts, used as normal target cells, were obtained by culturing PBLs with 1.5 ng/ml PHA (GIBCO BRL). Cells were tested for cytolytic activity in a 4-h 51 Cr-release assay as described previously 8 26 , either in the absence or presence of various mAbs. The concentrations of the various mAbs were 10 μg/ml for the masking experiments and 0.5 μg/ml for the redirected killing experiments. The E/T ratios are indicated in the text. Determination of [Ca 2+ ] i was performed as described previously 28 . Fura-2–labeled NK cells were incubated for 30 min at 4°C with saturating amounts of anti-NKp30 mAb (AZ20) or medium alone. Cross-linking of this receptor was obtained by adding into the cuvette 20 μg/ml of affinity-purified goat anti–mouse antiserum (GAM; ICN Biomedicals, Inc.). Integral NK cell membrane proteins 29 were prepared as follows: 25 × 10 6 cells were lysed in 100 μl TX buffer (20 mM sodium phosphate buffer, 1% Triton X-114, 10 mM EDTA, pH 8) for 30 min at 4°C, and centrifuged (5 min, 10,000 rpm). The supernatant was left for 10 min at 37°C, centrifuged, and the lower phase was resuspended 1:2 in TX buffer and left for 10 min at 4°C in order to clarify the lysates. The suspension was then left for 10 min at 37°C, centrifuged, and the lower phase was resuspended 1:3 in EB . Samples were analyzed in discontinuous SDS-PAGE, transferred to Immobilon P (Millipore Corp.), and probed with AZ20 mAb followed by rabbit anti–mouse horseradish peroxidase (HRPO; DAKO), or NKp30-specific antiserum followed by donkey anti–rabbit HRPO (Nycomed Amersham plc). The Renaissance Chemiluminescence kit (NEN Life Science Products) was used for detection. A 2.5-kg HY/Cr male rabbit (Charles River Laboratories) was immunized with 100 μg/100 μl of the 15 amino acid peptide WVSQPPEIRTLEGSC (Eurogentec) conjugated with KLH 13 . Four weekly treatments were performed, the first in association with 100 μl CFA and all others with 100 μl IFA. 1 wk after the last treatment, 10 ml of blood was drawn, and serum was tested and titered by ELISA against the immunizing and irrelevant peptides. NK cells (10 8 ) were stimulated or not with 100 μM sodium pervanadate 25 , and 1% digitonin lysates were precleared five times with sepharose protein A–coupled KD1 (anti-CD16) mAb. Lysates were then immunoprecipitated with sepharose-CNBr–coupled Z231 and BAB281 mAbs, or with sepharose protein A–coupled NKp30-specific rabbit antiserum and preimmune rabbit serum. Samples were analyzed in a 15% SDS-PAGE under reducing conditions (5% 2-ME), transferred to Immobilon P (Millipore Corp.), and probed with antiphosphotyrosine mAb (PY20-HRPO; Transduction Laboratories) or anti-CD3ζ mAb (2H2; Immunotech), followed by rabbit anti–mouse HRPO (DAKO). The Renaissance Chemiluminescence kit (NEN Life Science Products) was used for detection. The expression cDNA library was prepared in VR1012 plasmid (Vical Inc.) using RNA extracted from IL-2–activated polyclonal NK cells obtained from two healthy donors as described previously 24 25 . The library screening procedure was as described 24 25 30 . In brief, cDNA library was transiently transfected in COS-7 cells, and selection of positive pools was performed by immunocytochemical staining using the specific anti-NKp30 mAb A76 and sib-selection. DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing kit and a 377 ABI Automatic Sequencer (Applied Biosystems/Perkin-Elmer). COS-7 cells (5 × 10 5 /plate) were transfected with VR1012-NK-A1 (clone 5C) or with the vector alone by the DEAE-dextran or electroporation methods as described 13 . After 48 h, transfected cells were used for cytofluorimetric analysis. To analyze NKp30 transcript expression in different cell lines of hemopoietic origin, RNA was size fractionated by denaturing agarose gel electrophoresis and transferred onto a positively charged nylon membrane (NEN Life Science Products). In particular, 10 μg of total RNA prepared using CsCl gradient, or 2 μg of poly A + RNA prepared using oligo(dT) magnetic bead separation (Dynal AS), was loaded on each lane. Northern blots were performed under high stringency conditions as described 31 . The NKp30 421-bp cDNA probe was obtained by PCR amplification performed with 25 pmol of each primer for 30 cycles (30 s at 94°C, 30 s at 60°C, 30 s at 72°C), followed by a 7-min incubation at 72°C. The sequences of the primers are: CAG GGC ATC TCG AGT TTC CGA CAT GGC CTG GAT GCT GTT G (NK-A1 up) and GAC TAG GAT CCG CAT GTG TAC CAG CCC CTA GCT GAG GAT G (NK-A1 down). The cDNA fragment was 32 P-labeled by random priming 32 . Total RNA extracted using RNAzol (Cinna/Biotecx) from polyclonal NK and T cell populations and clones and from different hemopoietic cell lines were reverse transcribed using oligo(dT) priming. Primers used for cDNA amplification of NKp30 (606 bp) were the following: 5′ CAG GGC ATC TCG AGT TTC CGA CAT GGC CTG GAT GCT GTT G (NK-A1 up) and 5′ GAT TTA TTG GGG TCT TTT GAA G (A76-38 reverse). Amplification was performed with 25 pmol of each primer for 30 cycles (30 s at 94°C, 30 s at 60°C, and 30 s at 72°C), followed by a 7-min incubation at 72°C. The amplification products were subcloned in pCR2.1 by TOPO-TA Cloning kit (Invitrogen), and subsequently sequenced. Analysis of cross-species conservation of NKp30 gene was performed using a Zoo-Blot (CLONTECH). The Southern blot contained genomic DNA from humans, Rhesus monkey, Sprague–Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and Saccharomyces cerevisiae yeast. The hybridization probe was the same 421-bp cDNA fragment used to hybridize the Northern blot. Washes were carried out at low stringency conditions as described 32 . Mice were immunized with CD3 − , CD16 + , CD56 + NK cell clones or bulk populations. mAbs from different fusions were first selected according to their ability to induce lysis of the FcγR + P815 target cells in a redirected killing assay using polyclonal NK cell populations or clones as effector cells. Three mAbs, A76, AZ20, and Z25 (all of IgG1 isotype), were selected that induced a strong cytolytic activity similar to that elicited by other mAbs specific for known triggering NK receptors, including CD16, NKp46, and NKp44 22 23 26 . In Fig. 1 B, the NK cell cytotoxicity induced by graded amounts of AZ20 mAb is compared with that of isotype–matched anti-CD16 or anti-CD56 mAbs. The cytolytic response to AZ20 mAb paralleled that induced by anti-CD16 mAb, whereas anti-CD56 mAb had no effect. Moreover, as shown in Fig. 1 C, a sharp [Ca + ] i increase was detected in the representative clone 3M16 after stimulation with AZ20 mAb. Notably, [Ca 2+ ] i increments induced by this Ab occurred only in the presence of a goat anti–mouse second reagent, allowing efficient cross-linking of the activating receptor. Analysis of the cell surface distribution of the molecule(s) recognized by A76, AZ20, and Z25 mAbs, performed by indirect immunofluorescence and FACS ® analysis, revealed reactivity with various activated polyclonal or clonal NK cell populations derived from different donors (see below). These also included the infrequent CD16 − NK cell clones. On the contrary, no mAb reactivity was detected with PHA-induced polyclonal T cell populations or TCR-α/β and -γ/δ T cell clones (derived from different donors). Neither was any reactivity detected with EBV-induced B cell lines, monocytic and dendritic cell lines, and different hemopoietic and nonhemopoietic tumor cell lines, including HL60, U937, Eo/A3, THP-1, Daudi, Jurkat, IGROV, and all the various tumor cell lines used as target cells in this study (data not shown). We recently showed that polyclonal NK cell populations from some donors were characterized by a bimodal distribution of fluorescence intensity of NKp46 molecules (NKp46 bright and NKp46 dull ), and that NK clones derived from these individuals expressed a stable NKp46 bright or NKp46 dull phenotype 26 . Importantly, the cytolytic activity of NK cell clones against NK-susceptible target cells strictly correlated with their NKp46 phenotype 26 . We then analyzed the reactivity of the new mAbs on polyclonal NK cell populations and NK cell clones derived from individuals displaying different patterns of NKp46 expression. As shown in Fig. 2 A, the polyclonal NK cell population derived from the representative donor AM displayed a homogeneously bright phenotype when stained by either AZ20 or anti-NKp46 mAbs. On the contrary, in the polyclonal NK cells derived from donor CB, staining with the same mAbs resulted in a bimodal distribution of fluorescence. Notably, in donor CB, the same pattern of fluorescence intensity was also detectable in fresh purified NK cells . Moreover, the analysis of several clones derived from donor CB revealed that NKp46 bright clones were consistently AZ20 bright , whereas NKp46 dull clones always displayed an AZ20 dull phenotype . To further define the pattern of reactivity of the new mAbs in freshly isolated lymphocytes, PBLs derived from different individuals were assessed by double fluorescence analysis using informative mAbs. A representative donor is shown in Fig. 3 A: the surface molecule recognized by AZ20 mAb was selectively expressed on CD56 + cells. Moreover, most AZ20 + cells coexpressed CD16 molecules. However, AZ20 mAb did not stain CD3 + T lymphocytes or HLA-DR + B lymphocytes. It is of note that the CD56 + AZ20 − cell population detected in this donor also expressed surface CD3 molecules (not shown). Therefore, also in freshly derived lymphocytes, the reactivity of AZ20 mAb overlaps with that of anti-NKp46 mAb. A direct comparative analysis of the surface expression of NKp46 and AZ20 mAb–reactive molecules is shown in Fig. 3 A. The two molecules were clearly coexpressed by the same cell subset. However, no diagonal distribution could be detected in cells stained by AZ20 and anti-NKp46 mAbs, whereas this type of fluorescence distribution occurred when cells were stained simultaneously by two anti-NKp46 mAbs of different isotype. Notably, results identical to those described for AZ20 mAb were obtained with A76 and Z25 mAbs. These data suggested that the molecule recognized by the new mAbs may be distinct from NKp46. To directly evaluate this possibility, COS-7 cells transiently transfected with NKp46 cDNA 24 were analyzed for their reactivity with AZ20, A76, and Z25 mAbs. Cell transfectants, although reacting with different anti-NKp46 mAbs, were not stained by AZ20, A76, and Z25 mAbs (data not shown). Taken together, these data strongly suggest that A76, AZ20, and Z25 mAbs are specific for a novel surface molecule that defines all mature human NK cells, but is distinct from NKp46. To analyze the biochemical characteristics of the surface molecules recognized by AZ20, A76, and Z25 mAbs, NK populations were surface labeled with 125 I or biotin, immunoprecipitated with one or another mAb, and analyzed by SDS-PAGE. Under these conditions, no specific bands could be detected. Thus, integral membrane proteins were prepared from NK cells to further analyze a possible reactivity of the various mAbs in Western blot. As shown in Fig. 3 B, AZ20 mAb specifically reacted with an ∼30-kD molecule, thereafter termed NKp30. Under the same conditions, both A76 and Z25 mAbs displayed a poor reactivity (data not shown). Since NKp30 molecule, like NKp46, was expressed on fresh NK cells, we analyzed whether it could trigger the cytolytic activity of these cells as demonstrated previously for NKp46. As shown in Fig. 4 A, AZ20, A76, and Z25 mAbs induced a strong increase of cytolytic activity against P815 target cells, whereas the isotype–matched anti-CD56 mAb had no effect. This triggering effect was comparable to that obtained with anti-NKp46 mAb. Moreover, in these experiments, the use of AZ20 F(ab′) 2 fragments did not induce triggering of cytolytic activity, indicating that mAb-dependent NKp30 stimulation requires efficient cross-linking mediated by FcγR on target cells (data not shown). Previous data showed that mAb-mediated masking of NKp46 or NKp44 inhibited the non–MHC-restricted tumor cell lysis by activated NK cells 23 24 26 . Moreover, masking of NKp46 also inhibited the natural cytotoxicity mediated by freshly isolated peripheral blood NK cells 26 . We then evaluated whether masking of NKp30 could affect the cytolytic activity mediated by freshly derived NK cells or NK clones against a panel of FcγR-negative tumor target cells. As shown in Fig. 4 B, anti-NKp30 mAb, but not the isotype-matched anti-CD56 mAb, inhibited natural cytotoxicity mediated by fresh NK cells against the HLA class I–negative 1174 mel, AUMA, and FO-1 melanoma cell lines. In addition, a greater inhibitory effect occurred when anti-NKp30 mAb was used in combination with anti-NKp46 mAb. This suggests that NKp30 and NKp46 may represent receptors that act synergistically in triggering the natural cytotoxicity of fresh NK cells. In view of these data, we further analyzed the effect of mAb-mediated masking of NKp30 on the tumor cell killing by activated NK cells. Fig. 5 shows three representative NK cell clones analyzed in a cytolytic assay against different tumor targets, including two melanomas (MEL15 and M14), a hepatocarcinoma (SMMC), and a lung adenocarcinoma (A549). In previous studies, we showed that the cytolytic activity against the M14 melanoma was confined to NK clones displaying the NKp46 bright phenotype and could be inhibited by mAb-mediated masking of NKp46 receptor. On the other hand, NKp46 bright clones also killed MEL15. However, neither masking of NKp46 nor of NKp44 significantly inhibited their cytolytic activity 26 . These data strongly suggested the existence in these clones of additional triggering receptors responsible for the cytotoxicity against MEL15 target cells. As illustrated above, NKp30 is brightly expressed in NKp46 bright clones. Therefore, it is conceivable that it may play a role in the killing of MEL15 target cells. Indeed, as shown in Fig. 5 , anti-NKp30 mAb sharply inhibited the NK-mediated lysis of MEL15 cells (>50% of inhibition). Anti-NKp46 mAb exerted a minor effect, whereas an isotype-matched anti-CD56 mAb had no effect (data not shown). On the contrary, lysis of M14 melanoma was inhibited by anti-NKp46 mAb, whereas anti-NKp30 mAb had virtually no effect. Thus, while NKp46 appears as the major receptor involved in lysis of M14, NKp30 plays a central role in the killing of MEL15. Analysis of the same NK clones in cytolytic assays against other tumor target cells such as SMMC and A549 revealed a balanced contribution of NKp46 and NKp30 to the induction of cytotoxicity. Indeed, while mAb-mediated masking of NKp46 or NKp30 alone had a moderate inhibitory effect, the simultaneous masking of the two molecules resulted in a significant inhibition. These results indicate that the two receptors may exert an additive effect in the induction of cytotoxicity against certain target cells. Cooperation in NK cell triggering was previously demonstrated for NKp46 and NKp44 23 . Further analysis revealed that NKp30 could exert an additive effect in the induction of NK-mediated cytotoxicity, not only with NKp46, but also with NKp44. Fig. 6 A shows the cytolytic activity of the representative NK clone MIL69 against FO-1 or A549 tumor cells. Target cell lysis was only partially inhibited by mAb-mediated masking of NKp30, NKp44, or NKp46 receptors. However, the combined masking of two receptors resulted in a higher inhibitory effect, whereas the simultaneous masking of the three receptors gave the maximal inhibition. Isotype-matched anti-CD56 mAb had no inhibitory effect either when used alone or in combination with other mAbs (data not shown). We further analyzed the role of NKp30 alone or in combination with other receptors in cytolytic assay using PHA-induced T cell blasts as a source of normal target cells. In these experiments, lysis of autologous cells by NK cell clones was obtained by mAb-mediated masking of HLA class I molecules on target cells to disrupt the interaction with the HLA class I–specific inhibitory receptors expressed on NK cells. Also under these experimental conditions, the mAb-mediated masking of single receptors had only a partial inhibitory effect on cytotoxicity . On the other hand, the simultaneous masking of NKp30, NKp46, and NKp44 receptors strongly reduced (or virtually abrogated) target cell lysis (see the representative clones MX361 and P9). These data support the notion that the ligands recognized by these receptors are expressed not only in tumor, but also in normal cells. Finally, we analyzed the possible involvement of NKp30 in the recognition of murine target cells. Previous studies indicated that the NK-mediated killing of murine cells was abrogated by the mAb-mediated masking of NKp46 receptors, thus suggesting that NKp46 may represent the unique human NK receptor able to recognize ligand(s) expressed on murine cells 24 26 . This hypothesis was substantiated by the recent identification of a murine homologue that shared a high degree of identity with the human NKp46 receptor 33 . Although not shown, the mAb-mediated masking of NKp30 had no effect on the lysis of both BW1502 and YAC-1 murine thymoma target cells. Altogether, the above data indicate that NKp30 functions as a major receptor involved in the NK-mediated cytotoxicity against normal target cells and most, but not all, tumor cells. In addition, NKp30 may cooperate with NKp46 and NKp44, most likely depending on the expression of specific ligands by the target cell analyzed. In an attempt to identify the cDNA encoding the NKp30 molecule, a cDNA expression library was generated from the mRNA of human polyclonal NK cells 24 . COS-7 cells transfected with different cDNA library pools were stained with A76 mAb by an immunocytochemical detection method. A 674-bp cDNA was isolated that contained a single open reading frame (ORF) of 573 bp. Transfection of COS-7 cells with clone 5C cDNA construct resulted in the surface expression of a molecule that was recognized by all the various anti-NKp30 mAbs , but not by anti-NKp46 mAbs as assessed by cytofluorimetric analysis. As shown in Fig. 7 B, clone 5C ORF encoded a putative 190 amino acid polypeptide belonging to the Ig-SF, characterized by a signal peptide of 18 amino acids and by an extracellular region of 120 amino acids forming an Ig-like domain of the V type. The extracellular portion contains two potential N-linked glycosylation sites and no consensus sequences for O-linked glycosylation. A region rich in hydrophobic amino acids, potentially involved in protein–protein interactions, is connecting the Ig V-like domain with the transmembrane region. The 19 amino acid transmembrane region contains the positively charged amino acid, Arg, and the 33 amino acid cytoplasmic portion lacks typical immunoreceptor tyrosine-based activating motif (ITAM) consensus sequences. The presence of a charged amino acid in the transmembrane domain is a feature common to other triggering receptors expressed on NK cells 24 25 34 35 36 37 . These charged residues are usually thought to be involved in the association with ITAM-containing signaling polypeptides. Searching EMBL/GenBank/DDBJ databases revealed that the clone 5C cDNA was identical to a previously identified alternatively spliced form of the 1C7 gene . This gene has been mapped on human chromosome 6, in the TNF cluster of the MHC gene complex 38 . To date, however, owing to the lack of specific mAb, neither the function nor the surface distribution of the putative product of 1C7 gene could be identified. Moreover, the 1C7 transcript could not be revealed by Northern blot on different tissues and cell lines. On the other hand, by reverse transcriptase (RT)-PCR, the 1C7 transcript could be amplified by RNA isolated from spleen (but not from other tissues) or certain lymphoid and myeloid cell lines. These data suggested that 1C7 transcripts could be poorly represented, or could be expressed at substantial levels only in a narrow range of cell types 39 . Our present analysis of NKp30 expression by Northern blotting revealed a mRNA of ∼1 kb in polyclonal NK cell populations and NK cell lines, including NKL and NK3.3. On the contrary, consistent with the lack of reactivity with anti-NKp30 mAbs, no NKp30 mRNA could be detected in human monocytes or cell lines of different histotype, including U937, Jurkat, HL60, and LCL 721.221 cells . In some of these cell lines that were negative for mRNA expression by Northern blot (and for anti-NKp30 mAb surface staining), it has been possible to detect transcripts when analyzed by the RT-PCR technique. This finding is likely to reflect a low level of NKp30 transcription resulting in lack of NKp30 surface expression. Moreover, Northern blot analysis of multiple human tissues showed selective expression of NKp30 transcript only in spleen cells (data not shown). Altogether, these data are consistent with the notion that NKp30 expression is largely NK specific. Finally, the human NKp30 cDNA probe hybridized with genomic DNA from monkey, rat, mouse, dog, cow, and rabbit. These data suggest that the NKp30-encoding gene may be conserved in different species . An NKp30-specific antiserum was generated by immunizing rabbits with an NH 2 -terminal NKp30 peptide. As shown in Fig. 9 A, the antiserum recognized in Western blot a molecule identical to that previously detected by AZ20 mAb. Unlike the AZ20 mAb, the antiserum immunoprecipitates NKp30 molecules from polyclonal NK cell populations labeled with biotin (data not shown). Thus, a polyclonal NK cell population, treated or not with sodium pervanadate, was immunoprecipitated with the NKp30-specific antiserum and probed with antiphosphotyrosine mAb. To avoid nonspecific binding of rabbit Ig to CD16 molecules, cell lysates were extensively precleared with anti-CD16 mAb. Moreover, in all experiments, preimmune rabbit serum was used as negative control. In these experiments, no tyrosine phosphorylation of NKp30 receptor could be detected (data not shown). On the other hand, NKp30 receptor associated with a molecule that became tyrosine phosphorylated upon sodium pervanadate treatment and comigrated with the NKp46-associated CD3ζ chain. The identity between the NKp30-associated molecule and CD3ζ polypeptides was directly demonstrated by its reactivity with anti-CD3ζ mAb . Thus, NKp30, similar to other NK triggering receptors including CD16 34 35 and NKp46 23 , can transduce activating signals via association with the ITAM-containing CD3ζ polypeptides. These data are in agreement with the lack of ITAM in the NKp30 cytoplasmic tail, and with the presence of a charged residue in its transmembrane portion. In this study, because of the generation of specific mAbs, we identified and characterized NKp30, a novel triggering receptor that plays an important role in the natural cytotoxicity of both resting and activated human NK cells. Similar to NKp46, NKp30 is selectively expressed by all NK cells, both freshly isolated and cultured in IL-2, thus representing an optimal marker for NK cell identification. Although it belongs to the Ig-SF, NKp30 does not display any substantial homology with previously identified NK receptors. In many respects, NKp30 appeared similar to NKp46. Indeed, their parallel expression on all NK cells (including the rare CD16 − cells), the existence for both of a high or low density pattern of surface expression, together with their similar functional characteristics, led to the thought that the surface molecule recognized by the new mAbs could be identical or strictly related to NKp46. However, NKp30 and NKp46 displayed different molecular masses and, functionally, appeared to play a complementary role in the induction of natural cytotoxicity. Moreover, molecular cloning revealed that NKp30 is a protein with very limited homology with NKp46, as the two molecules display only 13% identity and 15% similarity, and are encoded by genes located on different chromosomes. The receptors responsible for the NK cell triggering during natural cytotoxicity and tumor cell lysis have remained elusive until recently. Available data were consistent with the hypothesis of the existence of multiple triggering NK receptors involved in natural cytotoxicity. In this context, we recently identified NKp46 and NKp44, two receptors involved in recognition and lysis of a variety of tumor targets. Both belong to the Ig-SF, but neither displays significant identity. They associate to different signal transducing polypeptides (CD3ζ/Fc∈RIγ and KARAP/DAP12, respectively) that become tyrosine phosphorylated upon NK cell activation. NKp46 and NKp44 were shown to cooperate in the process of tumor cell lysis by human NK cells. However, lysis of certain target cells was only marginally NKp46 and/or NKp44 dependent, since mAb-mediated masking of these molecules did not significantly interfere with cytotoxicity 26 . This finding strongly suggested the existence of additional triggering receptors that could induce cytotoxicity against these target cells. Moreover, although clearly NKp46 and/or NKp44 dependent, the cytolytic activity against other tumor cell lines could not be abrogated by mAb-mediated masking of both molecules, suggesting again the existence of additional receptor(s) cooperating with NKp46 and NKp44. Indeed, we show here that NKp30 represents a receptor that may cooperate with NKp46 and NKp44 in the induction of cytotoxicity against a variety of target cells. Perhaps more importantly, NKp30 represents the major receptor in inducing NK-mediated killing of certain tumor target cells, the lysis of which is largely NKp46/NKp44 independent. Remarkably, NKp30, similar to NKp46, is also involved in NK cell activation and target cell killing by fresh NK cells. As discussed above, the surface expression of NKp30 parallels that of NKp46. Indeed, NK cells displaying an NKp46 dull or an NKp46 bright phenotype were also characterized by NKp30 dull or NKp30 bright fluorescence. We showed previously that NK cell clones characterized by an NKp46 dull phenotype consistently express low amounts of NKp44 26 . The finding that NK cells express parallel densities of different triggering receptors may explain the existence of NK cell subsets displaying different “natural” cytolytic activity. For example, it was difficult to understand why the cytolytic activity against some target cells (such as MEL15), although largely NKp46 independent, was essentially confined to NK clones expressing the NKp46 bright phenotype. These results can now be explained by the finding that only NKp46 bright cells express a high density of NKp30 receptor. Thus, the previous demonstration of major differences in cytolytic activity of NKp46 dull and NKp46 bright cells can now be applied also to NK cells displaying different NKp30 phenotypes. Along this line, the cytolytic activity of NKp30 dull NK cell clones was markedly reduced compared with NKp30 bright clones (data not shown). NKp30, similar to NKp46, associates with CD3ζ, which is most likely involved in signaling via the receptor complex. However, CD3ζ does not appear to be necessary for the surface expression of both receptors, at least in COS-7 cells (this report, and reference 24). Molecular cloning revealed that NKp30 is the product of 1C7, a gene previously mapped on human chromosome 6 in the HLA class III region 38 39 . However, neither the function nor the cellular distribution of the putative product of 1C7 gene was known, and no indications existed of its role in natural cytotoxicity. In addition, the analysis of 1C7 transcript expression was limited to RT-PCR, whereas no detection had been possible by Northern blot analysis 39 . It should also be stressed that no correlation between transcript and surface expression could be established because of the lack of specific mAbs. In this study, we show that a precise correlation exists between the surface expression of NKp30, as determined by staining with three different mAbs, and mRNA expression, as assessed by Northern blot. On the contrary, the detection of 1C7 transcripts by RT-PCR does not allow prediction of the surface expression of the 1C7/NKp30 molecule. In conclusion, the NKp30 molecule represents a third member of an emerging family of receptors, termed natural cytotoxicity receptors (NCRs; 40 ), that are involved in NK cell triggering upon recognition of non-HLA ligands. These receptors appear to complement each other in the induction of target cell lysis by NK cells. The relative contribution of each receptor is likely to reflect the expression/density of their specific ligands on target cells. It has recently been shown that CD16 is also involved in natural cytotoxicity, thus suggesting that in addition to Fc binding and antibody-dependent cell-mediated cytotoxicity, CD16 may play a role in the regulation of NK cell function 41 . Besides CD16 and the different NCRs, several other surface molecules that can mediate NK cell triggering have been identified in humans and rodents. These include CD2 42 43 , CD69 44 , CD28 45 , 2B4 46 , and NKR-P1 47 . However, their actual role in natural cytotoxicity has yet to be clarified, since in most instances these activating structures are not NK restricted. Finally, although the identification of different NCRs constitutes a major step forward in our understanding of the NK cell physiology, both the nature and the distribution of the NCR ligands on target cells remain to be determined. Based on the available data, it is possible to envisage a novel mechanism of tumor escape consisting in the downregulation (on tumor cells) of ligand molecules specifically recognized by NK-specific triggering receptors. Thus, the identification of such ligands will allow the analysis of their distribution in normal versus tumor cells, and define whether a correlation exists between ligand expression and susceptibility to NK-mediated lysis by different tumor cells. | Study | biomedical | en | 0.999998 |
10562325 | The care of experimental animals was in accordance with National Institutes of Health guidelines. Single cell suspensions of lymph node cells and thymocytes were prepared from C57BL/6 or CD8β − mice thymi. Purified populations of CD4 + CD8 + thymocytes >96% pure were obtained by anti-CD8 panning of whole thymocyte populations and selecting the adherent cells, as described previously 17 . Cells (0.5–3 × 10 8 , as indicated) were lysed in ice-cold lysis buffer containing either 1% Triton X-100 or 60 mM octylglucoside. After clarification (10,000 g for 10 min), cell lysates were subjected to immunoprecipitation with the indicated antibodies. Immunoprecipitates were resolved on SDS-PAGE under reducing conditions. Antibodies used for immunoprecipitation were specific for: CD4 (GK1.5 or RM4.5; PharMingen), CD8α (53-6 7 ; PharMingen), or CD8β (53-5.8; PharMingen); and TCR-ζ (serum 551), Lck (serum 688), or LAT 14 . Antibodies used for immunoblotting were specific for: LAT ; Lck (serum 688); or phosphotyrosine (4G10; Upstate Biotechnology). For immunoprecipitations, mAbs were directly coupled to CnBr-activated Sepharose beads (Amersham Pharmacia Biotech), except when indicated otherwise. Pervanadate treatment (final concentration of 0.01 mM Na 3 VO 4 in the presence of 4.5 mM H 2 O 2 , extemporaneously prepared) was conducted for 10 min at 37°C. TCR cross-linking experiments were performed essentially as described 13 . Antibodies used for cross-linking were as follows: anti–TCR-β (H57-597 18 ), anti-CD4 (GK 1.5; PharMingen), and anti-CD8α (2.43 19 or 53-6.7 [PharMingen]). Thymocytes were cultured for 18 h at 37°C, pelleted, and resuspended at 10 7 /ml in ice-cold RPMI containing 1 mM Na 3 VO 4 and biotinylated antibodies (10 μg/ml). After 10 min at 4°C, the cells were pelleted and resuspended at 10 8 /ml in RPMI containing 20 μg/ml of streptavidin (Southern Biotechnology Associates) previously prewarmed at 37°C. After a 5-min incubation at 37°C, cells were pelleted and lysed in octylglucoside-containing buffer. cDNAs encoding mouse CD4, CD8α, and CD8β were provided by Dr. Jane Parnes (Stanford University, Stanford, CA 20 ). Fragments encoding each coreceptor domain, or mutant derivatives thereof, were prepared by restriction enzyme digestion, PCR amplification, or as double stranded oligonucleotides, and ligated to generate the indicated constructs. The CD8α AAM mutation, converting cysteines 227 and 229 to alanines, was introduced using PCR-mediated site-directed mutagenesis 21 . Amino acid sequences (single letter code) at the modified junctions were as follows: ABB, LDFACD/ITTLSL; AAT, LICYA/RSR; ABA, LDFACD/ITTLSL … VYFYCA/RSRKRVC; 4AA, GVNQTD/IYIWAPL; 4BB, GVNQTD/ITTLSL. The sequences of all PCR-amplified and oligonucleotide-encoded regions were verified by dideoxy sequencing for the presence of the desired modifications and the absence of additional mutations. Wild-type and mutant cDNAs were introduced in pcDNA3 (Invitrogen) for expression in 293T cells. Expression vectors for mouse and human LAT have been described 14 22 . 293T cells were transfected using the calcium phosphate coprecipitation method 23 . Total plasmid amount was kept constant among samples by adjusting the amount of empty expression vectors. Cells were harvested 36–40 h after transfection. Expression of the coreceptor derivatives in each sample was verified by cell surface staining with anti-CD4 (GK 1.5), anti-CD8α (53-6.7), and anti-CD8β (53-5.8) mAbs and cytofluorimetric analysis. To determine if coreceptor–LAT complexes existed on unstimulated murine T cells and thymocytes, we immunoblotted anti-CD4 and anti-CD8 immunoprecipitates for LAT . In lymph node T cells that express CD4 and CD8 coreceptors on separate cell populations, we found that LAT was associated with both CD4 and CD8 . In immature CD4 + CD8 + thymocytes, which express both CD4 and CD8 on individual cells, we found that LAT associated with CD8 in significantly greater amounts than with CD4 . Interestingly, the hierarchy of LAT binding in immature CD4 + CD8 + thymocytes (CD8 > CD4) is reciprocal to Lck, which binds CD4 > CD8 . We then analyzed CD8–LAT associations in thymocytes in greater detail. LAT molecules are largely localized to detergent-resistant areas of the plasma membrane referred to as glycolipid-enriched membrane microdomains (GEMs ), which also contain subpopulations of both CD4 and CD8 molecules 27 . Thus, it was conceivable that coimmunoprecipitation of LAT with CD4/CD8 coreceptors might simply reflect the presence of LAT and CD4/CD8 coreceptors in the same GEM. This was not the case, however, as LAT–CD8 associations were maintained in anti-CD8 immunoprecipitations of cells solubilized by the detergent octylglucoside, which solubilizes GEMs . In fact, detergent solubilization with octylglucoside increased the amount of LAT detected in anti-CD8 immunoprecipitates . Next, we assessed whether LAT association with CD8 required both components of CD8 (α and β). In normal mice, CD8 is expressed on thymocytes and thymic-derived T cells as an αβ heterodimer, whereas in CD8β − mice, CD8 is expressed as an αα homodimer 30 31 . Despite similar amounts of LAT in whole cell lysates from normal and CD8β − thymocytes , the amount of CD8-associated LAT was markedly greater in CD8β + thymocytes than in CD8β − thymocytes, although a small amount of LAT binding to CD8αα homodimers was evident in CD8β − thymocytes . Thus, CD8β contributes to CD8–LAT association, as it does to CD8–Lck association 32 33 . For Lck, CD8β is thought to increase accessibility to a binding site present in the cytosolic tail of CD8α, and this may be the case for LAT as well. To determine the molecular basis for LAT–coreceptor associations, we cotransfected 293T cells, a human transformed kidney cell line expressing SV40 T antigen, with murine LAT (mLAT) and murine coreceptor molecules (CD8α and β or CD4). The transfected coreceptor molecules were either wild-type or variants containing altered transmembrane or cytosolic domains . Surface expression of transfected coreceptor molecules was quantitated by immunofluorescence and flow cytometry and was found to be similar within all experimental groups ( Table ). Lysates from transfected 293T cells were immunoprecipitated by anti-CD8α mAbs or anti-CD4 mAb , and the immunoprecipitates were immunoblotted for LAT. Both CD8–LAT and CD4–LAT complexes were observed in 293T cells transfected with intact (AAA, 444) coreceptor molecules , demonstrating that coreceptor–LAT complexes could form in nonlymphoid cells and did not require Lck, which is not expressed in 293T cells. We further documented that LAT did not associate with CD4 or CD8α variants expressing either the cytosolic tail of CD8β (lanes 4 and 12) or lacking a cytosolic tail altogether (lanes 5 and 9). Thus, the cytosolic tails of CD4 and CD8α are the main determinants for LAT association, suggesting that a sequence common to both cytosolic tails is involved in LAT binding. Sequence homology between the cytosolic domains of CD4 and CD8α is restricted to a short region centered around a CxC cysteine motif that is involved in Lck binding 34 35 . To examine the possible role of this cytosolic CxC cysteine motif in LAT–coreceptor associations, we constructed a CD8α variant (AAM) in which both cytosolic cysteines C227 and C229 were mutated to alanines . We found that substitution with alanine of these two cysteines diminished CD8α associations with murine LAT by 70% . Thus, the cytosolic CxC cysteine motif important for coreceptor associations with Lck is also important for coreceptor associations with LAT. Because LAT molecules contain two molecular species that migrate at either 36 or 38 kD in SDS-PAGE 14 22 , we wished to determine if coreceptors preferentially associated with one or the other form of LAT. To address this issue, we used human LAT (hLAT) because the 36- and 38-kD forms of hLAT are easily distinguishable . We found that CD8 coreceptors were associated almost exclusively with the lower 36-kD form of LAT, and that that association was dependent on the cytosolic cysteines in the CD8α tail . However, appearance of the lower 36-kD form of hLAT is dependent on two juxtamembrane cysteines (C26, C29) in the cytosolic tail of hLAT, which are palmitoylated and responsible for targeting LAT molecules to GEMs 22 . Alanine substitution of both cysteines (C26/29A) removes the palmitoylation sites and results in migration of LAT as a single 38-kD (upper) band . Interestingly, despite the fact that the C26/29A mutant LAT molecule cannot be palmitoylated and targeted to GEMs, we found that the mutant LAT can still associate with surface CD8 coreceptor molecules . These results confirm that CD8–LAT associations are the result of specific protein–protein interactions and not simply of colocalization of both molecules in GEMs. However, we do not yet understand why CD8 normally preferentially associates with the lower band of LAT when it is also clearly capable of binding to the upper band. Since the cytosolic cysteine motif in CD8α promotes binding of LAT as well as Lck, we considered that LAT and Lck might compete for binding to individual coreceptor molecules. To address this issue, we transiently expressed CD8α, CD8β, and mLAT in 293T cells. In addition, we also transfected the cells with an excess of either Lck-containing vector or empty vector . Even though Lck did not affect the amount of LAT present in the cell lysates, Lck abrogated the ability of LAT to be immunoprecipitated by anti-CD8α . Thus, Lck protein, when present in excess, competes with LAT for binding to CD8. Since LAT function in TCR signal transduction depends on its tyrosine phosphorylation, we examined the ability of coreceptor-associated LAT molecules to be tyrosine phosphorylated. Treatment of thymocytes with pervanadate to induce activation of intracellular protein tyrosine kinases 36 resulted in tyrosine phosphorylation of CD8-associated LAT and recruitment of LAT-binding phosphoproteins previously identified to include phospholipase C (PLC)-γ1 and Cbl, among others 14 . Tyrosine phosphorylation of coreceptor-associated LAT molecules was also induced in thymocytes by antibody-mediated coengagement of TCR with either CD4 or CD8, but was not induced by TCR engagement alone . Coengagement of TCR with either CD4 or CD8 also resulted in the appearance of phosphoprotein bands indicative of PLC-γ1 and Cbl . Importantly, formation of oligomeric signaling complexes on coreceptor-associated LAT molecules occurred with a remarkable degree of specificity, preferentially forming on coreceptor-associated LAT molecules that had been coengaged with TCR, compared with LAT molecules that had not been coengaged with TCR. That is, PLC-γ1 and Cbl were preferentially recruited to CD4-associated LAT molecules by coengagement of TCR with CD4 compared with coengagement of TCR with CD8 ; and, reciprocally, PLC-γ1 and Cbl were preferentially recruited to CD8-associated LAT molecules by coengagement of TCR with CD8 compared with coengagement of TCR with CD4 . The small amount of phosphorylated Cbl that was immunoprecipitated by antibodies specific for the nonengaged coreceptor probably reflects the passive and inadvertent capture of some nonengaged coreceptor molecules within the TCR aggregate . Thus, coreceptors can promote LAT phosphorylation by TCR-associated ZAP-70 molecules and the subsequent recruitment of downstream signaling mediators. This study demonstrates that the LAT adaptor molecule associates with CD4 and CD8 surface coreceptors, and that such coreceptor associations are mutually exclusive with Lck. Indeed, the site on the cytosolic tail of CD4 and CD8α coreceptors to which LAT binds overlaps the site to which Lck binds, resulting in competition between Lck and LAT for coreceptor binding. Because of their association with coreceptor molecules, LAT molecules would be juxtaposed with TCR complexes upon coengagement of MHC–peptide complexes. In fact, we found that oligomeric complexes of downstream signaling mediators preferentially formed on those LAT molecules that were associated with coreceptors coengaged with the TCR. Thus, this study provides one solution to the problem of how TCR-associated ZAP-70 molecules can efficiently find their LAT substrate. Colocalization of LAT with TCR would be a second function performed by CD4 and CD8 coreceptor molecules in TCR signal transduction, with colocalization of Lck and TCR being the only previously known function. In fact, we think that the two coreceptor functions are analogous to one another, in that coengagement of TCR with CD4 or CD8 coreceptor molecules by MHC–peptide complexes serves to physically juxtapose both Lck and LAT with TCR . As a result, coreceptors promote both the initiation of TCR signaling and the activation of downstream signaling mediators. It is tempting to consider the implications of our present observations on the distinct but overlapping roles performed by CD4 and CD8 coreceptors in promoting TCR signal transduction in immature CD4 + CD8 + thymocytes during T cell development in the thymus, as CD4 engagement by intrathymic MHC class II molecules would preferentially promote Lck activation 25 , whereas CD8 engagement by intrathymic MHC class I molecules would preferentially promote LAT phosphorylation and activation of downstream mediators. It is conceivable that such coreceptor-induced differences in TCR signal transduction pathways influence lineage choices, but this possibility has not yet been examined. Unfortunately, LAT knockout mice have not been informative for this issue, as the absence of LAT arrests thymocyte development at an early CD4 − CD8 − stage of development that precedes expression of CD4 and CD8 coreceptors as well as the point at which CD4/CD8 lineage determination occurs 37 . The association of LAT with surface CD4 and CD8 coreceptor molecules provides one solution to the problem of how TCR-associated ZAP-70 molecules manage to contact and phosphorylate LAT molecules. Indeed, we found that association of LAT with CD4 and CD8 coreceptors was of functional significance for TCR signal transduction, as: (a) coreceptor-associated LAT molecules were tyrosine phosphorylated upon TCR–coreceptor coengagement, and, more importantly, (b) the scaffold for oligomerization of downstream signaling mediators preferentially formed on coreceptor-associated LAT molecules that were coengaged with TCR. Thus, coreceptor-induced colocalization of LAT with TCR promotes downstream TCR signaling events. We found that coreceptor–LAT associations are promoted by the same dicysteine motif in the coreceptor tails that promotes Lck binding. Nevertheless, we do not think that Lck and LAT bind to coreceptor molecules through identical mechanisms. Association of coreceptor molecules with Lck involves formation of a zinc-dependent complex between the dicysteine motif in the coreceptor tail and two cysteines in Lck 38 , and strictly requires those cysteines to be present in both the coreceptor tail and the Lck molecule 34 35 . In contrast, association of coreceptor molecules with LAT does not require cysteines in LAT, as CD8–LAT interactions were not abolished by mutation of LAT dicysteines to alanines. In fact, association of coreceptor molecules with LAT also does not strictly require the presence of the dicysteine motif in the coreceptor tail, as mutation of these coreceptor cysteines did not abrogate (but significantly reduced) association with LAT. Thus, we think that the molecular basis for LAT–coreceptor association is distinct from that of Lck–coreceptor association despite their overlapping binding sites. That Lck and LAT actually bind to overlapping sites in the cytosolic tail of CD8α is indicated by cotransfection experiments in which Lck was found to disrupt LAT binding to CD8. Such mutual exclusivity of binding may account for the opposite coreceptor binding preferences of LAT and Lck in CD4 + CD8 + thymocytes, such that LAT may be primarily associated with CD8 because Lck is primarily associated with CD4. However, we favor the possibility that Lck and LAT have intrinsically different binding preferences for CD4 and CD8 coreceptor molecules such that Lck binds CD4 > CD8 and LAT binds CD8 > CD4. LAT molecules are normally palmitoylated and, as a consequence, localized to GEMs 22 . Importantly, however, LAT–coreceptor associations are mediated through protein–protein interactions and do not simply reflect their colocalization in GEMs, as LAT–coreceptor associations occur even in the absence of LAT palmitoylation. Indeed, a nonpalmitoylated version of LAT (C26/29A LAT) which does not localize in GEMs still associated with CD8. Nevertheless, it should be appreciated that subpopulations of CD4 and CD8 coreceptor molecules are normally present in GEMs 27 , perhaps as a result of their association with palmitoylated LAT molecules. Consistent with such a possibility, we found that detection of CD8–LAT complexes was significantly increased by solubilization of cells with the detergent octylglucoside, which solubilizes GEMs far better than the detergent Triton X-100. Surface CD4/CD8 coreceptors are not strictly required for TCR signal transduction, as CD4 − CD8 − T cells, in the absence of either CD4 or CD8 coreceptor molecules, competently transduce TCR signals. Consequently, there must exist coreceptor-independent mechanisms for promoting LAT phosphorylation by ZAP-70, although they may be less efficient than the physical colocalization resulting from LAT association with surface coreceptor molecules. It is conceivable that trapping of GEM-associated LAT molecules between aggregated TCR complexes can induce LAT phosphorylation. Such a possibility is suggested by the finding that TCR stimulation results in the migration of TCR components into GEMs 39 40 . Importantly, however, it is not at all clear that ZAP-70 actually translocates to GEMs, a necessary event for ZAP-70 phosphorylation of GEM-associated LAT, as conflicting results have been reported 22 39 40 . In any event, the inadvertent trapping of LAT between TCR aggregates would be expected to be significantly less efficient than the juxtaposition of LAT and TCR induced by coengagement of MHC–peptide complexes. It has been suggested that LAT phosphorylation by ZAP-70 may be enhanced by proteins containing Src homology 2 (SH2) domains such as 3BP2, phosphatidylinositol-3 (PI-3) kinase p85 regulatory subunit, or PLC-γ1. 3BP2 is an SH2-containing adaptor protein that binds to tyrosine-phosphorylated forms of LAT and ZAP-70 41 . Because 3BP2 contains only one SH2 domain, it is not evident how it could couple activated ZAP-70 to LAT. PI-3 kinase p85 subunit is a protein that contains two SH2 domains and has been shown in platelets to couple tyrosine-phosphorylated LAT with other tyrosine-phosphorylated molecules such as FcR γ chain 42 . Similarly, PLC-γ also has two SH2 domains and could conceivably couple ZAP-70 to LAT 43 . However, because SH2 domain–containing linker proteins only bind tyrosine-phosphorylated substrates, they would be expected to promote ZAP-70 associations with already phosphorylated LAT molecules; they would not be expected to promote LAT's initial phosphorylation by ZAP-70. Consequently, the molecular basis for the initial phosphorylation of LAT by ZAP-70 in the absence of CD4 and CD8 coreceptors remains uncertain. In conclusion, this study documents the association of LAT with CD4 and CD8 coreceptor molecules in resting T cells and thymocytes, and documents that coengagement of coreceptor molecules with surface TCR results in tyrosine phosphorylation of LAT and recruitment of downstream signaling mediators. | Study | biomedical | en | 0.999999 |
10562326 | A partial clnk cDNA was initially cloned from a mouse primitive hemopoietic cell (EML-16) cDNA library, during a yeast two-hybrid system screen using the cytoplasmic domain of platelet-endothelial cell adhesion molecule (PECAM)-1 as a bait (our unpublished results). Several full-length cDNAs were subsequently isolated from a cDNA library made from day 16 fetal mouse thymus (provided by Dr. L. Matis, Alexion Pharmaceuticals, New Haven, CT), taking the partial cDNA as a probe. The 5′ end of clnk was also verified by 5′ rapid amplification of cDNA ends (RACE; data not shown). Both strands of a representative full-length cDNA clone were sequenced. The clnk cDNA sequence data are available from EMBL/GenBank/DDBJ under accession no. AF187819. The various hemopoietic cell lines used herein were described elsewhere 20 21 22 . Splenic T cells were isolated from 6–8-wk-old C57BL/6 mice using T cell columns (Cytovax Biotechnologies Inc.). More than 90% of cells obtained were CD3 + (data not shown). Splenic T cells were stimulated for 48 h with anti-CD3 mAb 145-2C11 (1 μg/ml 23 ) immobilized on plastic. They were then harvested, washed extensively, and replated for the indicated periods of time in the absence or presence of recombinant mouse IL-2. NK cells were obtained from splenic tissue of 6–8-wk-old Nude CD-1 mice (Charles River Canada), as described previously 24 . Resting spleen cells were expanded for ∼8 d in growth medium containing recombinant IL-2. After this period, nearly 100% of cells recovered were CD16 + and CD3 − (data not shown). Bone marrow–derived mast cells (BMMCs) were established from the femurs of 8-wk-old C57BL/6 mice by prolonged culture (>3 wk) of bone marrow–derived cells in IL-3–containing medium. Approximately 100% of cells obtained with this protocol were positive for Fc∈RI (data not shown). Ribonuclease protection assays were performed as described elsewhere 20 25 , using a radiolabeled antisense riboprobe corresponding to nucleotides 965–1231 of mouse clnk . The integrity of the various RNAs used in these assays was confirmed by electrophoresis of samples in agarose-formaldehyde gels, and subsequent staining of the gel with ethidium bromide (data not shown). Polyclonal antibodies against Clnk were produced by immunizing rabbits with a trpE fusion protein encompassing amino acids 199–301 of Clnk. These antibodies did not cross-react with SLP-76 (data not shown). Affinity purification was achieved by passing the crude serum over a column containing the immunogen immobilized on Affigel (Bio-Rad). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology. Rabbit antibodies directed against Vav have been reported elsewhere 26 . The IL-2–dependent mouse T cell line 5.32.10 22 was activated via the TCR by stimulation for 3 min at 37°C in the presence of anti-CD3 mAb 145-2C11 and rabbit anti–hamster (RAH) IgG. The IL-3–dependent mouse myeloid cell line B6SutA 1 21 was activated via FcγRI by incubation for 3 min at 37°C with mouse IgG 2a followed by F(ab′) 2 fragments of sheep anti–mouse (SAM) IgG. After stimulation, cells were lysed in TNE buffer (1× TNE: 50 mM Tris, pH 8.0, 1% NP-40, 2 mM EDTA), supplemented with protease and phosphatase inhibitors as detailed elsewhere 27 . For immunoprecipitation, postnuclear lysates were incubated with the indicated antibodies for 2 h. Immune complexes were then recovered by the addition of formalin-fixed Staphylococcus aureus (Pansorbin; Calbiochem-Novabiochem). After several washes, proteins were eluted in sample buffer and resolved by SDS-PAGE. For analysis of Clnk expression, cells were lysed directly in boiling SDS-containing sample buffer, and lysates corresponding to equivalent cell numbers were resolved by gel electrophoresis. Immunoblots were done according to a previously described protocol 28 , using either 125 I-labeled goat anti–mouse IgG (ICN Biomedicals) or protein A–horseradish peroxidase and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). For transfections, the mouse clnk and slp-76 cDNAs were individually cloned in the mammalian expression vector pXM139, which contains the adenovirus major late promoter and the SV40 origin of replication. Cos-1 cells were transfected by the DEAE-dextran method with either pXM139 alone or pXM139- clnk (4 μg), as outlined previously 29 . Jurkat TAg cells were transfected by electroporation with pXM139 alone, pXM139- clnk , or pXM139- slp-76 , in the presence of either pNFAT-luciferase, pAP-1–luciferase, or pIL2 promoter–luciferase, according to a protocol detailed elsewhere 15 . After 40 h, 10 6 viable cells were stimulated for 7 h with anti-CD3 mAb OKT3 (10 μg/ml) alone, OKT3 plus PMA (50 ng/ml), or PMA plus ionomycin (0.75 μg/ml). Cells were then lysed and assayed for luciferase activity using the luciferase reporter assay system (Promega) and a luminometer (EG&G Berthold). Results are presented as percentage of luciferase activity induced by PMA plus ionomycin. During an attempt to identify new ligands for the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of PECAM-1 30 31 using the yeast two-hybrid system, we cloned a novel mouse cDNA (see Materials and Methods). Preliminary sequence analysis indicated that this partial cDNA encoded a new SH2 domain–containing molecule (data not shown). Full-length cDNA clones were subsequently obtained through screening of a mouse thymus cDNA library and 5′ rapid amplification of cDNA ends (RACE). Although we were unable to show an association between PECAM-1 and this novel molecule in mammalian cells (our unpublished results), its characterization was pursued as it constituted a potentially interesting novel signal transduction molecule. The deduced amino acid sequence of a representative cDNA clone is shown in Fig. 1 A. This molecule, termed Clnk (see below), is predicted to be a 435 amino acid polypeptide. It contains, from the NH 2 to the COOH terminus: a basic domain, a portion rich in tyrosines and prolines, an SH2 domain, and a COOH-terminal tail . BLAST (available at http://www.ncbi.nlm.nih.gov/BLAST/) searches showed that the SH2 domain of Clnk is most closely related to those of SLP-76 and Blnk (∼40–53% identity, ∼62–69% similarity; references 1–4) . This degree of homology is typical for proteins belonging to the same family. While the overall structures of Clnk, SLP-76, and Blnk are also similar, it should be pointed out that the actual sequences outside the SH2 region of Clnk are quite distinct from those of the two other molecules. Despite this difference, it remains likely that Clnk represents a novel member of the SLP-76 family. Further support for this notion will require comparison of the exon–intron structures of the three slp-76 –related genes. The expression pattern of Clnk was analyzed by ribonuclease protection assay, as detailed in Materials and Methods . Analysis of various mouse tissues indicated that clnk RNA was low or undetectable in most tissues, including bone marrow (lane 3), lymph node (lane 4), spleen (lane 8), and thymus (lane 10). Slightly greater quantities were present in kidney (lane 11). Evaluation of a panel of mouse hemopoietic cell lines revealed that most T cell (lanes 2–5), B cell (lanes 6–11), and macrophage (lanes 12 and 13) cell lines did not contain clnk transcripts. However, a subset expressed easily appreciable amounts of clnk RNA. These included the IL-2–dependent T cell line HT-2 (lane 5), the IL-3–dependent pro-B cell line Ba/F3 (lane 6), the mastocytoma cell line P815 (lane 14), the IL-3–dependent myeloid cell lines B6SutA 1 (lane 15) and FDC-P1 (lane 16), and the primitive leukemia cell line L1210 (lane 17). clnk was also expressed in the IL-2–dependent T cell lines CTLL-2 and 5.32.10 (lane 7), and in the IL-3–dependent myeloid cell line 32D (lane 11). Strikingly, most of the cell lines expressing clnk were dependent on cytokines for sustained growth. On this basis, our novel cDNA was named clnk , for cytokine-dependent hemopoietic cell linker. The distribution of clnk was also characterized in normal hemopoietic cells. Considering our results with the factor-dependent cell lines, it was plausible that cytokine stimulation was required to induce accumulation of clnk in these cell types. To test this possibility, T cells were purified from mouse spleen, and the impact of IL-2 stimulation on clnk expression was evaluated. For these experiments, splenic T cells were pretreated with anti-CD3 antibodies in order to allow expression of functional IL-2 receptors. As expected, resting splenic T cells expressed no appreciable amount of clnk transcripts. In a similar way, cells preactivated with anti-CD3 antibodies for 48 h (lane 2) did not contain clnk , even though they exhibited robust thymidine incorporation (data not shown). However, additional stimulation with exogenous IL-2 (lane 3) induced strong expression of clnk , in a manner comparable to that seen in IL-2–propagated HT-2 cells (lane 4). Next, a time course of IL-2 stimulation was performed . After activation with anti-CD3 antibodies, splenic T cells were washed extensively and replated in growth medium with or without IL-2 for various periods of time. In the absence of exogenous IL-2 (lanes 2 and 3), there was a small increase in the levels of clnk RNA, which was only visible on longer autoradiographic exposures of this gel (data not shown). In the presence of IL-2 (lanes 4–11), though, there was a progressive induction of clnk RNA expression. The enhancement of clnk expression could be observed as early as 24 h after addition of IL-2 (lane 4), and was maintained for at least 10 d (lane 11). Coupled with the findings shown in Fig. 2 , these data strongly suggested that sustained IL-2 stimulation was necessary for induction of clnk expression in T cells. But it was also possible that a small subset of T cells constitutively expressing clnk expanded preferentially in the presence of IL-2. To help resolve this issue, the impact of IL-2 on clnk expression was ascertained in an established T cell line (HT-2). After depriving them of IL-2 for 12 h, HT-2 cells were restimulated with IL-2 for various periods of time, and the levels of clnk RNA were monitored by ribonuclease protection assay . First, this analysis showed that removal of IL-2 (lane 3) caused an approximately fourfold reduction in the abundance of clnk RNA in HT-2 cells, compared with cells grown in the continuous presence of IL-2 (lane 2). Furthermore, it demonstrated that reintroduction of the cytokine (lanes 4–8) provoked a rapid increase (approximately sixfold) in clnk expression, which was maximal after 6–9 h of stimulation (lanes 5 and 6). Thus, these observations were consistent with the idea that clnk expression in T lymphocytes was a consequence of IL-2 stimulation. Finally, the expression of clnk was measured in other cytokine-induced normal hemopoietic cell types . As was the case for IL-2–stimulated splenic T cells (lane 5), we found that clnk RNA accumulated in high amounts in IL-3–propagated BMMCs (lane 4) and IL-2–activated NK cells (lane 6). To identify the protein product of clnk , a polyclonal rabbit antiserum was generated against a bacterial fusion protein encompassing amino acids 199–301 of Clnk. When lysates from various hemopoietic cell lines were probed by immunoblotting with this antibody , we found that HT-2 (lane 1), CTLL-2 (lane 2), Ba/F3 (lane 3), and B6SutA 1 (lane 4), but not BI-141 T cells (lane 5), contained an ∼54-kD immunoreactive product consistent with Clnk. A similar polypeptide was observed in Cos-1 cells transfected with a clnk cDNA (lane 7), but not in control Cos-1 cells (lane 6). The presence of the Clnk protein was also examined in normal hemopoietic cells . This assay demonstrated that IL-3–propagated BMMCs (lane 2), IL-2–stimulated T cells (lane 3), and IL-2–activated NK cells (lane 4) contained easily appreciable amounts of the 54-kD Clnk protein. The nature of the additional immunoreactive products of ∼50 and 44 kD in clnk -expressing hemopoietic cells (lanes 2–4) remains to be determined. To obtain evidence for the participation of Clnk in immunoreceptor signaling, the impact of immunoreceptor stimulation on its state of tyrosine phosphorylation was examined . The IL-2–dependent mouse T cell line 5.32.10 was activated through the TCR by a combination of anti-CD3 mAb 145-2C11 and RAH IgG, and the changes in Clnk tyrosine phosphorylation were monitored by immunoblotting of anti-Clnk immunoprecipitates with antiphosphotyrosine mAb 4G10 . We found that TCR triggering (lane 2) induced an increase in tyrosine phosphorylation of a 54-kD protein (p54) consistent with Clnk. Interestingly, it also provoked the appearance of an ∼92-kD phosphotyrosine–containing molecule (p92) in Clnk immunoprecipitates. Neither p54 nor p92 was present in immunoprecipitates generated with normal rabbit serum (NRS, lanes 3 and 4). The regulation of Clnk was also studied in the IL-3–dependent cell line B6SutA 1 , which can be activated via its high-affinity receptor for IgG (FcγRI) by incubation with mouse IgG 2a followed by F(ab′) 2 fragments of SAM IgG . As seen in 5.32.10 T cells, Clnk became associated with a tyrosine-phosphorylated p92 in response to activation of B6SutA 1 cells (top panel, lane 2). However, it is noteworthy that, in contrast to 5.32.10, the Clnk protein found in B6SutA 1 was constitutively tyrosine phosphorylated (lane 1). While the basis for this difference is not known, it may reflect cell type–specific variations in Clnk regulation. Given the ability of SLP-76 and Blnk to associate with p95 vav 1 17 18 , we wanted to determine whether the Clnk-associated p92 represented Vav. For this purpose, parallel immunoprecipitates from the experiment depicted in Fig. 5 B were probed by immunoblotting with anti-Vav antibodies . Even though Vav could easily be seen in total cell lysates (lanes 5 and 6), we were unable to detect any amount of Vav in Clnk immunoprecipitates (lanes 1 and 2). Thus, it seemed probable that the 92-kD tyrosine-phosphorylated protein interacting with Clnk in response to immunoreceptor stimulation was distinct from Vav. Taking into consideration its relatedness to SLP-76 and Blnk, we wished to assess whether Clnk was able to impact on the outcome of immunoreceptor signaling. For this purpose, the effect of Clnk expression on antigen receptor–mediated activation of NFAT, AP-1, and IL-2 promoter was evaluated . Jurkat T cells were transiently transfected by electroporation with a construct encoding either Clnk or SLP-76, in the presence of NFAT-luciferase, AP-1–luciferase, or IL-2 promoter–luciferase reporter plasmids. 40 h after transfection, cells were stimulated with anti-CD3 mAb OKT3 in the absence or presence of the phorbol ester PMA. After cell lysis, changes in luciferase activity were determined using a luminometer. All results were normalized according to the luciferase activity induced by the combination of PMA and ionomycin. This experiment showed that, like SLP-76 , the Clnk protein markedly enhanced the activation of NFAT , AP-1 , and IL-2 promoter in response to stimulation with anti-CD3 antibodies. An analogous effect was seen in cells treated with anti-CD3 plus PMA. It is of note that both Clnk and SLP-76 were able to induce some extent of transcriptional activation of these promoters in the absence of CD3 stimulation. While the exact significance of this observation is unclear, it may reflect the high levels of protein expression typically achieved in these systems. In this manuscript, we report the identification of a novel SLP-76–related molecule which we have termed Clnk. At this time, the most unique feature of Clnk is its expression pattern. While SLP-76 is found in most, and perhaps all, T cells, NK cells, mast cells, and myeloid cells 5 , and Blnk is seemingly contained in all B cells 1 2 4 , the Clnk protein is absent from most hemopoietic cells. However, significant quantities were uncovered in IL-2–stimulated splenic T cells, IL-2–activated NK cells, and IL-3–propagated BMMCs. In a similar way, expression of Clnk was documented in a variety of IL-2–dependent and IL-3–dependent cell lines. By contrast, it was not found in a large number of cytokine-independent cell lines, with the exception of P815 and L1210. Interestingly, P815 is a mastocytoma cell line carrying an activated mutant of the Kit receptor protein tyrosine kinase 34 . Because this mutation mimics the effects of constitutive stimulation by the Kit ligand, this finding added further credence to the notion that Clnk expression is induced after sustained exposure to cytokines. Furthermore, it raises the possibility that growth factors other than IL-2 and IL-3 may have the capacity to induce Clnk expression. Future studies should be aimed at testing this possibility. The involvement of Clnk in immunoreceptor signaling was first implied by the observation that it became acutely associated with a tyrosine-phosphorylated molecule (p92) in response to stimulation of either TCR or FcγRI. Whereas the identity of p92 remains to be determined, it is likely that this molecule is an effector or a regulator of Clnk. Possibly, p92 allows Clnk to become functionally active in immunoreceptor-stimulated cells. More definitive evidence for the participation of Clnk in immunoreceptor-mediated signal transduction was lent by the finding that Clnk, like SLP-76, was capable of augmenting antigen receptor–induced activation of NFAT, AP-1, and IL-2 promoter in transiently transfected T cells. At first glance, this result may suggest that Clnk and SLP-76 actually have redundant functions in hemopoietic cells. The presence of Clnk could explain the lack of functional abnormalities noted in IL-2–activated NK cells from SLP-76–deficient mice 35 . Nonetheless, it should be pointed out that Clnk and SLP-76 are likely to have specialized roles. Clnk lacks the two DYESP motifs present in the NH 2 -terminal portion of SLP-76, which mediate binding to the exchange factor Vav and the adaptor molecule Nck 12 17 18 19 33 36 . Accordingly, we have been unable to show binding of Clnk to either Vav or Nck (this report; our unpublished results). Even though these interactions are not necessary for SLP-76–mediated activation of NFAT 33 36 , they appear to be required for proper reorganization of the actin cytoskeleton during T cell activation 12 . Likewise, the binding motif for the Gads adaptor molecule in SLP-76 (residues 224–244 ) is not strictly conserved in Clnk, raising the possibility that Clnk does not associate with Gads. Instead, Clnk possesses other sites of tyrosine phosphorylation and proline-rich motifs, as well as an SH2 domain, which presumably allow associations with an alternative set of partners. One of these molecules may be p92, which was tyrosine phosphorylated and became associated with Clnk in response to immunoreceptor stimulation. Clearly, a better understanding of the role of Clnk will come with the identification of these partners. In summary, we have identified a novel SLP-76–related adaptor molecule named Clnk. While Clnk is absent in most hemopoietic cells, it is abundantly expressed in a variety of hemopoietic cell types after sustained exposure to cytokines. Taking into consideration our finding that Clnk was able to regulate immunoreceptor signaling, these results suggest that Clnk may provide a mechanism that modulates immunoreceptor signaling in response to cytokine receptor stimulation. | Study | biomedical | en | 0.999997 |
10562327 | 8–12-wk-old female BALB/c, C57BL/6, and C57BL/6 β 2 -microglobulin–deficient mice were purchased from The Jackson Laboratory. Animals were maintained under a standard protocol with free access to food and water. The generation and characterization of 1D8 and 3E1 anti–mouse 4-1BB mAbs and murine 4-1BB–huIg soluble fusion protein has been previously described 8 , and both antibodies are rat IgG 2A molecules having identical functional properties. 6E9 is a rat IgG 2A anti–human CD40 ligand mAb that does not react with mouse CD40 ligand and was provided by Dr. Tony Siadak (Bristol-Myers Squibb). Female BALB/c mice (The Jackson Laboratory) were immunized intravenously with 10 8 SRBCs (Colorado Serum Co.) on day 0 and challenged 7 wk later in the same manner. In some experiments, mice received multiple challenges at varying time points following the same procedure. huIgG (Calbiochem Corp.) was administered in two doses of 50 μg each on days 0 and 6 and then challenged at varying time points depending on the nature of the experiment with 10 μg of huIgG injected intravenously. Mice were bled at indicated intervals, and total antibody response to solubilized SRBC membrane proteins was measured 11 . Humoral immunity to TNP–Ficoll (TNP-Ficoll-TNP -AGG-AECM-Ficoll), purchased from Biosearch Technologies, was established by injection of 50 μg of TNP–Ficoll intravenously on day 0 and again on day 14. Antibody responses to TNP were measured by ELISA using TNP-conjugated OVA as the substrate. 4-1BB Ig was bound to 96-well plates (Immunolon-2; Dynatech Labs, Inc.) at 0.1 μg/ml in PBS overnight at 4°C. Wells were washed and blocked by incubation for 1 h with specimen diluent (Genetic Systems, Inc.). Antibodies or antisera were diluted or solubilized in specimen diluent for 1 h at 22°C. Wells were washed and incubated with several different reagents, depending on the assay. For routine binding assays and hybridoma supernatant screening, wells were incubated with peroxidase-conjugated goat anti–rat IgG (Calbiochem Corp.). For mAb isotyping, wells were incubated with peroxidase-conjugated isotype-specific mouse anti–rat mAbs (Zymed Labs., Inc.). For pharmacokinetic assay, wells were incubated with biotinylated RG7 (mouse anti–rat κ chain), washed, and then incubated with streptavidin–HRPO (horseradish peroxidase; Amersham). After final washing, all assays were developed with TMB substrate (3,3′5,5′-tetramethylbenzidine; Kirkegaard & Perry Labs., Inc.). Reactions were stopped with the addition of 1 N H 2 SO 4 , and optical density was measured at 450–725. ELISAs for monitoring ligand blocking of 4-1BB–Ig were performed as described previously 8 . For anti-SRBC responses, mice were bled at indicated intervals, and total antibody response to SRBC membranes was measured in ELISA with HRPO-conjugated antibodies (Amersham). Solubilized SRBC membrane proteins were prepared as previously described and coated overnight onto Immunolon II plates (Dynatech Labs, Inc.) at 2.5 μg/ml PBS and washed five times before the mouse serum to be assayed was added. Serial dilutions beginning at 1:5 of each sera sample were performed in triplicate as described earlier. After a 30-min incubation at 4°C, the plates were washed five times with PBS before the addition of HRPO-conjugated anti-rat isotype-specific antibody . After a second 30-min incubation at 4°C, the plates were washed five times and then developed with TMB. We analyzed the serum half-life of all of our anti–4-1BB mAbs by pharmacokinetic analysis. 200 μg of the rat (IgG 2A ) mAb 1D8 was injected intravenously into the tail vein on days 0, 2, 4, and 6 into five mice. The mice were periodically bled, and serum levels of rat IgG 2A were determined in triplicate by ELISA for each mouse and expressed as the mean ± SD. The serum half-life of anti–4-1BB mAb 1D8 was found to be 7.5 d . Identical data were obtained for 12 other rat IgG 2A isotype anti–4-1BB mAbs tested. When injected into mice, cellular antigens such as SRBC or soluble proteins such as huIgG are strong, T cell–dependent antigens. To determine the effect of anti–4-1BB mAbs on the generation of humoral immunity to these antigens, we injected two groups of 15 female BALB/c mice with either SRBC (group I) or huIgG (group II). Each group was divided into three sets of five mice. One set was injected intravenously with 200 μg of 1D8 mAb in 200 μl of PBS beginning on the day of immunization and continuing every other day for four days (800 μg total). A second set received the isotype-matched negative control mAb 6E9, and the third set received PBS. In later experiments, as little as 50 μg of anti–4-1BB was injected with similar results. The mice were bled weekly, and their anti-SRBC or -huIgG serum titers were determined in triplicate by ELISA. At week 5, the mice were challenged with antigen. The results, repeated three times with similar data, are shown in Fig. 2 . The mice produced a primary and a vigorous secondary humoral response to SRBC and huIgG . However, when coinjected with anti–4-1BB mAb 1D8, the animals generated neither a primary nor secondary humoral response. By week 12, a small but significant anti-SRBC response occurred in the suppressed mice, and the response could be boosted with subsequent antigenic challenge. The isotype control mAb had no effect on the development of a primary or secondary humoral response to either antigen. To determine how long after antigen injection anti–4-1BB mAbs would block humoral immunity to SRBC, we injected two groups of mice with SRBC. One group of mice received a single injection of 200 μg of anti–4-1BB mAb 1D8 on day 0. The second group of mice was similarly treated, except that anti–4-1BB administration did not occur until day 10 after immunization. Fig. 3 demonstrates that antibody no longer inhibited the development of humoral immunity when the injection of anti–4-1BB mAbs was delayed by 10 d. In subsequent experiments, it was found that full suppressive activity of anti–4-1BB mAbs could be obtained 72–96 h after immunization (data not shown). TNP–Ficoll induces a type II TI humoral immune response. Type II TI responses do not require T–B cell cognate interactions but are greatly enhanced by T cell help 12 . Anti–4-1BB mAbs were injected into mice immunized with TNP–Ficoll, and the antibody response to TNP was measured by assaying serum samples for their reactivity with TNP–OVA. Fig. 4 demonstrates that injection of anti–4-1BB mAbs did not inhibit the ability of the mice to generate a humoral anti-TNP response. These results suggest that anti–4-1BB mAbs do not affect B cell function, an observation consistent with the fact that murine B cells do not express the 4-1BB receptor. We have previously shown that anti–4-1BB mAb is a potent costimulatory reagent for CD8 + T cells and, to a lesser degree, for CD4 + T cells 8 . To determine if CD8 + T cells are required to mediate anti–4-1BB inhibition of T cell–dependent humoral immunity, we immunized C57BL/6 β 2 -microglobulin–deficient mice with SRBC. These mice fail to develop normal CD8 + T cells during thymic selection due to their failure to express functional MHC class I molecules. Simultaneous injection of anti–4-1BB mAb completely blocked both primary and a secondary anti-SRBC response despite the absence of CD8 + T cells (not shown). Furthermore, T cell adoptive transfer into naive recipients could not transfer 4-1BB mAb–induced suppression. Splenic T cells (4 × 10 7 ) from SRBC-immune mice that were immunized and injected with either anti–4-1BB mAb or an isotype control mAb was injected intravenously into naive BALB/c mice together with SRBC. All of the recipient mice produced normal primary and secondary humoral responses to SRBC, showing no sign of adoptive suppression (not shown). We also wished to know if helper T cell function was affected by anti–4-1BB mAbs. C.B-17 SCID mice were reconstituted with BALB/c T and B cells in the following manner. Three groups of mice, five mice per group, were reconstituted with (a) T and B cells from naive untreated mice, (b) T cells from SRBC-immunized and anti–4-1BB–injected mice and B cells from untreated mice, or (c) T cells from untreated mice and B cells from SRBC-immunized, anti–4-1BB–injected mice. All recipient groups were injected with SRBC and challenged 5 wk later. In the total procedure, the donor mice received a single injection of 200 μg of antibody and two injections of SRBC. The recipient mice received two injections of SRBC only. The results shown in Fig. 5 demonstrate that adoptive transfer of T cells from SRBC-immunized and anti–4-1BB–injected mice along with B cells from untreated mice failed to generate an antiSRBC humoral response. This procedure was carried out under conditions in which T and B cells from untreated mice injected into C.B-17 SCID mice generated a primary and secondary response to SRBC. Likewise, adoptive transfer of T cells from untreated mice together with B cells from SRBC-immunized and anti–4-1BB–injected mice into C.B-17 SCID mice produced normal primary and secondary humoral responses to SRBC. Anti–4-1BB mAb is a potent costimulatory agent for T cells, especially CD8 + T cells 8 . We report here that anti–4-1BB mAbs effectively block the humoral immune response of B cells against T cell–dependent antigens. A series of experiments designed to explore the mechanism of 4-1BB–mediated suppression of humoral immunity is described in this paper. Antibody against the 4-1BB molecule, which is not expressed on B cells, does not inhibit the function of B cells directly, as demonstrated by several independent pieces of evidence. First, anti–4-1BB mAbs do not inhibit the T cell–independent antibody production by B cells. Second, B cells from suppressed animals are fully active in generating humoral immunity when T cell help from nonsuppressed animals is provided. Lastly, a 4-1BB–huIg fusion protein that binds to the 4-1BB ligand expressed by B cells cannot inhibit antigen-specific T cell–dependent humoral immunity to SRBC (data not shown). These observations point to suppression of T cell help by anti–4-1BB mAbs, the induction of helper T cell anergy by anti–4-1BB mAbs, or deletion. T cell help is provided by CD4 + T cells. As these cells express 4-1BB, it is conceivable that the antibody directly induces anergy of these cells. This possibility is consistent with our previously published findings demonstrating that although anti–4-1BB mAbs profoundly costimulated anti-CD3–activated CD8 + T cells to proliferate, they marginally activated CD4 + T cells 8 . In the same report, we demonstrated that there were marked qualitative and quantitative differences in protein tyrosine phosphorylation between CD8 + and CD4 + T cells. Most notably, phosphorylation patterns obtained with CD4 + T cells after stimulation with anti-CD3 and anti–4-1BB were similar to the pattern observed when T cells are stimulated with TCR/MHC-restricted, partially agonistic peptides 13 . Indirect induction of helper T cell anergy through suppressor cells is an alternative explanation for which our experiments did not provide convincing evidence. An exclusive involvement of CD8 + suppressor T cells can be ruled out by our observation that suppression was observed in mice that lacked CD8 + T cells. However, these animals may have developed alternative methods of helper T cell regulation, e.g., using CD4 + T cells or CD4/CD8 double-negative T cells for this regulatory purpose 14 . It may be informative to compare CD4 + T cell anergy induced with anti–4-1BB mAbs with CD4 + T cell anergy induced by SAg 15 16 . SAg has been shown to activate a majority of responding CD4 + T cells, with subsequent apoptotic death of most of them; a minority of responding T cells survive a primary SAg stimulus with long-term anergy 15 16 17 18 . Anergy is controlled by regulatory (suppressor) T cells, the removal of which restores the proliferative response of “anergic” CD4 + T cells to SAg stimulation 19 . The regulatory T cells appear to be idiotype specific, and they regulate the activity of preactivated (memory) T cells but not that of naive CD4 + T cells. In BALB/c mice, the regulatory T cells are found in the CD8 + T cell population. Such regulatory CD8 + T cells may function in two ways to inhibit CD4 + T cell activity: they may destroy their targets 20 or induce proliferative anergy 19 21 . It has been shown in mice that express the transgene for a SAg-reactive Vβ chain in T cells and therefore lack TCR diversity that anergy of preactivated CD4 + T cells is mediated by CD4/CD8 double-negative CD3 + T cells 14 . The assumption of a role for regulatory cells in mediating 4-1BB mAb–mediated suppression of helper T cell function in the humoral response to T cell–dependent antigens is compatible with the findings presented in this report. Regulatory T cells may express the CD8 + phenotype, but this is not an absolute requirement. Therefore, anti–4-1BB mAb–mediated suppression of humoral immunity in CD8 T cell–deficient mice is not contradictory to the concept. The fact that regulatory T cells affect preactivated (memory) CD4 + T cells but not naive T cells in the SAg system is consistent with the notion in this study that a 4-1BB mAb–suppressed T cell population is not conferring helper T cell anergy to CD4 + T cells. Taking all these considerations into account, we may conclude that 4-1BB mAb may induce helper T cell anergy in the humoral immune response by directly blocking helper T cells or by inducing regulatory T cells that block the activation as well as the reactivation of antigen-specific helper T cells on a long-term basis. This study introduces a unique agent to suppress the humoral immune response to T cell–dependent antigens in a lasting and exhaustive fashion. The antibody does not induce humoral immunity against itself and can therefore be repeatedly employed without concern for loss of efficacy due to antibody production against therapeutic mAbs and against other immunogenic biologics. The antibody may be considered for active suppression of antibody-mediated autoimmune reactions. | Study | biomedical | en | 0.999998 |
10562328 | Recombinant murine IL-12 was provided by Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan). FITC-conjugated anti–mouse T1/ST2 Ab (clone DJ8) was purchased from Morwell Diagnostics GmbH. Levels of IL-4, IL-5, IL-10, and IFN-γ were determined by ELISA kit (Genzyme). Lymphoid cell growth medium consisted of RPMI 1640 medium (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum, 100 μM 2-ME, 5 U/ml penicillin, and 50 μg/ml streptomycin. Bone marrow cell culture medium consisted of αMEM (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum, 5 U/ml penicillin, and 50 μg/ml streptomycin. The T1/ST2 locus on the mouse genome has been submitted to the EMBL/GenBank/DDBJ database . A targeting vector was designed to replace a 1,280-bp genomic fragment encoding amino acids 1–96 of the mature T1/ST2 with neomycin resistance gene ( neo ) from pMC1-neo (Stratagene). The targeting vector was flanked by the 5.8-kbp fragment at the 3′ end and the 1.0-kbp fragment at the 5′ end. A herpes simplex virus–thymidine kinase gene (HSV-tk) was inserted into the 3′ end of the vector. The targeting vector was linearized with SalI and electroporated into E14.1 embryonic stem (ES) cells. The clones resistant to G418 and ganciclovir were screened by PCR and confirmed by Southern blot analysis with the probe shown in Fig. 1 A for homologous recombination. An external primer (5′-AGGTGGATTATGACGTTGTGCTCATGG-3′) specific for the T1/ST2 gene upstream of the targeting construct and a neo primer (5′-ATCGCCTTCTATCGCCTTCTTGACGAG-3′) specific for neo were used in the PCR screening. Generation of chimeric mice and mutant mice was essentially as described previously 30 . Splenocytes were incubated with biotinylated anti–I-A b Ab (clone 25-9-17; PharMingen), biotinylated anti-CD8 Ab (clone 53-6.7; PharMingen), biotinylated anti–pan-NK cell Ab (clone DX5; PharMingen), biotinylated anti-CD45R Ab (clone RA3-6B2; PharMingen), and streptavidin microbeads (MACS; Miltenyi Biotec) followed by magnetic field separation to remove MHC class II, CD8, and B220-expressing cells and NK cells. Cell purity was analyzed by flow cytometry using FITC-conjugated anti-CD3 Ab (clone 145-2C11; PharMingen) and PE-conjugated anti-CD4 Ab (clone L3T4; PharMingen). Purified cells were cultured on 10 μg/ml anti-CD3 Ab–coated plates (clone 145-2C11; PharMingen) in the presence of exogenous cytokines and anticytokine Ab as indicated. Th2 differentiation was promoted in the presence of 1,000 U/ml IL-4 (PharMingen) and 10 μg/ml anti–IFN-γ Ab (clone XMG1.2; Endogen), whereas Th1 differentiation was promoted in the presence of 2 ng/ml IL-12. After the 5-d culture, cells were harvested and washed with HBSS. Cells (10 6 /ml) were stimulated on an anti-CD3 Ab–coated 96-well plate for 24 h. Supernatants were analyzed by cytokine ELISA. After 8 d of culture, cells were also analyzed for intracellular cytokines. Staining was performed by Cytofix/CytoPerm Plus kit (PharMingen). The following Abs were used: Cy-Chrome–conjugated anti-CD4 Ab (clone H129.19; PharMingen), PE-conjugated anti–IL-4 Ab (clone BVD4-1D11; PharMingen), and FITC-conjugated anti–IFN-γ Ab (clone XMG1.2; PharMingen). Samples were analyzed by three-color flow cytometry, and gates were set on CD4 + lymphocytes. 800 third-stage Nippostrongylus brasiliensis larvae were injected subcutaneously into 10-wk-old T1/ST2 −/− and wild-type control mice. Mice were killed 8 d after infection, and CD4 + T cells were enriched from the spleen by positive selection on LS + columns (MACS), according to the manufacturer's instructions (Miltenyi Biotec). CD4 + T cells were cultured at 10 6 /ml in 96-well plates coated with 10 μg/ml anti-CD3 Ab for 24 h. Supernatants were analyzed by cytokine ELISA. In a separate experiment, serum Ig levels were measured by ELISA. Bone marrow was aseptically flushed from femora of 8-wk-old mice. The cells were cultured at 10 6 /ml in bone marrow cell culture medium supplemented with 10% (vol/vol) PWM-stimulated spleen cell conditioned medium 31 for 3 wk. The bone marrow cultures were enriched for mast cells by repetitively transferring the suspension cell fraction into fresh culture flasks. After 3 wk of culture, culture medium was changed to bone marrow cell culture medium containing 10 ng/ml recombinant mouse IL-3 (Genzyme/Techne). After 1 wk of culture, mast cells were analyzed for T1/ST2 expression by incubating with IgE,κ (clone IgE-3; PharMingen), PE-conjugated anti–c-Kit (clone 3C1; Immunotech), and 0.8 μg/ml FITC-conjugated anti-T1/ST2 Ab, followed by biotinylated anti–mouse IgE (clone R35-92; PharMingen) and Cy-Chrome–conjugated streptavidin (PharMingen). After washing, cells were resuspended at 10 6 /ml and stimulated for 24 h with the polyclonal activator PMA at 50 ng/ml and 1 μM ionomycin. Supernatants were analyzed by cytokine ELISA. IgE cross-linking was performed by first incubating the cells for 30 min at 37°C in the presence of 3 μg/ml of monoclonal mouse IgE anti-dinitrophenyl group (DNP) (clone SPE-7; Sigma Chemical Co.). The mast cells were washed three times and then incubated with 10 ng/ml DNP 30–40 HSA (Sigma Chemical Co.) for 30 min at 37°C. After the treatment with antigens, supernatants were measured for histamine concentration by RIA (SRL, Inc.). Immunization and exposure of mice were carried out according to the method of Foster et al. 32 . In brief, 8- or 10-wk-old mice were sensitized by intraperitoneal injection with 50 μg OVA/1 mg aluminum hydroxide in 0.9% saline on days 0 and 12. Nonsensitized mice received 1 mg of aluminum hydroxide in 0.9% saline. On day 24, mice were placed individually in a chamber and inhaled aerosolized OVA (10 mg/ml) in 0.9% saline (sensitized, OVA-challenged group) for 30 min three times at 1-h intervals, and then every second day thereafter, for 8 d. Nonsensitized mice received saline only (nonsensitized, nonchallenged group). The aerosols were generated by ultrasonic nebulization. 24 h after the last aeroallergen challenge, the mice were anesthetized by intraperitoneal injection of pentobarbital (80 mg/kg body wt) and used for the following experiments. The blood was collected, and serum Ig levels were measured by ELISA. The trachea was cannulated, and the airway lumina were washed twice with 0.5 ml of PBS (Ca 2+ , Mg 2+ free) supplemented with 50 μM EDTA and 0.1% BSA. The bronchoalveolar lavage (BAL) fluid was immediately centrifuged (10 min at 4°C, 700 g ). The BAL cells were resuspended in 0.2 ml of rat serum and counted using a hemocytometer. For differential cell counts, 2–4 × 10 3 cells were spun onto glass slides, air dried, fixed with ethanol, and stained with Giemsa solution. The numbers of eosinophils, neutrophils, lymphocytes, and macrophages in 200 cells were counted based on morphology and staining characteristics. Serum IgG1 level was measured as described previously 33 , except that the following reagents were used: goat anti–mouse IgG1, mouse IgG1, biotinylated goat anti–mouse IgG1, and streptavidin-conjugated alkaline phosphatase (Southern Biotechnology Associates). Serum IgE level was measured according to protocol supplied by PharMingen. The mouse T1/ST2 gene was disrupted by homologous recombination in E14.1 ES cells. A targeting vector was designed to delete the first and second ATG codons of T1/ST2 with the neomycin resistance gene . 2 out of 733 clones screened were positive for homologous recombination. Two targeted ES clones were microinjected into C57BL/6 blastocysts, and successfully transmitted the disrupted T1/ST2 gene through the germline . T1/ST2 −/− mice were born at the expected Mendelian ratios (+/+: +/−: −/− = 55: 96: 49). The mice grew healthy and showed no obvious abnormalities until 20 wk old. Homozygous mutants of both sexes were fertile, and there was no significant difference in the birth rate between T1/ST2 −/− and wild-type mice. Thus, T1/ST2 was not required for mouse development or fertility. Reverse transcription (RT)-PCR analysis using total RNA from bone marrow cells of T1/ST2 −/− mice confirmed the absence of expression of T1/ST2 mRNA . T1/ST2 has been shown to be abundantly expressed on the cell surface of mast cells and Th2 cells 26 27 28 29 . When bone marrow cells were cultured in the presence of IL-3, these cells differentiated into mast cells expressing both high-affinity IgE receptor, Fc∈RI, and c-Kit receptor. Analysis by two-color dot plot revealed that >99% of the cells were IgE + c-Kit + double positive (data not shown). In wild-type mice, these bone marrow–derived mast cells expressed T1/ST2 on their cell surface . In contrast, T1/ST2 was not expressed on T1/ST2 −/− mast cells. When splenic CD4 + T cells were skewed for Th2 cell differentiation, several cells expressed T1/ST2 on their cell surface (data not shown). However, Th2-skewed CD4 + T cells from T1/ST2 −/− mice did not express T1/ST2 at all. Thus, targeted disruption of the T1/ST2 gene resulted in no expression of T1/ST2 protein in mice. FACS ® analysis of the expression of CD3, B220, CD4, CD8, I-A b , and IgM in thymocytes and splenocytes showed that lymphocyte composition was not altered in 6-wk-old T1/ST2 −/− mice compared with wild-type mice of the same age (data not shown). It has recently been shown that treatment of mice with anti-T1/ST2 Ab resulted in decreased Th2 cell–mediated functions, indicating that T1/ST2 might be functionally associated with Th2 cells 26 27 . Therefore, we analyzed in vitro and in vivo Th2 responses of T1/ST2 −/− mice. First, we explored in vitro differentiation assays to assess the effect of T1/ST2 deficiency on cytokine production from Th cells. CD4 + T cells were cultured for 5 d in the presence of IL-12 or IL-4 plus anti–IFN-γ Ab to develop into Th1 or Th2 cells, respectively. Th1-developing cells from both wild-type and T1/ST2 −/− mice produced almost equal amounts of IFN-γ . Th2-developing cells from wild-type mice produced increased levels of Th2 cytokines such as IL-4, IL-5, and IL-10. The cells from T1/ST2 −/− mice also produced the equivalent levels of Th2 cytokines. Th cells from wild-type mice developed into IL-4 producers in response to IL-4 or into IFN-γ producers in response to IL-12 . The cells from T1/ST2 −/− mice developed into IL-4 or IFN-γ producer cells as did the cells from wild-type mice. These results indicate that IL-4–induced in vitro Th2 cell differentiation was not affected in T1/ST2 −/− mice. Next, we examined in vivo Th2 response by infecting with the intestinal parasitic nematode N. brasiliensis . CD4 + splenic T cells were prepared 8 d after infection and stimulated with immobilized anti-CD3 Ab for 24 h. The supernatants were assayed for cytokine production by ELISA. Both wild-type and T1/ST2 −/− CD4 + T cells produced the same amounts of IL-4 . IFN-γ production from both CD4 + T cells was almost the same. IL-4 is shown to be required for Ig class switching of B cells to IgE and IgG1 producers 20 21 34 35 36 . Therefore, we measured serum Ig levels before and after N. brasiliensis infection. Total serum IgE and IgG1 levels after infection were almost equivalent in both wild-type and T1/ST2 −/− mice . These data indicate that T1/ST2 is not functionally involved in Th2 response in vivo. T1/ST2 has been shown to be expressed on bone marrow–derived cultured mast cells (BMCMCs) and peritoneal mast cells and to be regarded as a cell surface marker for the mast cell lineage 28 29 . Mast cells have been shown to secrete IL-4 and histamine when stimulated. We analyzed production of these mediators from BMCMCs. Both wild-type and T1/ST2 −/− BMCMCs secreted almost the same levels of IL-4 upon stimulation with PMA and ionomycin . Further, both wild-type and T1/ST2 −/− cells released almost equal levels of histamine in response to IgE and Ag . Mast cells were classified into two subtypes: the connective tissue–type mast cell, which is found in the skin, musculature, perivascular tissues, and peritoneal cavity, and the mucosal mast cell, which is widely distributed in mucosal tissues of the respiratory tract and the intestinal lamina propria 37 . The number of connective tissue–type mast cells in the back skin of T1/ST2 −/− mice was almost the same as that of wild-type mice. The number of mucosal mast cells in the lamina propria of the stomach was also equivalent between T1/ST2 −/− and wild-type mice, indicating that mast cell development is not affected in T1/ST2 −/− mice (data not shown). Thus, our data suggest that T1/ST2 expression does not contribute to development and function of mast cells. It has been shown that Th2-mediated eosinophilic lung inflammation of airways is attenuated by pretreatment with anti-T1/ST2 mAb 27 . Therefore, we examined whether T1/ST2 deficiency influences inflammatory responses in the murine model of Th2-dependent allergic airway inflammation. Wild-type and T1/ST2 −/− mice immunized with OVA were exposed to an aerosol of OVA. 24 h after OVA challenge, mice were bled, and serum concentrations of IgE and IgG1 were measured by ELISA. Serum concentrations of IgE and IgG1 were equally increased after OVA treatment in both wild-type and T1/ST2 −/− mice . We also analyzed the cells in BAL fluid 24 h after the exposure to OVA aerosol. The total cell number of BAL cells was dramatically increased after OVA exposure in both wild-type and T1/ST2 −/− mice . Furthermore, a dramatic increase in eosinophils was observed in both wild-type and T1/ST2 −/− mice. Histological analysis of lung taken from both wild-type and T1/ST2 −/− mice exposed to OVA displayed severe widespread inflammatory infiltration (data not shown). Thus, T1/ST2 −/− mice exhibited normal eosinophilic response in the mouse model of allergic airway inflammation. In this study, we generated T1/ST2 −/− mice and analyzed their phenotype. T1/ST2 has been shown to be expressed on the cell surface of Th2 cells, but not Th1 cells. Furthermore, administration of anti-T1/ST2 Ab resulted in reduced Th2-mediated functions in mice 26 27 . Thus, T1/ST2 has been implicated in Th2-mediated functions. However, T1/ST2 −/− mice displayed almost normal Th2 responses in both nematode infection and allergic airway inflammation. These results clearly demonstrate that T1/ST2 is not essential to Th2 cell functions. Since it has been shown that treatment of Th2 cells with anti-T1/ST2 Ab enhances complement-mediated lysis in vitro 26 , the reduced Th2-mediated functions observed in the studies with anti-T1/ST2 Ab administration might be a result of complement-mediated elimination of the T1/ST2-positive cell population. In addition to Th2 cells, mast cells express high levels of T1/ST2. However, development and activity of mast cells were unaffected in T1/ST2 −/− mice. Thus, our knockout study reveals that T1/ST2 does not play a critical role in development and function of Th2 cells and mast cells, although we cannot rule out the possibility that T1/ST2 may play some role in Th2 response. Alternatively, T1/ST2 may have an unknown function in these or other cell populations. At present, a ligand for T1/ST2 has not been identified. The identification of the ligand will reveal a novel function of T1/ST2 as well as its role in Th2 response. | Study | biomedical | en | 0.999995 |
10577025 | What is the relative size of the forces that ions experience while passing through a channel? To enter the channel ions have to become partly, if not completely dehydrated. Highly polar and even charged functional groups forming the channel walls compensate for this loss of hydration energy. In addition, there are two kinds of strong ion–ion forces: long range electrostatic forces and short range hard core interactions leading to volume exclusion effects. Considerable simplifications are necessary to compute current–voltage curves of physical model channels. At present only two theories of ion transport are sufficiently simple for direct comparison between theory and experiment. These two theories consider different parts of the relevant forces as the most strong ones. In reaction-rate theories, also designated barrier models, the interaction of the ion with its environment and volume exclusion effects among ions are considered to dominate. Neglecting electrostatic ion–ion interactions, the rates of barrier crossing can be computed from first principles . In contrast, Poisson-Nernst-Planck (PNP) theory assumes that electrostatic forces between the ions determine ion transport. Reaction-rate theories apply only if environmental forces surpass the electrostatic forces between ions. Otherwise, environmental interactions would not determine ion transport. Consequently, typical energies of electrostatic ion–ion interactions inside the channel represent lower limits for the energy differences between barriers and wells. Using a simple Coulomb law with a dielectric constant of 10, the energy required to bring two positively charged monovalent ions as close as 0.58 nm requires at least 250 milli electron volts (10 kT ). Thus, environmental forces only dominate if the barrier energies are much larger. Reaction-rate theories explain the saturation of the channel conductance with increasing external concentrations by hard core ion–ion interactions. To experience the short range volume exclusion interactions, the ions must come rather close to each other. The distances of closest approach between ions including a single intermediate water molecule are in the order of 0.3–0.5 nm. Consequently, reaction-rate theories predicting conductance saturation automatically involve strong electrostatic ion–ion interactions. Thus, even single ion channels require large barriers to dominate these electrostatic forces between ions. A more general discussion of the forces important for ion transport was published recently . The identification of the dominant forces is crucial for understanding ion transport. Only those models of ion permeation that include the strongest interactions can provide a reasonable picture of what is going on inside biological ion channels. In his editorial, Andersen 1999 questions the applicability of the mean field approximation for the situation of ions in the narrow pore of the channel: “Finally, notwithstanding the utility of the mean field approximation, is it appropriate for narrow channels that are occupied by only a few ions?” Similar doubts are mentioned in other Perspectives . Therefore, a critical inspection of the concepts of mean fields and mean field approximation in statistical physics applied to biological ion channels is timely. Whereas the introduction of mean fields generally does not involve any approximation, the mean field approximation is necessary to account for the nonlinear long-range electrostatic interaction between permeant ions. During a 10-pA, 1-s channel opening, 6.3 · 10 7 ions pass the channel. With a time resolution in the millisecond range, the experimental mean current samples >60,000 ions. Passing the bottleneck of the channel each of those ions will see different forces. Side chains at the channel wall may change their orientations between the passage of two ions, and sometimes they even block the path. Also, the position of other ions differs at different ion passages, resulting in different electrostatic forces. For instance, the forces seen by an ion entering a channel differ considerably whether the channel is occupied by another ion or not. However, these very different forces add up linearly. If the channel is 50% occupied by other ions, on the average the incoming ion sees a half-occupied channel. The average of 10 5 different configurations results in a mean force. The absolute value of the force seen at each passage is generally large compared with that of the mean force. What is important for the measured mean current is the mean force, the linear average over all those very different contributions. We do not measure mean forces, but their integrals over the paths of the ions. Current–voltage relations represent integral properties of the channel . Many particularities of the channel structure are averaged out. This explains why extremely simple theories, such as reaction-rate theories with few barriers or PNP equations without particular structural elements, often can reproduce experimental data. Since we are measuring integral channel properties averaged over many configurations, mean fields are the appropriate physical tools to mathematically describe ion transport through biological ion channels. Unfortunately, the introduction of mean forces is not sufficient to handle the strong, long-range electrostatic ion–ion interactions. To describe the behavior of plasmas, Vlasov 1938 approximated the mean conditional force seen by a single ion by the mean force, the electric force of the mean charge density. This approximation generalizes the Gouy-Chapman theory to nonequilibrium systems. What is the difference between the mean force and the conditional mean force? One of the oldest problems in physical chemistry is the nonlinear concentration dependence of the conductivity of electrolytes. In 1926, Onsager 1926 , Onsager 1927 explained this effect by the difference between the mean force (due to the electric field across the electrolyte) and the conditional mean force (the mean force seen by a single ion). The nonlinear deviation originates from the fact that the motion and distribution of ions in solution is correlated. This correlation leads to two mechanisms: electrophoresis and electropolarization. Each moving ion pushes a part of the solvent molecules, and thus induces hydrodynamic ion–ion forces. This electrophoresis results in a modified effective mobility of the ions in the solvent . The electric field deforms the counter ion cloud around each ion. This polarized counter ion cloud produces a local electric field that shields its host ion from the external electric field. Recently, Lehmani et al. 1997 included ion–ion correlations in the conductivity of ion-exchange membranes with large pores. Also within biological ion channels, one should expect effects because of ion–ion correlations. Solvent-mediated ion–ion interactions lead to single filing. This “electrophoretic” effect may be strong in channels such as the potassium channel. Consequently, PNP theories need to implement this mechanism, as for instance suggested by Conti and Eisenman 1966 . Also, electropolarization may influence ion transport. Because of long-range strong electrostatic interactions, the occupancy of the channel depends on whether an ion is placed at the channel entrance or not. Therefore, the electric field due to the other mobile ions seen by the ion ready to enter differs from the field of the mean ion distribution at the same position. How reliable are these mean field approximations? In physics, such mean field approximations are frequently employed with different success. Unfortunately, the comparison between the ion concentrations obtained from PNP and Brownian dynamics cannot be used to evaluate the PNP solution because the two ion channel models employed different forms of electrostatic interactions . However, excellent tools to test the quality of mean field approximations are the so-called sum rules . These are exact relations generally derived for equilibrium systems; any exact theory strictly obeys these rules. There are two rules that are particularly relevant for homogeneous and inhomogeneous electrolytes . The screening sum rule state that all charges, dipoles, and higher multipoles within an electrolyte system are screened by respective counter charges, dipoles, or higher multipoles. It has been shown that the PNP theory fulfills this rule exactly. The other important sum rule is the contact theorem. It relates the electric field at a certain boundary to the respective ion concentrations. PNP follows this rule only approximately . The agreement becomes almost exact in the case of strong electric fields. Because both sum rules apply to equilibrium, they justify to some extent the neglect of the counter ion cloud polarization. However, they say nothing about the importance of single filing. Thus, in accord with the estimates of the size of the forces inside the channel, the PNP theory, using the mean field approximation for the electrostatic interactions, is the method of choice for modeling ion channels dominated by strong electrostatic fields. This theory obeys two important sum rules derived in statistical physics. The fact that the current–voltage relations used to compare theory with experiment are integral properties of the channel renders the judgment of the sum rules particularly valid. In contrast, commonly used reaction-rate theories obey none of these rules. Therefore, they cannot account correctly for strong, long range electrostatic interactions. McCleskey 1999 and Miller 1999 discuss the value of reaction-rate and PNP2 theories according to their ability to reproduce particular experimental current–voltage curves. They neglect the question of whether the basic physical assumptions of those theories are satisfied or not. As shown above, the applicability of any of the two theories depends critically on the size of electrostatic interactions compared with environmental forces. To study the fundamentals of ion permeation, simpler channels than the potassium or calcium channel should also be considered. For instance, the kind and position of mutations in the acetylcholine receptor channel has no correlation with the obtained barrier energy profile. Such correlation, however, would be expected if this profile is assumed to represent the interactions of the ions with their environment. Random changes of the parameters of reaction-rate theories generally lead to nonlinear current–voltage curves . If reaction-rate theories represented the physics of ion channels such as the acetylcholine receptor channel, most single point mutations should result in nonlinear current–voltage curves. In contrast, nearly all mutations in this channel lead to fairly linear current–voltage curves. Such behavior is characteristic for ion channels dominated by electrostatic interactions . Today, the formulation of the Gouy–Chapman theory is considered as an important first step in understanding the behavior of strong, inhomogeneous electrolytes. Since then, electrolyte theories have considerably improved . In the same sense, PNP theory should be considered as a first step to describe strong, long range electrostatic interactions in ion channels. The inclusion of ion size effects such as single filing must be one of the next steps. | Other | biomedical | en | 0.999995 |
10577028 | Several statements about molecular dynamics (MD) indicate that the approach is misunderstood. The approach consists of constructing detailed atomic models of the macromolecular system of interest and, having described the microscopic forces with a potential function, use Newton's classical equation, F = MA, to literally “simulate” the dynamical motions of all the atoms as a function of time . The calculated trajectory, though an approximation to the real world, provides detailed information about the time course of the atomic motions, which is difficult to access experimentally. Despite its limitations, MD provides arguably the best available representation of biomolecular systems. Even though the trajectories are typically on the order of nanoseconds, MD simulations are not limited to rapid processes occurring within that time scale. If a well-defined slow process can be identified (e.g., an allosteric gating transition, or the ion movement across a free energy barrier), one can fully characterize such processes using special computational techniques. These well-developed techniques are routinely used by computational chemists and physicists . For example, such methods were used to compute kinetic gating transition rates for dioxolane-linked gramicidin that were on the order of a millisecond . It is wrong to state that numerical integration of Newton's equation of motions is not reliable for times longer than several picoseconds because the calculated trajectories are very sensitive to initial conditions and round-off errors, and therefore diverge exponentially. This question was debated among theoretical chemists and physicists in the late 1960's or so, and it was shown that these concerns are perfectly solvable . The resolution is based on the fact that imperfections in any single numerical trajectory will cancel when considering average value. This is because molecular memory is very short term, meaning that the system forgets rapidly its past. The decay time of the relevant correlations (i.e., the time after which randomization sets in) are therefore very short, which is also one of the reasons why the response of a molecular system to a small time-dependent perturbation is linear . Kinetic models are constructed on the basis of two assumptions: first, it is assumed that the total configurational space of the whole system comprises a complete collection of distinct subspaces (the states), and, second, it is assumed that the system possesses no dynamical memory when it leaves one state to enter another (the Markov assumption). While it is always possible to define a complete collection of discrete states for any system, the Markov assumption is not necessarily valid (e.g., if there are no free energy barriers between different regions and the movement is purely diffusive). A kinetic rate model is valid as long as the long-time behavior of a molecular system can be described by a finite number of states whose lifetimes are exponentially distributed. Equilibrium properties do not rely on the Markov assumption, as it can be shown rigorously that the equilibrium probabilities for occupancy of multiply occupied channels have the familiar algebraic form for saturation behavior that is obtained from kinetic models with discrete states corresponding to a specific number of ions inside the pore . The concept of a rate constant has been used and discussed for a long time , but it was only in the 1970's that its fundamental microscopic basis was clarified . Perhaps for this reason, there remain many misconceptions about the physical significance of kinetic rate models. First, rate models do not assume that ions literally jump over tens of angstroms. Second, rate models do not neglect ion–ion electrostatic interactions. Third, rate models do satisfy Coulomb's law (or Poisson's equation)—if properly constructed. Fourth, rate models do not assume that proteins are rigid. Fifth, the validity of rate models do not hinge on assumptions about the prefactor k B T / h , where k B , T , and h are Boltzmann's constant, the temperature in Kelvin, and Plank's constant, respectively. This prefactor should never be used for dense systems such as aqueous solutions of membrane-spanning pores. Even if the Markov assumption holds, an experimentally determined rate constant is a single number; without further information, there is no way to determine a unique dynamical prefactor and a unique activation free energy. The best aspect of kinetic rate models is their flexibility; one can adjust the rate constants constituting the model to fit most complex observed behaviors. The worst aspect of kinetic rate models is also their flexibility. The lack of internal constraints between the various rate constants makes it virtually impossible to guarantee a unique interpretation of what is going on at the microscopic level. The most sensible choice is thus to keep the models as simple as possible, even though a complex reality is not necessarily best described by a simple model. There are many misconceptions about the transmembrane potential and its role in ion permeation. For example, in rate models, the transmembrane potential is usually treated as an external (constant?) field independent of the free energy profile . The statistical mechanical basis for such a separation has been clarified recently . The total electrochemical free energy profile of an ion along the channel axis can be rigorously expressed as an intrinsic ion-pore free energy profile (independent of the applied voltage) and other contributions that arise from the transmembrane potential. According to this analysis, the transmembrane potential arises from the electrostatic potential of the ions that are in the bulk solution, but not in the immediate vicinity of the pore. Detailed calculations, based on an atomic structure of the gramicidin channel in a DMPC bilayer, show that the transmembrane potential is in fact quite linear over the length of the channel, thus providing validity to the concept of electric distance . PNP electrodiffusion is an approximate theory combining the diffusion equation under the influence of an electric field, which itself is evaluated based on a continuum electrostatic approximation using the average (mean) charge density of the diffusing ions. Hence its “mean-field” characteristic. Although the earlier form probably dates back to Planck, a more complete description was developed by L. Onsager in the 1940's . Initially, a one-dimensional reduced model was used to describe ion permeation , but full three-dimensional (3D-PNP) theories are now available . In the absence of ion flux, the 3D-PNP theory reduces to the standard nonlinear equilibrium Poisson-Boltzmann equation. Such an equivalence cannot be made for the 1D-PNP theory. For example, the significance of the 1D charge density profile used to fit experimental data is not known . Thus, the 1D-PNP theory involves even further approximations that are difficult to assess and the 3D-PNP theory is preferable. I will limit my comments to 3D-PNP. The best aspect of PNP is that it aims at doing everything at once: ion–ion, ion–channel, ion–water interactions, and the transmembrane potential are all treated in a consistent way. The worst aspect of PNP is that, while it aims at doing everything at once, it leaves out much of the atomic reality that we know is important at the microscopic level (e.g., van der Waals interactions, core repulsion, induction, hydrogen bonding, solvation structure, and protein flexibility). In practice, PNP is based on several simplifications: rigid channel structure, structureless dielectric solvent, and mean-field ion–ion interactions. If one is to adopt a continuum electrodiffusion approach, such simplifications are necessary to have partial differential equations that can be solved numerically. Interestingly, the debate about PNP often hinges on the use of the mean-field approximation to represent ion–ion interactions. While this is a nontrivial approximation (see below), the most fundamental problems with PNP are related to the approximations about channel rigidity and the representation of the solvent in terms of continuum electrostatics. Let us examine the physical significance of those approximations. The flexibility of ion channels, as any proteins, plays an important role in its function ; atomic fluctuations are usually on the order of 0.5–1.0 Å root-mean squared. Ion–protein interactions are very large . Although continuum electrostatics is successful in treating processes taking place in bulk solution; i.e., Born model of solvation , Debye-Hückel theory of electrolytes , finite-difference Poisson-Boltzmann calculations , there are significant effects arising from the granularity of water molecules and their ability to form hydrogen bonds. Continuum electrostatic models depend on empirical parameters (e.g., Born radii) that must be fitted to yield quantitatively accurate results . In bulk solution, continuum dielectric behavior is observed only at distances larger than a few water diameters , the effective ion–ion interaction energy has some microscopic structure (wells, barriers, bumps, and crevasses) and deviates from the smooth and simple Coulomb's law q 1 q 2 /∈τ 12 ; the interaction energy between two anions or two cations in bulk water are different, while continuum electrostatics is unable to make that distinction. In single file channels, the deviations from the continuum behavior are expected to be even more significant; e.g., ion–ion interactions in the gramicidin channel are species dependent even at a distance of 20 Å . From that point of view, the mean-field ion–ion approximation is not the main problem of PNP. This approximation may or may not hold depending on the situation. The Poisson-Boltzmann equation (the equilibrium equivalent of PNP) works well at a low ion concentration. In a channel with a high probability of occupancy, the situation essentially corresponds to that of an effective high concentration, where the Poisson-Boltzmann equation could have problems. PNP is a consistent but approximate theory. It may, or may not, provide a useful picture of ion permeation because it relies on several physical approximations (rigid channel, continuum electrostatics, and mean-field ion–ion interactions) that are of unknown validity in the context in which they are used. Ultimately, the significance of that picture should not be expected to exceed that of the physical approximations upon which it is built, as is the case of the Born model of solvation, Debye-Hückel, or Poisson-Boltzmann theories. It would be useful to determine the validity of those physical approximations, but that would require more than algebraic mathematical considerations. It requires a comparison between the results from atomic models with explicit molecules and experimental data. Prediction of experimental results alone cannot reveal the limitations of PNP at the microscopic level. Rather than focussing on the narrow question, “Which is the best: kinetic rate models or electrodiffusion?,” one should ask the deeper question, “Where do these theoretical models stand within modern biology and ion channel science?” It is necessary to step back and try to address more fundamental questions about the role of theory in biology. Understanding the function of biological systems is one the greatest scientific challenges of our times. To this day, biology remains primarily an experimental science, as it should. Complex biological systems are broken down in isolated elementary constituents, which are then analyzed for their structural and functional properties. These efforts represent more or less what one might (rather pompously) call the enterprise of modern scientific reductionism that has been so successful in deciphering the laws of physics during the last two centuries. Historically, theory played a huge role in the development of physics. Should one expect that theory will contribute to biology in an equivalent way? What is the role of theory and theoretical models in biology? When there is no three-dimensional atomic structure available, the goal of theory in biology is to help formulate plausible and reasonable models to help organize the information from experimental data (e.g., current–voltage-concentration relation). Simple models with a limited number of adjustable parameters are most desirable. There is no reason to complicate theoretical models when the atomic structure is not known. When a three-dimensional atomic-resolution structure is available, the goal of theory is to analyze all the details that play an important role (van der Waals, electrostatic and hydrogen bonding interactions, protein flexibility, and hydration/dehydration processes) on the observed properties (permeation, selectivity, and gating). Can one predict observed properties such as a channel conductance from the atomic structure using only the fundamental laws of physics? At the present time, this is not really possible. The complexity of biological systems requires a hierarchy of inter-related levels of descriptions, the laws governing each level emerging from the fundamental behavior of the lower level: electrons and atoms obey, more or less, the laws of quantum mechanics as described by the Schrödinger equation, the forces acting on atoms and molecules are, more or less, described by the Born-Oppenheimer approximation, the dynamical trajectory of molecules follows, more or less, the laws of classical mechanics, the complexity of dense molecular systems leads, more or less, to chaotic diffusive motions guided by some sort of free energy potential surface, variations in the local composition may (or may not) follow a Markovian kinetic rate process, and so on. One should be able to continue like this all the way to the macroscopic physiological level . These multiple levels become an absolute necessity to describe biological systems because of their complexity. Such a construct is not as necessary in physical sciences, and this is a fundamental difference between theoretical physics and theoretical biology. Useful contributions in theoretical biology should clearly acknowledge their relationship to the other levels of description (above and below). One level below connects with a more fundamental basis, one level above connects to the macroscopic laws. The need to consider and incorporate several levels of description is an important methodological aspect of theoretical biology. Good theory teaches us all (experimentalists and theoreticians) how to better understand the behavior of biological systems by providing an integration of the information from different levels of description. For example: “How does an atomic model of an ion channel relate to a continuous electrodiffusion or to the kinetic rate model?” or “Can one make such a relation?” Detailed computations based on atomic models can contribute by helping in assessing quantitatively the relative importance of microscopic factors. Theoretical models, at any level, are approximations. Excessive criticism of a theoretical model therefore becomes irrelevant when it is taken out of context. For example, kinetic rate models have been criticized extensively by the proponents of continuum electrodiffusion. But many of the criticisms of kinetic rate theory are largely unjustified and reflect misunderstandings and misconceptions about fundamental molecular statistical physics. The danger becomes that such criticism deters experimentalists from doing quantitative analysis of experimental data because they fear that they cannot attain the required level of theoretical rigor. Such an outcome would be very unfortunate because a quantitative characterization of biological systems (a painstaking task that is not always glamorous) is very important. The ultimate goal should be to understand mechanisms better, not to develop a black-box that spits out numbers. A useful calculation can reveal important aspects of the function of an ion channel while it fails to reproduce exactly the conductance of the channel. In that sense, accurate reproduction of experimental data is desirable, but not necessarily the absolute criteria because no one at this point can reproduce the observed macroscopic behavior of a biological system starting from the most fundamental level. One must be patient and not ask too much too soon. | Study | biomedical | en | 0.999995 |
10578012 | A fundamental property of cells is the capacity to regulate cell volume. The water permeability of most cell membranes is sufficiently high that cell volume is determined by the total amount of intracellular solute. Regulation of intracellular solute takes place by way of negative feedback mechanisms in which the synthesis, degradation, influx, or efflux of various solutes is increased or decreased to correct perturbations in cell volume. Mechanisms for increasing total cell solute include regulated osmolyte synthesis and shrinkage-activated Na + –H + exchange or Na + –K + –2Cl − cotransport . Mechanisms for decreasing cell solute include swelling-activated Cl − channels , K + –Cl − cotransport , K + –H + exchange , and organic osmolyte transporters or channels . Although the identities of many volume-sensitive transporters are known , the mechanisms by which cell volume causes an increase or decrease in transport activity are not well understood. The vertebrate red blood cell is a simple system in which to investigate mechanisms of cell volume regulation. The mechanisms of volume regulation in red cells are limited to post-translational events, and some aspects of osmoregulation , cannot be studied in red cells. Nonetheless, red cell volume regulation is a physiologically important phenomenon in its own right, and a large body of information is available about volume regulatory mechanisms in red cells of various species . In addition, an understanding of red cell volume regulation could lead to new approaches to the treatment of sickle cell disease . In red cells of many species, cell swelling activates K + –Cl − cotransport (KCC) 1 . In addition to cell swelling, many other interventions are known to activate red-cell KCC, including N -ethylmaleimide , low intracellular pH , inhibitors of protein kinases , urea , high hydrostatic pressure , high temperature , oxidizing agents , and low Mg 2+ . To understand the mechanisms by which cell volume affects transport, it will be important to identify, in kinetic and biochemical terms, the sequence of events associated with activation and inactivation of transport. An early attempt to analyze the kinetics of activation and inactivation of KCC was based on a simple two-state model in which KCC exists in either a resting or an activated state. In rabbit red cells, the rate of activation in swollen cells is much slower than the rate of inactivation in cells of normal volume . According to the two-state model, these data indicate that cell swelling activates transport by causing a decrease in the inactivation rate constant k 21 rather than an increase in the activation rate constant k 12 . Parker and co-workers have found similar kinetics of KCC regulation in dog red cells. In LK sheep red cells, the lag times for volume-sensitive activation and inactivation are similar, indicating that cell volume can affect both the forward and the reverse rate constants in these cells . In human SS and AA red cells the rates of KCC activation and inactivation are similar to those in rabbit red cells, but in CC red cells the rate of inactivation is very slow . The two-state model for KCC regulation is undoubtedly an oversimplification. Even if the transporter does exist in only two main functional states, the rate constants for activation and/or inactivation themselves are very likely regulated by forward and reverse rate processes . An additional complexity is the presence of more than two functional states of the transporter under some conditions . One of the difficulties in obtaining mechanistic information about transport activation and inactivation is that the measured rate in general depends on more than one elementary rate constant. In the simplest two-state model, the measured relaxation rate is the sum of the forward and reverse rate constants. In a three-state model the observed rates of activation and inactivation are affected not only by the rate constants for the rate-limiting events, but also by the equilibrium constants for rapid events. The purpose of the present work is to obtain quantitative estimates of the rate constants for the rate-limiting activation and inactivation events that regulate KCC. Rabbit red cells were chosen as an experimental system because they exhibit a relatively large, volume-dependent KCC flux ; the resultant regulatory volume decrease is sufficiently slow that the volume does not change significantly in the time needed to measure the KCC flux . Therefore, rabbit red cells are well suited for detailed kinetic studies of KCC regulation. We show that, for stimulation of transport by low intracellular pH, low Mg 2+ , cell swelling, or NEM, the kinetics of activation of transport are consistent with the presence of a single rate-limiting event. The rate-limiting step for transport activation was examined in cells pretreated with NEM at 0°C and was found to be highly dependent on temperature (E a ∼ 32 kCal/mol), but independent of cell volume. The kinetics of inactivation of transport by cell shrinkage indicate that the main volume-dependent event is the rate-limiting inactivation process, which is stimulated by cell shrinkage in the physiological range of volumes. Blood was obtained from healthy New Zealand white rabbits by either venipuncture or cardiac puncture, the latter in animals that were being killed for the purposes of obtaining other tissues for study in other laboratories. All animal procedures were in compliance with American Physiological Society guidelines. Some of the experiments were carried out using rabbit blood purchased from Pel-Freez; results obtained with blood from Pel-Freez were indistinguishable from those with blood from laboratory animals. Most experiments were performed using blood that had been stored <3 d at 4°C. Okadaic acid and ionophore A23187 were purchased from Calbiochem Corp. 86 Rb + was purchased as RbCl from DuPont NEN. All salts and buffers were purchased from Sigma Chemical Co. or Fisher Chemicals. For experiments involving transport activation by swelling, low pH, or Mg 2+ depletion, cells were separated on Percoll-Renograffin as previously described to select the least dense one third of cells. The lower-density fractions are enriched in younger cells, which have a higher volume-dependent KCC activity . The NEM activation experiments in Fig. 6 Fig. 7 Fig. 8 Fig. 9 used unseparated red cells; comparable experiments with density-separated cells gave indistinguishable results. If blood had been stored more than a few hours, cells were washed three times and incubated 60–90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na-phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state. Cells were washed in HPS, suspended at 5% hematocrit in HPS, and chilled until the temperature of the suspension was <2°C. NEM was then added from a freshly prepared 1-M stock solution in dimethylformamide to a final concentration of 2 mM. Control suspension received dimethylformamide at the same final concentration (0.2%). The suspensions were incubated 15 min on ice, washed once, and resuspended in HPS on ice. For some aliquots of cells, the NEM treatment was repeated once or twice more. After all NEM treatments were complete, the cells were washed twice in ice-cold HPS before further incubations. In some experiments, 0.1% β-mercaptoethanol was included to remove all traces of remaining NEM; results were identical whether or not β-mercaptoethanol was included in the wash medium. Cells were suspended at 2% hematocrit in “standard flux medium” (155 mM NaCl, 5 mM KCl, 10 mM HEPES hemisodium, pH 7.5 at 25°C, 10 −4 M ouabain). Depending on the design of the experiment, 86 Rb + (0.5–1 μCi/ml) was present at the outset or was added after incubating the cells in flux medium for various intervals . At timed intervals after exposure of cells to 86 Rb + , the intracellular radioactivity was determined as described previously . The influx of 86 Rb + (assumed to be an ideal tracer for K + ) is expressed as micromoles *K + per milliliter cells and was calculated from the cpm in each sample, the volume of original cells (milliliters) in the sample, and the extracellular specific activity (cpm 86 Rb + per micromole K + ). The time course of activation of KCC by low pH was determined by adding MOPS from a 1-M stock solution to a final concentration of 15–18 mM. Within a short time after extracellular acidification, the intracellular pH reaches Donnan equilibrium with the extracellular pH . We estimated intracellular pH in lysed pellets (in 10 vol water, with ionic strength then returned to 100 mM with KCl). This method does not accurately determine the absolute value of intracellular pH, but it is adequate for estimating the time course of changes in pH. We found that the half time for pH equilibration under the conditions of the flux experiment in Fig. 3 (25°C, ambient CO 2 ) is ∼0.8 min in rabbit red cells. At higher temperature , the time course of pH equilibration was not measured, but is believed to be considerably faster, not only because of the high temperature dependence of pH equilibration , but because 4–5 mM NaHCO 3 was added to facilitate pH equilibration by the Jacobs–Stewart cycle . Intracellular Mg 2+ was estimated colorimetrically in cells that were prepared as in the flux experiments. Cells were incubated at 2% hematocrit at 25°C in 160 mM NaCl, 10 mM HEPES, pH 7.45, 1 mM EDTA. Two aliquots (1 ml) were removed before addition of ionophore A23187 and mixed with 10 ml of cold 160 mM KCl/10 mM HEPES, centrifuged 2 min at 4,000 rpm, and the supernatants were removed. A23187 (10–20 μM final) was then added, and further aliquots were removed, centrifuged, and supernatants removed. The cell pellets were lysed in 0.5 ml water, heated for 2 min in boiling water, and allowed to cool. The tubes were then centrifuged, and 0.1 ml of the supernatant was mixed with 0.9 ml of Mg 2+ color reagent containing calmagite (1-[1-hydroxy-4-methyl-2-phenylazo]-2-naphthol-4-sulfonic acid; Sigma Diagnostics). Absorbances were compared with those of standards, and the results were expressed as micromoles Mg 2+ per milliliter cells. In all these experiments, the main measured parameter is the lag time for the transition from one steady state to another. The rate of this transition (inverse lag time) was calculated as follows. The time course of accumulation of intracellular 86 Rb + [*K + ] after a step change in conditions at t = 0 is given by the following expression : 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*} \left \left[^{{\mathrm{*}}}{\mathrm{K}}^{\;+\;}\right] \right _{in}=J_{1}t+ \left \left(J_{0}-J_{1}\right) \right \left \left({\mathrm{{\tau}}}\right) \right \left \left[1-{\mathrm{exp}} \left \left(-{t}/{{\mathrm{{\tau}}}}\right) \right \right] \right + \left \left[^{{\mathrm{*}}}{\mathrm{K}}^{\;+\;}\right] \right _{in}^{t=0}{\mathrm{,}}\end{equation*}\end{document} where J 0 is the initial flux (micromoles per milliliter cells minute −1 ), J 1 is the flux in the new steady state, τ is the lag time for establishing the new steady state, and [*K + ] in t = 0 is the amount of intracellular tracer at the time of the step change in conditions. In most experiments, the change in conditions is at the time of first exposure of cells to 86 Rb + ; in these cases, [*K + ] in t = 0 represents the small amount of tracer that is not removed by the washing procedure. For the activation experiments in this paper, J 0 and [*K + ] in t = 0 were determined directly in a parallel suspension in isosmotic medium at physiological pH. The influx data were fit (Sigma Plot; Jandel Scientific) to , with two adjustable parameters, τ and J 1 , for most experiments. In the NEM activation experiments, the steady state flux J 1 was determined independently in a parallel suspension that had been incubated 15–20 min at 37°C to allow KCC to activate. In these experiments, the only adjustable parameter in the curve fits was the lag time τ, which can be estimated accurately when all the other parameters are determined independently. In the inactivation experiments , the initial flux was estimated in swollen cells, and the lag time was determined in a two-parameter fit (τ and J 1 ). The estimate of τ is reasonably accurate in an inactivation experiment (despite the two-parameter fit) because the final steady state flux is small. The purpose of these experiments is to obtain quantitative estimates of the rate constants for the rate-limiting activation and inactivation events in the regulation of rabbit red cell KCC. Experimentally, under a wide variety of conditions, the time course of activation by cell swelling or NEM is not distinguishable from a single exponential in red cells from rabbit , dog , LK sheep , mouse , and human , suggesting that one of the activation steps is much slower than any other step. To test further the idea that there is a single rate-limiting step in the activation of KCC, the kinetics of activation by step decreases in intracellular pH or Mg 2+ were compared with activation by hypotonic swelling. It is known that low intracellular pH (e.g., pH 6.9) activates KCC in human and LK sheep red cells . Extracellular pH in the range 6.8–7.5 does not have major effects on KCC . The time course of activation of KCC by low intracellular pH in rabbit red cells is shown in Fig. 1 . Cells were initially in an isosmotic medium at pH 7.5, and the influx of 86 Rb + was measured for about 20 min. At t = 23.5 min (arrow), 16 mM MOPS and 4 mM NaHCO 3 were added. The HCO 3 − facilitates rapid equilibration of intracellular and extracellular pH by the Jacobs–Stewart cycle . Based on data at lower temperatures and lower HCO 3 − concentration (see materials and methods ), Donnan equilibrium should be reached in roughly 1 min at 37°C. After acid addition, the 86 Rb + influx rises over the next 10 min with a single exponential time course. The increased flux is inhibited by the presence of the protein phosphatase inhibitor okadaic acid, as is true for activation of KCC by cell swelling or NEM . Fig. 2 represents another acid activation experiment and also shows the time course of decrease in K + influx when the pH is suddenly returned to an alkaline value. As in Fig. 1 , sudden acidification causes activation of transport, with a lag time of 14 min, which is somewhat longer than in Fig. 1 . However, we do not have sufficient data to determine whether the lag time at pH 6.8 is actually different from that at pH 7.0. Fig. 2 (top) shows cells that were preincubated at pH 6.8 to allow KCC activation before addition of 86 Rb + . The initial flux is large in these cells, and addition of NaOH to return the pH to alkalinity causes a rapid decrease in the flux. The time course of inactivation was not studied in detail because the rate may depend in part on the rate of intracellular alkalinization, but it is clear that inactivation of the flux by alkaline pH is more rapid than activation by acidic pH, just as inactivation by shrinkage is more rapid than activation by swelling . Fig. 3 shows the time course of acid activation at 25°C. For comparison, the flux was also activated by hypotonic cell swelling in the same preparation of cells. The flux activates much more slowly at 25° than at 37°C for both modes of activation. The lag time at 25°C is 60–80 min for activation by swelling and low pH. This lag time is in the same range as that measured previously (average 55 min) for swelling-activated KCC at 25°C , but there is considerable uncertainty in the lag times for activation experiments because the final steady state flux is not known precisely. In human and LK sheep red cells, depletion of Mg 2+ at normal cell volume activates KCC . The time course of activation after a step decrease in Mg 2+ has not been reported. We found that Mg 2+ -depleted rabbit red cells do not tolerate incubation at 37°C for more than ∼30 min; accordingly, Mg 2+ depletion experiments were performed at lower temperature, either 25° or 30°C. Fig. 4 (top) shows that it is possible to deplete rabbit red cells of Mg 2+ in <5 min by addition of ionophore A23187 and EDTA, in agreement with the work of Flatman and Lew 1980 on human red cells. Sudden Mg 2+ depletion causes activation of KCC with a time course similar to that observed after stimulation by cell swelling or low pH. The 86 Rb + flux stimulated by low Mg 2+ is inhibited by replacement of Cl − with NO 3 − and is also inhibited by preincubation with okadaic acid (data not shown). Fig. 5 shows that, in cells that have been depleted of Mg 2+ , there is only a very minor effect of cell volume on the KCC flux. A slight volume dependence of the flux in Mg 2+ -depleted cells is observed even in NO 3 − medium. Varying the osmolality from 410 to 185 mosmol/kg (2.4–5.4 [osmol/kg] −1 ) caused the Cl − -dependent flux to change by <20%. The same results were observed in two other experiments. In one earlier experiment, there appeared to be a decrease in the flux in very hypertonic solutions (>400 mosmol/kg), but in the range between 200 and 400 mosmol/kg there is essentially no effect of volume on KCC in Mg 2+ -depleted rabbit red cells. In contrast, KCC is still volume sensitive in LK sheep red cells, though less so than in cells with normal Mg 2+ . It is impossible to determine whether there is a lag time in the response of KCC to cell swelling in low Mg 2+ rabbit red cells, because the flux is not volume dependent. The above experiments indicate that the time course of activation of KCC is similar after step changes in cell volume, pH, or Mg 2+ , suggesting that the same event is rate limiting for all three modes of activation, and it is of interest to try to measure the rate constant for this event. The measured lag time for transport activation in general depends not only on the rate-limiting activation event, but also on the rate constant for inactivation. The most direct way to estimate the rate constant for activation is to devise conditions in which the rate of inactivation is negligible. Under these conditions, the measured rate of activation is very nearly equal to the rate constant for the rate-limiting step in the activation process. One approach to measuring activation kinetics under conditions of maximal activation would be to measure transport at very high cell volume. However, extreme cell swelling may cause prelytic leaks or other abnormalities. Instead of trying to activate transport maximally by cell swelling, we used NEM, which has long been known to activate KCC . Activation of KCC by NEM exhibits a lag time similar to that observed after cell swelling . In our previous studies , NEM was added at 37°C and allowed to react for 2 min before residual NEM was removed by adding cysteine. This protocol made it possible to observe a lag time for transport activation after NEM, but precise rate measurements were difficult because transport activation and NEM reaction with its target protein (identity unknown, but possibly a protein kinase) were taking place simultaneously. To characterize the kinetics of activation by NEM more precisely, cells were incubated with 2 mM NEM for 15 min at 0°C, and residual NEM was removed by washing at 0°C. Transport was then activated by incubating for 15–20 min at 37°C. Fig. 6 shows that activation by NEM is nearly maximal after two NEM treatments (2 mM; 15 min) at 0°C. Further treatments with NEM at low temperature do not cause significant further activation or inhibition. Therefore, as originally shown by Lauf and Adragna 1995 , treatment with NEM at low temperature produces only activation of KCC; the inhibitory effects of high concentrations of NEM are only observed when NEM treatment is at higher temperature. The data in Fig. 6 indicate that two exposures to 2 mM NEM at 0°C are sufficient to activate KCC to at least 80% and probably >90% of maximal activity. The fluxes shown in Fig. 6 were measured by adding 86 Rb + after incubating NEM-pretreated cells for 15–20 min at 37°C. If pretreated cells were exposed to 86 Rb + without preincubation at 37°C, the influx was initially small but increased with a single exponential time course to the same steady state level as preincubated cells . The lag time for KCC activation at 37°C was estimated in five separate preparations of cells that had been treated twice with 2 mM NEM on ice. The lag time was 9.2 ± 1.4 (SD) min. The lag time was also measured in five preparations of cells that had been treated once with 2 mM NEM. The lag time in this case was slightly shorter (7.6 ± 0.6 min), as expected if the flux is not quite maximally activated and the reverse rate constant is not completely inhibited. From these experiments, we conclude that the rate constant for the rate-limiting forward step in transport activation in NEM-treated cells is ∼0.11/min at 37°C. The temperature dependence of the activation rate was measured by treating with NEM at 0°C, and then measuring the time course of 86 Rb + influx at 25°C. The flux in the fully activated state at 25°C was measured by pretreating cells with NEM at 0°C, incubating 15–20 min at 37°C to activate >90% of the transporters , and then shifting the temperature back to 25°C for the flux measurement. Fig. 8 shows that the lag time for activation at 25°C is much longer than at 37°C. In four experiments (with either one or two pretreatments with NEM), the activation lag time was 75 ± 13 (SD) min, which is a factor of about eight longer than at 37°C. The apparent activation energy of the rate-limiting activation process is ∼32 kCal/mol in this temperature range. Fig. 9 shows that the activation rate constant is not detectably dependent on cell volume in NEM-pretreated cells. Cells were preincubated with NEM at 0°C, and the activation rate was measured as in Fig. 7 in isotonic and hypotonic media. The activation rate in swollen cells is indistinguishable from that in cells of normal volume. In agreement with earlier data obtained under different conditions of NEM treatment , the steady state KCC flux in NEM-treated cells is not dependent on cell volume. The above experiments indicate that the rate-limiting kinetic step in transport activation is not dependent on cell volume, in agreement with our previous proposal that the main volume-dependent process is the inactivation step . To examine the effect of cell volume on KCC inactivation more directly, the rate of transport inactivation was examined under conditions in which, in the final steady state, nearly all the transporters are inactivated. Cells were preincubated 15–20 min in 200 mosmol/kg medium at 37°C to activate a substantial fraction of the transporters. The preincubation is long enough to activate KCC, but not long enough to allow a significant regulatory volume decrease . Cells were then resuspended at 25°C in 86 Rb + -containing media of varying osmolalities. At all osmolalities, the temperature shift causes a decrease in the steady state number of activated transporters because of the very high temperature dependence of the activation rate constant (see above). Although the flux inactivates rapidly, it is possible to get a reliable estimate of the inactivation rate because the initial flux is known (from that in the most swollen cells), and the final flux can be measured reasonably accurately. Fig. 10 shows that the rate of inactivation of the flux increases as cell volume decreases, even in the range of cell volumes in which the vast majority of the transporters are inactivated in the steady state. The results of the experiment in Fig. 10 plus two additional experiments are summarized in Fig. 11 . A single experiment with fluxes at 37° instead of 25°C is shown in Fig. 12 . Again, the rate of inactivation continues to increase as cell volume decreases, in a volume range where the steady state KCC flux is very small. Therefore, the rate of inactivation does not reach a limiting value as cell volume decreases. This finding is evidence that the major volume-dependent step in transport regulation is the rate-limiting inactivation event (see below). The experiments described above have provided the most quantitative information to date on the rate-limiting events in activation and inactivation of a volume-regulatory transporter. The rates were measured under conditions where, in the final steady state, KCC is either fully activated by NEM or nearly fully inactivated by shrinkage . Kinetic analysis is much simpler under these conditions than it is when an intermediate (and unknown) percentage of transporters are in the activated state. The data will be discussed below in terms of specific current models for activation and inactivation of KCC. However, it is useful first to summarize the main experimental findings without reference to particular models. (a) Activation of KCC by sudden acidification, depletion of Mg 2+ , cell swelling, or NEM takes place with a time course that is consistent with a single rate-limiting activation event. (b) In contrast to LK sheep red cells , there is very little effect of cell volume on KCC in Mg 2+ -depleted rabbit red cells. (c) In cells pretreated with NEM at low temperature, KCC does not activate until the temperature is raised; using NEM-pretreated cells, it is possible to measure the rate of KCC activation under conditions of maximum steady state activation. (d) The activation rate constant in NEM-treated cells is very dependent on temperature (E a ∼ 32 kCal/mol), but is not detectably dependent on cell volume. (e) The inactivation rate can be measured by shifting the osmolality to produce a final steady state in which nearly all transporters are inactivated. Under these conditions, the rate of inactivation increases as cell volume decreases over the entire range of cell volumes studied, including physiological cell volume. The acidification experiments in Fig. 1 Fig. 2 Fig. 3 show that activation of KCC by acidification has a similar time course to activation by hypotonic swelling. It should be pointed out that, in these experiments, there was slight cell swelling in addition to the acidification. Acidification of red cells is accompanied by a net influx of Cl − , which causes cell swelling in an isosmotic medium . In our experiments, MOPS and NaHCO 3 were added from 1-M stock solutions, which increased the osmolality of the medium and minimized cell swelling during acidification. Nonetheless, there is slight swelling under these conditions. Rabbit red cell water at pH 7.4 (in 310 mosmol/kg medium) is 1.76 ± .025 g H 2 O/g solids (mean ± SD, four preparations). In cells acidified as in Fig. 2 , cell water increases to 1.89 ± 0.03 g H 2 O/g solids (three preparations). This cell water is the same as in cells at pH 7.4 in 290 mosmol/kg H 2 O medium. At this cell water content, there is detectable activation of KCC in young rabbit red cells , but the activation is much less than that observed after acidification. A complete study of the relationship between acid activation and swelling activation in rabbit red cells was not performed because it is already clear from the work of Brugnara et al. 1985 and Lauf et al. 1994 that pH affects the volume dependence of red cell KCC (and vice versa). The important point for present purposes is that, after acidification, under conditions of only slight cell swelling, the time course of activation is similar to that after hypotonic (200 mosmol/kg) swelling, suggesting that the volume and pH signaling pathways converge on a single rate-limiting event. Earlier data on activation and inactivation rates were analyzed in terms of a simple two-state mechanism, which is almost certainly an oversimplification, and it is important to consider the implications of the current data in the context of more complex models. Dunham et al. 1993 proposed a three-state model, based in part on the finding that Mg 2+ depletion of LK sheep red cells causes partial activation of KCC and that subsequent cell swelling causes further activation without a time lag. Moreover, Mg 2+ depletion increases the V max for KCC without changing the apparent K + affinity, whereas cell swelling increases the K + affinity . Swelling of inside-out vesicles also increases apparent K + affinity for KCC . Dunham et al. 1993 proposed that there is an intermediate state (not fully activated because affinity is still low) that is observed in Mg 2+ -depleted LK sheep red cells. Cell swelling converts this intermediate into the fully activated state. In rabbit red cells there is very little effect of cell volume on KCC in Mg 2+ -depleted cells . Instead, the cells behave similarly to NEM-treated cells; KCC is activated and is not strongly affected by volume. The influx was measured at an extracellular K + concentration of 5 mM, which is well below the apparent Michaelis constant for extracellular K + ; therefore, an increase in substrate affinity would have been detected as an increased flux. Cell swelling, therefore, does not appear to raise the substrate affinity of KCC in Mg 2+ -depleted rabbit red cells. Irrespective of the differences between rabbit and LK sheep, we feel that it is worthwhile to discuss the present results in reference to a three-state model, because it is quite possible that rapid events take place in series with the rate-limiting activation/inactivation events, and it is important to know whether the volume dependence of KCC is a consequence of effects of volume on rapid events or on the rate-limiting events. The three-state model of Dunham et al. 1993 proposes that the activation process involves a rate-limiting step (A to B) followed by a fast step (B to C) . To discuss relaxation rates, no assumptions are necessary about the detailed kinetic properties of the three states, other than that the flux is much higher in the C than in the A state. In this model, the relaxation rate for approach to any new steady state is (see ): 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}^{-1}=k_{12}+{k_{21}}/{ \left \left(1+K_{{\mathrm{BC}}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where K BC is the equilibrium constant k 23 / k 32 for the rapid second step. In rabbit red cells, τ −1 is smaller in swollen cells than in cells of normal size , suggesting that the rate-limiting inactivation event k 21 is very dependent on cell volume. However, it is possible in principle that the actual volume dependence is entirely in the fast step; that is, K BC could be strongly increased by cell swelling. If so, the measured relaxation rate would be small in swollen cells, as observed. Therefore, published data do not rule out the possibility that a rapid second step rather than the slow step is the main volume-dependent event. The inactivation experiments in Fig. 10 Fig. 11 Fig. 12 provide a way to address the question of whether the slow or the rapid step is the major volume-dependent step. Suppose, for example, that the rapid (B to C) step were the only volume-dependent step. If so, then K BC must decrease as cell volume decreases. As K BC becomes small, the steady state number of C states ([C] ss ) decreases in proportion to K BC , but the relaxation rate τ −1 should reach a limiting value ( and ): 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{{\mathrm{BC}}}{\rightarrow}0{\mathrm{:}} \left \left[{\mathrm{C}}\right] \right _{{\mathrm{ss}}}=k_{12}{K_{{\mathrm{BC}}}}/{ \left \left(k_{12}+k_{21}\right) \right }\end{equation*}\end{document} and 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{{\mathrm{BC}}}{\rightarrow}0{\mathrm{:{\tau}}}^{-1}=k_{12}+k_{21}{\mathrm{.}}\end{equation*}\end{document} In other words, if the major volume-dependent process is the rapid B to C transition, then the rate of inactivation should become independent of cell volume in the limit of low cell volume. The data in Fig. 10 Fig. 11 Fig. 12 show that, experimentally, the rate of inactivation continues to increase as cell volume decreases in a range of cell volumes where nearly all the transporters are inactivated in the steady state. This indicates that the rate constant k 21 for the rate-limiting inactivation event is strongly dependent on cell volume. These data are the best evidence to date that the rate-limiting inactivation event is also the main volume-dependent event. It is significant that the KCC inactivation rate is strongly dependent on cell volume at physiological cell volumes. This indicates that, even though most of the transporters are inactivated under physiological conditions, modulation of KCC by small changes in cell volume is a real physiological mechanism for maintaining normal cell volume over the long life of the cell. In the context of the three-state model, NEM could activate transport by increasing k 12 , decreasing k 21 , or increasing K BC . NEM probably does not have a large stimulatory effect on k 12 , because the rate of activation in NEM-pretreated cells is relatively slow. Accordingly, NEM must cause a large decrease in k 21 and/or increase in K BC . In either case, the measured rate of activation τ −1 is approximately equal to the forward activation rate constant k 12 under conditions of maximum NEM activation, because the second term in is very small when activation is maximal. Experimentally, cell swelling has no detectable effect on the activation step k 12 . This is consistent with our earlier data , but the current method for estimating k 12 is more accurate. It is of course possible that NEM removes a volume effect on k 12 that is normally present, but we have no evidence for an effect of volume on k 12 in rabbit red cells. Although volume has very little effect on the activation rate constant, temperature has a very large effect. Earlier data on the effect of temperature on rabbit red cell KCC indicated that the rate constant for activation is more temperature dependent than that for inactivation. The present data, with improved methods for measuring the forward rate constant, show that the temperature dependence of the rate-limiting activation event is extraordinarily high. The rate increases by a factor of ∼8 between 25° and 37°C, which corresponds to an activation energy of ∼32 kCal/mol. The transport process itself is much less dependent on temperature; the flux varies by a factor of <2 in the same temperature range , corresponding to an activation energy of <10 kCal/mol. However, it should be noted that the flux was measured at low extracellular K + concentrations, and it is possible that the V max for KCC actually has a higher activation energy than 10 kCal/mol. The high temperature dependence of k 12 is in agreement with the recent work of Willis and Anderson 1998 , who showed that guinea pig red cell KCC is activated by warming. Willis and Anderson 1998 also showed that the large effect of temperature is on a regulatory process rather than on the cotransport process itself. Our results are in complete agreement with this idea. The most probable reason for the activation by high temperature is that, at all cell volumes, the forward rate constant for activation is far more temperature dependent than the reverse rate constant. We have been able to estimate the temperature dependence of k 12 reasonably accurately, but it is much more difficult to do the same for k 21 (inactivation), because k 21 depends strongly on cell volume, and the volume dependence of k 21 may be affected by temperature. We therefore do not know the temperature dependence of k 21 , but we are confident that the inactivation process is much less temperature dependent than the activation process. The fact that elevated temperatures activate KCC , without NEM treatment, indicates that the high temperature dependence of k 12 applies not only to NEM-treated cells but also to normal cells. Inhibitors of serine-threonine protein phosphatases prevent activation of KCC , indicating that a dephosphorylation event is necessary for KCC activation. It is not known whether the dephosphorylation event is on KCC itself or on a modulatory protein. The effects of okadaic acid and calyculin A are consistent with the involvement of protein phosphatase 1 (PP1), although the okadaic acid dose response is complicated by its slow permeation and adsorption to cellular constituents . It is of interest to compare the temperature dependence of the activation rate constant k 12 with those for known protein phosphatases. Mitsui et al. 1994 have shown that the activities of smooth muscle phosphatase IV (SMP-IV) and myosin-associated phosphatase (MAP) have much higher temperature dependence (Q 10 of 5.2–5.3) than several other phosphatases, including the catalytic subunits of PP1 and PP2A, which have Q 10 of ∼2 . The temperature dependence of SMP-IV and of MAP is nearly as high as that measured for the KCC activation rate constant k 12 (Q 10 ∼ 6 between 27° and 37°C), and it is possible that a similar phosphatase mediates the rate-limiting activation step for KCC. However, not enough is known about the properties of red cell protein phosphatases to draw firm conclusions on this point. In fact, it is not really established that a protein phosphatase mediates the rate-limiting activation event. The effects of phosphatase inhibitors on the activation rate suggest that k 12 represents a phosphatase activity, but it is nonetheless possible that protein dephosphorylation is not the rate-limiting activation event, but rather plays a permissive role before the activation event. In resealed human red cell ghosts, protein serine/threonine phosphorylation and dephosphorylation do not appear to be in the signal transduction pathway for regulation of volume sensitive KCC ; the relationship between regulation of KCC in intact cells and in resealed ghosts remains unclear, however. If k 12 does represent a protein dephosphorylation event, then k 21 most likely represents phosphorylation mediated by a serine-threonine protein kinase, as suggested previously . If so, the kinase activity is inhibited by cell swelling, possibly by a mechanism involving the relief of cytoplasmic crowding . The identity of the swelling-inhibited protein kinase, if such a kinase exists, is unknown. However, the data presented here should perhaps stimulate the search for such a kinase, because there is now better evidence for a volume-sensitive, rate-limiting inactivation event that could be kinase mediated. The relatively low temperature dependence of k 21 is also consistent with the idea that k 21 represents a protein kinase activity . It is of interest to ask whether the measured rate constants for activation and inactivation are consistent with the observed effects of volume on the steady state fluxes. In the two-state model, the steady-state flux is related to the inverse lag time as shown in : 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}J_{{\mathrm{ss}}}={J_{{\mathrm{Max}}}k_{12}}/{ \left \left(k_{12}+k_{21}\right) \right }=J_{{\mathrm{Max}}}k_{12}{\mathrm{{\tau}.}}\end{equation*}\end{document} Therefore, if the only volume-dependent parameter is k 21 , then the flux should be directly proportional to the lag time. In Fig. 11 and Fig. 12 , both the flux and the lag time are strongly dependent on cell volume, but the flux varies somewhat more rapidly than does the lag time. Therefore, the volume dependence of the inactivation rate constant k 21 , by itself, does not appear to be sufficient to account for the entire volume dependence of the flux. As discussed above, we have no direct evidence for volume dependence of the activation rate constant k 12 , although the estimates of k 12 were necessarily made in NEM-treated cells, and it is possible that, without NEM treatment, k 12 is volume dependent. Another possibility is that a rapid step in the activation is volume dependent, as found in LK sheep red cells by Dunham et al. 1993 . In rabbit red cells depleted of Mg 2+ , the flux is not very dependent on cell volume , but in normal cells there could be a rapid, volume-dependent activation event that takes place before or after the rate-limiting event. Our data indicate that the volume dependence of k 21 can account for much, but not all, of the volume dependence of the steady state flux. We currently have no information on what other KCC regulatory events depend on cell volume in rabbit red cells. Although there are differences between the current results and those obtained in LK sheep red cells , there are also points of agreement. For example, the rate constant k 12 for the rate-limiting activation event estimated here (0.11/min) is quite similar to that (0.09/min) derived by Dunham et al. 1993 . The estimates of the inactivation rate constant k 21 (∼1/min in cells of normal volume) are also quite similar in rabbit and LK sheep cells. Our determinations of the effect of volume on k 21 indicate a lower volume dependence than that proposed by Dunham et al. 1993 , but both laboratories agree that k 21 is strongly volume dependent. The main difference in the two species is the much higher volume dependence of the flux in Mg 2+ -depleted cells in LK sheep than in rabbit. The basic mechanism of volume dependence is very likely quite similar in the two species, but clearly there are differences in some aspects of the regulation. The three-state model of Dunham et al. 1993 is among the simplest mechanisms containing more than one event in the activation–inactivation process. However, many other relatively simple mechanisms involving multiple steps are possible. For example, a cascade mechanism of the kind described recently by Lytle 1998 for duck red cell Na-K-2Cl cotransport could, in principle, describe the data shown here. In this kind of mechanism, the rates of forward and reverse transitions between active and inactive states are not directly affected by cell volume, but rather are modulated by an enzyme (e.g., kinase) whose activity is sensitive to cell volume. For example, the reverse rate constant k 21 could represent a kinase activity that is, in turn, controlled by a separate phosphorylation/dephosphorylation cycle . This kind of modulation of rate constants by a separate phosphorylation/dephosphorylation cycle could of course be applied to either a two- or three-state mechanism, but, for simplicity, we consider only the two-state case. In this model, cell volume could in principle affect k 34 and/or k 43 , and thereby affect k 21 . That is, k 21 could be inhibited by cell swelling, but the parameter directly affected by cell volume could actually be k 34 or k 43 . Our data cannot rule out a model of this type. However, it should be pointed out that the inactivation rate constant k 21 increases without a detectable lag time when cells are suspended in media of increasing osmolality . The measurements do not have the time resolution to make quantitative estimates of the rate of change of k 21 , but the data can be fit very easily by assuming that, when the cells first shrink to the new volume, k 21 changes much more rapidly than the rate of change of transport. The rapid change in k 21 implies that, if the inactivation rate constant is regulated by a volume-dependent phosphorylation/dephosphorylation cycle, then that cycle must be able to reach a new steady state in much less than 1 min. Many variations on simple cascade-type models are possible, including those in which a tyrosine kinase modulates the activity of a serine/threonine phosphatase . This kind of model can explain the interesting finding of de Franceschi et al. 1997 that deficiencies in Src family kinases Fgr and Hck cause activation of KCC in mouse red cells. Tyrosine phosphorylation is known to inhibit the activities of PP1 and PP2A . Bize et al. 1998 recently showed that H 2 O 2 and staurosporine stimulate protein phosphatase (probably PP1) in LK sheep red cell membranes. The stimulation (∼25%) is smaller than the severalfold activation of KCC, but it is possible that there are multiple phosphatase activities in the membrane and that only the one associated with KCC is activated by staurosporine. In the context of the present work, the high temperature dependence of the activation rate constant k 12 could be a consequence of the fact that the activating phosphatase is itself regulated by a temperature-dependent process. If so, the high apparent E a of k 12 could reflect the E a of the catalysis of dephosphorylation, augmented by the temperature dependence of events that regulate the phosphatase. Although a regulated phosphatase activity may mediate the rate-limiting activation event, much more needs to be learned about the actual enzyme activities associated with regulation of KCC and the interdependencies among these activities before the biochemical correlates of the rate-limiting rate constants for activation and inactivation of KCC are understood. | Study | biomedical | en | 0.999997 |
10578013 | Stimulation of insulin secretion by glucose is linked to the generation of electrical activity in pancreatic β cells. This electrical activity, first documented more than 30 yr ago , consists of oscillations in membrane potential between depolarized plateaus, on which action potentials are superimposed, and repolarized electrically silent intervals . The cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) varies in synchrony with electrical activity and correlates with pulsatile insulin secretion . Patch-clamp experiments on isolated β cells have enabled the characterization of many of the membrane conductances participating in glucose-induced electrical activity . The processes that underlie the bursting pattern remain undetermined, although a number of hypotheses have been proposed . These include activation of large-conductance Ca 2+ -activated K + channels , inactivation of the voltage-gated Ca 2+ current , cell–cell coupling , cyclic activation of a store-activated cation conductance , and opening of Ca 2+ -activated K + channels by mobilization of intracellular Ca 2+ . However, it has not been possible to establish which of these mechanisms (if any) participates in the generation of the glucose-induced oscillations in membrane potential. This is because dissociated β cells maintained in tissue culture, the standard preparation for patch-clamp recordings, only rarely exhibit oscillatory electrical activity reminiscent of that seen in the intact islets . We have now applied the patch-clamp technique to β cells within intact islets. These cells retain the bursting pattern of action potential firing. Here we describe a Ca 2+ -activated K + current that activates gradually during electrical activity and exhibits several features consistent with a role in the regulation of the rhythmic firing of action potentials. Unless otherwise indicated, the electrophysiological experiments were carried out on β cells in intact islets. NMRI mice were purchased from a commercial breeder (Moellegaard). The mice were stunned by a blow against the head and killed by cervical dislocation and the pancreas quickly removed. Collagenase (2 mg) was dissolved in Hank's buffer and injected into the pancreatic duct. Pancreatic islets were isolated by gentle collagenase digestion (25 min, 37°C). Islets thus isolated were subsequently maintained in short-term tissue culture (<16 h) in RPMI 1640 containing 5 mM glucose and 10% (vol/vol) fetal calf serum (Flow Laboratories) and supplemented with 100 μg/ml streptomycin and 100 IU/ml penicillin (both from Northumbria Biologicals, Ltd.). The experiments in Fig. 1C and some of those displayed in Fig. 5 were carried out on dispersed β cells. These were prepared by shaking islets in Ca 2+ -free solution. The resultant cell suspension was plated on glass cover slips (diameter: 22 mm) or Nunc plastic petri dishes and maintained in tissue culture for up to 48 h using the tissue culture medium mentioned above. Pancreatic islets were immobilized by a wide-bore (diameter: 50–100 μm) suction pipette. The measurements were performed using an EPC-9 patch-clamp amplifier (HEKA Electronics) and the software pulse (version 6.2 and later). Patch pipettes were pulled from borosilicate glass (tip resistance: 3–7 ΜΩ when filled with the pipette solution). Pancreatic β cells were functionally identified by the generation of oscillatory electrical activity in the presence of 10 mM glucose. Cells thus identified exhibited electrophysiological characteristics similar to those previously described for β cells maintained in tissue culture . Using these criteria, the β cells can safely be distinguished from the α and δ cells; the latter cell types being equipped with a large voltage-gated Na + (Göpel, S.O., T. Kanno, S. Barg, J. Galvanovskis, and P. Rorsman, manuscript submitted for publication). In the intact islet, the β cells are electrically coupled and electrical activity in neighboring cells spreads into the voltage-clamped cell via the gap junctions . To allow voltage-clamp measurements without interference by currents originating from the neighboring cells, the glucose concentration was usually lowered to 5 mM to suppress glucose-induced electrical activity. Electrical activity was then simulated by application of a sequence of voltage-clamp pulses. This consisted of depolarization from −70 to −40 mV for 5 s, followed by a series of 26 simulated “action potentials.” The latter consisted of a voltage ramp between −40 and 0 mV (100 ms) followed by a ramp from 0 to −40 mV (100 ms). The action potential waveform was applied at a frequency of 5 Hz. This voltage range, frequency, and duration approximate the β cell action potential. Subsequent to the train of voltage-clamp pulses, the cell was held at −40 mV for 10 or 20 s to facilitate the observation of K + currents. The interval between two successive stimulation series was normally 0.5–2 min to allow complete recovery from inactivation. All experiments were carried out using the perforated patch whole-cell configuration and were conducted at 30–32°C. During the experiments, the islet was continuously superfused with extracellular medium at a rate of 1–2 ml/min. The standard extracellular medium consisted of (mM): 140 NaCl, 3.6 KCl, 2 NaHCO 3 , 0.5 NaH 2 PO 4 , 0.5 MgSO 4 , 5 HEPES, pH 7.4 with NaOH, 2.5 CaCl 2 , and d -glucose at the indicated glucose concentration. The pipette solution was composed of (mM): 76 K 2 SO 4 , 10 NaCl, 10 KCl, 1 MgCl 2 , and 5 mM HEPES, pH 7.35 with KOH. Whole-cell Ca 2+ currents were recorded with the same solutions with the exception that K 2 SO 4 in the pipette-filling solution was replaced by an equimolar amount of Cs 2 SO 4 . In all recordings, electrical contact with the cell interior was established by addition of the pore-forming antibiotic amphotericin B to the pipette solution (Sigma Chemical Co.). Perforation required a few minutes and the voltage-clamp was considered satisfactory when G series exceeded 20 nS. Charybdotoxin and apamin were from Alomone and dissolved in water. Tolbutamide was obtained from Sigma Chemical Co., and nifedipine was supplied by Research Biochemicals, Inc. Whenever DMSO was used as the solvent of the test compounds, the final concentration in the experimental solution was ≤0.1%. The [Ca 2+ ] i measurements were made using an Axiovert 135 inverted microscope equipped with a Plan-Neofluar 100×/1.30 objective (Carl Zeiss, Inc.) and an fluorescence imaging system (Ionoptix) as described elsewhere . Excitation was effected at 340 and 380 nm and emitted light recorded at 510 nm with a video camera synchronized to the excitation light source and a computer interface. The experiments were conducted using the perforated-patch whole-cell configuration using the pipette-filling solution specified above. Before the experiments, the cells were loaded for 20 min with 0.2 μM of fura-2/AM (Molecular Probes, Inc.). Calibration of the fluorescence ratios was performed by using the standard whole-cell configuration to infuse fura-2 with different of Ca 2+ -EGTA mixtures of known [Ca 2+ ] i . Data are presented as mean values ± SEM. Statistical significances were evaluated using Student's t test. Fig. 1 A shows a patch-clamp recording of the membrane potential in a β cell within an intact pancreatic islet. In the presence of 10 mM glucose, the β cell exhibited regenerative electrical activity consisting of bursts of action potentials. Confocal microscopy reveals that ≈30% of the superficial cells are insulin-containing β cells . The number of cells exhibiting the characteristic glucose-induced electrical activity, a hallmark of pancreatic β cells , amounted to 52 cells from total of >200 during a period of 2 yr. In 21 randomly selected cells exposed to 10 mM glucose, the duration of the depolarized plateau and the silent interval between two bursts averaged 7.8 ± 0.8 and 14.2 ± 2.1 s, respectively. To identify the current that terminates the burst, the glucose concentration was lowered to 5 mM and the amplifier switched into the voltage-clamp mode . Electrical activity was then simulated by a pulse train of voltage-clamp depolarizations. The voltage pulses evoke inward as well as outward currents. The latter reflect activation of delayed rectifying K + channels and, in agreement with previous results , exhibited significant inactivation during the train . However, close inspection of the current responses revealed that electrical stimulation was also associated with the gradual development of an outward holding current . The activation of the current could in most cases be described by a single exponential. The time constant of activation (τ a ) was determined as 2.3 ± 0.3 s ( n = 27). In a series of 30 experiments, the amplitude of this current measured at the end of the train amounted to 28 ± 2 pA. Fig. 1 D shows a representative example of two bursts of action potentials on an expanded time base. The membrane potential was maximally negative immediately after termination of the burst, and then gradually returned towards the threshold potential from which a new burst of action potentials originated. Comparison of the mean values of τ a (2.3 s) and the burst duration (7.8 s for glucose-induced electrical activity or 5 s for the train of simulated action potentials) suggests that the current has reached ≥90% of its maximal amplitude at the end of the burst. After cessation of stimulation, the current deactivated and returned to the prestimulatory level within 10 s. The correlation between the deactivation of the current and the depolarization between two bursts was analyzed in a subset of 18 cells in which the current returned to the baseline. In these cells, the time constant of current deactivation (τ d ) averaged 6.4 ± 1.8 s and the interval between two bursts in the same cells amounted to 13 ± 3 s ( n = 18). During the latter period, the measured deactivation of the current amounted to 94 ± 2% ( n = 18), close to the 87% predicted from τ d . Consistent with previous reports , electrical activity in intact islets differs from that recorded from β cell clusters maintained in short-term (24 h) tissue culture. Electrical activity in the latter preparation consists of very long bursts (often lasting several minutes) of action potentials. Fig. 1 E shows an example of electrical activity evoked by 10 mM glucose in a small cluster of cultured β cells. It can be seen that action potential firing continues uninterruptedly for at least 2 min, and that removal of glucose leads to termination of electrical activity. It is unlikely that the current in Fig. 1 C is attributable to the induction of regenerative electrical activity in neighboring β cells. As shown in Fig. 1 F, action potential firing in neighboring unclamped β cells in an islet exposed to 10 mM glucose gives rise to an inward current, whereas the current elicited by the train of simulated action potentials is outward. Also shown in Fig. 1 F is the current recorded in the same cell after lowering the glucose concentration to 5 mM. This suppresses electrical activity in the neighboring cells and allows the slowly activating and deactivating current to be studied in isolation. It is clear that reduction of the glucose concentration from 10 to 5 mM is associated with a 50% increase in the resting (K ATP ) membrane conductance (compare step current responses when stepping the membrane potential from −70 to −40 mV). It is also evident, however, that the current elicited by the train of depolarizations has the same amplitude, activation, and deactivation properties at both glucose concentrations. We next determined the ionic selectivity of the current elicited by the train of action potentials. Fig. 2 A shows the currents recorded after the train when the membrane potential was subsequently clamped at −50 mV in the presence of 3.6 and 15 mM extracellular K + ([K + ] o ). It is clear that whereas the current is outward at normal [K + ] o , it becomes inward at the supraphysiological concentration. Fig. 2 B summarizes the relationship between membrane potential and peak current amplitude. With 3.6 mM extracellular K + , the current reversed at a membrane potential of −73 ± 1 mV ( n = 4). After elevation of [K + ] o to 15 mM, the reversal was observed at −36 ± 2 mV ( n = 3). This shift of 37 mV for a 4.2-fold change of [K + ] o is precisely that predicted by the Nernst equation for a K + -selective conductance. Because the current activates and deactivates slowly and flows through K + -selective channels, we will henceforward refer to it as the K slow current. Fig. 3 shows recording of glucose-induced electrical activity in a β cell in an intact islet (A) and the variations of the holding current subsequently measured in the same cell under voltage-clamp conditions (B, top). It can be observed that the holding current oscillates in a way reminiscent of inverted bursts of action potentials. This is because the electrical activity in the neighboring cells spreads into the voltage-clamped cells via the gap junctions, giving rise to oscillations in the holding current . Voltage pulses (±10 mV, 200-ms long, 2 Hz) were applied to monitor changes in the membrane conductance (B, bottom). The input conductance was the same during the silent intervals and the periods of action potential firing and averaged 1.0 nS. When the same series of pulses were applied after a train of depolarizations, the membrane conductance (measured in the same β cell after lowering of the glucose concentration from 10 to 5 mM) was greatest at the end of the train and subsequently declined to a new steady state level , from a starting value of >2.1 nS to a steady state value of ≈1.3 nS . The latter value is higher than that measured in the presence of 10 mM glucose because the K ATP conductance increased when the concentration of the sugar was lowered to 5 mM. Collectively, the data presented in Fig. 3 suggest that, whereas electrical activity in the neighboring β cells spreads into the voltage-clamped cells via the gap junctions and thus gives rise to oscillations of the holding current, activation of ion channels or passive charging of the membrane in the unclamped cells does not contribute to the measured membrane conductance measured in the voltage-clamped cell. Tolbutamide, an inhibitor of K ATP channels , had no effect on the current , but reduced the current step obtained when stepping the membrane potential from −70 to −40 mV. The latter observation indicates that some K ATP channels remained active in 5 mM glucose. The K slow current was likewise unaffected by both charybdotoxin (100 nM) and apamin (1 μM), blockers of large- and small-conductance Ca 2+ -activated K + channels, respectively (not shown). By contrast, the broad spectrum K + channel blocker tetraethylammonium (TEA) 1 reduced the amplitude of the K slow current in a concentration-dependent manner. Fig. 4 B shows an example where 20 mM TEA reduced the slowly deactivating current by ≈70%. The concentration dependence of the inhibition is summarized in Fig. 4 C. Inhibition was half-maximal at ≈5 mM TEA and ≈30% of the current was resistant to TEA. The effect of TEA on the amplitude of the current evoked by the train was associated with dramatic changes of the action potential firing pattern and the bursts of action potential were replaced by large overshooting action potentials that were either generated singly or as groups of 5–10 spikes . We next investigated the relationship between K slow current activation and [Ca 2+ ] i . These experiments had to be conducted on isolated β cells rather than intact islets as it is not possible to voltage-clamp an entire islet with the patch electrode. Moreover, fluorescence originating from the unclamped neighboring β cells an be expected to contribute to the overall signal and obscure that derived from the voltage-clamped cell. Fig. 5 A shows simultaneous recordings of [Ca 2+ ] i and K slow current. It is clear that both the activation and deactivation of the current reasonably correlate with the observed changes of [Ca 2+ ] i . In this particular cell, the K slow current amplitude at the end of the train was 14 pA. This is considerably larger than the typical value and the average, determined without intracellular fura-2, was 4 ± 1 pA ( n = 18). This amplitude in the isolated β cells is only 15% of the K slow current observed in intact islets . Given the correlation between [Ca 2+ ] i and K slow current activation, we tested the effects of inhibiting the voltage-gated Ca 2+ channels. The Ca 2+ channel antagonists nifedipine (10 μM, not shown), Co 2+ (5 mM, not shown), and Cd 2+ all prevented activation of the K slow current. Because the K slow current is dependent on Ca 2+ influx, and its activation and deactivation echoes changes of [Ca 2+ ] i , it seems plausible that it flows through some sort of Ca 2+ -activated K + channel. We next considered the possibility that the K slow current is smaller in dispersed cultured β cells because the Ca 2+ current is reduced in these cells. Indeed, the Ca 2+ current elicited by 100-ms depolarizations was significantly (40%) smaller in dispersed cells than in β cells within intact islets at all voltages ≤0 mV . We finally compared the amplitude of the K slow current with the changes of the K ATP conductance associated with the transition from oscillatory into uninterrupted electrical activity. Fig. 6 shows parallel recordings of membrane potential and K ATP conductance in a β cell within an islet. In the absence of glucose, the membrane potential was approximately −80 mV. The membrane conductance under these conditions (determined by switching the amplifier into the voltage-clamp mode, holding at −70 mV, and applying ±10-mV voltage pulses) exceeded 5 nS . Addition of 15 mM glucose resulted in membrane depolarization and the induction of oscillatory electrical activity. This was associated with a >75% reduction in resting membrane conductance, which fell to ≈1.2 nS . Subsequent addition of 100 μM tolbutamide, to inhibit remaining K ATP channel activity, evoked continuous spiking and a further reduction of the membrane conductance to ≤1 nS . In a series of six experiments, the membrane conductance measured in the absence of glucose averaged 3.9 ± 1.0 nS. In the presence of 10 (five cells) or 15 (four cells) mM glucose (i.e., when the β cells generated oscillatory electrical activity), the membrane conductance dropped to 1.4 ± 0.2 nS ( n = 9; P < 0.001 vs. that observed in the glucose-free solution). The corresponding value in the simultaneous presence of 10 or 15 mM glucose and 100 μM tolbutamide was 1.0 ± 0.1 nS ( n = 7). The decrease in membrane conductance obtained by addition of tolbutamide (100 μM) to islets already exposed to 10 or 15 mM glucose thus amounted to 0.4 ± 0.1 nS ( n = 7; P < 0.025). This value is smaller than the 0.8 ± 0.1 ( n = 30) that can be derived for the K slow current from its amplitude and reversal potential (28 ± 2 pA at −40 and −73 mV, respectively). We describe here a Ca 2+ -activated K + current that turns on gradually during electrical activity (K slow current). A number of considerations argue that the K slow current is recorded from single β cells in the islet and that it cannot be attributed to regenerative electrical activity in neighboring unclamped cells. First, the measured cell capacitance for β cells in the intact islets is only 40% larger than that of the isolated cells . Second, the amplitude of the K ATP conductance in intact islets is close to that measured in isolated cells . Third, regenerative electrical activity in neighboring cells does not give rise to detectable changes of the membrane conductance in the voltage-clamped cell . Fourth, a current with similar properties can be recorded from a small fraction of dispersed β cells . We considered the possibility that depolarization of the voltage-clamped cell influences the membrane potential of neighboring cells and that the slowly deactivating current we observe reflects the reversal of this process. The time constant (τ) of the latter process would be represented by the product R j C m , where R j is the gap junction resistance and C m is the cell capacitance. The gap-junction resistance has been estimated as 0.7 GΩ and the cell capacitance is ≈7 pF . These values predict that the time constant for passive charging of the voltage (and its reversal) is ≈5 ms; i.e., three orders of magnitude from the value of τ actually observed. This argument is simplistic in assuming that the value of R j reflects the coupling to a single cell. Nevertheless, these considerations make it obvious that the voltage-clamped β cell needs to be directly coupled to ≥1,000 cells to give rise to the experimentally observed time constant of current decay that is clearly unreasonable. The slowly activating current is small, but it may nevertheless play an important role in the shaping of oscillatory electrical activity in the β cell. For example, the gradual decline in action potential frequency and slight repolarization of the plateau potential that is observed during the burst can be attributed to the gradual turn-on of a repolarizing K + current. The time course of the deactivation of the current is similar to that of the pacemaker depolarization between two bursts and may echo the gradual return of [Ca 2+ ] i to the resting concentration. The amplitude of the whole-cell K slow conductance (G K,slow ) attained at the end of the burst is equivalent to 0.8 nS. This is 20% of the whole-cell K ATP conductance (G K,ATP ) in the absence of glucose. It is important to emphasize, however, that the magnitude of G K,slow is actually larger than the decrease in G K,ATP when the tolbutamide is added to β cells already exposed to 10 or 15 mM glucose; i.e., when the β cell goes from oscillatory to continuous electrical activity . This serves to illustrate that small changes in the whole-cell conductance are capable of exerting marked effects on the pattern of action potential firing . The fact that the K slow current we now describe is not affected by selective inhibitors of large- and small-conductance Ca 2+ -activated K + channels such as charybdotoxin and apamin makes it difficult to unequivocally demonstrate that it is responsible for the repolarization terminating the burst of action potentials. However, it is of interest that TEA at high concentrations (≥10 mM) reduces the current by ≈70% and suppresses the normal oscillatory pattern . The interpretation of the effects of TEA is naturally complicated by the nonselective properties of this compound and TEA affects all K + conductances characterized in the β cell, including delayed rectifying K + channels and large-conductance Ca 2+ -activated K + channels . The finding that TEA abolishes the normal oscillatory pattern can therefore not be used to argue that the Ca 2+ -dependent K + current we describe here (the K slow current) participates in the generation of the bursting pattern. Indeed, the fact that the action potentials remain grouped in the presence of TEA seemingly contradict such a role, but we point out that a fraction (>30%) is resistant to TEA. The overshooting and long-lasting action potentials generated in the presence of TEA are associated with massive Ca 2+ influx and large elevations of [Ca 2+ ] i that can be expected to result in great activation of the K slow current. It is therefore possible that 30% of the current activated by the large action potentials in terms of the absolute current amplitude is greater than that activated by a normal burst of action potentials. The failure of high concentrations of TEA to completely inhibit the current may be explained if it flows through two types of K + channel, one of which is resistant to TEA. K ATP channels show little sensitivity to TEA and it is possible that the Ca 2+ -influx taking place during repetitive stimulation lowers the ATP/ADP ratio sufficiently to produce some activation of the K ATP channels . However, ≈70% of the current is TEA sensitive and the K slow current is unaffected by tolbutamide, making the contribution of the K ATP channels questionable. As to the molecular identity, a Ca 2+ -activated K + channel clearly represents an attractive candidate despite the failure of both charybdotoxin and apamin to be effective. However, we point out that the slow activation and deactivation kinetics as well as resistance to apamin conform with the properties of SK1 channels . With regard to the future pharmacological and molecular characterization of the K slow current, it is of interest that a Ca 2+ -activated K + current, which seemingly shares many properties with the K slow current in β cells, has been documented in clonal βTC3 cells . The finding that dispersed β cells generate an atypical electrical activity combined with the observation that the K slow current is much reduced in such cells provide circumstantial evidence for its involvement in the termination of the bursts of action potentials. Interestingly, rapid membrane potential oscillations can be induced in isolated β cells under experimental conditions associated with InsP 3 -dependent mobilization of Ca 2+ from intracellular stores . The associated increase in [Ca 2+ ] i leads to the activation of a Ca 2+ -activated K + current that, like the K slow current described in this study, is resistant to both apamin and charybdotoxin , and is little affected by TEA at concentrations ≤5 mM. Given these similarities, it seems possible that it is identical to the K slow current we now describe in situ. The whole-cell conductance of the current measured with physiological ionic gradients in dispersed cells is 0.25 nS ; i.e., ≈30% of the K slow current amplitude in the islet. It remains to be determined why this current is not activated by the Ca 2+ influx associated with a train of action potentials (or voltage-clamp depolarizations). However, it may be of relevance that the amplitude of the Ca 2+ current in dispersed cells is only 60% of that observed in β cells within intact islets . Another possibility is that the architecture of the β cell changes during cell isolation so that the K slow and Ca 2+ channels become separated from each other. The domain of elevated Ca 2+ beneath the Ca 2+ channel may consequently not extend sufficiently to activate the K slow channels. The latter possibility is suggested by the observation that although they could not be activated by Ca 2+ influx during action potential firing, they remained activatable by InsP 3 -dependent mobilization of intracellular Ca 2+ . We propose that control of the membrane potential oscillations by two distinct K + conductances, one regulated by glucose metabolism (K ATP channels) and one determined by electrical activity via increases in [Ca 2+ ] i (K slow channels), provides the β cell with the means of responding to glucose in a graded fashion. The generation of oscillatory β cell electrical activity can be explained in the following way: increasing the glucose concentration from 0 to 10 mM produces a decrease in G K,ATP from 4 nS to ≈1.5 nS . This results in membrane depolarization and induction of electrical activity. The Ca 2+ entry associated with electrical activity leads to a gradual increase in K slow channel activity (G K,slow ) that echoes the change of [Ca 2+ ] i . When the total K + conductance is sufficient to overcome the depolarizing influence of the voltage-gated Ca 2+ current, the β cell starts repolarizing, leading to regenerative closure of the voltage-gated Ca 2+ channels and a reduction of [Ca 2+ ] i . The decrease in [Ca 2+ ] i in turn leads to the deactivation of G K,slow . In this model, the background G K,ATP defines how much G K,slow may increase before initiating membrane repolarization. The concentration-dependent glucose-induced decrease in G K,ATP thus allows for progressively greater activation of the K slow current before repolarization occurs and the duration of the burst accordingly increases. Continuous electrical activity is elicited at glucose concentrations approaching 20 mM when G K,ATP is sufficiently suppressed to allow electrical activity to continue even when G K,slow is maximally activated. This model is compatible with the observation that tolbutamide converts oscillatory electrical activity to continuous action potential firing and the latter observation should not be used as an argument that the K ATP channels are directly involved in the burst repolarization. We acknowledge that an increase in K + conductance may not be the sole mechanism participating in the generation of oscillatory electrical activity in the β cell. For example, inactivation of the voltage-gated Ca 2+ current has also been implicated in the process The scenario outlined above predicts that [Ca 2+ ] i should increase throughout the burst of action potentials, reaching a peak at the time repolarization commences . However, not all studies have documented such behavior; for example, Santos et al. 1991 report that [Ca 2+ ] i quickly rises to a plateau at the beginning of the burst of action potentials but subsequently remains fairly stable. It should be kept in mind, however, that the relationship between [Ca 2+ ] i and indo-1 fluorescence (the dye used in the above study) is nonlinear and does not increase much at concentrations >1 μM . In this context, it should also be pointed out that the Ca 2+ channels and the K slow channels are likely to be spatially separated. This is suggested by the fact that glucose-induced electrical activity consists of bursts of action potentials. If the two types of ion channels were situated in the immediate vicinity of each other, then the β cell would be expected to exhibit fast afterhyperpolarizations after every action potential as documented in, for example, hippocampal neurons . The finding that such afterhyperpolarizations are not observed instead argues that Ca 2+ needs to diffuse over some distance within the β cell to activate the K slow channels. During this process, the Ca 2+ signal may be subject to various modulatory influences. For example, it may be amplified by Ca 2+ release from intracellular stores . This scenario is supported by the finding that exposure of pancreatic islets to thapsigargin, an inhibitor of the Ca 2+ -ATPase in smooth endoplasmic reticulum, abolishes the bursting pattern and results in uninterrupted action potential firing . A recent study also demonstrates that a train of voltage-clamp depolarizations evoke a slowly decaying component of [Ca 2+ ] i increase that disappears after pretreatment with thapsigargin . The time constant of this [Ca 2+ ] i component was reported to be 13 s, not too different from the 6.5 s we observe for the deactivation of the K slow current and very close to the 13-s interval between two successive bursts. It is therefore of interest that preliminary evidence suggests that the K slow current is sensitive to thapsigargin and that its amplitude is reduced after pretreatment with this ATPase inhibitor . | Study | biomedical | en | 0.999996 |
10578014 | Neutrophils generate superoxide (O 2 · 2 ) during phagocytosis of opsonized bacteria, immune complexes, and other bodies . Oxygen is reduced to O 2 · 2 at the exterior of the cell, while H + is liberated from NADPH at the cytoplasmic surface. It has been proposed that, to maintain the activity of the membrane-bound oxidase, there is an efflux of H + through what are assumed to be H + -selective ion channels. This would not only prevent a substantial fall in intracellular pH (pH i ), but also compensate for the separation of charge, which would otherwise generate a large positive membrane potential. Analysis of genetic lesions in patients with chronic granulomatous disease (CGD) 1 has facilitated the identification of the components of the phagocytic NADPH oxidase ( phox ). There is considerable evidence that one component, gp91- phox , a product of the X-linked CGD gene , acts as an H + -selective pathway. Not only do CGD cell lines in which gp91- phox is absent have a deficient H + permeability, but suspensions of a Chinese hamster ovary (CHO) cell line expressing gp91- phox (CHO91 cells) show a marked H + membrane flux in the presence of sodium arachidonate . A likely candidate for the H + -selective pathway is the voltage-gated H + conductance first described in giant molluscan neurons and subsequently reported in human neutrophils and other phagocytes including microglial cells and osteoclasts . Its expression in microglial cells, osteoclasts, and HL60 cells is associated with the differentiation and expression of NADPH oxidase. This correlation between NADPH oxidase expression and the presence of voltage-gated H + currents suggests that gp91- phox may form a voltage-gated H + pathway in phagocytic cells. Here we report a positive correlation between the expression of human gp91- phox and the presence of a voltage-gated H + current in a CHO cell line studied using the whole cell configuration of the patch clamp technique. Studies of cells containing mutated versions of gp91- phox suggest that its voltage sensitivity resides in the NH 2 -terminal 230 amino acids and that histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H + permeation. The stable CHO cell line expressing the full-length gp91- phox (CHO91) was constructed and cultured as described previously . In brief, it was constructed by transfecting CHO-K12 cells with the full-length cDNA for gp91- phox , placed behind the inducible human metalothionein IIa promoter . In the studies presented here, expression was induced by incubating cells in 10 μM CdCl 2 for 24 h before assay. Cells were studied in the whole cell configuration under conditions designed to maximize the amplitude of H + currents present . The main aims were to exclude currents carried by ions other than H + and to acidify the cell contents to shift activation of the H + conductance towards experimentally accessible membrane potentials. The patch pipette filling solution contained 119 mM tetramethylammonium (TMA) hydroxide, to block potassium currents, a small quantity of calcium buffer (3.7 mM EGTA, 0.74 mM CaCl 2 ) and was adjusted to pH 6.5 with Mes so that its final concentration was ∼120 mM. Pipettes had a resistance of 2.5–4 MΩ and the seal resistance was in the order of 1–3 GΩ at the outset. Cells were bathed in a saline that contained 110 mM TMA methane-sulphonate, 2 mM Ca(OH) 2 , 2 mM Mg(OH) 2 , 5 mM glucose, and 100 mM pH buffer. The pH was adjusted to 8.0 or 7.5 with N -[2-hydroxyethyl]-piperazine- N ′-[3-propane-sulphonic acid] (EPPS) or 7.0 with HEPES. Other recordings were made from cells bathed in 120 mM NaCl, 10 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 50 mM HEPES, 1 mM NaH 2 PO 4 , 5 mM glucose, pH 8.0. Cells were superfused with up to seven different solutions using an in-house superfusion system . Except where indicated, low amplitude prepulses in the linear current–voltage range (−60 to −100 mV) were scaled by PCLAMP 6 software (Axon Instruments) and used for online subtraction of linear capacitive and leakage currents . To produce a transient 1-U change in the pH of cell cytoplasm, it is necessary to inject at least 10 mmol H + /liter ; to hold pH i at a steady level, it is necessary to overcome membrane-bound regulatory mechanisms as well as a complex system of endogenous fixed and mobile pH buffers. Here, CHO cells were studied in the whole cell configuration under conditions designed to exclude currents carried by ions other than H + . The bathing solution was Na + free and contained no added HCO 3 − so that known mechanisms of proton exchange in the plasma membrane should be inactive. Nevertheless, we were uncertain as to the effect on the cell cytoplasm of its coming into contact with a patch pipette filled with pH buffer. In experiments on squid axons, the intra-axonal pH was controlled by a perfusion solution containing 45 mM buffer , but much of the axoplasm had been removed by enzymatic digestion. In intact molluscan neurons dialyzed against the contents of a macropipette, buffer concentrations as high as 120 mM were inadequate to control pH i if the pipette diameter was less than one third of the cell diameter . In the work reported here, 25-μm-diameter CHO cells were dialyzed against 2-μm-diameter patch pipettes and it was necessary to establish the time course of the exchange between pipette and cell and the effect on pH i . The time taken for the pipette filling solution to equilibrate with the cell cytoplasm was assessed using a confocal optical scanning microscope (MRC 600; Bio-Rad Laboratories). Because the microscope collects emitted fluorescent light only from within the plane of focus of the objective lens, it performs noninvasive optical sectioning . Patch pipettes were filled with the inert fluorescent dye, Lucifer yellow (500 μM; Molecular Probes, Inc.), and seals formed to CHO91 cells mounted on the microscope stage. Images focused midway through the cell (excitation at 488 nm; Kalman average of three scans), were collected before perforation of the cell membrane and at 60-s intervals in the whole cell configuration. Images showing the fluorescence of the pipette contents were taken at the end of the experiment by adjusting the focal plane of the microscope. Average fluorescence intensities of regions of interest were obtained using COMOS software (Bio-Rad Laboratories). Confocal images were collected as above with the pH sensitive fluorescent probe, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 50 μM) in place of Lucifer yellow. Images of patch pipettes filled with 50 μM BCECF dissolved in pH 6.1, 6.5, and 7.0 pipette solutions were used to calibrate the BCECF fluorescence intensity. NH 2 -terminal mutants of gp91- phox were constructed with three tandem copies of the hemagglutinin (HA) epitope on the COOH terminal of the protein . CHO cell lines were cultured on glass coverslips and treated with 10 μM Cd 2+ 24 h before immunostaining to induce expression of the protein. The cells were fixed (4% formaldehyde, 10 min) and permeabilized (0.2% Triton X-100) before staining with anti–HA epitope monoclonal antibody (1 h). Cells were incubated with FITC-labeled anti–mouse (1 h) and imaged on a confocal microscope. In the work reported here, 25-μm-diameter CHO cells were dialyzed against 2-μm-diameter patch pipettes. In view of the difficulty in controlling pH i (see materials and methods ) preliminary experiments were designed to (a) establish the time course of the exchange between pipette and cell, and (b) determine the final level of pH i . The time course of the exchange between the contents of a small cell and the contents of a patch pipette in the whole cell configuration has been studied both experimentally and theoretically . If the contents of both pipette and cell remain homogenous, exchange at the pipette tip is rate limiting. Other influential factors are the cell volume and the size of the diffusing molecule (see materials and methods ). In Fig. 1 , a patch pipette filled with Lucifer yellow was used to record from a 25-μm-diameter CHO91 cell in the whole cell configuration. Although the intensity of the cytoplasm was undetectable 60 s after perforation of the cell membrane, fluorescence increased rapidly over the first 10 min . Measurement of the average fluorescence within the boundary of the cell membrane showed an increase that followed an approximately exponential time course with a time constant of 150 s . This is consistent with the findings of Pusch and Neher 1988 , who found that molecules the size of Lucifer yellow (∼600 D) should transfer though a 5-MΩ patch pipette to the cytoplasm of a 25-μm-diameter cell with a time constant of between 85 and 169 s. A comparison between the maximum intensity of the cell (mean 136, n = 3) and the fluorescence of the pipette (mean 133, n = 3) showed that the dye was evenly distributed between cell and pipette . There was no change in cytoplasmic fluorescence intensity if the cell membrane remained intact (not shown). To examine the time course of the change in pH i , we used patch pipettes filled with the pH indicator BCECF buffered with 120 mM pH buffer (Mes). The molecular weight of BCECF (520) is similar to Lucifer yellow, while Mes (213 D) is somewhat smaller and should equilibrate with the cell more rapidly than the dye. The emitted fluorescence intensity of BCECF decreases with decreasing pH and so the cytoplasm should register only a small overall increase in fluorescence if its pH followed that of the pipette solution (pH 6.2). Fig. 2A and Fig. B , shows that the time course of the fluorescence increase was similar to that for Lucifer yellow, fluorescence reaching a maximum in 15–20 min. However, unlike Lucifer yellow, the fluorescence intensity of the cell and the pipette were not equal once the system had reached a steady state and remained different even after 60 min in the whole-cell configuration. The average fluorescence intensity of six cells was 163 U compared with 91 U for the pipette contents. Assuming that BCECF, like Lucifer yellow, was equally distributed between cell and pipette, and also that there was no interaction between BCECF and the cytoplasm, the inequality of fluorescence must arise from a difference in pH between the pipette solution and the cell contents. From the calibration curve , the average value for pH i was near 6.9. As this was significantly different to the pipette solution (buffered to pH 6.2), it seemed that pH i was largely determined by endogenous cellular regulation. Nevertheless, the level of acidification achieved by using pipettes filled with 120 mM pH buffer was sufficient for the purposes of the present experiments. In control cells loaded with BCECF, pH i at rest is estimated to be 7.2 . In previous experiments, suspensions of CHO91 cells have been used to demonstrate that gp91- phox functions as an H + pathway when activated by sodium arachidonate . To determine whether the gp91- phox pathway was voltage gated, we recorded membrane currents from CHO91 cells using the whole cell configuration of the patch-clamp technique. There are low levels of arachidonate-activated H + conduction in both untransfected CHO cells and transfected but uninduced (i.e., nonexpressing) CHO91 cells but, in keeping with the report by Cherny et al. 1996 , any voltage-gated H + currents present were within the noise level of our recordings. Fig. 3 A shows data from a typical untransfected CHO cell bathed in pH 8.0 saline, under conditions designed to exclude currents other than those carried by H + . The cell was depolarized by a series of voltage commands, as shown in the protocol at top. In this and the four other untransfected cells tested, the whole-cell currents were all within the noise levels shown. Similar records were obtained for nonexpressing CHO91 cells under the same conditions ( n = 6). Fig. 3 B shows a typical example. Under identical conditions, CHO91 cells expressing full-length gp91- phox generated large time- and voltage-dependent currents. The outward currents were not recorded immediately upon going whole cell, but increased with time, reaching a maximum after 10–25 min. This is in good agreement with the time course of exchange between the cell and the patch pipette shown in Fig. 1 and Fig. 2 and suggests that the amplitude of the current was not just a property of the transfected cells, but depended on the degree of acidification of the cell cytoplasm. This was confirmed by experiments in which the pipette contents were buffered to pH 7.5 with EPPS buffer (not shown). In this case, there was no change in the whole cell current during a prolonged period of recording. Small differences in the expression of gp91- phox may explain the observed variation in current amplitude, but there were also differences in time course. Fig. 3 C shows a cell in which the current steadily increases throughout each 800 ms command step, while in Fig. 6 A the currents rapidly achieve a steady level. In a population of cells with a steadily rising outward current, the mean amplitude after 800 ms at +80 mV was 3.4 nA ( n = 9; SD 1.1 nA; range 1.7–4.6 nA); in a population of cells with steady outward currents the mean amplitude at +80 mV was 4.2 nA ( n = 9; SD 2.1 nA; range 1.5–7.0 nA). Voltage-gated proton currents in other cell types show similar steadily increasing currents, but at present only a tentative explanation can be put forward to account for them; it is possible that during prolonged depolarizing commands negatively charged buffer molecules contribute to the pipette current by leaving the cytoplasm. The resulting acidification at the membrane near the pipette tip will produce a progressive, local shift in activation towards more negative membrane potentials. The currents were sustained during command pulses of 2 s and showed no inactivation (not shown), which is another characteristic of voltage-gated proton currents. The H + pathway associated with NADPH oxidase , like that of gp91- phox and the voltage-gated H + pathway in human neutrophils , is activated by low levels of sodium arachidonate. Fig. 4 shows a similar effect on the voltage-gated currents observed in CHO91 cells. Currents elicited by control commands to 20, 40, and 100 mV are shown as solid lines , whereas those recorded during the application of 20 μM sodium arachidonate are shown as data points. The scale on the right refers to the test data; the control currents having been increased to match. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Thus, although sodium arachidonate produced a significant increase in the amplitude of the voltage-gated currents, the time course of activation remained quite unaffected. The effect on current amplitude was rapidly reversible (not shown). As shown in Fig. 4 B, sodium arachidonate appeared to shift the voltage dependence of activation towards more negative membrane potentials. The membrane conductance at the end of each command pulse was calculated assuming −55 mV for the equilibrium potential (see later for tail current reversal potential) and the maximum value normalized to 1. The Boltzmann curve (solid line) fitted to the data obtained in 20 μM sodium arachidonate has a slope factor of 20 mV with half-activation (V 1/2 ) at −2 mV. The control data when normalized to the same maximum value were described by a second Boltzmann curve with the same slope factor and a V 1/2 of 21 mV. The maximum conductance in the control was 0.87× that found in the presence of sodium arachidonate. It appears that the effect of arachidonate is both to increase the maximum conductance available and shift the activation curve to more negative values. The shift of the activation curve to more negative voltages in the presence of arachidonate as shown in Fig. 4 B should permit activation of proton currents at membrane voltages more negative than the H + equilibrium potential (E H ) and make it possible to observe them flowing inward across the activated pathway. To test this, membrane currents were recorded in pH o 6.8 bathing solution before the equilibration of cytosol and pipette solution. Under these conditions, pH i would be higher than pH o . Currents in response to a series of depolarizing command pulses to −20 mV are shown in Fig. 4 C. A sustained inward current was recorded from the cell perfused with 20 μM arachidonate that was not observed in its absence and was reversible upon its removal ( n = 3). As in suspensions of CHO91 cells , the direction of H + flux through the NADPH oxidase-associated H + pathway in the presence of arachidonate is either inward, if the pH o is less than the pH i , or outward, if the pH o is greater than the pH i . As originally described in snail neurons, voltage-dependent H + currents are inhibited in a readily reversible manner by 1 mM Cd 2+ , Zn 2+ , Ni 2+ , and other divalent ions . In human neutrophils, low concentrations of Zn 2+ reversibly inhibit both the arachidonate-activated and voltage-gated H + pathways. Zn 2+ also reversibly inhibited the gp91- phox –mediated pathway in CHO91 cells. Fig. 5 A shows the amplitude and time course of membrane currents before and after addition of 200 μM Zn 2+ to the bathing solution. The outward current was significantly reduced in the presence of Zn 2+ and showed partial recovery when examined after washing. Fig. 5 B shows the time course of the effect. A series of depolarizing command pulses was used to monitor the outward current. At the point shown, the normal bathing solution was replaced by one containing 200 μM Zn 2+ . The pronounced inhibition of the large time- and voltage-dependent outward current recovered rapidly once the perfusion solution was returned to the Zn 2+ -free control solution. Studies on preparations as different as snail neurons and human neutrophils have shown that the voltage dependence of the H + conductance is shifted to more negative potentials by low pH i and high pH o . The amplitude of the time- and voltage-dependent currents recorded from CHO91 cells also depended on the pH of both the bathing and pipette solutions. At pH o 8.0, there was a significant outward current with depolarizing commands to values as negative as −20 mV , while at pH o 7.0 time-dependent currents were generally absent at commands below 0 mV. In Fig. 3 and Fig. 4 , the time-dependent outward currents were rapidly deactivated after each command step, and this was seen as an outward “tail” when the cell was repolarized to −40 mV. To establish whether H + was the charge-carrying species, tail currents were measured in different external solutions and their reversal potential was determined. For this series of experiments, the pipette solution was buffered to pH 6.5. For Fig. 6 A, pH o was set to 8.0 and the cell was depolarized to 0 mV to activate the outward conductance. Repolarization to −100 and −80 mV resulted in a marked inward tail current, but with the potential at −60 mV the current was clearly outward. When repolarized to –40 mV, some deactivation was evident, but there was also a small maintained outward current . Note that measurements at potentials more positive than approximately −40 mV were precluded because of the voltage activation range of the pathway. The decline in tail current measured over 200 ms was plotted against potential in Fig. 6 D (□). When trials were repeated with cells bathed in pH 7.5 saline, inward tail currents were observed not only at −100 and −80 mV, but also at −60 and −40 mV, and so it was clear that the reversal potential had moved in the positive direction . As before, the decline in tail current was measured over 200 ms and plotted against potential in Fig. 6 D (○). CHO91 cells in pH 7.0 buffered saline generally had a high leakage conductance after 30 min recording and their time-dependent currents were small so that it was necessary to depolarize the cell to 120 mV for significant outward current. Activation was markedly slower in this solution. The decline in tail current amplitude was measured as before and plotted as in Fig. 6 D (▵). In both pH 7.5 and 7 solutions, it was not possible to generate outward tail currents because of the voltage-activation range of the pathway. A large decrease in the rate of activation with acidification of the external medium has been reported previously . In Fig. 6 D, the lines through the data were drawn according to a constant field equation with pH i taken as 6.9. The H + permeability was chosen to fit the pH o 8 data; with the pH o 7.5 and 7 data scaled up by factors of 3.5 and 7.5. Filled symbols represent data from other cells in the same external solutions: pH 8.0 (▪), scaling factor, 1; pH 7.5 (•), scaling factor, 3.5; pH 7.0 (▴), scaling factor, 11. The reversal potential in each solution was estimated by simple extrapolation and in pH o 8 solution its mean ± SD was −55 ± 6 mV ( n = 18). In pH o 7.5 solution, the average reversal potential was −20 ± 3 mV ( n = 9) and, in pH o 7 solution, it was 1 ± 5 mV ( n = 6). The good correspondence between this data and the 58 mV change/pH unit predicted by the Nernst equation confirms that H + (or an H + equivalent) is the probable conducting species. Chloride ions are unlikely to make a contribution because neither the amplitude of the outward current nor the tail current reversal potential was affected by bathing solutions containing different concentrations of chloride (0, 1, 2, 4, and 120 mM; not shown). However the Goldman-Hodgkin-Katz equation predicts larger inward currents as the pH of the bathing solution is made progressively more acid. In practice, because of the shift in the activation curve, the currents were significantly smaller in acid bathing solution and increasing degrees of scaling were necessary to fit the data. A number of CHO cell lines have been constructed that express mutated forms of gp91- phox . Suspensions of the cell line CHO-N, which expresses only the NH 2 -terminal 230 amino acids, exhibit a fully functioning arachidonate-activated H + flux, but this flux is absent in CHO-N3Leu, a cell line that express the same NH 2 -terminal region with leucine in place of the three histidine residues in positions 111, 115, and 119. The flux is also greatly reduced in cells with a single histidine residue, 115, mutated to leucine . Examination of whole-cell currents from cells containing the mutated versions of gp91- phox suggests that voltage as well as arachidonate sensitivity is retained by cells with only the NH 2 -terminal amino acids. Furthermore, the three histidine residues, 111, 115, and 119, are functionally important for H + permeation. The whole-cell current traces in Fig. 7 A show that in transfected but not induced CHO-N cells, as in CHO91 cells under similar conditions, any voltage-gated current present is concealed within the noise level of the recording. Fig. 7 A shows data (typical of six observations) from a single cell bathed in pH 8.0 saline and depolarized by a series of voltage commands . CHO-N cells induced to express the NH 2 terminus, generated large time- and voltage-dependent currents in response to depolarizing voltage commands in the range 0 to +120 mV. Just as for CHO91 cells, the outward currents were not recorded immediately upon going whole cell, but increased progressively over a 10–25-min period ( n = 10). The mean current amplitude at the end of a 400-ms command to +80 mV was 2.3 nA (±0.53 nA; n = 9). Outward currents from CHO-NLeu–expressing cells, like those from CHO-N cells, became activated by depolarizing commands to potentials in the range 0 to +120 mV, but they were of a reduced amplitude and slower rise time . In these cells, the mean current amplitude at the end of a 400-ms command to +80 mV was 0.61 nA (±0.4 nA; n = 6). In CHO-N3Leu–expressing cells, the outward currents had an even lower amplitude in part because activation was shifted to more positive voltages; in most cases, little or no current was observed at potentials more negative than +80 mV. At this voltage, the mean current amplitude at the end of a 400-ms command was <0.01 nA ( n = 6); at +140 mV, the mean current was ∼0.3 nA. The tail currents recorded at −40 mV from both CHO-NLeu and CHO-N3Leu cells, in pH 8.0 bathing solution, were inwardly directed, unlike those recorded from CHO- and CHO-N–expressing cells, which were outward. In CHO-N3 cells, the mean reversal potential was 23 mV (±9 mV; n = 3). This apparent shift in tail reversal potential suggests an alteration in the selectivity of the conducting pathway when histidine 115 was replaced by leucine. From the fluorescence intensity of immunostained cells, we were able to compare levels of expression in different mutated forms of gp91- phox . The cDNA constructs have three tandem repeats of the hemagglutinin epitope attached to their COOH-terminal ends. In Fig. 8 , confocal images of Cd 2+ induced CHO-N (A and B) and CHO-N3Leu (D and E) cells immunostained with antihemaglutinin antibody gave an annular pattern of fluorescence that was not observed in uninduced cells (C and F). This pattern of staining implies that the antigen is not only expressed, but that it is located at or in the plasma membrane and it is similar to that already observed in CHO cells expressing full-length gp91- phox . The levels of staining do not differ greatly; expression in CHO-N3Leu being slightly greater than that in CHO-N. In this paper, we conclude that gp91- phox , the product of the X-linked CGD gene and component of the phagocytic NADPH oxidase, functions as a voltage-dependent H + conductance. Transfected CHO cells expressing gp91- phox exhibit large time- and voltage-dependent outward currents not present in untransfected or nonexpressing cells . The currents were observed under conditions that minimized a contribution from the smaller ions normally present in physiological saline. The main cation present both internally and externally was tetramethylammonium ion, while the main anion was methane-sulphonate. The tail current that followed a depolarizing command had a reversal potential that depended on the pH gradient across the cell membrane, changing by ∼58 mV for a unit change in pH o . Consequently, the charge carrier for these outward currents is likely to be either H + or an H + equivalent. Voltage-gated H + conductances have been studied in a wide range of different tissues and animal species that include molluscan neurons, amphibian eggs, human neutrophils, and other phagocytic cells . Their properties have been found to be remarkably consistent: the voltage dependence is shifted by both pH i and pH o , they are inhibited by low levels of divalent ions with a special sensitivity to Zn 2+ , and in some cases they are amplified by arachidonate. The inhibition of the gp91- phox –associated voltage-gated current by Zn 2+ and its augmentation by arachidonate suggests that it flows through the NADPH oxidase–associated H + pathway previously studied in cell suspensions by Henderson et al. 1995 . The voltage-gated H + conductance described by DeCoursey and Cherny 1993 in human neutrophils closely resembles that found in other cells: its voltage dependence is shifted to more positive potentials by external H + and it is inhibited by external Cd 2+ or Zn 2+ ; however, its activation kinetics are significantly slower than those described in molluscan neurons or in Ambystoma oocytes . In neutrophils , the currents are amplified by arachidonic acid, but it is not known whether this property is characteristic of the class of voltage-gated proton conductances. Although we would expect the gp91- phox –associated H + conductance to have properties broadly similar to those found in vivo in neutrophils, the CGD gene product that we are using is one subunit of the oxidase complex, which normally has four or five protein components. Consequently, even if these other subunits do not directly contribute to the proton pathway, we would expect the environment of the expressed product in CHO cells to be significantly different from that in the natural state. This may account for their activation kinetics, which is the most marked difference between the gp91- phox– associated currents and the proton currents recorded from neutrophils . In neutrophils at room temperature, the outward current continued to rise even with commands that lasted 3–4 s, and there was no initial rapid phase as found here. However, the neutrophil pathway has a higher temperature sensitivity than most ion channels , and this may also be reflected in a sensitivity to its membrane environment. Fig. 4 shows that the voltage dependence of the gp91- phox –associated H + conductance could be described by a Boltzmann distribution with a slope factor of 20 mV. The effect of 20 μM sodium arachidonate was to shift the voltage dependence of the conductance by 19 mV to more negative potentials and also to increase (1.2×) the maximum H + conductance available. Both effects took place without appreciably changing the slope of the voltage relationship. In neutrophils, 50 μM sodium arachidonate has a comparable effect, although here a change in the slope factor from 14.7 to 7.5 mV was reported. The activation curve shifts by 14–23 mV and there is an approximately threefold increase in the maximum conductance . Under normal experimental conditions, the relative positions of the activation curve and the position of the tail current reversal potential (equivalent to the H + equilibrium potential, E H ) means that a maintained inward proton current is impossible to record. The large shift in the voltage dependence of activation produced by arachidonate, which is seen in both neutrophils and transfected CHO cells, means that the pathway may be activated even when the membrane is more negative than the E H . The effect of arachidonate on the gp91- phox current differs somewhat from its effect in peritoneal macrophages, however. In macrophages, arachidonate not only shifts the activation curve to more negative membrane potentials and increases the maximum conductance, but also accelerates the rate of rise of the outward current . This effect would not be evident at the most positive membrane potentials shown in Fig. 4 A because activation is close to maximum in this range, but even at +20 mV there appears to be little or no effect. It is possible that any change in kinetics is concealed by whatever process is responsible for the progressive increase in outward current that is seen in some cells. The inhibition by Zn 2+ of the H + conductance in neutrophils occurs in the same concentration range as we report here for the gp91- phox –associated conductance . In electrophysiological experiments on single cells, almost full inhibition is reported at ∼100 μM , although in experiments on cell suspensions slightly higher concentrations (1 mM) were required . Fig. 5 is consistent with the higher figure; 200 μM Zn 2+ produced ∼60% inhibition. In Fig. 3 , the data are fitted by a Boltzmann function with a 20-mV slope factor and half-activation voltage under control conditions (pH o 8, pH i 6.9) of ∼21 mV. In human neutrophils, DeCoursey and Cherny 1993 used slope factors of ∼15 mV to fit their control data and half-activation values of ∼60 mV (pH o 7, pH i 6). The more positive value found in the neutrophil may be attributed to the effect of the more acid pH o because, in a similar pH o 7 solution, the gp91- phox– associated conductance showed little activation at 0 mV. It was difficult to obtain a full activation curve under such conditions because the maximum appeared to occur at potentials greater than ∼120 mV, at which level the seal between patch pipette and cell membrane often became unstable. The 230 NH 2 -terminal amino acids of gp91- phox when expressed in CHO cells exhibit an arachidonate-activated H + flux that is significantly reduced when leucine is used to replace the histidine residue in position 115 . The flux is abolished when the three histidine residues in positions 111, 115, and 119 are changed to leucine . All three residues are in a putative membrane-spanning region of the molecule. The outward current recorded from the CHO-NLeu expressing cells at pH o 8.0, demonstrates a reduced current amplitude, slower activation time, and a shifted tail current reversal potential when compared with currents from expressing CHO91 or CHO-N cells. The precise role that histidine-115 plays in H + conduction is not clear, but the change in tail current reversal potential suggests that the mutated pathway is less selective for H + , as if the presence of this protonated site within the pathway is essential to maintain its selectivity for protons. The alteration in properties of the outward currents observed in CHO-N3Leu–expressing cells suggest that the histidine residues on either side of histidine-115 also contribute to H + permeation. If, as the shift in the voltage dependence of activation suggests, one or the other histidine contributes to the voltage sensitivity of the proton conductance, the large shifts in the activation curve with changes in either pH o or pH i become understandable. An alkaline shift in the pH of the intracellular fluid would have the effect of reducing the charge on any histidine residue to which there is access. The observed positive shift in the activation curve corresponds to the effect of substituting the uncharged leucine for the charged histidine. Yet to be explained is the effect of changes in external pH, which is in the opposite direction. Neutrophils provide the first cellular immune response of the body to invading micro-organisms. They are attracted to a site of infection where they engulf antibody-coated bacteria, killing and digesting them. That the generation of superoxide by the NADPH oxidase is a major contributor to the process is evident from the susceptibility to infection demonstrated by CGD patients. In vivo, gp91- phox probably functions as a charge compensator for the electron efflux generated upon production of superoxide. It also prevents a large and rapid fall in pH i caused by the coincident release of H + internally. NADPH oxidase activity is stimulated by a number of physiological and nonphysiological stimuli such as phorbol esters, unsaturated fatty acids (such as arachidonic acid), and formyl-Met-Leu-Phe. Thus, the large pH gradients and positive voltages used experimentally here were necessary to activate a significant H + current only in the absence of arachidonate. After the oxidase is activated in vivo, an outwardly directed electron flux, measured by Schrenzel et al. 1998 will produce a rapid depolarization of the membrane. Estimates, based on dye partition, which may be inaccurate, suggest that the membrane potential of activated neutrophils is approximately −10 mV . This, together with the appearance of H + at the internal surface of the membrane and the interaction with arachidonate combine to activate the H + pathway so that H + becomes passively distributed across the cell membrane with pH i close to its resting value. This is the first voltage-gated H + conductance to be described at the protein level. The amino acid sequence for gp91- phox is unlike any other protein , but its hydropathy plot suggests that there may be multiple (four or six) transmembrane domains at the NH 2 terminus of the protein. There is also a large hydrophilic domain that, as it contains the predicted FAD and NADPH binding sites, is assumed to be on the cytosolic side of the plasma membrane. Within one putative transmembrane domain there is a sequence of three histidine residues (positions 111, 115, and 119) in an “H-X-X-X-H-X-X-X-H” motif that appear to play an essential role in H + conduction. Stationary noise analysis of the voltage-gated H + current in cultured human muscle suggests that the elementary conductance is <0.1 pS . An even lower value has been reported for human neutrophils . In molluscan neurons, the estimated unitary proton current at +10 mV is <0.004 pA . We can derive an estimate of the number of NADPH oxidase molecules (and therefore the number of gp91- phox ) in each neutrophil cell from the concentration of cytochrome b 558 present in cell suspensions. As there are ∼5 pmol heam/10 5 cells and each molecule probably has two heam moieties, there must be ∼10 6 molecules/cell. Of these, ∼20% are in the cell membrane. Assuming that the single channel H + current for a 100-mV driving force is <1 fA at pH 6 , whole cell currents of less than ∼0.2 nA are to be expected for neutrophils, which is of the order observed experimentally. Unfortunately, CHO cells do not exhibit the characteristic cytochrome b 558 spectrum, and so we were unable to carry out the same analysis on them. Such low conductances follow naturally from the low H + concentrations on either side of the cell membrane and are consistent with the pathway being either a continuous channel across the cell membrane or a carrier site becoming exposed to first one and then the other membrane surface. In the latter case, it is possible that the mechanism of H + flux through gp91- phox may involve a cycle of protonation/deprotonation with histidine-115 being exposed alternately to the interior and exterior faces of the cell membrane, as described by Starace et al. 1997 for the S4 segment of a histidine-containing mutant of the Shaker K + channel. Among other alternatives, the H-X-X-X-H-X-X-X-H motif may line a continuous channel pore so that protons “hop” along the membrane-spanning helix. The role of Zn 2+ in inhibiting the H + conductance is under investigation because, in proteins that bind zinc, the Zn 2+ is held by residues in an H-X-X-X-H-X-X-X-H motif. The extent to which this motif occurs in the structure of other proton pathways has yet to be established. | Study | biomedical | en | 0.999996 |
10578015 | In mammalian heart, the process of excitation–contraction (E-C) 1 coupling is mediated by calcium-induced Ca 2+ release . It has been postulated that Ca 2+ entering through single L-type Ca 2+ channels (dihydropyridine receptor channels, DHPRs) locally controls the activity of the release units, composed of Ca 2+ release channels/ryanodine receptors located in the membrane of the sarcoplasmic reticulum (SR) across a 20-nm wide junctional gap. Single DHPR activity is characterized by brief openings (∼0.2 ms) separated by relatively long closures . When the channel opens, Ca 2+ in its vicinity immediately rises to levels above 10 μM; when the channel closes, the local Ca 2+ gradient dissipates rapidly (microseconds) due to Ca 2+ diffusing away . Thus, according to the local control concept of CICR, the physiological trigger of calcium release must be a rapid and transient elevation of Ca 2+ to above 10 μM lasting <0.5 ms. The gating properties of the RyRs have been studied after reconstitution of the channels into lipid bilayers. All these studies have been performed under stationary Ca 2+ conditions , or during sustained changes in Ca 2+ produced by photolysis of “caged calcium” or mechanical solution exchange . These studies, although yielding important information on channel behavior, do not reveal how the RyR responds to the brief Ca 2+ stimuli that are likely to initiate E-C coupling in vivo. In the present study, we used the photolabile Ca 2+ chelator DM-nitrophen (DMN) to produce brief Ca 2+ elevations that mimic the waveform of Ca 2+ changes associated with openings of single DHPRs. Photolysis liberates Ca 2+ from the DMN-Ca complex much faster then free DMN binds Ca 2+ . Thus, Ca 2+ released from the photolyzed DMN will be free for some time until it rebinds to unphotolyzed DMN, producing a brief (<1 ms) Ca 2+ overshoot. Our results show that application of such brief “Ca 2+ spikes” to RyR channels in bilayers results in rapid and transient activation of the channels. The probability that a single RyR will be activated is determined by the amplitude and duration of the Ca 2+ trigger signal. These results support the possibility that activation of release units is triggered by single DHPR events, and that DHPR–RyR coupling can be the subject of physiological modulation and pathological failure. Heavy SR microsomes were prepared from canine left ventricles by standard procedures . Single SR Ca 2+ release channels were reconstituted by fusing heavy SR microsomes into planar lipid bilayers as described previously . The experimental solution contained 400 mM CsCH 3 SO 3 , 10 mM CsHEPES, and 1 mM glutathione, pH 7.4. The bilayer chamber was designed to minimize the background current noise during recordings with high temporal resolution. The bilayer aperture had a diameter of 0.1 mm, resulting in bilayer capacitance of 50–70 pF. Single-channel currents were measured using a patch-clamp amplifier (Axopatch 200A; Axon Instruments), filtered at 2–10 kHz, and digitized at 5–100 kHz. Data acquisition and analysis were performed using pClamp (version 6.0.1; Axon Instruments) and Origin (version 5.0; Microcal Software). The experimental variance was estimated according to Landau and Páez 1997 . Peak open probability was defined as the intersection of the fits of the ascending and descending parts of the ensemble open probability (by exponential association function raised to the power n a , and by monoexponential function, respectively). Fast changes of the Ca 2+ concentration in the microenvironment of the reconstituted channel were performed by flash photolysis of DM-nitrophen (Calbiochem Corp.) as described previously . Intense, 9-ns long UV laser flashes produced by a pulsed, frequency-tripled, Nd:YAG laser (Spectra-Physics) were applied through a fused silica fiber optics (450 μm diameter) positioned perpendicular to the bilayer surface (100 μm diameter) so that the whole volume between the fiber optics and the bilayer was illuminated evenly and instantaneously. The amplitude and time course of Ca 2+ after the flash were determined from the concentration of total and free DMN and Ca 2+ , and from the proportion of DMN photolyzed during the flash according to the reaction scheme shown below in Fig. 1 A. The total concentration of DMN was kept at 3 mM. The concentration of steady state free Ca 2+ was determined with a Ca 2+ -selective minielectrode . The local Ca 2+ changes near the bilayer were calibrated by transforming the bilayer aperture into a Ca 2+ electrode, using Ca 2+ ionophore resin . The potential of the Ca 2+ electrode was measured with 0.2 mV precision using the patch-clamp amplifier in current-clamp mode. The increase in free steady state Ca 2+ after photolysis was plotted as a function of flash intensity and free Ca 2+ before the flash to construct a calibration curve . The proportion of DMN photolyzed at a given free Ca 2+ and flash intensity was calculated from the pre- and post-flash steady state free Ca 2+ , using parameters taken from the literature . The time course of Ca 2+ concentration changes in a particular experiment was reconstructed from the above data, using the published set of differential equations and kinetic parameters of DM-nitrophen complexation and photolysis . Computations were performed with a program written in Mathematica (version 3.0; Wolfram Research). To simulate the RyR response to Ca 2+ spikes, we used our previously published minimal gating model of RyR with one Ca 2+ binding step . As alternative models, we used extensions of Model 1Ca, incorporating consecutive binding of two to five Ca 2+ ions. It was assumed that Ca 2+ binding sites are identical and behave independently. Subsequent gating steps are possible only if all calcium binding sites are occupied ( Table , Model 2Ca–Model 5Ca). The rate constants of transitions not involving Ca 2+ binding were unchanged. In models with multiple Ca 2+ binding steps, the ratios of the on and off rates for calcium binding were calculated from the apparent peak and steady state calcium sensitivities of the channel P o to provide a mean value identical to that of Model 1Ca. The on rates were optimized for best description of the rate of RyR activation. Single-channel activity in response to Ca 2+ stimuli was simulated using the program SCESim . Channel kinetics were described by a matrix of transition rates between individual channel states . The time course of theoretical Ca 2+ spikes for selected initial DMN saturation and percentage of DMN photolyzed were first calculated with 10-μs resolution, and then used as input for channel gating simulations. The theoretical time course of channel open probability during and after the Ca 2+ spike was calculated in Mathematica (Wolfram Research) by combining the differential equations for DMN complexation and photolysis with those describing channel kinetics . The analysis of statistical significance of differences between models was performed by χ 2 tests, according to the procedure described by Landau and Páez 1997 . The values of χ 2 were determined from the sum of squares of differences between experimental data and model prediction, and from the experimental variance. The models that did not pass the χ 2 test at P = 0.01 were rejected. The apparent calcium sensitivity of peak open probability in response to a Ca 2+ spike was described by a general equation : 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{{\mathrm{o}}}^{{\mathrm{max}}}=\frac{ \left \left[{\mathrm{Ca}}\right] \right ^{n_{{\mathrm{H}}}}}{ \left \left(K_{Ca}\right) \right ^{n_{{\mathrm{H}}}}+ \left \left[{\mathrm{Ca}}\right] \right ^{n_{{\mathrm{H}}}}}{\mathrm{,}}\end{equation*}\end{document} where K Ca is apparent calcium sensitivity of the channel, and n H is the apparent Hill slope. In general, the apparent Hill slope may not necessarily correspond to the actual number ( n ) of Ca 2+ binding sites. Specifically, n H < n when the activation path contains a Ca 2+ -independent closed state (as with our models of RyR), even if the binding sites are equivalent and independent. Single cardiac RyR channels were reconstituted at a steady state Ca 2+ concentration of 20 μM. After incorporation of a single RyR, DMN (3 mM) was added to the cytoplasmic (cis) side of the channel. The free Ca 2+ was titrated to 75–150 nM. Identical precalibrated photolytically induced Ca 2+ spikes were applied to the channel. After each UV pulse, resting conditions were reestablished by stirring the solution in the cis chamber for at least 30 s. The laser flash-induced Ca 2+ spike is too fast to be directly measured by any available method, including measurements using the fastest Ca 2+ indicators . Therefore, the amplitude and time course of photolytic Ca 2+ changes were reconstructed from the pre- and post-flash steady state free Ca 2+ (see methods ). In 21 independent experiments, the calculated free Ca 2+ rose virtually instantaneously (τ on = 6–18 μs) to 9–30 μM, and then decayed with a τ off = 106–200 μs to a final level of 105–190 nM. Ca 2+ was elevated to over 5 μM for 0.1–0.4 ms and to over 1 μM for 0.3–0.7 ms. A typical example of such a Ca 2+ spike is shown in Fig. 1 C. The amplitude and duration of this Ca 2+ stimulus is similar to that expected to occur near a RyR channel during a single brief opening of an adjacent DHPR channel . We recorded single RyR channel activity in response to such brief free Ca 2+ stimuli . The required temporal resolution was achieved by recording at a sampling rate of 100 kHz and cut-off filter setting ≥5 kHz. Before the flash, the channels exhibited essentially no activity. The channels responded to the Ca 2+ stimulus in ∼25% of the episodes. The activity evoked by DMN photolysis consisted mostly of single openings, after which the channel stayed closed until the end of the episode . To quantify the time course of channel activity, at least 32 single channel records obtained from an individual channel were combined to generate ensemble averages . Channel open probability transiently increased upon photolysis of DMN. The time course of activation was best fit by a single exponential association function raised to the power n a . The rising portions of P o on expanded time scale are shown in B (○) along with the fits (solid lines). At 2 kHz bandwidth, the rise of P o was relatively slow (τ a = 0.22 ms, n a = 1.4). Expanding the bandwidth to 5 kHz resulted in a significant decrease in the rise time of P o (τ a = 0.10 ms). In addition, a notable delay between the application of the laser flash and the ascent of P o became evident ( n a = 3). Increasing the filter cutoff frequency to 10 kHz had no further impact on the observed rate of channel activation (τ a = 0.09; n a = 2.8). Therefore, the temporal resolution of our measurements at 5 and 10 kHz was adequate to resolve the kinetics of RyR activation. The rising phase of P o at both 5 and 10 kHz was best fit by an exponential function with a power close to 3 (solid line), strongly suggesting that binding of several Ca 2+ ions must occur before the channel can open. To further quantify the kinetics of the channel response, we analyzed the distribution of the first latencies of channel openings induced by the laser flash . The cumulative first latency distributions (•) closely corresponded to the time courses of the rising phases of the respective ensemble open probabilities at each bandwidth (dashed lines). This confirms that the delay observed in the open probabilities at ≥5 kHz reflects channel behavior and is not merely an artifact introduced by experimental noise. Consistent with the observed time course of ensemble open probabilities, the distributions of first latencies showed no delay at 2 kHz bandwidth. At both 5 and 10 kHz, they exhibited a prominent rising phase and peaked at ∼0.2 ms. The average channel open time was 1.9 ± 0.2 ms at 2–10 kHz. Deactivation of the channel after the Ca 2+ spike had a monoexponential time course (τ d = 3.2 ± 0.4 ms). It was much slower than channel activation or the decay of the Ca 2+ spike and was independent of the bandwidth. Previous studies of RyR activation by photolysis of DMN showed only sustained RyR responses decaying (i.e., adapting) with a time constant of ∼1 s and displayed no brief responses demonstrated in the present study. To explore the relationship between the rapid and sustained responses, we performed measurements of RyR activity during two sequential laser flashes of equal intensity . It can be seen that while the first flash elicited predominantly single openings (Ca 2+ spike response), the second pulse triggered mostly multiple openings (adaptation response). The corresponding changes in free [Ca 2+ ] (continuous line) calculated from the Ca 2+ electrode response (dashed line) using published parameters of complexation and photolysis of DMN are also presented . The first flash elicited a Ca 2+ spike followed by a small steady [Ca 2+ ] elevation; the second flash elicited a similar Ca 2+ spike, which was followed by a steady [Ca 2+ ] elevation to a significantly higher level. The increase in steady component of the Ca 2+ signal during successive flashes is due to a gradual increase in the saturation of DMN by Ca 2+ , leaving less DMN for rebinding of Ca 2+ after the flash. These results clearly show that the adaptation behavior is determined by the steady component of the [Ca 2+ ] signal. To further characterize the activation of RyRs by Ca 2+ spikes, we measured RyR activity in response to laser flashes of different intensities. Fig. 4A–C , shows channel responses to laser flashes of low, intermediate, and high intensity along with the corresponding calculated free [Ca 2+ ] spikes in a representative experiment. In this experiment, the amplitude of the Ca 2+ spike was estimated to be 9.3, 18.3, and 27.4 μM for low, intermediate, and high intensity pulses, respectively. The Ca 2+ spikes decayed with time constants of 0.17, 0.18, and 0.20 ms, respectively. Ca 2+ was elevated to over 5 μM for 0.13, 0.27, and 0.34 ms, and to over 1 μM for 0.4, 0.6, and 0.7 ms, respectively. As can be seen, low-intensity flashes caused channel openings only in relatively few occasions (peak P o ∼ 0.06); increasing flash energy increased the probability of activation (peak P o ∼ 0.25 and 0.50, respectively). Interestingly, in all cases the responses were composed of isolated openings with a similar duration. The time constants of activation, determined by fitting single exponential association function raised to the power n to the ensemble averages, progressively decreased with increasing the energy of the laser pulse . Similar results were obtained in five other experiments. These results are summarized in Fig. 6 F, which plots the peak P o of the channel as a function of spike amplitude. The [Ca 2+ ] dependence of P o could be described by with a K Ca value of 29 ± 1 μM and an apparent Hill slope of 2.5 ± 0.2. The high values of the activation exponent and of the Hill slope further indicate that activation of the RyR channel requires binding of several calcium ions. To better understand the mechanisms of activation and deactivation of RyR in response to Ca 2+ spikes, we performed single channel simulations using our published minimal model of RyR gating with two open and three closed states and one Ca 2+ binding step . Similar to experimental observations, the simulated responses consisted mostly of single, ∼2-ms long openings . However, other features of simulated channel activity were at odds with the experimental data. For example, contrary to real channels, simulated channels exhibited substantial basal activity. In addition, the ensemble P o or the distribution of the first latencies of the simulated responses showed no delay after the Ca 2+ spike, seen with experimental data. The excessive background activity and a lack of delay between the Ca 2+ spike and RyR activation could be ascribed again to the possibility that binding of more than one Ca 2+ ion is required to produce channel opening. Considering the tetrameric organization of the RyR, we extended our minimal RyR model by including four sequential Ca 2+ binding steps . The ensemble P o generated using the extended model showed essentially no spontaneous openings before the Ca 2+ spike. After the Ca 2+ spike, it exhibited a significant delay, similar to the experimentally observed behavior . Furthermore, the first latency distribution yielded a peak near 0.25 ms, close to the experimentally observed value of 0.2 ms. These results suggest that activation of the RyR by Ca 2+ spikes may indeed involve binding of multiple, perhaps as many as four, Ca 2+ ions to the channel. To further elucidate how many Ca 2+ binding steps are involved in channel activation, we carried out theoretical simulations using models with different numbers of Ca 2+ binding sites. We compared the abilities of the models with different numbers of Ca 2+ binding sites to reproduce the experimentally observed kinetics of RyR activation. This approach is illustrated in Fig. 6A–E , for the experiment shown in Fig. 4 and for models with one to five Ca 2+ binding steps, respectively. Differences between the models were statistically analyzed by the χ 2 test, applied to the whole data set of six experiments. The χ 2 values were determined from the sum of squares of differences between experimental data and predictions of the particular model, and from the experimental variance . We obtained χ 2 values of 17,120, 9,821, 6,712, 4,667, and 4,711 (4,510 degrees of freedom) for models with one, two, three, four, and five Ca 2+ binding sites, respectively. Models with less than four calcium binding sites have failed the χ 2 test at the significance level P = 0.01, while models with four and five Ca 2+ binding sites passed the test and can be considered, therefore, compatible with the data. These tests strongly suggest that binding of at least four Ca 2+ ions are necessary for RyR activation. Fig. 6 F shows theoretical Ca 2+ – P o dependence curves obtained from the above series of models along with the Ca 2+ – P o dependence curve obtained from experimental data. The apparent Hill slopes of the theoretical [Ca 2+ ]– P o relationships yielded by the models with one, two, and three Ca 2+ binding steps (0.97 ± 0.15, 1.69 ± 0.02, and 2.09 ± 0.01, respectively) were significantly different from those derived from experimental data (2.5 ± 0.2) at significance levels of 0.0001, 0.001, and 0.05, respectively. Therefore, these models are not compatible with the experimental results. Models with four and five Ca 2+ binding steps (apparent Hill slopes 2.6 ± 0.1 and 2.6 ± 0.1, respectively) were not significantly different from the experimental data even at P = 0.5. Therefore, the response of the RyR to Ca 2+ spikes can be described by our minimal model of the RyR modified by including a total of four Ca 2+ binding steps. The chemistry of DMN limits flash-photolysis experiments to a rather narrow range of amplitude-duration characteristics of Ca 2+ spikes. In contrast, the parameters of local Ca 2+ signals associated with the activity of DHPRs vary widely. Therefore, to gain further insight into the dependence of the channel activation on the characteristics of the trigger signal, we performed simulations in response to a broad range of rectangular Ca 2+ pulses using Model 4Ca with four Ca 2+ binding sites described above. The properties of the Ca 2+ pulse in the physiological range of durations and amplitudes had a profound effect on peak open probability of the RyR, as illustrated in Fig. 7 . Calcium elevations lasting <10 μs had negligible probability to open the RyR in the whole amplitude range. To increase the peak open probability from 5 to 95%, the amplitude of the calcium pulse has to be increased by ∼10-fold for any pulse duration. Prolongation of the Ca 2+ pulses above 1 ms was not effective in increasing peak P o of the RyR. In the high Ca 2+ pulse amplitude range (>10 μM), the dependence of peak P o on pulse duration was very steep for short pulse durations (0.1–0.5 ms). In the present study we measured the kinetics of activation of cardiac SR Ca 2+ release channels/RyRs using fast Ca 2+ concentration spikes produced by photolysis of DM-nitrophen. The Ca 2+ spikes mimic the profile of Ca 2+ produced by openings of single DHPRs in the vicinity of the RyRs. Thus, our results show, for the first time, how single RyRs might respond to a physiological trigger signal. Under our experimental conditions (∼100 nM resting Ca 2+ and 3 mM DMN), the reconstructed Ca 2+ spikes were characterized by an activation time constant of ∼15 μs, a duration of ∼0.1–0.4 ms (at 5 μM Ca 2+ ) and a peak amplitude of 10–30 μM . Application of such Ca 2+ pulses resulted in activation of the RyR with 5–50% probability, depending on spike magnitude. The activity of RyR was characterized by isolated single openings with duration of ∼2 ms. It is important that in our experiments we used Cs + instead of Ca 2+ as the charge carrier. Besides improving the signal-to-noise ratio, this allowed us to determine the parameters of channel kinetics without potential side effects related to “feed-through” influences of luminal Ca 2+ at the cytosolic activation and inactivation sites . Previous studies using caged Ca 2+ did not yield channel activation in response to Ca 2+ spikes . These negative results can be ascribed to lower concentrations of the calcium cage, low time resolution of the measurements, and the presence of a laser flash artifact that could have concealed the occasional, brief channel openings in response to the flash. In the above studies, the effective trigger signal consisted of both a transient (i.e., spike) and a sustained component. The reported time constants of channel activation were 1–2 ms. Our present experiments with improved time resolution showed that rapid Ca 2+ spikes can activate the channel with much faster kinetics (activation time constant ∼0.15 ms). We believe that our measurements yield the true response time of the channel because channel activation displayed a distinct delay, and the kinetics of the RyR response were unaffected by increasing the filter cutoff frequency from 5 to 10 kHz. The lifetime of isolated RyR channel openings induced by Ca 2+ spikes ( t o ∼ 2 ms) was substantially longer than the average channel open time (∼1 ms) reported under similar conditions at steady state . However, it was similar to the average channel open time within the high activity (H) gating mode . The deactivation rate obtained from ensemble averages of the channel responses to the Ca 2+ spike was ∼3 ms, and it corresponded approximately to the average channel open time. These results provide further evidence for the idea that the H-mode activity is the preferred initial regime of channel operation upon activation . In previous studies with photolysis of DMN and NP-EGTA, the RyRs activated rapidly, and then the P o decayed slowly, by a process termed adaptation . It has been argued that adaptation might simply be a result of the spontaneous deactivation of the RyR after its activation by the rapid Ca 2+ spike . Our direct measurements of the RyR response to Ca 2+ spikes indicate that the deactivation of the RyR after a Ca 2+ spike is too fast to account for the adaptation phenomenon. Further, our results with double flashes that induce Ca 2+ waveforms with similar transient but different steady components showed that the adaptation response is evoked only by the Ca 2+ signal with a large steady component. Thus, it appears that the type of response of the RyR (i.e., rapid or prolonged) is determined by the steady component of the photolytic Ca 2+ change. The kinetics and [Ca 2+ ] dependence of the response of the RyRs to Ca 2+ spikes could be well described by our minimal model of RyR with two open and three closed states modified by including three additional (a total of four) Ca 2+ -dependent closed states . We have shown previously that the minimal model reproduces reasonably well the main aspects of channel behavior, including modal gating activity, under both stationary and nonstationary conditions . This model consists of three sets of states (i.e., gating modes) connected by slow transitions. The results of our present study with improved time resolution allowed us to refine the state structure of the high activity mode corresponding to the activation path of the channel. The existence of multiple Ca 2+ binding steps in the RyR activation path is consistent with the results of analysis of closed time distributions of steady state recordings at low [Ca 2+ ], yielding at least five closed states . Importantly, the four-Ca 2+ binding site model is also consonant with the molecular structure of the RyR, a protein composed of four homologous subunits with each monomer carrying at least one Ca 2+ binding site . Based on our model simulations, we suggest that the response of the RyR to a Ca 2+ spike includes the following steps. (a) Sequential binding of four Ca 2+ ions to the channel promotes transition from closed states (R–C4) to an open state (O1). The need for binding of four Ca 2+ ions to open the channel accounts for the delay in channel activation, for the negligible P o at basal [Ca 2+ ], and for the fact that spikes do not always cause channel opening. (b) After termination of the spike, Ca 2+ dissociates from the channel and the channel deactivates by returning first to the closed states (C4–C1) and eventually to the resting state. Transitions between states C4–O2 and O1–C2 are very slow ; consequently, the probability of the channel entering these late states during brief Ca 2+ spikes is low. Thus, as we have previously predicted , the channel has just enough time to enter the fast access states of the H-mode, but not the slow access states of the L-mode when challenged by brief, calcium spike–like stimuli. The slow transitions between states C4–O2 and O1–C2 can only occur when Ca 2+ remains elevated in the vicinity of the channel . In this respect, our gating model could be simplified by omitting the slow access states (O2, C5, and I) and still be able to account for most results with brief Ca 2+ spikes. However, such a truncated model would clearly become inadequate for describing channel behavior in response to sustained Ca 2+ elevations when the initial passage to rapid access states is followed by a transition to slow access states, accounting for the phenomenon of RyR adaptation . Neither would the truncated model be able to describe steady state activity characterized by modal behavior; i.e., random transitions between periods of high and low activity . Our results have important ramifications for understanding CICR in vivo. It has been suggested that during E-C coupling Ca 2+ entering through single L-type Ca 2+ channels locally controls the activity of the Ca 2+ release channels, presumably arranged into functionally independent release units . One important premise of the local control theory is that the RyR must be fast enough to track the fast Ca 2+ changes associated with single DHPR openings (see introduction ). The results of the present study show that brief (<0.5 ms) trigger Ca 2+ signals are adequate to activate RyRs and are consistent with the possibility that RyR channels are controlled by single DHPR events. Such rapid activation could also provide a means for effective cross-activation of neighboring Ca 2+ release channels within a single release unit, thus accounting for the synchronization of multiple RyRs during a Ca 2+ spark . At the same time, the presence of four Ca 2+ binding sites that must be occupied for channel opening would tend to reduce activation by global background Ca 2+ while still enabling the local Ca 2+ increase in the diadic cleft to activate the channels efficiently . We showed that Ca 2+ spikes with an estimated amplitude of 10–30 μM, which mimic single DHPR-related signals, have a 5–50% probability of inducing RyR activation. The results of our simulations in a wider range of amplitudes and durations of the Ca 2+ elevations demonstrate that the probability of activation of a single RyR is graded with the amplitude as well as duration of the triggering Ca 2+ pulse. These results are consistent with a DHPR–RyR coupling arrangement that could be the subject of physiological modulation and pathological failure in the heart . The relatively low efficiency of activation of single RyRs by brief Ca 2+ stimuli could also reflect the importance of clustering of RyRs in the junctional gap, which would be expected to improve responsiveness of the RyRs . While our data suggest that the lower and shorter Ca 2+ elevations produced by DHPR openings trigger RyR activation with a relatively low probability, the resulting longer RyR openings of higher amplitude can be expected to activate the neighboring RyRs with a much higher probability, giving rise to the stereotypical spatio-temporal shape of a calcium spark . | Study | biomedical | en | 0.999997 |
10578016 | The cystic fibrosis transmembrane conductance regulator (CFTR) channel functions as a PKA-activated ion channel that is predominantly expressed in epithelial cells. The channel selects for anions over cations ( P Na / P Cl ≈ 0.03) and exhibits modest discrimination among anions as judged either from reversal potential measurements or the block of Cl conduction by other permeant ions . Within the framework of a two-barrier, one-site permeation scheme, permeability selectivity may be thought of as a reflection of the relative ease with which anions enter the channel (barrier height) while blockade of conduction measures the tightness of anion binding (well depth) within the channel . The selectivity patterns for both anion permeability and anion binding fall in the so called “lyotropic” sequence. Anions that are more readily dehydrated than Cl experience a reduced barrier to entry into the pore and also stick more tightly inside the pore than Cl (i.e., they see a deeper well). These parallel changes in peak height and well depth are evident for an anion like SCN, which is three times more permeant than Cl ( P SCN / P Cl < 3.4), but also blocks Cl flow . Anion binding differs from anion entry, however, in that it is highly sensitive to changes in pore structure, whereas permeability ratios are much less so . To gain insight into the nature of the physical interactions that are reflected in the peak and well energies that characterize anion permeation, we compared the selectivity of CFTR to that of a well-characterized, synthetic anion-selective membrane composed of plasticized poly(vinyl chloride) (PVC) doped with tridodecylmethylammonium chloride (TDMAC) for which the physical basis for the response to anion substitution is more readily apparent . The selectivity patterns for CFTR and the synthetic membrane differed by a multiplicative constant and both could be predicted by a continuum electrostatic model, based on the Born charging energy, that modeled anion–channel interactions as the energy of a charged sphere in a polarizable medium. The continuum electrostatic approach provides a unified, quantitative interpretation of the observed energetics of permeation and block and offers a plausible explanation for the differential effects of mutations on these two processes that may be useful in understanding the physical nature of the conduction path, and in evaluating proposed structural models for the pore domain. To estimate anion dimensions and surface area, each of the ions used was modeled using the PC Spartan molecular modeling program from Wavefunction, Inc. The equilibrium geometry of each of the polyatomic species was optimized using ab initio molecular orbital calculations. For those ions with a regular geometry, all additive and trigonometric calculations were done using nucleus-to-nucleus measurements from the model, and then the overall dimensions were approximated by adding the appropriate van der Waal radii to the terminal atoms. For those ions with an irregular geometry, the solid dimensions were estimated from the smallest “box” into which the ion would fit. The equivalent radius was determined by taking the surface area of the model and determining the radius of a regular sphere with the same surface area (where surface area = 4πr 2 ). PC Spartan lacks basis values for gold so we could not perform the full set of calculations for Au(CN) 2 ; however, we were able to model Ag(CN) 2 , which should have approximately the same dimensions. One of the advantages of the molecular orbital calculation method is that it permits an assessment of the charge distribution within the molecule. A hallmark of the halides and pseudohalides is that the negative charge is uniformly distributed , whereas for some other polyatomic anions the charge is concentrated on a particular group. For example, 98% of the charge on gluconate is associated with the carboxyl group (-COO, −0.984 e ), while the other end of the molecule has a slight cationic character (-CH 2 OH, +0.032 e ). The physical properties of the halides, pseudohalides, and polyatomic anions used in this study are summarized in Table . In the Born model, the free energy of ion-solvent interaction is equated with the work required to move a charged sphere of radius, r, from a vacuum into a structureless continuum characterized by a dielectric constant, ∈ . For each ion listed in Table , we obtained the equivalent radius from the molecular model (applying the correction of Latimer et al. 1939 (0.1 Å for anions and 0.85 Å for cations) and calculated the work of transfer from a vacuum to water (∈ = 80). There is reasonable agreement between the hydration energy calculated this way and the measured values for the halides and pseudohalides , but very poor correspondence for a number of the polyatomic anions. Gluconate, for example, is much more difficult to dehydrate than predicted by the Born analysis based on its equivalent radius, presumably due to the increased local charge density resulting from nonuniform charge distribution . This point is demonstrated in Fig. 1 , which shows values of |Δ G hyd | plotted versus reciprocal ionic radius. It is apparent that the polyatomic anions exhibit thermodynamic behavior consistent with an equivalent “Born thermochemical radius” that is much smaller than any actual dimension of the molecule ( Table ). The plasticized PVC-TDMAC membrane studied here is employed in an ion-selective electrode designed to detect small anions via a dissociated ion-exchange mechanism . The ionophore, positively charge TDMA + , renders the membrane selective for anions over cations by a Donnan mechanism (see ). Because there is little or no ion pairing in the membrane phase, the relative anion selectivity of the TDMAC electrode is determined solely by the relative anion partition coefficients between water and the PVC membrane. Hence the membrane exhibits the so called “Hofmeister” or “lyotropic” selectivity sequence [i.e., C(CN) 3 > SCN > I > Br > Cl > F] . The electrode polymer membrane was composed of 1 wt% TDMAC, 33 wt% PVC, and 66 wt% ortho-nitrophenyloctyether (o-NPOE), a plasticizer. The ions were tested at a 10-mM concentration (as a Na or K salt) in distilled/deionized water, as well as in a 10-mM HEPES solution buffered at pH 7.4. The values were calculated from the average of six electrodes. The selectivity coefficient was calculated using the separate solution method as follows ( ): 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{log}}K_{{\mathrm{A,B}}}^{{\mathrm{pot}}}= \left \left(\frac{E_{{\mathrm{B}}}-E_{{\mathrm{A}}}}{{2.3RT}/{z_{{\mathrm{A}}}}F}\right) \right {\mathrm{,}}\end{equation*}\end{document} where log K A,B pot is the potentiometric selectivity coefficient, E A and E B are the experimentally determined potentials of the cells, R is the gas constant, T is temperature in degrees Kelvin, F is the Faraday constant, and z is the valence of the ion. The physical significance of K pot and its relation to the free energy change associated with the transfer of an anion from water to the PVC membrane is presented in the and discussion sections. Dielectric constants of a number of electrode membranes were determined using an electrochemical impedance analyzer as described by Buck 1982 . The applied sinusoidal voltage amplitude was 20 mV. The measured potentiometric selectivity coefficients (log K pot ) for the ions used in this study are presented in Table . For ions like dicyanamide and tricyanomethanide, for which values for the free energy of hydration (Δ G hyd ) were not available, we used the measured log K pot to estimate Δ G hyd values by fitting a line through the data for ClO 4 , SCN, I, NO 3 , Br, and Cl using reported values of Δ G hyd and measured K pot values. The extrapolated values are given in Table . Human wild-type CFTR (wtCFTR) in a pBluescript vector (Stratagene Inc.) was linearized with XhoI and used as template for the generation of cRNA using the mMessage Machine protocol (Ambion, Inc.). The cRNA was resuspended in DEPC-treated water and maintained at −70°C before injection into oocytes. Female Xenopus (Xenopus-1) were anesthetized by immersion in ice water containing 3-aminobenzoic acid ethyl ester (Tricaine, 3 mg/ml; Sigma Chemical Co.) and oocytes were removed through an abdominal incision. The follicular membrane was removed by mechanical agitation (2–3 h) in a nominally Ca 2+ -free collagenase solution containing (mM): 82.5 NaCl, 2 KCl, 1 MgCl, 10 HEPES, pH 7.5, and 2.5 mg/ml collagenase (GIBCO BRL). Defolliculated oocytes were maintained in a modified Barth's solution (MBSH) containing (mM): 88 NaCl, 1 KCl, 2.4 NaHCO 3 , 0.82 MgSO 4 , 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 10 HEPES, pH 7.5, and 150 mg/liter gentamicin sulfate. Oocytes were maintained at 18°C in a humidified incubator. 1 d after isolation, oocytes were injected with cRNA (diluted to give 50–250 μS of stimulated conductance: ∼0.15 ng/oocyte in a 50-nl volume) using a microinjector (Drummond Scientific Co.) and beveled injection needles (∼10-μm tip diameter). Injected oocytes were maintained in MBSH and used for electrophysiological analysis 2–6 d after injection. Individual oocytes were perfused with an amphibian Ringer's solution containing (mM): 100.5 NaCl, 2 KCl, 1.8 CaCl, 1 MgCl, and 5 HEPES, pH 7.5. The oocyte was impaled with two microelectrodes with tips pulled (P-97; Sutter Instruments Co.) to give 0.5–1.5 MΩ of resistance when filled with 3 M KCl. The open circuit membrane potential was continuously monitored on a strip chart recorder (Kipp & Zonen), and periodically the membrane was clamped (TEV-200; Dagan Corp.) and using a computer-driven protocol (Clampex; Axon Instruments), ramped from −120 to +60 mV at a rate of 100 mV/s for most analyses, although a step protocol (from −120 to +40 mV in 10-mV steps, 200 ms/step) was also used to check for time-dependent currents. The membrane conductance was calculated using the slope conductance over a 20-mV range centered on the reversal potential, and using chord conductances at various voltages. For ramp data, a correction for the capacitive transient was estimated by comparing the current measured at the holding potential to that determined at the same potential within the ramp, and the difference was subtracted from the entire event. The data was analyzed using an Excel (Microsoft Co.) spreadsheet, and secondary analyses were performed using Sigmaplot (SPSS Inc.). After the oocyte recovered from impalement, CFTR was stimulated by adding a cocktail containing 10 mM forskolin and 1 mM 3-isobutyl-methylxanthine (IBMX) (Research Biochemicals, Inc.) to the perfusate. For ion substitution protocols, the basic amphibian Ringer's was modified to reduce interference from the endogenous Ca 2+ -activated Cl channel and contained (mM): 98 Na-anion, 2 K-aspartate, 1 Mg-aspartate, 1.8 Ba-acetate, and 10 HEPES, pH 7.5. Some of the anions used are only available as K salts, in which case the solution was modified to contain 98 K-anion and 2 Na-aspartate; otherwise identical (all salts were from Aldrich Chemical Co., Sigma Chemical Co., or Strem Chemicals, Inc.). Typically, an uninjected, mock-injected, or CFTR-expressing, but unstimulated, oocyte exhibited a background conductance of 0.5–1.5 μS, with a slightly higher conductance at depolarized potentials due to the endogenous Ca 2+ -activated Cl channel in parallel with an endogenous voltage-gated Ca 2+ channel. Three of the substitute anions [Au(CN) 2 , C(CN) 3 , and N(CN) 2 ] produced a current in uninjected, mock-injected or CFTR-expressing, but unstimulated, oocytes. If the background current was >2% of the current in the stimulated condition, then the background currents were subtracted. The CFTR-independent currents seen in the presence of Au(CN) 2 , C(CN) 3 , and N(CN) 2 were completely reversible and the magnitude could be faithfully reproduced with repeated exposure. The CFTR-independent current for Au(CN) 2 , C(CN) 3 , and N(CN) 2 were moderately sensitive to flufenamic acid (∼50% inhibition with 250 μM flufenamic acid), suggesting that at least a portion of the conductance is through an endogenous, anion-selective pathway. A portion of the residual current may be due to partitioning into the bilayer, as suggested by the lipophilicity of these compounds . There were no apparent “toxic” effects as judged by the ability of the CFTR conductance to completely recover after exposure to Au(CN) 2 . Ag(CN) 2 appeared to be somewhat toxic to the oocytes and, therefore, its permeation was not characterized. Permeability ratios were calculated using the Goldman-Hodgkin-Katz equation ( ) as follows: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{P_{{\mathrm{sub}}}}{P_{{\mathrm{Cl}}}}= \left {\mathrm{exp}} \left \left(\frac{{\mathrm{{\Delta}}}E_{{\mathrm{rev}}}}{{RT}/{zF}}\right) \right - \left \left(\frac{ \left \left[{\mathrm{Cl}}\right] \right _{{\mathrm{0}}}}{ \left \left[{\mathrm{Cl}}\right] \right {\mathrm{^{\prime}}}_{0}}\right) \right \right {\cdot} \left \left(\frac{ \left \left[{\mathrm{Cl}}\right] \right {\mathrm{^{\prime}}}_{{\mathrm{0}}}}{ \left \left[{\mathrm{sub}}\right] \right _{{\mathrm{0}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where Δ E rev is the measured shift in zero current potential after Cl o is replaced with the substitute ion, sub o , [Cl]′ o is the bath concentration of Cl, [Cl] o is the residual Cl in the substituted solution, [sub] o is the concentration of the substitute ion, and R, T, z, and F have their usual meaning. The central goal of the analysis presented here was to use measurements of relative anion permeability and relative anion blockade to estimate the energies associated with transferring an anion from water into the channel. This required that we adopt a model (or models) for the anion translocation process that would permit us to estimate the energetic significance of differences in anion permeability or binding. It is important to note that the primary aim was not to arrive at absolute values for these energies, but rather to determine the trend in the change in the energies from one anion to another so that this trend could be compared with the change in anion size. The relation used to interpret permeability ratios in terms of energy differences can be obtained using either of two complimentary approaches. Rate theory models for permeation predict that permeability ratios are determined by the difference in peak height for the two ions. For example, for Cl and a substitute anion the permeability ratio is given by : 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{P_{{\mathrm{sub}}}}{P_{{\mathrm{Cl}}}}= \left \left[\frac{-{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{peak}}}}{RT}\right] \right {\mathrm{,}}\end{equation*}\end{document} where Δ(Δ G ) peak is the difference in free energy between Cl and the substitute ion. This calculation applies to any number of barriers as long as the difference in barrier height, Δ(Δ G ) peak , is the same for all barriers . A recognized limitation of the rate theory approach is ambiguity in the value of the “prefactor” in the rate equations , but in the present calculation we are concerned exclusively with the ratios of permeabilities so that, as long as the permeation process is similar for all anions, ambiguities in the absolute value of the prefactor should have a small effect. A second route to the relation described by is to use a lumped Nernst-Planck model in which the permeability is expressed ( ): 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{{\mathrm{i}}}=\frac{{\mathrm{{\beta}}}_{{\mathrm{i}}}D_{{\mathrm{i}}}A}{{\mathrm{{\Delta}}}x}{\mathrm{,}}\end{equation*}\end{document} where β i is the water-channel equilibrium partition coefficient, D i is the diffusion coefficient within the channel, A is the cross-sectional area, and Δ x is the length of the channel. If it is assumed that the long-range, anion–channel interaction is reflected in the apparent value of β; and that D is approximately equal for different anions, then ( ): 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{P_{{\mathrm{sub}}}}{P_{{\mathrm{Cl}}}}=\frac{{\mathrm{{\beta}}}_{{\mathrm{sub}}}}{{\mathrm{{\beta}}}_{{\mathrm{Cl}}}}= \left \left[\frac{-{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{eq}}}}{RT}\right] \right {\mathrm{,}}\end{equation*}\end{document} where −Δ(Δ G ) eq is the difference in the equilibrium transfer free energy between Cl and the substitute ion. In the discussion, a simple model is used to show how these two approaches, one focusing on peak energy and the other on an equilibrium transfer energy, can yield the same result; i.e., Δ(Δ G ) peak = Δ(Δ G ) eq . Blocking efficacy was determined by exposing the oocyte to a 5-mM concentration of the substitute anion (some of the salts are only available as K salts and, for those experiments, a 5-mM KCl control was added to the protocol). Percent block was determined by measuring the decrease in the slope conductance (+/− 10 mV) at the reversal potential. Each anion was characterized by a half-maximal inhibition constant, K i 1/2 , calculated by assuming blockade to be a unimolecular binding event that can described by Michaelis-Menten kinetics ( ): 6 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{{1}/{2}}^{{\mathrm{inhib}}}= \left \left[{\mathrm{x}}\right] \right {\cdot} \left \left(\frac{g_{{\mathrm{b}}}}{g_{0}-g_{{\mathrm{b}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where [x] is the concentration of added blocker, g b is the conductance measured in the presence of the blocker, and g o is the conductance measured in the absence of the blocker. To convert the relative values of K i 1/2 to differences in well depths, we assumed that all blocking anions bound at the same site and that blockade was the result of competition between the blocking anion and Cl for that site . The values of K i 1/2 were, therefore, assumed to be related to the actual binding constants of the blocking anion and those of Cl by a relation of the form expected for simple, competitive inhibition , where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{{1}/{2}}^{{\mathrm{inhib}}}=K_{{\mathrm{A}}} \left \left(1+\frac{ \left \left[{\mathrm{Cl}}\right] \right }{K_{{\mathrm{Cl}}}}\right) \right \end{equation*}\end{document} and [Cl] is the concentration of Cl at the site, K Cl is the binding constant for Cl at the site and K A is the binding constant for the blocking anion at the site. If it is further assumed that the value of [Cl] and K Cl are independent of the nature of the blocking anion, then the ratio of any two values of K i 1/2 is given by : 7 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{K_{{1}/{2}}^{{\mathrm{inhib,A}}}}{K_{{1}/{2}}^{{\mathrm{inhib,B}}}}=\frac{K_{{\mathrm{A}}}}{K_{{\mathrm{B}}}}= \left \left[\frac{-{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{well}}}}{RT}\right] \right {\mathrm{,}}\end{equation*}\end{document} where Δ(Δ G ) well is the difference in well depth between the two ions, Δ G A well − Δ G B well . We note two qualifications as regards this approach to determining relative well depth. First, the blocking anions used were all permeant to varying extents and were expected, therefore, to contribute to the measured current. Errors due to permeation of the blocking anion were minimized by using a blocker concentration (5 mM) that was ∼5% that of Cl (105 mM). Second, multiple anion occupancy of CFTR could in principle affect the estimation of relative peak heights and well depths, but under the conditions used in the present experiments (external substitution), only monotonically decreasing conductance was seen with increasing concentration of the blocking anion , so we assumed that channels were singly occupied at all times. The experimentally derived parameters that comprise the selectivity patterns for CFTR and the PVC-TDMAC membrane are presented in Table . Permeability ratios are tabulated in two ways; using either the least permeant anion, Cl, or the most permeant anion, C(CN) 3 , as a reference. The latter were used to calculate Δ(Δ G ) peak so that each number this column reflects the increase in peak height seen by each anion over that seen by C(CN) 3 . The table also contains the K 1/2 for blockade of Cl currents for each anion and the ratio of each K 1/2 to that measured for C(CN) 3 . The latter values were used to calculate Δ(Δ G ) well , which represents the change in well depth for each anion relative to C(CN) 3 , so that positive values indicate a shallower well and negative values a deeper well. Also tabulated in Table are values for log K pot , obtained as described in materials and methods from anion substitution protocols using the PVC-TDMAC membrane and expressed relative to Cl and to C(CN) 3 . As with permeability ratios and inhibitory constants, the values for log K pot were used to calculate Δ(Δ G ) trans (see ), which represents the increase in transfer free energy from water to the synthetic membrane for each of the anions with respect to C(CN) 3 . The values for permeability ratios and relative values of K i 1/2 span a range from 1 to 8 for the former and 1 to 14 mM for the latter, and the energies associated with these indices of anion permeation and binding range from ∼0.4–5 kJ/mol. As expected , the differences in peak heights and well depths associated with ion translocation through the CFTR channel, as well as the energies associated with anion partitioning into the synthetic membrane, are small with regard to an absolute reference such as the hydration energies for the anions shown in Table . It is also apparent, however, that the energies associated with the responses of CFTR and the synthetic membrane to anion substitution are highly correlated. In Fig. 2 , the energy differences associated with anion permeation (A) and block (B) are plotted versus the corresponding energies derived from the response of the PVC-TDMAC membrane. The high correlation of these values (with the exception of iodide, see below) indicates that the selectivity pattern exhibited by CFTR, as judged by either relative permeability or relative binding, is qualitatively identical to that of the synthetic membrane, differing in each case only by a multiplicative constant. Anions that see a barrier height that is increased relative to that of C(CN) 3 also experience a more positive (less favorable) transfer free energy between water and the synthetic membrane. Similarly, anions that bind less tightly than C(CN) 3 are those for which the water-synthetic membrane transfer free energy is less favorable. It is apparent from Fig. 2 that the peak and well energies change in a parallel fashion. SCN, for example, sees an energy barrier to entering the CFTR channel that is lower than that of Cl, and also sees an equilibrium free energy associated with partitioning into the synthetic membrane that is more favorable than that of Cl. Similarly, the tighter binding of SCN (relative to Cl) is correlated with ease of partitioning into the synthetic membrane. These results are, perhaps, not surprising in that the selectivity patterns for both CFTR and the PVC-TDMAC membrane have both been previously identified as being consistent with the “lyotropic” or Hofmeister series, which is ordered according to relative free energy of hydration . Anions that are more readily dehydrated than Cl exhibit higher permeability ratios and bind more tightly within the CFTR pore, and also partition more readily into the PVC-TDMAC membrane. To understand the physical basis of the selectivity patterns common to CFTR and the PVC-TDMAC membrane, it was necessary to relate the energy differences associated with anion permeability ratios, relative anion binding affinities, and anion partitioning into the synthetic membrane to some physical property of the anions. As exemplified by the seminal work of Eisenman 1962 and the analysis of the acetylcholine receptor by Lewis and Stevens 1983 , the natural choice for this parameter is ion size, expressed as the reciprocal of ionic radius, 1/r. This is so because the dominant contribution to the electrostatic free energy of a spherical ion varies with 1/r . Fig. 3A and Fig. B , shows the results of such analysis for the PVC-TDMAC membrane and CFTR, respectively. In Fig. 3 A, the energy differences associated with anion partitioning into the PVC-TDMAC membrane obtained from Δ(Δ G ) trans (see ) are plotted versus the reciprocal of ionic radius ( Table ). In Fig. 3 B, the relative heights of the energy barriers associated with entering the CFTR channel obtained from permeability ratios ( Table ) are plotted versus the reciprocal of the anionic radius. Because C(CN) 3 , the largest and most permeant ion, was chosen as the reference anion for both plots, for each anion, either the increase in equilibrium transfer energy (synthetic membrane) or the increase in barrier height (CFTR) relative to that seen by C(CN) 3 is plotted versus 1/r. In both cases, the energy difference increases linearly with 1/r. Lewis and Stevens 1983 pointed out that the slope of this type of plot provides a quantitative measure of selectivity, and by that standard it is apparent that the synthetic membrane is approximately five times more selective than CFTR. The physical significance of the plots of energy difference versus reciprocal radius can be appreciated by expressing the total free energy of transfer as the sum of two components; one due to the difference in hydration energy and the other the difference in the energy of solvation of the anion within the synthetic membrane or CFTR. The former measures the energy of interaction of the anion with water, while the latter is a measure of the energy of interaction of the anion with the channel and its contents. The relative free energy of transfer, Δ(Δ G trans ), associated with either differential anion partitioning into the synthetic membrane (see ) or the barrier to entry into the channel, can be written as in : 8 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{trans}}}={\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{solv}}}-{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G\right) \right _{{\mathrm{hyd}}}{\mathrm{,}}\end{equation*}\end{document} where Δ(Δ G hyd ) is the relative hydration energy and Δ(Δ G solv ) the relative solvation energy in the membrane, both calculated using C(CN) 3 as a reference (i.e., and ): 9 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G_{{\mathrm{hyd}}}^{{\mathrm{A}}}\right) \right ={\mathrm{{\Delta}}}G_{{\mathrm{hyd}}}^{{\mathrm{A}}}-{\mathrm{{\Delta}}}G_{{\mathrm{hyd}}}^{{\mathrm{C}} \left \left({\mathrm{CN}}\right) \right _{{\mathrm{3}}}}\end{equation*}\end{document} and 10 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\Delta}}} \left \left({\mathrm{{\Delta}}}G_{{\mathrm{solv}}}^{{\mathrm{A}}}\right) \right ={\mathrm{{\Delta}}}G_{{\mathrm{solv}}}^{{\mathrm{A}}}-{\mathrm{{\Delta}}}G_{{\mathrm{solv}}}^{{\mathrm{C}} \left \left({\mathrm{CN}}\right) \right _{{\mathrm{3}}}}{\mathrm{,}}\end{equation*}\end{document} where Δ G hyd A ,Δ G hyd CCN 3 ,Δ G solv A , andΔ G solv CCN 3 are the energies of hydration and solvation, respectively for anion, A, and C(CN) 3 expressed with respect to a vacuum phase. The solid lines in Fig. 3A and Fig. B , represent the values of |Δ(Δ G hyd )| relative to C(CN) 3 calculated as the Born charging energy (see ) using corrected radii as described in materials and methods ( Table ). Note that, although the values of Δ(Δ G hyd ) are negative, they are plotted here as absolute values for convenience of comparison with Δ(Δ G trans ). The hydration component of the energy difference will be identical for any channel or membrane, as the necessity to remove an ion from bulk water is a universal constant for any permeation process . The linear, monotonic behavior of Δ(Δ G t rans ) and Δ(Δ G hyd ) suggested a straightforward calculation of Δ(Δ G solv ) as the difference between these two functions, and the predicted behavior of the solvation energy is indicated by the dotted lines in Fig. 2A and Fig. B . The selectivity patterns exhibited by the PVC-TDMAC membrane and CFTR can be readily understood in terms of the differences in the relative interaction energy of the anions with water, Δ(Δ G hyd ), and with the membrane, Δ(Δ G solv ). In the synthetic membrane, the work required to transfer an anion from water to the plasticized PVC decreases with increasing anion radius because Δ(Δ G hyd ) is a steeper function of 1/r than is Δ(Δ G solv ). In other words, anions larger than Cl experience weaker interactions with water and with the synthetic membrane, but they partition into the membrane more readily because they see the smallest difference between these two energies. The linear relation between the apparent solvation energy for the PVC-TDMAC membrane and reciprocal anionic radius suggests that Δ G solv , the anion-membrane interaction energy for the PVC-TDMAC membrane, behaves precisely as predicted by the Born energy (see ) for a spherical anion contained within a polarizable medium having a dielectric constant somewhat less than that of water. The slope of the plot predicts an effective dielectric constant, ∈ eff , for the synthetic membrane of 4.1. The measured dielectric constant of the PVC-TDMAC membrane was ∈ = 11.5 ± 0.6, as compared with a published value of ∈ = 14 for o-NPOE plasticized PVC membranes constructed without TDMAC . In other words, the synthetic membrane is somewhat more selective than would be predicted by the bulk dielectric constant determined from the apparent capacitance. Fig. 3 A (inset) provides some appreciation of the impact on Δ G solv of changes in the dielectric constant. Δ G solv for a 1-Å sphere increases rapidly as ∈ increases from 1 to ∼20, but changes are minimal for ∈ > 20. From the perspective of the continuum analysis of solvation energy, a medium having a dielectric constant >20 is effectively as polarizable as water (∈ = 80). Fig. 3 B shows the behavior of Δ(Δ G peak ), Δ(Δ G hyd ), and Δ(Δ G solv ) for CFTR. It is immediately apparent from Fig. 3 B that the modest permeability selectivity of CFTR can be attributed to the fact that the energies of hydration and solvation differ very little over the range of anion sizes examined. In other words, a visiting anion is solvated within the CFTR pore nearly as well as it is in bulk water. Accordingly, the solvation energy predicts an effective dielectric constant within the pore of ∼19. The near identity of the value of Δ G hyd and Δ G solv justifies treating the energies associated with anion entry as a near equilibrium process. The point is made more explicitly in Fig. 4 , in which are shown the predicted values expressed with respect to a vacuum reference phase for Δ G peak , Δ G hyd , and Δ G solv , calculated using a value of 19 for the effective dielectric constant within the channel. This plot predicts a peak energy for Cl of 14.5 kJ/mol (5.86 RT ), which agrees well with the values derived by Linsdell et al. 1997a for a multi-site model, and is about half the value of 27.8 kJ/mol (11.2 RT ) predicted from a symmetric two-barrier, one-site model using a well depth of 8.2 kJ/mol (3.3 RT ) and constraining the single channel conductance to 10 pS . Although these values are likely to be significantly affected by the ambiguity as to the appropriate value for the prefactor in the Eyring rate equations , it is apparent that the absolute barrier heights predicted from continuum analysis fall in a range consistent with observed transport rates. The error in the prefactor incurred by using kT / h has been estimated, using a discrete approximation to a continuum model, to be of the order of 10 2 , which would translate into an error in the calculated value of Δ G of ∼4.6 RT . Fig. 5 A contains data taken from the analysis of Bormann et al. 1987 of relative anion permeability in the GABA receptor (GABAR). The pattern of permeability selectivity for the ligand-gated channel also conforms to the predictions of a continuum electrostatic model, where the anion–pore interaction is modeled as the stabilization of an anion by a polarizable medium. The effective dielectric constant is predicted to be somewhat lower than that of CFTR, ∼12.4, because the selectivity is somewhat higher. The absolute barrier for Cl is predicted to be 19.6 kJ/mol (7.9 RT ), which agrees fairly well with the estimates of Bormann et al. 1987 of a barrier height of 24 kJ/mol (9.7 RT ) for a three-barrier, two-site model. A similar analysis of the glycine receptor and the outwardly rectifying chloride channel (ORCC) from T-84 cells is shown in Fig. 5B and Fig. C , respectively. Fig. 6A and Fig. B , shows values for the binding energies derived from the comparative analysis of blockade of CFTR by halides and pseudohalides, which we presume to reflect the presence in the permeation path of at least one energy well. Fig. 6 A shows that the relative energies of binding, like the barriers to permeation, decrease with increasing values of 1/r. The implications of the variation in well depth depicted in Fig. 6 A are more readily apparent from a plot of estimated values for the anion–channel interaction energies calculated at the binding site with respect to a vacuum reference . The values plotted in Fig. 6 B were calculated by fixing the well depth for SCN at 12.5 kJ/mol (5.1 RT ) on the basis of the dissociation constant of 6.4 mM reported by Tabcharani et al. 1993 , and adding the calculated hydration energy. The points for C(CN) 3 , Au(CN) 2 , N(CN) 2 , and N 3 were determined by taking their values relative to SCN, and then adding the calculated hydration energy. The dashed line is the best fit to the data points. The predicted well depth for Cl is ∼6.5 kJ/mol, which agrees well with the value of 8.2 kJ/mol calculated from the dissociation constant of 38 mM reported by Tabcharani et al. 1997 , which is shown as an open circle on the plot. The anion–channel interaction energy derived from an analysis of anion binding behaves as if it consists of two components: a constant, negative energy that is similar for all anions and a radius-dependent portion that increases with decreasing anion size. Well depth, the difference between the anion–channel interaction energy (dashed line) and the hydration energy (solid line), increases with increasing anion size because, as with the barrier height, the change in hydration energy is a steeper function of 1/r than is the apparent anion–channel interaction energy at the binding site. As anionic size increases, the hydration energy decreases more rapidly than does the anion–pore energy so that the net effect is to deepen the well. This result appears to be counter intuitive, because the anion that is most tightly bound experiences the smallest anion–channel interaction energy. However, relative well depth always reflects changes in both anion–channel and anion–water interactions. The dotted line in Fig. 6 B represents the anion–channel interaction energy derived from relative permeability measurements taken from Fig. 4 , plotted for comparison and to emphasize an important point, namely that the radius-dependent portion of the anion-channel interaction energy is similar, regardless of whether it is defined by relative permeability (peak height) or relative blocking affinity (well depth). This plot shows why larger anions experience not only a reduced barrier to entering the channel, but also a deeper energy well within the channel. In terms of permeability selectivity, iodide stands out as an anomaly. In Fig. 2 A, it can be seen that the value of P I / P Cl determined for human wtCFTR expressed in Xenopus oocytes is well below that predicted for an ion that is easier to dehydrate than Cl. Tabcharani et al. 1997 provided evidence that CFTR can exhibit a higher value of P I / P Cl , but that the ion causes a rapid modification of CFTR that leads to the lower value of 0.2–0.4 that is most often reported . The higher value of P I / P Cl would place iodide in its predicted position on the 1/r plot . Using human wtCFTR expressed in Xenopus oocytes, we have not detected any evidence of this higher P I / P Cl using a panel of various mole fractions of I:Cl, as well as various voltage clamping protocols (data not shown). It may be, however, that the conversion is simply too fast to resolve in this experimental setting. In accord with the hypothesis of Tabcharani et al. 1997 that I is inducing a modification of CFTR, we have observed a block of CFTR by I that is qualitatively distinct from the block seen with the other permeant ions . Block by a small amount of I in the external bath is weakly voltage dependent in the negative quadrant, as shown in Fig. 7 , whereas the block seen with most permeant ions is largely voltage independent . The efficacy of block is enhanced if the external concentration of Cl is lowered (by substitution with aspartate), as shown in Fig. 7 B, suggesting that Cl and I ions are competing for the same site(s). Iodide has a tendency to form interhalogens, particularly triiodide, a reactive species known to attack cysteine, tyrosine, and histidine residues within proteins, but the addition of sodium thiosulfate, which reduces I 3 , did not alter the observed effects. Furthermore, increasing concentrations of I 3 (made by adding I 2 to NaI solution) appeared to be quite toxic above 500 μM I 3 , leading to uncontrollable increases in oocyte conductance. While in solutions near or below 100 μM, the I 3 appeared to be unstable in our standard amphibian Ringer's (as judged colorimetrically by clarification of the yellowish-red color of the I 3 ), it was possible to make a fresh I 3 solution and feel relatively confident that the oocyte was seeing 10–100 μM I 3 , in which case the effect on CFTR was an irreversible, voltage-independent block. However, we cannot rule out the possibility that I 3 may be forming within the lumen, leading to a mild chemical modification of CFTR. Data from mutagenesis experiments support the view that iodide permeation is most properly thought of as a special case in that mutations that affect P I / P Cl do not appear to dramatically alter the permeability ratios for other permeant ions . The measurements and analysis presented here provide a plausible explanation for the anion selectivity pattern often referred to as the lyotropic or “Hofmeister” sequence that is seen in a variety of Cl channels and in synthetic, anion-selective membranes. In both cases, the selectivity pattern can be predicted by assuming that the energy of interaction of the anion with the channel or synthetic membrane is dominated by the electrostatic energy associated with the stabilization of a charged sphere in a dielectric medium. The analysis of selectivity by Eisenman and co-workers laid the foundation upon which most analysis of ion selectivity is based, namely that the relative tendency of an ion to enter or associate with a channel is dependent on the balance of two energies, one resulting from the ion–water interactions and the other reflecting the ion–channel interactions. In its original form, Eisenman's formulation of the selectivity problem relied on the assignment of a variable “field strength” to presumed sites of interaction in the channel that could be occupied by visiting ions. By varying the field strength, and hence the predicted energy of the ion–channel interaction, it was possible to generate different selectivity patterns. The limiting patterns were those associated with very high or very low field strength, respectively, in which either ion–channel or ion–water energies would dominate the ion transfer process. The analysis presented here provides a physical basis for “weak field strength” selectivity. In the continuum electrostatic model, the field strength is that due to the polarization of dipolar entities that are subject to the field of the visiting anion. The magnitude of the ion–channel interaction energy depends on the size of the anion and the effective dielectric constant experienced by the anion when it resides within the channel. It is important to point out that the value of the effective dielectric constant would not be expected to be governed solely by the properties of amino acid side chains that might line the pore. Although the polarizability of such entities would contribute to ∈ eff , other contributions would be expected from the remainder of the protein, including side chains and the peptide backbone, water molecules that may reside within the channel, the surrounding lipid, and even water bathing the membrane. The term “effective dielectric constant” embraces this notion . It is clear from this analysis that the implication of the term “weak field strength” is not that the anion–channel energies are small. They are, in fact, predicted to be approximately equal to the anion–water energies, but to increase slightly less steeply with decreasing anion size. The Born-type model employed here is, from both a conceptual and computational perspective, the simplest approach to accounting for the apparent solvation energies of anions that traverse the pore. The model represents the heterogeneous ensemble of components that comprise the environment of an anion as an equivalent continuum of infinite extent characterized by an effective dielectric constant, ∈ eff . The most important result of the analysis is not the value of ∈ eff , however. Rather, it is the fact that the lyotropic selectivity pattern can be predicted by presuming that anions interact with the channel much as they do with water, such that the stabilization energy is linearly related to reciprocal anion radius. Attempts to measure or predict the value of the apparent dielectric constant seen by an anion inside a channel have produced widely varying results. In a large bore (minimum diameter ∼10 Å), anion-selective porin (phoE, for example), Gutman et al. 1992 used time-resolved fluorometry to determine a value of ∈ eff for the pore of 24, whereas Karshikoff et al. 1994 predicted a value for the same channel of 30 using a macroscopic, multidielectric model. The analysis conducted by Sham et al. 1998 and Sansom et al. 1997 , however, suggests that the definition of a unique ∈ eff may be elusive because the value is likely to depend on the type of electrostatic interaction used to define it. As indicated in materials and methods , the value of ∈ eff could also depend on the approach taken to define the radius of the ion . It is probably most prudent to consider ∈ eff , determined here, as an empirical parameter that provides a measure of the relative ability of the channel to solvate a permeating ion that offers a useful first approach to conceptualizing the electrostatic origins of the selectivity pattern. The behavior of ions in physical or biological systems has been analyzed previously using an approach similar to the one adopted here. An analysis of the swelling and shrinking of gels by Büchner et al. 1932 led to a relationship between the action of salts on colloidal systems and reciprocal ion radius not unlike that used here. In addition, Lewis and Stevens 1983 presented essentially similar findings for cation permeation in the acetylcholine receptor. The Born analysis, based on their permeability ratios, would predict ∈ eff = 55. More recently, Aqvist and Warshel 1989 used two methods, free energy perturbation and protein dipoles Langevin dipoles simulations, to analyze the polarizable interior of the gramicidin channel to predict the energy barrier to permeation for Na, deriving values of 9.6 (3.9 RT ) and 16.7 (6.7 RT ) kJ/mol, respectively. Using the data of Urban et al. 1980 , the analysis used here would predict an ∈ eff of 21.5–27 for gramicidin A, and an absolute barrier for Na entry of 10–14 kJ/mol. Partenskii et al. 1994 reported that, within the framework of a three-dielectric, continuum model in which the dielectric constant of the pore region was restricted to between 2 and 5, it was difficult to account for the apparent energy barrier that characterizes cation flow through the gramicidin channel. As regards the structure of the CFTR pore domain, the most important implication of the analysis presented here is the prediction that the permeability selectivity pattern characteristic of CFTR, as well as several other nonhomologous anion channels, does not depend on the interaction of the permeating anion with some specific component of the channel. The basic selectivity pattern, common to anion permeability and anion binding, can be viewed as the result of the interaction of the anion with a volume that exhibits the generalized property of dielectric polarizability. This type of environment could presumably be provided by a configuration of the membrane spanning segments of CFTR which, along with the resident water molecules, forms a polarizable “tunnel” through which the anions can pass . The present results do not speak to the location of any “selectivity filter” and, in fact, suggest that the very idea that the anion permeability selectivity of CFTR is associated with any specific structure, as in the K channel visualized by Doyle et al. 1998 , may be moot. The barrier to anion entry (or the energy associated with equilibrium partitioning) is predicted to be a generalized feature of this dielectric tunnel that is not highly dependent on the details of the structure of the transmembrane segments. This prediction has, in fact, been borne out by the results of mutational analysis, which have shown that permeability ratios are largely insensitive to mutations . In addition, the observation that the same analysis predicts the relative pattern for anion channels that are likely to be divergent in their structure lends further support to the notion that the anion–channel interaction energy is the result of a generalized interaction of the anion with the pore. The selectivity of anion binding by CFTR differs from relative permeability in at least two ways. First, in contrast to permeability, binding is highly sensitive to point mutations, particularly in transmembrane segments 5 and 6 . Second, the analysis of energetics presented here suggests that, in order to predict the selectivity pattern associated with anion binding, the underlying anion–channel interaction energy must be envisioned as comprising two components: one due to the same radius-dependent, dielectric stabilization that appears to determine barrier height, and a second that is similar for all anions, roughly independent of size. These two attributes, sensitivity to structural perturbations and two-component energetics, are easily reconciled by assuming that anion binding, represented by the energy well in the rate theory description, is dependent on the existence within the conduction path of a narrow region in which the residues that line the channel are able to come into a more intimate, inner sphere and contact the visiting ion. If this region were relatively rigid so that it could not change conformation in the presence of different permeant ions, then the energy associated with residing there could be roughly independent of anion size. In contrast, the contribution to the anion–channel energy due to the region of the protein surrounding the cavity would exhibit the dependence on anion size predicted for a polarizable medium. Mutations could alter the nature of this cavity by changing its size or, equally likely, by changing the charge or orientation of amino acid side chains that are required for a favorable anion–channel interaction . Reduction or elimination of the anion–channel contact would leave the underlying, radius-dependent anion–channel energy intact so that permeability ratios would not be greatly affected even though anion binding was nearly lost. The analysis of permeation and binding energetics undertaken here provides the basis for a working model of the anion conduction process in CFTR and perhaps the GABAR, GlyR, and T84-ORCC as well. In the case of CFTR, it is possible to envision two sorts of CFTR pores: those that bind anions, exemplified by the wild-type channel, and those that do not, exemplified by mutant CFTRs like G314E or Q and R347D . This structural dichotomy suggests that it is reasonable to treat these two facets of the conduction process as representing two distinct types of physical interaction of the anion with the channel. Consider first a channel that does not bind anions . It is useful to envision permeant anions in the bulk solution as coordinated by an inner sphere of water molecules and surrounded by an outer sphere or shell that is the remainder of the bulk solution . Upon colliding with the mouth of the channel, the anion and channel form a transition state complex that leads to the anion, along with most of its inner shell water, residing within the channel. In this state, the channel, to a first approximation, has replaced the outer shell waters (i.e., the bulk solution), but the inner sphere waters remain associated with the anion. The energy profile expected for the process is depicted as the trapezoidal line in Fig. 8 D and represents an equilibrium transfer free energy (reflected in the partition coefficient, β) that, for Cl, would be unfavorable by ∼6 RT (see results ) due to the fact that the effective dielectric constant of the channel protein, the lipid membrane, and surrounding water is somewhat less than that of the outer sphere of water molecules in bulk solution. This represents the energy barrier seen by an anion entering the channel. To arrive at a model for anion binding, we begin by envisioning the lining of the wild-type pore as a “thin-walled” tube immersed in bulk water . When the anion encounters the channel in this (albeit hypothetical) condition, it enters with little or no energy cost as it retains its inner sphere water, and the outer sphere energy within the thin-walled tube is virtually identical to that experienced by the anion in bulk water. As the anion moves along the lining of the wild-type channel, however, it encounters a narrowing where some portion of the inner sphere water molecules are replaced by interactions with the polar or charged groups that line this region of the pore. These inner-sphere anion–channel interactions with the wall of the pore are somewhat more favorable than those with water so that an energy well is created. The profile for this hypothetical thin-walled anion-binding channel residing in water is depicted as the dashed line in Fig. 8 D. This binding might be imagined as being analogous to that seen with anion inclusion compounds, like the katapinates, that form inner sphere interactions with Cl and Br in aqueous solution . One of these, a diprotonated diazabicyclic encloses a Cl that is stabilized by two ionic hydrogen bonds with amino nitrogens. The stability constant for this compound predicts a “well depth” of ∼11.4 kJ/mol (4.6 RT ) that is of the same order as that seen in the weakly selective channels considered here. If these two profiles are summed , they give rise to the familiar two-barrier, one-well profile that is depicted as the dotted line in D. Although the shape of the profile as depicted in Fig. 8 D is largely arbitrary, the diagram makes the point that it is possible, in principle, to account for the energetics of anion permeation through CFTR in a relatively straightforward way and illustrates how the summing of an equilibrium transfer energy with a single, localized energy well could give rise to the familiar two-barrier, one-site channel model. Fig. 8 E illustrates how radius-dependent variation in the outer sphere anion energy could vary the peak heights and well depths in a parallel fashion if the inner-sphere contribution in the binding region of the channel was roughly size independent. Large anions that experience a reduced barrier height, and enter the channel more readily, also see a deeper energy well and bind more tightly because of the reduced, radius-dependent equilibrium transfer energy. The permeability selectivity exhibited by CFTR, and shared by GABAR, GlyR, and T84-ORCC, represents the most primitive type of ion discrimination, characteristic of a permeation path that functions as a polarizable tunnel that can stabilize a partially dehydrated ion as it passes through. This situation is a striking contrast to that envisioned for the bacterial K channel that selects for K by means of a clearly identifiable structure, the selectivity filter, consisting of a tetrahedral array of oxygen ligands . In a recent review of the physical factors that govern anion separations, Moyer and Bonnesen 1997 suggested the utility of viewing selectivity within the framework of two limiting types. In one, exemplified in biology by the K channel, the host (channel) is structurally specialized to recognize the guest (ion). In the other, here exemplified by CFTR, selectivity is based simply on a physical bias that is imposed by the physics of hydration and a primitive form of solvation of the guest by the host molecule. This approach to categorizing selectivity types provides a useful framework for interpreting selectivity patterns characteristic of different channel families . The physical forces that produce a bias-type selectivity must impinge on any ion–channel interaction, but channels may be expected to vary in the extent to which this bias is overshadowed by a specific recognition component. The muscle Cl channel, ClC-1, may be a case in point. It has been reported to exhibit a distinctly nonlyotropic pattern of permeability selectivity (Cl > SCN > Br > NO 3 > I) that may be an indication of some structural specialization that has evolved to recognize the Cl anion. It is of interest in this regard that mutation of several residues in ClC-1 resulted in a reversion of the selectivity sequence to the more primitive, lyotropic order . What is striking in the case of CFTR (and, likely, GlyR, GABAR, and T84-ORCC) is that the compound effects of bias-based permeability selectivity and anion binding result in channels that are “optimized” Cl filters. The analysis employed here emphasizes one aspect of the relation of anion size to permeation, namely that halides and pseudohalides having an equivalent sphere radius larger than that of Cl enter the pore more readily due to lower anion-water interaction energies. There is clearly a limit, however, to any “larger is better” theory of permeation. As anion size increases, the physical dimension of the pore, its effective diameter, must become limiting. Several of the “larger” molecules that were the focus of this study are roughly cylindrical in shape and the actual physical diameter of the cylinder is less than that of the diameter of the equivalent sphere (dicyanoaurate, for example, has a cylindrical diameter of ∼3.4 Å at its widest point, while the equivalent sphere diameter of the molecule is ∼6 Å; Table ). On the other hand, the trimmest right cylinder into which tricyanomethanide, which has flattened pyramidal geometry, could fit would be ∼7.4 Å in diameter, its widest dimension, due to the fact that there is no way to “twist” the pyramid to fit it into a smaller cylinder. This may seem to be inconsistent with the effective pore diameter of ∼5.5 Å determined by Linsdell et al. 1997b , Linsdell et al. 1998 ; however, if one imagines that the shape of the pore is elliptical, such that the widest part of the pore is on the order of 7.5 Å, then the narrow portion of the pore could be on the order of 5.5 Å, and thereby accommodate tricyanomethanide. It may be necessary, however, to exercise some caution in imputing effective pore size from the behavior of poorly permeant ions. As indicated in Fig. 1 , polyatomic molecules like gluconate are characterized by a hydration energy that is much larger than their physical size would predict, an effect that is presumably due in part to a nonuniform charge distribution . On the basis of Fig. 1 , it is possible to assign to gluconate an apparent “thermochemical radius” of ∼1.65 Å. Using the value of ∈ eff = 19 for the CFTR pore predicts a peak barrier height of 15.9 kJ/mol (6.4 RT ), and the difference in this value and that for Cl [14.5 kJ/mol (5.86 RT ), see results ] predicts a permeability ratio ( P gluconate / P Cl ) of ∼0.58. This may be compared with the experimentally determined values of 0.071 and 0.013 reported by Linsdell and Hanrahan 1998 , determined by substitution of the solution on the cytoplasmic and extracellular side of the patch, respectively. The larger of these two values (0.071) predicts a peak energy of 21.1 kJ/mol, 5.2 kJ/mol greater than the simple electrostatic model. This increased barrier height could point to a size exclusion effect. Marcus 1997 , however, points out that asymmetric charge distribution could have other consequences that are not directly related to size, because the polar and nonpolar portion of the molecule can experience very different interactions with the immediate environment. | Study | biomedical | en | 0.999995 |
10578017 | Voltage-gated proton channels differ from other voltage-gated ion channels, not only in their extreme selectivity for H + , but also in the regulation of their gating by pH o and pH i . The mechanism of permeation is believed to differ radically from traditional ion channels, which comprise water-filled pores through which ions diffuse: proton channels appear to conduct H + by a Grotthuss-like mechanism of hopping across a hydrogen-bonded chain spanning the membrane . If ion channels are defined narrowly as water-filled pores, then proton channels are not ion channels, although they conduct protons passively down their electrochemical gradient, and independently of other ionic species. In spite of these fundamental differences, it is remarkable how closely proton channels resemble other voltage-gated channels. H + channels activate upon depolarization with a sigmoidal time course, deactivate exponentially, and exhibit a Cole-Moore effect practically indistinguishable from the behavior of delayed rectifier K + channels . Low pH o shifts the voltage-activation curves of both H + channels and other ion channels toward positive potentials and slows activation at a given voltage. Voltage-gated proton channels characteristically are inhibited by extracellular polyvalent cations. The transition metals Zn 2+ and Cd 2+ have been used most frequently , but Cu 2+ , Ni 2+ , Co 2+ , Hg 2+ , Be 2+ , Mn 2+ , Al 3+ , and La 3+ have similar effects . To the extent that each has been explored, all of these metal cations shift the voltage dependence of activation (channel opening) to more positive potentials and slow the opening rate . These effects resemble those of polyvalent cations on other ion channels . Closer examination reveals differences, however. The H + channel is much more sensitive to external ZnCl 2 than are voltage-gated K + channels, which require 1,000-fold higher concentrations to produce comparable effects in squid , 10–100-fold higher concentrations in frog skeletal muscle , and 100-fold higher concentrations in Shaker . In addition, the effects of ZnCl 2 on squid axon K + channels are similar for addition to either side of the membrane and internally applied ZnCl 2 is quite potent . In contrast, we find that ZnCl 2 has qualitatively different effects on H + channels depending on the side of application and thus binds to distinct external and internal sites. The effects of metal cations on H + currents have been characterized variously as voltage-dependent block, voltage shifts induced by electrostatic effects on the voltage sensor, and specific binding to the channel. These interpretations invoke different mechanisms. Voltage-dependent block suggests that the metal ion enters the channel and crosses part of the membrane potential field to reach its block site in the pore. Here we explore the effects of ZnCl 2 , one of the more potent inhibitors of H + channels, as a prototypical metal inhibitor. We find that voltage-dependent block is not a viable mechanism. Prominent effects of Zn 2+ reflect specific binding that allosterically alters gating. A key feature of the inhibition of H + currents by Zn 2+ is a profound pH dependence, which has not been described previously. Lowering pH o decreases the effectiveness of ZnCl 2 . Competition between Zn 2+ and H + has been noted previously for other channels, including Cl − and K + . We consider whether the pH o dependence indicates that (a) the active form is not Zn 2+ but ZnOH + , (b) Zn 2+ and H + compete for the same binding site, or (c) there is noncompetitive inhibition; i.e., protonated channels have a lower affinity for Zn 2+ . We conclude that the external Zn 2+ receptor is formed by three or more protonation sites, perhaps comprising His residues, that together coordinate one Zn 2+ . Type II alveolar epithelial cells were isolated from adult male Sprague-Dawley rats using enzyme digestion, lectin agglutination, and differential adherence, as described in detail elsewhere , with the exception that we now use elastase without trypsin to dissociate the cells. The rats were anesthetized using sodium pentobarbital. In brief, the lungs were lavaged to remove macrophages, elastase was instilled, and then the tissue was minced and forced through fine gauze. Lectin agglutination and differential adherence further removed contaminating cell types. The preparation at first includes mainly type II alveolar epithelial cells, but after several days in culture the properties of the cells are more like type I cells. H + currents were studied in approximately spherical cells up to several weeks after isolation. Solutions contained 100 mM buffer supplemented with tetramethylammonium (TMA) methanesulfonate (TMAMeSO 3 ) to bring the osmolarity to ∼300 mOsm. One exception was the pH o 7.0 solution made with 70 mM PIPES. External solutions contained 2 mM CaCl 2 or 2 mM MgCl 2 . Internal solutions contained 2 mM MgCl 2 and 1 mM EGTA. Solutions were titrated to the desired pH with TMA hydroxide (TMAOH) or methanesulfonic acid (solutions using BisTris as a buffer). A stock solution of TMAMeSO 3 was made by neutralizing TMAOH with methanesulfonic acid. TPEN ( N,N,N ′ ,N ′-tetrakis(2-pyridylmethyl)ethylenediamine) was purchased from Sigma Chemical Co. The following buffers were used near their negative logarithm of the acid dissociation constant ( pK a ) (at 20°C) for measurements at the following pH: pH 5.0, Homopipes (homopiperazine- N,N ′-bis-2-(ethanesulfonic acid), pK a 4.61); pH 5.5–6.0 Mes ( pK a 6.15); pH 6.5 BisTris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pK a 6.50); pH 7.0 PIPES ( pK a 6.80); pH 7.5–8.0 HEPES ( pK a 7.55). Buffers were purchased from Sigma Chemical Co., except for Homopipes (Research Organics). Buffers such as Tricine and BES that reportedly complex strongly with transition metals were avoided. We could not find information in the literature on the Zn 2+ or Cd 2+ binding properties of the buffers used. Therefore, we measured the binding constants for a number of buffers, according to the method described by Good et al. 1966 . This consisted of titrating the buffer alone, and then together with an equimolar amount of the metal salt (usually 10 mmol in a 100-ml vol). The binding constant was calculated from the relationship ( ): 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K{\mathrm{^{\prime}}}_{{\mathrm{M}}}=\frac{2 \left \left(\displaystyle\frac{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }{K_{{\mathrm{a}}}}-1\right) \right }{ \left \left[{\mathrm{B}}\right] \right \left \left(\displaystyle\frac{K_{{\mathrm{a}}}}{ \left \left[{\mathrm{H}}_{{\mathrm{M}}}^{+}\right] \right }+1\right) \right }{\mathrm{,}}\end{equation*}\end{document} where K ′ M is the metal binding constant, K a is the proton dissociation constant defined in Fig. 3 (− pK a value), [H + M ] is the H + concentration at the midpoint of the titration curve in the presence of the metal being tested and [B] is the total buffer concentration. The higher the affinity of the buffer for metal, the greater the shift in the titration curve. Table gives the results. Good et al. 1966 reported that the affinity of several buffers for Ca 2+ was generally about five log units weaker than that for Cu 2+ . A notable exception is Mes, which binds Ca 2+ weakly but Cu 2+ negligibly . We find that Zn 2+ is bound roughly two log units more weakly than Cu 2+ , consistent with the lower affinity binding of Zn 2+ than Cu 2+ to various ionizable groups on proteins . One exception to this rule is that PIPES did bind Zn 2+ weakly, whereas Cu 2+ was bound negligibly . All buffers bound Cd 2+ detectably and to roughly the same extent that they bound Zn 2+ . It should be noted that Table lists log metal dissociation constant ( K M ) values, and that a value <1.3 indicates that >50% of the total metal remains unbound. Thus, much of the binding indicated is rather weak and does not preclude using these buffers in studies of metals. An upper limit to the concentration of ZnCl 2 is set by the limited solubility of Zn(OH) 2 . The maximal soluble concentrations: ∼40 μM at pH 8, ∼4 mM at pH 7, and ∼400 mM at pH 6, were not approached during experiments. We encountered solubility problems when titrating the buffers to test for metal binding (above). For this purpose, we usually used 10 mM ZnCl 2 , and in fact the solutions began to precipitate just above pH 7. To extend the pH range, buffers with higher pK a were titrated at 1 instead of 10 mM. The dihydroxide of Cd 2+ is somewhat more soluble than that of Zn 2+ , and the maximal attainable concentration is ∼5 mM at pH 8, so solubility was less of a problem. However, when the metal titrations exceeded pH ∼8, precipitation commenced. Conventional whole-cell, cell-attached patch, or inside-out patch configurations were used. Inside-out patches were formed by lifting the pipette into the air briefly. Micropipettes were pulled using a Flaming Brown automatic pipette puller (Sutter Instruments, Co.) from EG-6 glass (Garner Glass Co.), coated with Sylgard 184 (Dow Corning Corp.), and heat polished to a tip resistance ranging typically from 3 to 10 MΩ. Electrical contact with the pipette solution was achieved by a thin sintered Ag-AgCl pellet (In Vivo Metric Systems) attached to a Teflon-encased silver wire. A reference electrode made from a Ag-AgCl pellet was connected to the bath through an agar bridge made with Ringer's solution. The current signal from the patch clamp (List Electronik) was recorded and analyzed using a Laboratory Data Acquisition and Display System (Indec Corp.). Seals were formed with Ringer's solution (mM: 160 NaCl, 4.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.4) in the bath, and the zero current potential established after the pipette was in contact with the cell. Bath temperature was controlled by Peltier devices, and monitored by a resistance temperature detector element (Omega Scientific) in the bath. Because the voltage dependence of H + channel gating depends strongly on ΔpH, the threshold for activation ranging from −80 to +80 mV at ΔpH 2.5 and −1.5, respectively , the holding potential, V hold , must be adjusted appropriately. V hold was set sufficiently negative to the threshold of activation at each ΔpH to avoid Cole-Moore effects , but positive enough to avoid unnecessarily large voltage steps. We refer to pH in the format pH o //pH i . In the inside-out patch configuration, the solution in the pipette sets pH o , defined as the pH of the solution bathing the original extracellular surface of the membrane, and the bath solution sets pH i . Currents and voltages are presented in the normal sense; that is, upward currents represent current flowing outward through the membrane from the original intracellular surface, and potentials are expressed by defining the original bath solution as 0 mV. Current records are presented without correction for leak current or liquid junction potentials. The time constant of H + current activation, τ act , was obtained by fitting the current record by eye with a single exponential after a brief delay ( ): 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I \left \left(t\right) \right = \left \left(I_{0}-I_{{\mathrm{{\infty}}}}\right) \right {\mathrm{exp}}\frac{- \left \left(t-t_{{\mathrm{delay}}}\right) \right }{{\mathrm{{\tau}}}_{{\mathrm{act}}}}{\mathrm{,}}\end{equation*}\end{document} where I 0 is the initial amplitude of the current after the voltage step, I ∞ is the steady state current amplitude, t is the time after the voltage step, and t delay is the delay. The H + current amplitude is ( I 0 − I ∞ ). No other time-dependent conductances were observed consistently under the ionic conditions employed. Tail current time constants, τ tail , were fitted to a single exponential ( ): 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I \left \left(t\right) \right =I_{0}{\mathrm{exp}}\frac{-t}{{\mathrm{{\tau}}}_{tail}}+I_{{\mathrm{{\infty}}}}{\mathrm{,}}\end{equation*}\end{document} where I 0 is the amplitude of the decaying part of the tail current. Data are presented as mean ± SD or SEM, as indicated. Significance of differences between groups was calculated by two-tailed student's t test. The inhibition of H + currents by external ZnCl 2 is illustrated in Fig. 1 . The H + current elicited by a pulse to +10 mV is reduced in a concentration-dependent manner by ZnCl 2 . The rate the current turns on during a depolarizing voltage pulse is slower, as seen more clearly in Fig. 1 B, where the currents are scaled to the same value at the end of the pulse. Another effect (explored below) is to shift the voltage dependence of H + channel gating to more positive voltages. To some extent, the reduced H + current amplitude and slower activation can be attributed to this voltage shift. One implication is that any attempt to quantitate the apparent “block” of H + currents by ZnCl 2 by measuring the current at the end of a pulse will be arbitrary because the result depends strongly on the length of the pulse and the voltage selected for the measurement. The apparent extent of block at the end of the pulses in Fig. 1 would be greatly reduced if longer test pulses were applied and especially if a more positive test potential were selected. If ZnCl 2 binds with rapid kinetics to a site in the H + channel within the membrane electrical field, this should manifest itself in the instantaneous current–voltage relationship. The control instantaneous current–voltage (I-V) relationship in Fig. 2 A (•) exhibits moderate outward rectification, consistent with previous studies . The instantaneous I-V relationship in the presence of 10 μM ZnCl 2 (▪) is also plotted. The currents are reduced even though the prepulse was 40 mV more positive. After both sets of currents are scaled to match at +100 mV , the currents superimpose, indicating that there is no rapid voltage-dependent block. In some experiments with CdCl 2 , there was a suggestion that the inward currents were reduced preferentially, but this effect was too small to be sure of, even with data spanning 200 mV. Thus, metals have negligible effects on the instantaneous I-V relation of H + channels. The effects of ZnCl 2 and other metals might reflect voltage-independent interaction of the metal with the channel or nearby membrane. By binding to or screening negative charges near the external side of the H + channel, metals could bias the membrane potential sensed by the channel's voltage sensor . In the simplest scenario, the voltage-dependent properties of the channel will simply shift along the voltage axis. Fig. 3 A illustrates proton chord conductance ( g H )–V relationships in one cell in the absence (dashed lines) or presence of 100 μM ZnCl 2 (⋄), 10 mM NiCl 2 (▵), or several concentrations of CdCl 2 (solid symbols). When shifted along the voltage axis, the g H -V relationships appear quite similar , consistent with this mechanism. These metals may reduce the limiting g H ( g H,max ) slightly, although for the data shown here this effect was smaller than the variability in the control measurements. At higher metal concentrations, some reduction in g H,max usually became evident, but was difficult to measure accurately. In Fig. 3 C, the g H -V relationships are plotted on linear axes, scaled to the same g H,max to illustrate their similar shape and slope. The predominant effect is a simple voltage shift. Even though there is no rapid voltage-dependent block , the apparent voltage shift might conceivably reflect a slow block/unblock process. If we estimate the steady state voltage dependence of this apparent ZnCl 2 block in the usual manner by plotting the ratio I H (ZnCl 2 )/ I H (control), the apparent block is quite steep. Fig. 3 D shows the ratios for the same experiment as in other parts of this figure. These curves have similar slopes: a simple Boltzmann fit gives slope factors 8–13 mV. However, if the actual effect is a simple voltage shift of the g H -V relationship, then the apparent steepness of the “voltage-dependent block” will be identical to the steepness of the Boltzmann relationship in the absence of Zn 2+ . This being the case, the data in Fig. 3 D strongly suggest that metals shift the voltage sensed by the channel rather than binding to the channel in a voltage-dependent manner. A prominent effect of ZnCl 2 is to slow the activation of H + currents. We quantified this effect by fitting the turn-on of current during depolarizing pulses to a single exponential, after a delay. This procedure provides a reasonable fit under most conditions. In the presence of ZnCl 2 , both the delay and τ act were increased by roughly the same factor. We focussed mainly on metal effects on τ act , which are illustrated in Fig. 4 for the same cell shown in Fig. 3 . Because the τ act -V relationship is nearly exponential (linear on semi-log axes), it is not possible to distinguish whether τ act is slowed or its voltage dependence is shifted, or both. In the simplest case of a Huxley-Frankenhaeuser-Hodgkin voltage shift, all kinetic parameters should be shifted equally along the voltage axis. To explore the extent to which this model might apply, the τ act data in Fig. 4 B were “corrected” by the voltage shift determined for the g H -V relationship . To a rough approximation, the τ act effect in CdCl 2 and NiCl 2 appears to be explainable by this simple voltage shift. Closer examination of Fig. 4 B and other data (not shown) at high CdCl 2 concentrations indicates that CdCl 2 slows activation somewhat more than is accounted for by the shift of the g H -V relationship, consistent with a previous study of CdCl 2 on H + currents . In contrast, ZnCl 2 slows channel opening dramatically, and far beyond its shift of the g H -V relationship. The effects of ZnCl 2 are dominated by an interaction with the H + channel that results in τ act slowing, beyond a simple voltage shift of all parameters. The tail current decay seemed faster in the presence of external ZnCl 2 or CdCl 2 . However, attempts to evaluate metal effects on H + channel closing were hampered by the tendency of metals to reduce H + currents and by the weak voltage dependence of the closing rate . The latter property (τ tail changes e-fold in ∼50 mV) means that a 35-mV shift of the τ tail -V relationship would change τ tail at a given voltage by a factor of only two. Examination of data on ZnCl 2 and CdCl 2 in a number of cells under different conditions gave the impression that the τ tail -V relationship may have been shifted in the positive direction at most by roughly the amount that the g H -V relationship was shifted, but little effect was seen in some experiments. Fig. 5 illustrates the effects of ZnCl 2 on H + currents at three pH o . ZnCl 2 reduces the H + current at each voltage, slows activation, and shifts the voltage dependence of activation to more positive voltages. At each pH o , the effects are similar, but the concentration of ZnCl 2 required to produce these effects is much greater at low pH o . In this sense, lowering pH o decreases the efficacy of ZnCl 2 . To quantitate the effects of ZnCl 2 , we measured τ act and calculated the ratio of τ act in the presence of ZnCl 2 to that in its absence in the same cell at the same voltage. In most cells, this ratio was the same at all voltages, thus the effect of ZnCl 2 is a uniform voltage-independent slowing. Average ratios at several pH o are plotted in Fig. 6 and can be thought of as reflecting the “apparent potency” of ZnCl 2 at various pH o . The concentration required to slow τ act twofold is (μM) 0.22 at pH o 8, 0.46 at pH o 7, 5.4 at pH o 6, 89 at pH o 5.5, and 1,000 at pH o 5. The apparent potency of ZnCl 2 (estimated for a fourfold slowing of τ act where the curves are parallel) decreased only 2.3-fold between pH o 8 and 7, 10-fold between pH o 7 and 6, and 103-fold between pH o 6 and 5. Most of the buffers used bind Zn 2+ detectably ( Table ). In Fig. 6 B, the data from Fig. 6 A are replotted after correcting the metal concentrations for binding by buffer. The correction factors are given in the legend. The main effect is to reduce the shift in apparent potency between pH o 7 and 8. After correction, the concentration required to slow τ act twofold is (μM) 0.22 at pH o 8, 0.27 at pH o 7, 4.3 at pH o 6, 80 at pH o 5.5, and 1,000 at pH o 5. The apparent potency of ZnCl 2 (again estimated for a fourfold slowing of τ act where the curves are parallel) decreased 1.3-fold between pH o 8 and 7, 14-fold between pH o 7 and 6, and 129-fold between pH o 6 and 5. Measurements made in the same external solutions with different pipette pH gave no indication that pH i affects the interaction between externally applied ZnCl 2 and τ act . As illustrated in Fig. 6 , there was no obvious difference in the effects of ZnCl 2 at constant pH o in cells studied with pH i 5.5 (solid symbols and continuous lines) or at pH i 6.5 (open symbols and dashed lines). This result is consistent with externally applied ZnCl 2 exerting its effect at the external side of the membrane. Besides slowing activation, metals also shift channel opening to more positive voltages. This voltage shift was estimated from graphs of the g H -V relationships in the absence or presence of metal and is plotted in Fig. 7 . This parameter was somewhat arbitrary and less well defined than τ act , because it required extrapolating the fitted time course of H + current and measuring V rev in each solution (whenever pH o was changed). Nevertheless, the pH o sensitivity of the g H -V relationship to ZnCl 2 (solid symbols) qualitatively resembles that of τ act . In fact, the interaction between ZnCl 2 and pH o manifested in the g H -V relationship appears to be somewhat stronger than that for the τ act -V relationship. The concentration of ZnCl 2 required to produce a 20-mV depolarizing shift of the g H -V relationship was 0.13 μM at pH o 8.0, 0.77 μM at pH o 7.0, 54 μM at pH o 6.0, 470 μM at pH o 5.5, and 12.4 mM (by extrapolation) at pH o 5.0. The apparent potency of ZnCl 2 thus decreased sixfold between pH o 8 and 7, 70-fold between pH o 7 and 6, and 230-fold between pH o 6 and 5. The larger difference between the effective potency of ZnCl 2 between pH 7 and pH o 8 requires a higher pK a for the steady state conductance measurement than for the kinetic τ act measurement (see discussion ). Effects of internally applied ZnCl 2 were studied in the whole-cell configuration and in inside-out patches. Fig. 8 illustrates families of H + currents in cells studied at pH 6.5//6.5 without (A) and with (B) 2.5 mM ZnCl 2 added to the pipette solution. The H + currents appear generally similar, although closer inspection reveals that the tail currents decayed more slowly in the cell with internal ZnCl 2 . The pipette solution contained 1 mM EGTA and BisTris buffer (which will bind ∼90% of the Zn 2+ under these conditions, Table ), so the addition of 2.5 mM ZnCl 2 results in a free [Zn 2+ ] ∼170 μM. Fig. 8 C illustrates that addition of the pH 6.5 ZnCl 2 containing pipette solution to the bath dramatically reduced the H + current at +50 mV. This result makes it clear that ZnCl 2 applied externally is much more effective than when applied internally. Several cells were studied with 2.5 mM ZnCl 2 in the pipette at pH i 7.5. HEPES buffer does not bind ZnCl 2 detectably ( Table ), hence the free [ZnCl 2 ] was ∼1.5 mM. In these cells, the H + currents also appeared normal (data not shown). Our impression was that there was nothing unusual in the behavior of the g H in these experiments. H + currents were studied after allowing at least 5–10 min equilibration of the ZnCl 2 -containing pipette solution. The amplitude of I H did not change consistently during the experiment. The mean I H normalized to the input capacity was reduced significantly ( P < 0.05) at +80 and +100 mV in cells studied with 2.5 mM ZnCl 2 in the pipette, on average after 29 min in whole-cell configuration. Internal ZnCl 2 at high concentrations reduces I H , but this effect is not very pronounced. Fig. 9 illustrates mean τ act values in cells studied at pH 6.5//6.5 with (▪) and without (□) 2.5 mM ZnCl 2 in the pipette solution. No difference in the kinetics of H + current activation was detected. However, channel closing was significantly slower in cells studied with internal ZnCl 2 . Fig. 9 shows mean values of τ tail in cells studied with internal ZnCl 2 (•) and in control cells (○). The deactivation rate on average was 3.1-fold slower with internal ZnCl 2 (measured between −50 and +10 mV). In three cells studied with 2.5 mM CdCl 2 added to the pipette solutions, the average slowing of τ tail was 1.8-fold at 10 voltages from −80 to +20 mV ( P < 0.05 at each voltage) (not shown). Applied internally, ZnCl 2 thus slows closing without affecting activation. In contrast, externally applied ZnCl 2 slowed activation and, if anything, accelerated deactivation. Clearly the internal and external sites of action of ZnCl 2 are functionally quite different. A concern during these experiments was the extent to which ZnCl 2 in the pipette solution actually diffused into the cell. ZnCl 2 diffusion into the cell will be slowed by binding to cytoplasmic proteins, acting as fixed buffers. That measurable effects on τ tail were seen is evidence that the ZnCl 2 diffused into the cells to a significant extent. The mobility of ZnCl 2 is not unusually small . V rev values were consistent with the applied ΔpH (pH 6.5//6.5, Nernst potential = 0 mV), suggesting that buffer from the pipette solution diffused into the cell. In 11 cells studied with 2.5 mM ZnCl 2 in the pipette, V rev averaged −1.3 ± 2.4 mV (mean ± SEM). To confirm that ZnCl 2 entered the cell, we used TPEN, a membrane-permeant metal chelator with a high affinity for Zn 2+ . Shortly after addition of 250 μM TPEN to cells studied with ZnCl 2 -containing pipette solutions, the tail current kinetics became more rapid. On average, the ratio of τ tail before/after TPEN was 1.65 ± 0.32 (mean ± SD, n = 7), measured at pH 6.5//6.5 at −20 or −40 mV. This is in qualitative agreement with the threefold slowing of τ tail observed in the groups of cells studied with or without internal ZnCl 2 . Addition of TPEN to three cells studied with metal-free pipette solutions did not affect τ tail detectably. Inside-out patches were studied at pH o 7.5 or 6.5 (pipette pH) and pH i 6.5 (bath pH). Addition of 2.5 mM ZnCl 2 to the bath (∼170 μM free Zn 2+ ) reduced the H + current amplitude . This effect of ZnCl 2 was reversible upon washout . The reduction of H + currents was similar to that observed in whole-cells dialyzed with ZnCl 2 containing pipette solutions , suggesting that similar concentrations were reached in the whole-cell experiments. There was no clear shift of the voltage dependence of gating. If anything, there was sometimes a small shift to more negative voltages. A small hyperpolarizing shift might be explainable by the slight lowering of pH after addition of ZnCl 2 to the solution (0.023 U calculated, 0.05 U measured), due to displacement of protons from buffer. In some inside-out patches, the H + currents decreased progressively and gradually after addition of ZnCl 2 . Spontaneous rundown may account for this largely irreversible loss of H + current. In summary, the inside-out patch data support the conclusion that effects of internally applied ZnCl 2 differ qualitatively as well as quantitatively from those of externally applied ZnCl 2 . Internal application of high concentrations of ZnCl 2 produces only modest effects. Although we intended to study ZnCl 2 as a prototype for the effects of all polyvalent cations on H + currents, there were subtle differences between the effects of ZnCl 2 and CdCl 2 . Both metals slowed activation and shifted the g H -V relationship to more positive voltages. However, to a first approximation, the effects of CdCl 2 could be viewed as a simple shift of all parameters to more positive voltages. “Correction” of the τ act –V relationships in Fig. 4 according to the shift observed in the g H -V relationship in Fig. 3 normalized the data for CdCl 2 , but not for ZnCl 2 . In other words, ZnCl 2 has a pronounced additional slowing effect. Examination of τ act data in individual cells revealed that ZnCl 2 effects could usually be approximated as uniform slowing at all voltages, whereas the relative slowing by CdCl 2 sometimes decreased for larger depolarizations. As a result of this subtle difference, there was not a unique “slowing factor” for CdCl 2 , and we did not try to plot CdCl 2 data in Fig. 6 . The slowing of τ act by CdCl 2 was strongly pH o dependent, however. To a first approximation, the pH o dependence of CdCl 2 was similar to that of ZnCl 2 . Another difference between metals is evident in Fig. 7 . The shifts of the g H -V relationships indicate that CdCl 2 is ∼30× less potent at either pH o 7 or 6. In contrast, the slowing of τ act by 100 μM ZnCl 2 exceeded that by 10 mM CdCl 2 over most voltages , and thus there is a >100-fold difference in potency for this effect. Thus the relative potency of the two metals for slowing τ act and shifting the g H -V relationship differs. Perhaps distinct binding sites are involved in these effects, and the relative affinities of the metals for the sites differ. ZnCl 2 has a high affinity for the site that slows activation, whereas most of the effects of CdCl 2 are consistent with binding to a “nonspecific” site that shifts the apparent membrane potential sensed by the H + channel. Polyvalent cations and protons have similar effects on many ion channels , perhaps because they bind to similar sites. It has been postulated that the function of voltage-gated proton channels requires at least two distinct types of protonation sites. Conduction likely occurs via a hydrogen-bonded chain , in which case the entryway of the “channel” is a protonation site, where H + must bind to initiate permeation. The second type of protonation sites are allosteric regulatory sites that govern the strong ΔpH (ΔpH = pH gradient = pH o − pH i ) dependence of gating; i.e., the 40 mV/U shift in the voltage-activation curve with changes in either pH o or pH i . The ΔpH-dependent gating mechanism was explained economically by assuming identical internally and externally accessible regulatory protonation sites . More recent evidence suggests the internal and external sites have distinct chemical properties . Given this background, H + channels might be affected by Zn 2+ in several ways. (a) Binding at or near the entry to the channel should inhibit H + current by preventing H + binding or reducing the local [H + ] available to enter the channel. The attenuation of g H,max at high metal concentrations might reflect local H + depletion by this mechanism. However, most of the effects of metals are not compatible with metal binding to and occluding the channel entry. (b) Binding to a site remote from the entry but which is sensed by the voltage sensor of the channel could shift the position of the voltage dependency of gating, the most simple mechanism of which would result in all voltage-dependent parameters shifting equally along the voltage axis. This mechanism is consistent with most of the effects of Cd 2+ and Ni 2+ . (c) Binding near the allosteric sites on either side of the membrane might reduce the local [H + ] electrostatically, and hence affect gating in the same manner as an increase in pH. The effects of metals are in the wrong direction for this mechanism to apply. (d) Finally, metal binding to the allosteric protonation sites might have a similar effect on gating as protonation of these sites, and might thus mimic the effects of low pH near the site. The details of the effects in this case are hard to predict, because due to differences in binding kinetics and steric factors, Zn 2+ can hardly be expected to mimic a single H + , or even two H + . Nevertheless, most of the effects of Zn 2+ can be explained by assuming that it binds to the same regulatory sites as protons, and has the same effects as protons in our model . Thus, Zn 2+ (or H + ) binding at the external site prevents channel opening, and Zn 2+ (or H + ) binding at the internal site prevents channel closing. Although polyvalent cation effects on H + currents in various cells are quite similar, some authors have characterized these effects as modification of the voltage dependence of gating , whereas others describe the effects as voltage-dependent block . These views are not equivalent. The voltage dependence of ionic block is generally assumed to arise from the entry of the blocker into the channel pore partway across the membrane potential field, where it gets stuck, physically occluding the pore. Interpreted in terms of voltage-dependent block, metal binding affinity depends strongly on voltage , whereas effects due to binding to a modulatory site can be explained with a fixed K M . Because the instantaneous I-V relation was simply scaled down by ZnCl 2 with no detectable voltage dependence , we ruled out the possibility of rapidly reversible binding of Zn 2+ to a site within the membrane potential field. Even though there is no rapidly reversible block, the more obvious effects of ZnCl 2 could be due to a slow time-dependent block/unblock. Five arguments oppose the idea that the slow activation of H + current in the presence of Zn 2+ reflects voltage-dependent unbinding of Zn 2+ from the channel. (a) If τ act in the presence of metals (several seconds) reflects the unblock rate, then block must have very slow kinetics. If we assume that pK M = 6.5 and that the binding rate of Zn 2+ is 3 × 10 7 M −1 s −1 , a characteristic rate of complex formation between Zn 2+ and proteins , then the unbinding rate is 9.5 s −1 . Thus, Zn 2+ probably binds and unbinds in a fraction of a second. If the kinetics are rapid, effects should have been manifested in the instantaneous I-V relation. (b) In normal drug-receptor reactions, the unblock rate is independent of concentration. However, increasing the concentration of ZnCl 2 slowed H + current activation progressively. There was no indication that two populations of gating behavior resulted, as would be predicted if ZnCl 2 modified a fraction of channels that then opened slowly, with the remaining channels opening at the normal rate. A single exponential (after a delay) continued to fit the data at all [ZnCl 2 ]. Thus it appears that ZnCl 2 binds and unbinds the channel repeatedly during a single pulse, with the slowing effect related to the fraction of time ZnCl 2 is bound to the channel. (c) The steady state voltage dependence of this apparent Zn 2+ block, defined as the ratio I H (Zn 2+ )/ I H (control), is quite steep: a simple Boltzmann fit gives slope factors 8–13 mV . In terms of traditional voltage-dependent block mechanisms , if z is the charge on the blocking ion and δ is the fraction of the membrane potential sensed by the ion at the block site, then z δ ≥ 2.0, which implies that Zn 2+ , Cd 2+ , and Ni 2+ traverse ≥100% of the membrane field to reach the block site. Several examples of δ > 1.0 for ionic blockade exist in the K + channel literature and are traditionally explained by interaction between permeant ions in a multiply occupied channel . Because it is unlikely for a hydrogen-bonded–chain conduction mechanism to support multiple protons simultaneously, especially at physiological pH , explaining the high z δ observed for divalent cation “blockade” is problematic. (d) If ZnCl 2 simply shifted the g H -V relationship along the voltage axis, then the apparent steepness of the block, defined as the ratio I H (Zn 2+ )/ I H (control), will be precisely identical to the steepness of the g H -V relationship in the absence of Zn 2+ . The slopes of the fractional block curves, 8–13 mV , and control g H -V relationships, 8–10 mV , are the same, consistent with a simple voltage shift. (e) Finally, any part of the H + channel conductance pathway comprised of hydrogen-bonded chain would not allow Zn 2+ passage; thus the possibility for voltage-dependent block by Zn 2+ could exist only in an aqueous vestibule. We conclude that polyvalent cations do not exert their effects by entering into the pore, but instead bind to sites on the channel that are accessible to the solution and outside of the membrane potential field. Binding must be specific because different divalent cations have very different concentration dependencies. For example, effects of micromolar concentrations of Zn 2+ are seen in the presence of millimolar [Ca 2+ ] o or [Mg 2+ ] o . Byerly et al. 1984 proposed that divalent cations bind specifically to a site on the external side of H + channels, based on their observation that the g H -V relationship was shifted less by CdCl 2 than was the τ act –V (actually t 1/2 -V) relationship. For epithelial H + channels, the disparity in effects on channel opening compared with the g H -V relationship was even more pronounced for ZnCl 2 than for CdCl 2 . A similar sequence of voltage shifts by ZnCl 2 (τ act -V > g H -V > τ tail -V) was seen for K + channels . Their interpretation was that Zn 2+ binds to a site on the external side of the channel that is exposed to the bath solution only when the channel is closed. This is precisely the nature of the proposed external modulatory protonation site in our model of H + channel gating . The site must be deprotonated before the channel can open, and during the opening process the site “disappears” and the same site (or a distinct site) appears on the internal side of the membrane. It was necessary to assume that the protonation sites were not accessible to both sides of the membrane at the same time to account for the ΔpH dependence of gating. In solution, zinc exists as several chemical species, whose relative proportions depend strongly on pH. One plausible explanation for the increased apparent potency of ZnCl 2 at higher pH is that ZnOH + , rather than the divalent form, is the species acting on H + channels. As pH is increased, the proportion of ZnCl 2 in monohydroxide form, ZnOH + , increases 10-fold/U, up to ∼pH 8 . The absolute concentration of ZnOH + is a small fraction of the total, and >90% of ZnCl 2 is divalent at pH < 8.0, hence [Zn 2+ ] remains relatively constant . Spalding et al. 1990 concluded that ZnOH + was the active form for Cl − currents in muscle. The “consensus” potency sequence for inhibiting H + currents by divalent metal cations, Cu < Zn > Ni > Cd > Co > Mn > Ba, Ca, Mg < 0 , is intriguingly similar to the tendency of these cations to hydrolyze (indicated by their pK a ): Cu (8.0) > Co (8.9), Zn (9.0) > Ni, Cd (9.9) > Mn (10.6) > Mg (11.4) > Ca (12.6) > Ba (13.4). The pK a sequence generally reveals the proportion of total metal in monohydroxide form at a given pH. If the monohydroxide form were active, then the apparent potency for all of these metals should increase ∼10-fold per unit increase in pH, and the pH dependence should saturate around the pK a . We show here that the apparent potency of both ZnCl 2 and CdCl 2 increase at higher pH, and the pH o dependence saturates for ZnCl 2 . However, saturation occurs at a pH that is too low by ∼1 U, and the change at low pH o is at least 100-fold/U , both inconsistent with the hypothesis that the monohydroxide form is active. If ZnOH + were the active form, an additional mechanism (e.g., competition with H + ) would be required to enhance the pH sensitivity of its effects. Several polyhydroxide forms of zinc (with net negative charge) also are increasingly represented at high pH, but we rule these out as candidates for interaction with the H + channel because (a) it seems unlikely that anions and protons would compete for the same sites, and (b) the fraction of all of these forms combined at pH 5 is <10 −12 of the total ZnCl 2 present . In conclusion, the most probable form of ZnCl 2 active on H + channels is the divalent form. The explores the predictions of several possible mechanisms of competition between Zn 2+ and H + for hypothetical binding sites on H + channels. The pH o dependence of Zn 2+ effects on τ act are reasonably compatible with Models 4, 5, or 6 . These models assume that the external Zn 2+ receptor on proton channels is formed by multiple protonation sites that are accessible to the external solution and that coordinate the binding of a single Zn 2+ . If H + and Zn 2+ compete directly for the same site(s), then at least two to three protonation sites must exist. If H + and Zn 2+ bind to different sites, then there must be substantial interaction between them, and the range of the pH dependence indicates that protonation of one site lowers the affinity of the remaining site(s) for Zn 2+ by a factor ∼30. Similar binding constants reproduce the pH dependence of Zn 2+ effects using any of several models: pK M is 6.5 and pK a is 6.2–6.6 and is somewhat model dependent. To apply the model equations in the to real data, it is necessary to define the effect that metal binding has on channel behavior. By making one assumption, we can define the entire body of τ act data in Fig. 6 . We assume that when Zn 2+ is bound to its receptor on the H + channel, the channel cannot open. For the simplest case of a two-state channel, with Fig. 1 : where α is the opening rate and β is the closing rate, the time constant is (α + β) −1 . Because the slowing of τ act was voltage independent, β evidently is negligibly small in the voltage range measured, hence τ act ≈ α −1 . The opening rate will be slowed by the factor (1 − P Zn ), where P Zn is the probability that the receptor is occupied by Zn 2+ . Thus the observed time constant will be ∼[α (1 − P Zn )] −1 , and the ratio of τ act in the presence of ZnCl 2 to that in its absence will be simply (1 − P Zn ) −1 . Given these assumptions, τ act is slowed by a factor of 2.0 at the K M of Zn 2+ . In Fig. 11 , the τ act data from Fig. 6 are replotted with smooth curves superimposed that assume Model 6, in which the Zn 2+ receptor is formed by three protonation sites and protonation of each site reduces the affinity of the receptor for Zn 2+ by a factor a . We selected Model 6 because it comes closest to embodying the pH dependence observed. The entire set of theoretical curves is determined by the assumption that a Zn 2+ -bound channel cannot open, and by pK M = 6.5, pK a = 6.3, and cooperativity factor a = 0.03. Setting a to 0.03 produces an ∼100-fold change in apparent Zn 2+ potency between pH 5 and 6 that resembles the τ act data. With a = 0.01, the shift was too large, and at a = 0.1 the shift was too small. We could not collect data at pH 4, which might have revealed whether the saturation of the effect at very low pH predicted by this model ( ) occurs. The agreement is generally excellent, although the slope of the data appears shallower than that defined by the theory. Expressed in terms of Zn 2+ activity rather than concentration, calculated with to the Davies equation at the ionic strength of all solutions used, ∼0.13 M, the pK M is 7.0. The effects of ZnCl 2 on the g H -V relationship were modeled in a similar manner as the effects on τ act . Fig. 12 shows the predictions of Model 6 , with parameters adjusted to match the ZnCl 2 data from Fig. 7 , which are superimposed. Several differences in the g H -V data compared with the τ act data required different parameters. It was necessary to assume that more than two protonation sites were involved because the shift between pH o 6 and 5 was ∼230, whereas 100 is the maximum possible shift for a two-site model. pK M in all equations is defined by the metal concentration–response relationship at high pH o , where binding is unaffected by pH. pK a is somewhat model dependent, and is defined by the pH o at which the interaction between metal and H + saturates. For a given model, this is set by the size of the shift in the high pH o region. Thus, in Fig. 12 , pK a is 7.0 because this produces a sixfold shift between pH o 8 and 7, as observed in the data. Finally, it was necessary to assume some interaction between binding sites, because pure competition in a three-site model predicts too large a shift at low pH o . The value of the interaction factor, a , is established by the entire shift over the pH o range from 8 to 5. This shift was 10 5 in the data, and a = 0.01 matched this value. Setting a = 0.02 reduced the range to 3 × 10 4 and at a = 0 (pure competition) the range was too large, 4 × 10 5 . The mechanistic interpretation is that protonation of one of the sites lowers the affinity of the Zn 2+ receptor 100-fold. Assuming the same model, the affinity of Cd 2+ for the external metal receptor is lower than that of Zn 2+ by ∼2 U (roughly pK M 6). A depolarizing shift in the g H -V relationship at low P open can be approximated as a reduction of P open by a constant fraction. Because the slope factor of the g H -V relationship fit by a simple Boltzmann function is ∼10 mV , an e-fold reduction in P open should produce a 10-mV depolarizing shift. In Fig. 12 , we use Model 6 under the assumption that the channel cannot open when Zn 2+ is bound. The voltage shift is given by ln (1 − P Zn ) −1 × 10 mV. The g H -V data were best described by pK M = 8.0, pK a 7.0, and a = 0.01. Expressed in terms of Zn 2+ activity rather than concentration, pK M is 8.5. The g H -V data were best described by pK M = 8.0, pK a 7.0, and a = 0.01 , whereas the optimal values for the same model of τ act data were pK M = 6.5, pK a 6.3, and a = 0.03 . The different parameter values describing the interactions observed for the g H -V relationship and τ act may reflect that the former is a steady state parameter and the latter a kinetic one. Alternatively, distinct metal binding sites may be involved in slowing τ act and shifting the g H -V relationship, as suggested by the greater relative potency of ZnCl 2 (compared with other metals) for the slowing effect. If so, the “nonspecific” site at which polyvalent metals shift all voltage-dependent parameters simply has a higher pK a than the site that regulates τ act . Another possibility is that the Zn 2+ receptor has a higher affinity for both protons and Zn 2+ when the channel is open. This idea is incompatible with the external Zn 2+ receptor being comprised of the regulatory protonation sites that govern gating in our model, because these sites become inaccessible to the external solution when the channel is open . We saw no evidence of decay of H + current in the presence of metals, which would be expected if metals bound (with resolvable kinetics) preferentially to open channels. A final possibility is that fitting the τ act and g H -V data simply provides two ways to estimate the binding parameters of the Zn 2+ receptor. The modeling exercise indicates that protons and polyvalent cations (at least Zn 2+ and Cd 2+ ) compete for a common site at the external surface of the H + channel. Furthermore, the metal receptor can also bind two or more H + , and protonation inhibits metal binding. The best fit was achieved with the assumption that three protonation sites coordinate one Zn 2+ . We propose that metal binds to the same external modulatory sites at which extracellular protons regulate the gating of H + channels. Extracellular metals and protons have qualitatively similar effects on channel gating. Both slow activation (increase τ act ), shift the voltage-activation curve ( g H -V relation) to more positive potentials, and have relatively small effects on the channel closing rate. In our model , the ΔpH dependence of gating arises from the requirement that three externally accessible sites must be deprotonated for the channel to open. The agreement between the numbers of protonation sites involved in gating and Zn 2+ binding may be serendipitous, but lends support to both models. Although metals produce dramatic effects on H + currents at quite low concentrations for external application, internally applied ZnCl 2 or CdCl 2 also altered H + currents. Deactivation was slowed with no effect on τ act , and H + current amplitude was reduced. Because internally applied ZnCl 2 had relatively weak effects, we could not study them in as much detail, and could not determine whether Zn 2+ and H + compete for internal sites. Nevertheless, in our model , the first step in channel closing is deprotonation at internally accessible sites. Thus, the slowing of deactivation by internal ZnCl 2 with no effect on the opening rate is qualitatively consistent with the idea that Zn 2+ binds to the same internal protonation sites that help regulate gating. Because the effects of internal and external addition were qualitatively different, distinct metal binding sites must exist at the inner and outer surfaces of the channel. In contrast, ZnCl 2 has similar effects whether applied externally or internally to K + channels, on ionic currents as well as on gating currents , leading Spires and Begenisich 1995 to conclude that Zn 2+ can reach its binding site in the channel from either side of the membrane. The dissimilarity of effects on H + channels leads us to conclude that there are distinct internal and external sites and, furthermore, that negligible quantities of these metals applied internally reach the external binding site. Not only is there no evidence that ZnCl 2 can cross the membrane, the lack of effects of pH i on external ZnCl 2 effects indicates that intracellular protons do not affect the local pH near the external Zn 2+ receptor. Thus, H + channels are less promiscuous than are K + channels. In turn, this conclusion supports the concept that voltage-gated proton channels are not water-filled pores that might conduct detectable amounts of Zn 2+ or Cd 2+ (or perhaps ZnOH + or CdOH + ), but instead comprise a hydrogen-bonded chain. The extremely high selectivity of H + channels is another argument for this conduction mechanism . To account for the ΔpH dependence of the voltage activation curve of the H + channel, we originally proposed identical external and internal protonation sites with pK a 8.5 . Deprotonation at the external site was the first step in channel opening, and deprotonation at the internal site was the first step in channel closing. That deuterium substitution slowed activation threefold with negligible effects on closing suggested that the external and internal sites were chemically different, with the external site likely composed of His, Lys, or Tyr residues and the internal site possibly a sulfhydryl group, presumably Cys . A classical example of His forming a Zn-binding site is carbonic anhydrase, in which zinc is coordinated between three His residues (and one OH − ) to form the catalytic site of this metalloenzyme . Chelators can remove this zinc and it can then be replaced by various other ligands, which bind with a relative potency Hg >> Cu > Zn > Cd, Ni > Co > Mn , a sequence similar to that reported for metal inhibition of H + currents (see above). The data presented here are compatible with the idea that the Zn 2+ binding site is the same site at which external protons regulate gating. In this regard, it is intriguing that pH o acting on extracellular His residues shifts the voltage dependence of the gating of a plant K + channel . Protonation of this stomatal guard cell channel shifts the activation curve toward more positive voltages, just as the g H -V relation of voltage-gated proton channels is shifted to the right at lower pH o . Because this K + channel is activated by hyperpolarization, it is activated by low pH o , whereas the voltage-gated proton channel is activated by depolarization and thus is inhibited by low pH o . Zinc binding sites have been created in α-hemolysin channels by introducing His residues . The external Zn 2+ receptor on H + channels binds Zn 2+ with a substantially higher affinity, pK M ∼6.5, than the “normal” association constant for 1:1 binding of Zn(II) to His, pK M 2.5 . The higher affinity is compatible with our conclusion that multiple His (or other ionizable groups) coordinate the binding of a single Zn 2+ . The Zn 2+ dissociation constant for carbonic anhydrase, in which three His coordinate one Zn 2+ , is 4 pM . The typical pK a of His in proteins ranges from 6.4 to 7.2 , encompassing the pK a values derived from most of the models tested here. Thus, many types of evidence point to His as a likely candidate for forming the external Zn 2+ receptor. Henderson 1998 demonstrated recently that mutation of any of three His residues to Leu in a putative transmembrane domain abolished the H + conductance associated with NADPH oxidase in neutrophils. This intriguing result may support the identity of the external modulatory site as His. However, epithelial and phagocyte H + channels differ significantly , and some phagocyte H + channels have a higher sensitivity to ZnCl 2 . Furthermore, the role of one or more of the His might be in conduction, forming part of the hydrogen-bonded chain, rather than in regulation of gating. The much weaker deuterium isotope effect on H + channel closing than on opening led to the suggestion of Cys as a candidate for the internal regulatory protonation site because sulfhydryl groups typically have smaller pK a shifts in D 2 O . The weak effects of internal ZnCl 2 reported here, however, must be reconciled with the typically high affinity binding of Zn 2+ to Cys . If Cys does help form the internal site, steric constraints may allow proton or deuteron binding, but disfavor close approach by Zn 2+ . Because they are more sensitive to polyvalent metal cations than most other ion channels, H + channels would be among the first to register effects of metal poisoning. Human plasma zinc levels are maintained at ∼15 μM , most of which is complexed with plasma proteins or phosphates. However, the g H -V relationship is quite sensitive to Zn 2+ at physiological pH with a distinct shift at <0.1 μM ZnCl 2 . ZnCl 2 or CdCl 2 suppress the respiratory burst—the release of bactericidal reactive oxygen species—in human neutrophils in vitro, presumably by inhibiting H + currents . Inhalation of zinc oxide produces metal fume fever, apparently by elevating plasma interleukin-6 (a pyrogen produced by granulocytes) levels . Voltage-gated proton channels in alveolar epithelium may contribute to CO 2 extrusion by the lung . Volume regulation of alveolar epithelial cells is inhibited by high concentrations of ZnCl 2 . This evidence is circumstantial, but worth worrying about. | Study | biomedical | en | 0.999996 |
10579710 | What happened to the third region accumulating stain in the holo complex, which is seen only rarely in the core complex? One explanation is that stain was accumulating in the tetratricopeptide repeat (TPR) proteins, Tom70 and Tom20. The single TPR unit of Tom20 is required for interaction with Tom70, which has seven TPR units . Based on the 3D structure of the TPR domains of protein phosphatase 5, Das et al. 1998 demonstrated that eight TPR units would form a tall helix surrounding a central channel. Intriguingly, the radial dimensions of the TPR twist are similar to the size of the third region accumulating stain in the TOM holo complex. Another possibility is that the presence of the Tom20 and Tom70 subunits in the holo complex induces a docking or rearrangement of the Tom40 subunits to form three channels in the plane of what would be the mitochondrial membrane. Beyond this study of a mitochondrial outer membrane complex, EM has been applied to structural analysis of a host of biologically important complexes. Structures of ribosomes, nuclear pore complexes, kinesin–microtubule complexes, and clathrin coats are being solved to ever higher resolution. This limited list of some of the beautiful structures becoming available already highlights the impact that EM is having for molecular cell biologists. In addition to revealing the structure of the eukaryotic ribosome, images have been reconstructed that depict how the ribosome docks with the Sec61 complex . The 3D structure of the complex provides a framework in which to understand the multiple and sequential interactions made by a nascent polypeptide as it leaves a cytoplasmic ribosome to be translocated through an intracellular membrane. Like the structure of the TOM complex, the ribosome–Sec61 structure prompts new experiments and new ideas on the function and mechanics of the system. The key to solving large macromolecular structures that are not amenable to 3D crystallization has been the development of cryo-EM and image processing techniques. Methodologies exist for analysis of two-dimensional crystals or sheets (e.g., many membrane proteins); helical structures (e.g., microtubules and flagella); icosahedral structures (e.g., viruses); and other objects with inherent symmetries. Single-particle image processing techniques applicable to objects lacking symmetries were pioneered by Frank and coworkers , and recently, such methods have begun to reveal features of secondary structure at resolutions approaching 10 Å. Images recorded from specimens that assume either random or preferred orientations within a layer of vitreous ice on a specimen grid can be aligned, classified, and combined mathematically to give a 3D structure of the macromolecule in its native conformation, without the distortions induced by air-drying or heavy metal-contrasting. For images obtained by cryo-EM, optical densities within the reconstructed volume are proportional to actual mass density in the original object. Indeed, the next logical step for the solubilized TOM complex will be cryoimaging and 3D reconstruction by single-particle methods. Even where the structural detail obtained from EM can be pushed to resolutions near 10 Å, individual protein subunits usually cannot be visualized within a complex. One approach towards pinpointing individual subunits is to label the isolated complexes with mAbs or with large peptide tags, or to use natural ligands to lock the complex in two discrete conformations, and use difference mapping of the two forms of the complex. An elegant example of what can be done is seen in recent work with gold cluster labeling of precise segments of kinesin motors docked onto microtubule lattices, providing much needed detail in the conformational changes that occur during the ATP-driven conformational changes in the motor protein, kinesin (R. Milligan, personal communication). Ultimately, the atomic-resolution structures obtained with X-ray diffraction from crystallized protein and ribonucleoprotein domains can be mapped onto either a moderate resolution EM map or a high, but not quite atomic, resolution map from X-ray crystallography. The lower resolution map provides a framework or context in which the fine structure of the domain can be interpreted. This divide and conquer strategy is being applied to solve the structure of the prokaryotic ribosome, where individual subunits and subcomplexes have been crystallized, and their structures mathematically fitted or docked into 11.5 Å electron microscopic maps (Gabashvili, I.S., R.K. Agrawal, C.M.T. Spahn, R.A. Grassucci, J. Frank, and P. Penczek, manuscript in preparation) or 5.0–7.8 Å resolution crystallographic maps of the intact ribosome or ribosomal subunits . Now that several laboratories are progressing towards atomic resolution domain structures for components of the TOM complex, the same strategy could well provide a means to visualize the intact protein translocation machinery in three dimensions, with the phospholipid bilayer stripped away. | Study | biomedical | en | 0.999997 |
10579712 | An 8.6-kb fragment containing exons 2–12 was isolated from a 129/Sv mouse liver genomic library. To produce the targeting vector, an NsiI-BamHI fragment was deleted, removing exons 8 to part of 11, and replaced with the Pgkneo neomycin resistance cassette in reverse orientation to the Lmna gene. The targeting vector was linearized with ClaI and electroporated into W9.5 ES cells. Clones were picked, expanded, and screened for homologous recombinants, after digestion with EcoRI, using a probe to exon 2. Two clones were injected into C57Bl/6 blastocysts, and chimeras were derived and bred to produce germline offspring as described . Homozygotes and heterozygotes were distinguished from wild-type sibs by EcoRI digestion of tail DNA. A human lamin A cDNA was subcloned into the pTracer-CMV vector (Invitrogen Corp.). The linearized vector was transfected into lamin A/C −/− mouse embryonic fibroblasts (MEFs). Stable clones were selected using Zeocin, according to the manufacturer's instructions and the clones pooled. Subsequent analysis showed that the cells in the pool were heterogeneous with regard to lamin A expression. The antibody to mouse emerin was provided by Dr. Glenn Morris (NE Wales Institute, UK). Dr. Erich Nigg (University of Geneva) provided the antibody against lamin B2. Dr. Larry Gerace (Scripps Institute) provided both antibodies against LAP2 and an antibody against lamin B. The antibodies SA1 (specific for Nup153) and XB10 (against the lamin A/C central rod domain) have been described previously . Dr. Frank McKeon provided the 1E4 antibody to the amino terminal region of lamins A and C. The rhodamine and FITC-conjugated secondary antibodies were from Tago, Inc. Tissues and cells were fixed in 10% buffered formalin or 3% paraformaldehyde in PBS respectively. Tissues for immunohistochemical analysis were embedded in OCT and snap frozen. Tissues for histological analysis were dehydrated, cleared, embedded in paraffin, sectioned at 6 microns, and stained in hematoxylin/eosin. MEFs were processed for immunofluorescence microscopy as previously described . The in situ processing of cultured cells was modified from a previously described method . Epon sections (50 nm), stained with uranyl acetate and lead citrate, were examined and photographed at 75 kV. To mutate the mouse lamin A/C ( Lmna ) gene, we deleted a region extending from exon 8 to the middle of exon 11. This removed 114 codons as well as the 3′ untranslated sequence, including the polyadenylation signal of lamin C, whereas 152 codons were eliminated from the lamin A coding region. The deletion was introduced by homologous recombination into ES cells and six homologous recombinant clones were identified . After blastocyst injection of two clones, chimeric offspring were derived with subsequent germline transmission of the mutated allele. Heterozygotes were intercrossed to derive viable homozygous offspring. At birth, these were indistinguishable from their heterozygous or wild-type siblings. Loss of lamin A/C expression was determined by Western blot analysis of cell extracts, nuclei, or NEs prepared from the livers of weaned offspring or from embryonic fibroblasts (MEFs) established from day 13 embryos. Whereas NEs could be readily prepared from the livers of mice heterozygous and wild-type for the Lmna gene, they could not be isolated in an intact form from the lamin null mice. Instead, the −/− NEs fragmented, leading to poor recovery. Using two independent antibodies against epitopes within either the first 250 amino acids or the central rod domain of lamin A/C (1E4 and XB10, respectively) , proteins of the appropriate molecular masses were undetectable in any of the samples prepared from tissues homozygous for the mutated gene. Neither was there any evidence for truncated forms of the two proteins. Lamin B1 levels in all of the cell types remained unaltered . Northern blot analysis of poly(A) + mRNA from −/− livers revealed two faster migrating faint bands at levels 10-fold lower than the wild-type lamin A and C transcripts . Taken together, these results indicate that the partial deletion of the Lmna gene resulted in the absence of both full-length transcripts and stable lamin A/C proteins. These results were confirmed by immunocytochemistry since neither of the two anti–lamin A/C antibodies labeled the NEs of the Lmna −/− MEFs . In contrast, lamins B1 and B2 were readily detected . These labeling experiments also revealed dramatic changes in nuclear morphology. Whereas nuclei of wild-type MEFs are roughly circular or slightly ovoid, those of Lmna −/− MEFs are often highly elongated or irregular and exhibit loss of B-type lamins from one pole . Although the nuclei remain intact, the overall impression is of large-scale herniation of the nuclear membranes. This abnormality, evident in >80% of the −/− MEF nuclei, is apparent in Fig. 2 e, where Nomarski and lamin B immunofluorescence images are superimposed. Similar results were obtained with antibodies against both the inner nuclear membrane protein LAP2 and the nuclear pore complex (NPC) protein Nup153 . The latter also revealed a slight degree of NPC clustering within some −/− nuclear envelopes. An intermediate phenotype was observed for +/− MEFs with frequent elongation of nuclei, but largely normal distribution of nuclear envelope proteins (data not shown). Ultrastructural examination of −/− MEFs and hepatocytes revealed a thinning or loss of heterochromatin at discrete regions of the nuclear face of the INM. These segments of the nuclear envelope, which also lack morphologically identifiable NPCs, likely correspond to the herniations observed in the light microscope . Thus, the integrity of NEs in −/− cells is profoundly compromised and shows conclusively for the first time that A-type nuclear lamins are essential for the maintenance of normal nuclear architecture. This complements a previous study in Drosophila showing that a B-type lamin is also essential for nuclear integrity . At birth, Lmna null mice were indistinguishable from their heterozygous or wild-type sibs. However, within 2–3 wk a reduction in their growth rate was noted and by ∼4 wk, despite normal tooth development and the continued ability to eat, their growth had ceased. At this time their mean body weight was roughly 50% that of their wild-type or heterozygous littermates. At ∼3–4 wk, the homozygotes began to display an abnormal gait with a stiff walking posture, characterized by splayed hind legs and an inability to hang onto structures with their forepaws. Their overall posture became progressively more hunched, exhibiting distinct scoliosis/kyphosis. By the eighth week, all of the homozygotes had died. The heterozygotes are also apparently normal and have not exhibited any premature mortality when compared with their wild-type sibs. Histological analysis of homozygotes revealed that the majority of their internal organs were normal, although some thymic atrophy and a reduction in spleen size was evident as was an absence of white fat, possibly as a secondary consequence of physiological stress. The wild-type and heterozygous mice exhibited no overt abnormalities. Examination of the musculature of the homozygote nulls revealed that the perivertebral muscles and those surrounding the femur (rectus femoris and semimembranous) were dystrophic. The involvement of individual fibers within each muscle was not uniform with those proximal to the bone being the most severely impaired. Many of these were atrophic with others exhibiting signs of degeneration with hyalin or flocculent cytoplasm. The dystrophic muscle fibers also exhibited variations in diameter, plus an increase in the number of nuclei with some being centrally located within the fibers . Muscles of the head, tongue, and diaphragm were largely unaffected. In the heart, the ventricular muscle was most severely compromised although myocyte involvement was nonuniform. Some were of normal size, but had degenerated with condensed or flocculent eosinophilic or vacuolated cytoplasm . These were often associated with patchy mineralization. Other cardiac myocytes were clearly atrophic. The Lmna null mice did not exhibit elevated serum creatine kinase levels, a feature associated with some but not all forms of muscular dystrophy (data not shown) . Overall, the Lmna −/− mice develop a cardiac and skeletal myopathy bearing a striking resemblance to human EDMD . EDMD was originally described as an X-linked disorder that mapped to the gene for emerin, a ubiquitous INM protein . While the function of emerin is unknown, the finding that A-type lamin defects result in an EDMD-like disorder suggests these proteins might functionally interact. Consistent with this, a recent study by Bonne et al. 1999 revealed that in humans an autosomal variant of EDMD maps to the lamin A/C ( LMNA ) gene. Since the localization of integral proteins to the INM is thought to involve a process of selective retention , it is possible that A-type lamins serve to immobilize emerin within the INM. Therefore, we examined the distribution of emerin in wild-type and lamin A/C −/− MEFs. While the overall levels of emerin in these cells is identical , immunofluorescence microscopy revealed dramatic differences in emerin subcellular localization. In wild-type cells, emerin is concentrated within the nuclear envelope . In the −/− cells, there is partial loss of NE-associated emerin with a more general cytoplasmic distribution identical to that observed for resident ER proteins . Presumably, emerin is no longer retained within the INM, and is free to access the outer nuclear membrane and ER via the membrane continuities at the periphery of NPCs . MEFs from heterozygous embryos exhibited intermediate levels of cytoplasmic emerin . These results indicate that at least in MEFs, correct emerin localization is contingent upon A-type lamin expression. In contrast, LAP2, which shares some sequence homology with emerin but which is known to interact with both chromatin and B-type lamins , still concentrates at the nuclear periphery in −/− cells . To further address the role of A-type lamins in emerin localization, we transfected a human lamin A cDNA into Lmna null MEFs and followed the distribution of emerin by double label immunofluorescence microscopy. Fig. 5e and Fig. f , shows a representative example of two −/− MEFs, only one of which is expressing the heterologous LMNA . While emerin in the nonexpressing cell is distributed between both the NE and the cytoplasm, it is restricted almost exclusively to the nuclear periphery in the cell expressing human lamin A . These data strongly support a role for A-type lamins in the correct localization of emerin. Immunocytochemical examination of emerin in several tissues from the lamin A/C null mice revealed that its normal nuclear envelope localization was affected in a cell type–specific manner. In tongue epithelium, NE-associated emerin was completely lost , whereas in skeletal muscle NE-associated emerin was still detectable, albeit at a greatly reduced level . In the longitudinal fibers of the ventricular cardiac muscle, the loss of A-type lamin expression had more heterogeneous effects with ∼20% of nuclei showing a marked polar clustering of emerin staining that was particularly apparent in those cells sectioned along their longitudinal axis. In heterozygous mice, <0.1% of the ventricular nuclei exhibited emerin polarization and none were detected in wild-type mice. These observations reveal a clear role for A-type lamins in emerin localization. However, NE-associated emerin is not uniformly lost in tissues from Lmna null mice, suggesting the involvement of additional factors. This is supported by our findings that in P19 embryonal carcinoma (EC) cells, which do not express A-type lamins at any detectable level , emerin is, nevertheless, associated with the nuclear periphery . This implies that there is at least one other nuclear envelope component with which emerin must be able to interact, and that the level of expression of this component is likely to be cell type–specific. EDMD arises from either loss of emerin protein or mutations resulting in its subcellular mislocalization , with the rarer autosomally inherited form being linked to dominant acting mutations in the LMNA gene . From this study, it was not clear whether the NE was disrupted and emerin localization affected. However, this is a possibility as some lamin A-type mutants do act in a dominant form, since their injection or transfection into cells causes severe perturbations in nuclear envelope structure and organization . Here, we have shown that an EDMD-like phenotype, albeit more severe and earlier acting, arises in mice after ablation of A-type lamin expression. This is associated with the mislocalization of emerin to varying degrees in different cell types. Previous observations on the fragility of purified NEs from P19 EC cells, which don't express A-type lamins, indicated a role for the lamins in NE integrity . In this study, we observed a similar, pronounced effect on hepatocyte NEs lacking A-type lamins with gross structural changes to the NEs and emerin distribution occurring in a variety of −/− cells and tissues. These observations indicate that in addition to the lamins, emerin itself may represent another important determinant of interphase NE organization, possibly as a link between the INM and lamina. However, the relative contributions of the A-type lamins, and emerin either alone or in combination, to NE integrity remain unclear at the present. From our studies, loss of the A-type lamins clearly affects NE integrity. Whether it does so alone or whether the phenotype is exacerbated by loss/mislocalization of emerin, possibly in conjunction with some other tissue-specific and/or developmentally regulated factors will have to await the derivation of emerin-deficient mice. Intermediate filament proteins have long been recognized in providing structural integrity to a variety of cells and tissues. In particular, mutations in members of the cytokeratin gene family are associated with numerous pathological conditions, most notably those of epidermal and neurological origin . Here, we have shown cells lacking A-type lamins, members of the nuclear branch of the intermediate filament protein family, also develop a characteristic pathology. However, while these proteins are expressed in the majority of adult tissues, this pathology is manifest primarily in the form of muscular dystrophy. A possible explanation for this is that in the absence of functional lamins and/or emerin, muscle nuclei may be unable to withstand the mechanical stresses to which they are continually subjected. In other tissues, which do not experience the same contractile forces, while nuclear envelope integrity may still be compromised, the effects may be less deleterious. The derivation of mice with tissue-specific deficiencies in lamina-associated proteins will now allow us to test this suggestion. | Study | biomedical | en | 0.999996 |
10579713 | The open reading frame of human ECT2 was introduced into the mammalian expression vector pCEV32F3 to express a FLAG–ECT2 fusion protein. COS-7 cells were plated in 100-mm dishes and transfected with 10 μg of plasmid DNA with Lipofectamine (GIBCO BRL). Transfected cells were cultured for 48 h, harvested, and lysed in 1 ml of cold lysis buffer (25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 20 μg/ml each of leupeptin and aprotinin, and 100 μg/ml PMSF). FLAG–ECT2 fusion proteins were immunoprecipitated from the lysates with 9 μg/ml of anti-FLAG mAb (Sigma Chemical Co.) and protein G–Sepharose beads (Amersham Pharmacia Biotech). Guanine nucleotide exchange assays were performed essentially as described using these immunoprecipitates. In brief, 3 μg of GDP-loaded recombinant GTPases were incubated with 5 μM [ 35 S]GTPγS (0.25 mCi mmol −1 ) and 10 μl of protein G beads suspension in 190 μl of exchange buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2, 0.5 mM DTT, 100 mM NaCl, 0.5 mg/ml BSA). At the indicated times, 30 μl of the reaction was removed and passed through nitrocellulose filters. Filters were washed and then counted in a liquid scintillation counter. For phosphatase treatment, immunoprecipitates were incubated with recombinant VHR protein or λ phosphatase (New England Biolabs) for 30 min at 30°C, and then used for exchange assays. GST–Ect2 fusion protein was expressed in Escherichia coli and used for immunizing rabbits. The NH 2 -terminal half (ECT2-N; amino acids 1–421) or Dbl homology domain (DH; amino acids 414–639) of human ECT2 was expressed as fusion proteins with thioredoxine and oligohistidine using the pET-32 vector (Novagen). Anti-ECT2 antibodies were prepared by passing antiserum through affinity columns coupled with the corresponding human ECT2 proteins using AminoLink Plus Immobilization Kit (Pierce). Anti–ECT2-DH recognized a single ECT2 protein of ∼100 kD. Anti–ECT2-N also recognized the endogenous ECT2 protein, although some additional bands were weakly detected. HeLa cells were grown in DMEM (GIBCO BRL) supplemented with 10% FCS in 7% CO 2 at 37°C. Cells were synchronized at the G1/S boundary by a thymidine-aphidicolin double block . In brief, cells were incubated with 2 mM thymidine for 14 h, released from arrest, and then arrested at G1/S again with aphidicolin (1 μg/ml) (Sigma Chemical Co.). Cells were then placed under normal growth conditions (time 0). After 6 h, nocodazole (final concentration 100 ng/ml) was added to arrest the cells at prometaphase. Mitotic cells were collected by mechanical shake-off and replated in normal growth medium. Following trypsinization, samples were analyzed by flow cytometry using FACS ® II instrument (Becton Dickinson). For phosphatase treatment, G1 or M phase arrested cells were lysed in TNE buffer (10 mM Tris-HCl, pH 7.8, 1% NP-40, 0.15 M NaCl, 1 mM EDTA pH 7.0, 10 μg/ml aprotinin) and incubated with 1 μg/ml of recombinant VHR protein for 30 min at 30°C. Samples were resolved by 6% SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and analyzed by immunoblotting using affinity-purified anti-ECT2 antibodies. To determine the localization of endogenous ECT2, HeLa cells were grown on coverslips, fixed for 10 min with 3.7% formaldehyde in PBS, permeabilized for 10 min with 0.2% Triton X-100 in PBS, and then incubated with 1% BSA in PBS for 10 min. For costaining of endogenous ECT2 and tubulin, coverslips were incubated with affinity-purified anti–ECT2-DH antibody and anti–β-tubulin (TUB2.1; Sigma Chemical Co.) mAbs for 1 h at 4°C. After washing, samples were incubated with FITC-conjugated anti–rabbit IgG and Cy3-conjugated anti–mouse IgG (Jackson ImmunoResearch, Inc.) for 1 h at room temperature. To express green fluorescent protein (GFP)-fused ECT2 proteins in U2OS cells, ECT2-N, ECT2-C, and ECT2-F were introduced into pEGFP-C1 (Clontech). After transfection, cells were fixed and proteins visualized by green fluorescence using a Zeiss Axiovert microscope equipped with a Photometrics digital camera and IPLab software (Signal Analytics Co.). HeLa cells were grown on poly- l -lysine–coated glass coverslips for 18–24 h until the cultures reached ∼50% confluency. The coverslips were transferred into fresh medium containing 10 mM Hepes, pH 7.6, before injection. Antibodies were dialyzed and concentrated to 1 mg/ml using a Centricon-30 concentrator (Amicon). Rabbit IgG (Sigma Chemical Co.) was used as a control. Microinjection into the cytoplasm was performed using Eppendorf semiautomatic microinjection apparatus. Immediately after injection, coverslips were washed with PBS and incubated in growth medium for the indicated time period. To examine the function of ECT2 as a Rho GEF, we expressed ECT2 as a FLAG epitope-tagged protein in COS cells immunoprecipitated with anti-FLAG mAb, and used immunoprecipitated protein for exchange assays. Full-length ECT2 efficiently stimulated nucleotide exchange on three representative members of the Rho GTPases, RhoA, Rac1, and Cdc42 in vitro, whereas similar immunoprecipitates from vector transfectants showed no significant activity . In contrast to the activity on Rho GTPases, ECT2 did not exhibit significant exchange activity on two Ras family GTPases, H-Ras and Rap1A (data not shown). The predicted ECT2 protein also contains a novel domain homologous to yeast S phase cyclin Clb6 , and two tandem repeats of the recently identified BRCA1 COOH-terminal repeat (BRCT) motif that is widespread throughout checkpoint and repair proteins . Since these structural similarities suggest the possibility that ECT2 is involved in cell cycle control, we examined in detail the expression pattern of ECT2 during the cell cycle. HeLa cells were synchronized at the G1/S boundary, and the expression of ECT2 protein was examined by immunoblotting after the release from G1 arrest. Although we did not observe significant changes in the overall expression level of ECT2 protein during progression of the cell cycle, a shift of the mobility of ECT2 was detected by anti-ECT2 antibody . A slowly migrating protein appeared at 9 h as cells entered G2 phase. At 14 h, as cells entered M phase, the slowly migrating band predominated. The fraction of slowly migrating protein gradually decreased as cells entered G1 phase . To determine whether the shift of mobility of ECT2 was due to protein phosphorylation, we treated M phase–arrested cells with the tyrosine/serine/threonine phosphatase VHR. No mobility shift was detected in M phase cells after VHR treatment . Therefore, ECT2 appeared to be phosphorylated specifically at G2 phase, and this state was manifested through M phase. To further examine the effect of phosphorylation on the exchange activity of ECT2, we treated FLAG–ECT2 immunoprecipitates with various concentrations of VHR. As shown in the right panel of Fig. 1 b, the exchange activity of ECT2 was inhibited by VHR in a dose-dependent manner, and the incubation for 1 h with 0.5 μg/ml of VHR decreased the activity of FLAG–ECT2 to background levels. Inhibition of exchange activity was also observed by treatment of FLAG–ECT2 with λ phosphatase, and this inhibitory effect was strongly prevented by a phosphatase inhibitor vanadate (data not shown). These results suggest that phosphorylation is required for the exchange activity of ECT2 in vitro. Next, we examined the subcellular localization of endogenous ECT2 during cell cycle progression by immunofluorescent analysis of HeLa cells. Interestingly, in interphase cells ECT2 was located predominantly in the nucleus, where no expression of Rho family proteins has been reported . After nuclear membrane breakdown in prometaphase, ECT2 spread into the cytoplasm . During metaphase, ECT2 accumulated in the regions where the mitotic spindle is present . Costaining of ECT2 and tubulin generated the yellow colocalization signal. As cells entered anaphase, ECT2 appeared to concentrate in the central region of the spindle . In late anaphase and telophase, ECT2 was mainly located in the midzone, where the cleavage furrow is formed . During cytokinesis, ECT2 accumulated at the midbody, a region in the middle of the bridge that connects the two daughter cells . When the cells exited mitosis and the nuclear envelope reassembled, ECT2 translocated to the nucleus again (data not shown). To locate the region of ECT2 responsible for midbody localization, we transfected cells with plasmids containing the NH 2 -terminal half (ECT2-N), the COOH-terminal half (ECT2-C), or full-length (ECT2-F) ECT2 as GFP fusion proteins. GFP proteins were visualized 72 h after transfection . Like endogenous ECT2, GFP-tagged ECT2-F was detected in the midbody of dividing cells. Although ECT2-N localized in the cytoplasm of interphase cells (data not shown), it accumulated in the midbody of dividing cells. In contrast, ECT2-C was detected in the cytoplasm of mitotic cells. These observations indicate that the NH 2 -terminal half of ECT2 is required for midbody localization. The nuclear localization of ECT2-F in interphase cells may be attributed to nuclear localization signals located in the central region of ECT2 . The dynamic change of subcellular localization and G2/M-specific modification of ECT2 suggest a possible role of ECT2 in cell division. If ECT2 controls cell division through the regulation of Rho GTPases, expression of ECT2 mutants containing the domain that is required for localization, but lacking the catalytic domain, may induce a dominant negative phenotype. Therefore, we closely examined the morphology of cells expressing ECT2-N. Surprisingly, ∼60% of cells expressing ECT2-N became multinucleated, with most cells containing two nuclei 72 h after transfection . Division of ECT2-N–expressing cells appeared to proceed to a stage just before the separation of two daughter cells with single nuclei , suggesting that a very last step of cytokinesis was inhibited by ECT2-N expression. In contrast, cells expressing ECT2-F, ECT2-C, or GFP expression vector alone did not exhibit such phenotypes. Since most of the cells expressing ECT2-N underwent normal nuclear division , it is unlikely that these multinucleated cells were generated as a consequence of aberrant nuclear division. These results suggest that ECT2-N can function as a dominant negative mutant to inhibit cytokinesis. To further examine the involvement of ECT2 in cytokinesis, we inhibited ECT2 function by microinjection of affinity-purified anti-ECT2 antibodies into asynchronously growing HeLa cells . Whereas cells injected with control IgG divided normally, ∼60% of cells injected with anti–ECT2-DH, which recognizes the catalytic domain of ECT2, became larger with double nuclei 24 h after injection. Since 25% of the injected cells had not divided at this stage, ∼80% of the cells were multinucleated upon completion of mitosis. After 48 h, 29% of anti–ECT2-DH–injected cells contained three or four nuclei. The presence of cells with more than two nuclei may indicate inhibition of multiple rounds of cytokinesis with anti–ECT2-DH. In contrast, very few control IgG-injected cells were multinucleated. To rule out the possibility that anti-ECT2 cross-reacted with other molecules that regulate cytokinesis, we prepared a second affinity-purified antibody that specifically recognizes the NH 2 -terminal domain of ECT2. Microinjection of this antibody (anti–ECT2-N) also strongly inhibited cytokinesis . These results strongly suggest that ECT2 plays a critical role in cytokinesis. The results from this study provide details about the biochemical and biological functions of the ECT2 protooncogene product. We detected a mobility shift of ECT2 at G2/M phases. Treatment of the ECT2 immunoprecipitates with VHR dual specificity phosphatase resulted in shift back of the band to the normal size. This strongly suggests that ECT2 is modified by phosphorylation. We found that dephosphorylation of ECT2 by phosphatases reduced the exchange activity in vitro in a dose-dependent manner. Therefore, ECT2 appears to be activated by phosphorylation, which occurs specifically in M phase. Since we could not detect phosphorylated ECT2 with antiphosphotyrosine antibodies (our unpublished results), ECT2 may be phosphorylated by serine/threonine kinases. Candidate protein kinases that can activate ECT2 may include Cdc2-regulated kinases and Cdc2 itself. The predicted amino acid sequence of ECT2 contains two consensus Cdc2 phosphorylation sites (amino acids 327–330 and 814–817). Further studies on ECT2 phosphorylation, including determination of phosphorylation sites and generation of phosphorylation-deficient mutants, will clarify the activation mechanisms of ECT2. Rho proteins were found to regulate actin polymerization in studies on cytoskeletal organization of fibroblasts . During cytokinesis in animal cells, an actin-based contractile ring divides the cell into two daughter cells. We found that ECT2 catalyzes nucleotide exchange on Rho proteins and this activity appears to be dependent on G2/M phase-specific phosphorylation. ECT2 was released to the cytoplasm after the nuclear membrane breakdown and then accumulated in the midbody during cytokinesis. Since ECT2-N localized in the midbody, but ECT2-C did not, ECT2-N appears to determine the midbody localization of ECT2. We found that expression of ECT2-N strongly inhibited cytokinesis. Because ECT2-N localizes in the midbody but cannot catalyze nucleotide exchange, it appears to function as a dominant negative mutant. Moreover, microinjection of affinity-purified anti-ECT2 antibody also inhibited cytokinesis. Therefore, ECT2 might play an important role in cytokinesis. The nuclear localization of ECT2 may suggest another role of ECT2 in mitosis. This possibility is under investigation. However, the inhibition of cytokinesis with ECT2-N or anti-ECT2 might not be attributed to the inhibition of mitosis, because ECT2-N, which lacks nuclear localization signals, was expressed in the cytoplasm and anti-ECT2 was injected into the cytoplasm. The presence of cell cycle regulator-related domains and G2/M phase-specific phosphorylation of ECT2 suggest the regulation of ECT2 activity by cell cycle machinery. Therefore, ECT2 might be an important molecule linking cell cycle machinery to Rho signaling pathways involved in the regulation of cytokinesis. In Xenopus , multiple Rho proteins are involved in cytokinesis: whereas local activation of Rho is important for proper constriction of the contractile furrow, Cdc42 plays a role in furrow ingression . In Drosophila , cytokinesis is developmentally controlled during embryogenesis and is omitted during the initial nuclear division cycles. It starts in somatic cells with mitosis 14, but is blocked with mutations in the pebble gene . Recently, it was reported that pebble encodes an ECT2-related protein, which can interact with Drosophila Rho1 GTPase , suggesting that ECT2 is also involved in cytokinesis in Drosophila . In mammalian cells, Rho GTPases also regulate cortical actin remodeling . Inhibition of RhoA in normal rat kidney cells revealed that Rho regulates the completion of cytokinesis possibly through modulating the mechanical strength of the cortex . Although the role of Rho in division of mammalian cells is not well-documented, recently it was reported that two Rho effectors, Rho-associated kinase and Citron kinase, play a role in cytokinesis . Further studies on ECT2, Rho GTPases, and their effectors should clarify the signal transduction pathways involved in the control of cell division. | Study | biomedical | en | 0.999996 |
10579714 | N1E-115 neuroblastoma cells (obtained from Dr. M. Niremberg, National Institutes of Health, Bethesda, MD) were grown and loaded with the calcium indicator fura-2 as previously described . Images were acquired using a high speed Photometrics CCD camera (frame transfer EEV37 chip) on an inverted Zeiss microscope, controlled by Inovision software on a Silicon Graphics workstation. Cells were bathed in pH-controlled EBSS and maintained at 37°C by means of a plexiglass housing unit built onto the microscope stage. Ratio pairs of 340- and 380-nm excitation were collected for some experiments, with emission passed through a >530-nm filter. Using this acquisition protocol, ratio pairs could be collected approximately once a second. For faster imaging, a single wavelength (380 nm) excitation protocol was used, in which images could be obtained every 65 ms. Relative changes in calcium concentrations were calculated from the fluorescence changes as in Fink et al. 1999 . All image analysis was performed on Silicon Graphics workstations using the programs Ratiotool (Inovision Corp.), used for calcium analysis of dual wavelength experiments, and ISee (Inovision Corp.), on which graphical analysis programs were constructed to do the single wavelength analysis. For quantitative InsP 3 uncaging, we used NPE-InsP 3 and calcium green-1 (CG-1)–loaded cells on an inverted NORAN confocal microscope with UV flashes from a xenon arc lamp attached to a computer-controlled shutter. The procedure is detailed in Fink et al. 1999 . All experiments were performed at 37°C. InsP 3 mass was calculated using a competitive radioligand binding assay with canine cerebellar microsomes, 10 nM 3 H-InsP 3 , and various concentrations of nonradioactive InsP 3 . Effects of inhibitors were calculated by Scatchard analysis. Intracellular volume measurements were performed by determining excluded volumes with 3 H-insulin (Pharmacia Biotech, Inc.) relative to total volume determined with 14 C-urea. Full details of the model and citations to the origins of all the parameters are available at http://www.nrcam.uchc.edu . Throughout, corrections to the two-dimensional simulations are made to account for true three-dimensional surface to volume ratios by approximating the soma as a hemisphere and the neurite as a hemicylinder . Methods for antibody staining, analysis of relative intracellular antigen densities, and determination of cytosolic indicator concentrations are detailed in Fink et al. 1998 . Glass pipettes were filled with a solution containing 500 nM bradykinin (BK) and 5 μM fura-2 (pentapotassium salt) in EBSS buffered to pH 7.4 with 10 mM Hepes. Cells were loaded with fura-2 and mounted on a specially constructed chamber that provides a steady flow of EBSS buffer solution across the cell. The loaded pipette was directed with a micromanipulator such that the ejected solution was oriented in the flow so that only a chosen segment of the cell (soma, neurite, or distal neurite) was briefly exposed to BK. The ejected puff was visualized by the fluorescence of the coejected fura-2. Ratio images were then collected and calibrated as described. When stimulated with a saturating concentration of BK, a nonapeptide neuromodulator, N1E-115 neuroblastomas show a highly reproducible calcium response . After a brief latency (mean ± SEM; 2.97 ± 0.23 s; n = 16), a calcium increase started in the neurite and propagated bidirectionally as a wave towards the soma and growth cone. The wave typically traversed the soma with an average velocity of 39.2 ± 3.7 μm/s ( n = 16), and peak calcium concentrations approximating 1 μM . The calcium wave propagated more quickly through the neurite although the amplitude of the calcium response was not significantly different than observed in the soma . [Ca 2+ ] cyt relaxation to basal levels occurred within 30 s of stimulation. Subsequent stimulations with BK did not evoke new calcium signals even after BK washout. This calcium response is known to be mediated by InsP 3 generation and subsequent release of calcium from ER stores. The amplitude of the calcium response is not dependent upon extracellular calcium, indicating that the elevation of [Ca 2+ ] cyt comes strictly from intracellular stores. In addition, all calcium efflux from the ER is through InsP 3 -sensitive channels, since this cell type lacks ryanodine receptors . To determine the calcium response to an InsP 3 signal with well-controlled spatial and temporal characteristics, caged NPE-InsP 3 was microinjected into the cell (along with CG-1 for calcium imaging) and photoreleased by exposing the entire cell to a brief flash of UV light. As shown in Fig. 1 b, calcium levels increased throughout the cell. However, the levels of calcium increase were substantially higher in the soma than in the neurite. This contrasts sharply with the responses to BK stimulation in Fig. 1 a, where the calcium signal has a uniform 1-μM amplitude throughout the cell. Further, to determine the dependence of the [Ca 2+ ] cyt signal on [InsP 3 ] cyt in each region of the cell, we performed quantitative InsP 3 uncaging experiments , followed by application of BK to the same cells. A dose-response relationship can be observed between calcium release and [InsP 3 ] cyt , which could be fit with the Hill equation . This is consistent with other measurements on the [InsP 3 ] cyt dependence of calcium release . Subsequent to the uncaging flashes, the BK-evoked calcium response in the soma and neurite was compared with the dose-response. Note that the BK-induced calcium signal in the soma corresponds to uncaged [InsP 3 ] of 2.1 ± 0.1 μM ( n = 11 cells). However, BK-induced [Ca 2+ ] cyt signals at the initiation point in the neurite could only be matched by uncaging InsP 3 in the range of 5–10 μM . This result is consistent with the lower amplitude of the calcium response to a uniform pulse of uncaged InsP 3 in the neurite compared with the soma , and indicates that the neurite requires three to four times as much InsP 3 as the soma to produce the calcium response evoked by the physiological stimulus of Fig. 1 a. Competitive radioligand binding techniques were employed to approximate the time course of the InsP 3 signal during the BK-stimulated calcium wave for a population of differentiated N1E-115 neuroblastoma cells . These values have been corrected for cellular volume and the presence of inhibitory factors in the cytosol. The basal level of InsP 3 was determined to be 0.16 μM, rising to a peak concentration of 2 μM within 10 s after stimulation with BK. InsP 3 returned to baseline after an additional 10 s. The amplitude of this InsP 3 signal is consistent with the quantitative uncaging measurements . The time course of [InsP 3 ] cyt parallels that of [Ca 2+ ] cyt , and is indicative of a rapid (half-life <10 s) degradation time for cytosolic InsP 3 , in agreement with previous estimates . To understand how the neurite produces a higher InsP 3 signal than the soma, and why it requires this higher concentration of InsP 3 to set off a calcium wave, we used a computational system for cell biological modeling to construct a model based on experimental data for geometrical, electrophysiological, and biochemical components of the system. In Fig. 3 a, the geometric distributions of critical receptors (BK, InsP 3 , and SERCA) are mapped onto a geometry based on the cell in Fig. 1 a. This information was compiled through analysis of confocal micrographs of immunofluorescence distribution, using the quantitative procedures developed by Fink et al. 1998 . The other inputs to the model comprised the individual biochemical and electrophysiological processes contributing to the BK-induced calcium wave. These included: flux of InsP 3 into the cytosol from the plasma membrane; rate of InsP 3 degradation; calcium uptake rate of SERCA pumps; [InsP 3 ] cyt and [Ca 2+ ] cyt binding to the InsP 3 -receptor and the consequent activation and inactivation of calcium efflux from the ER; calcium buffering in the cytosol by mobile and fixed buffers; and diffusion coefficients for InsP 3 , mobile buffers, and calcium. The results of the simulation can then be displayed as time-dependent maps of any of the variables. Fig. 3 b shows the results of the simulation for the response of InsP 3 and Ca 2+ to BK (first four columns). The first two columns represent the results for the average receptor distributions . The prediction for [Ca 2+ ] cyt can be directly compared with the experiment in Fig. 1 a. Consistent with experiment, the initial calcium increase is observed in the middle of the neurite after 2.2 s, spreading bidirectionally to the soma and growth cone, and reaching a peak [Ca 2+ ] cyt everywhere of ∼1 μM. It should be noted that the simulations in Fig. 3 b were carried out with the average receptor distributions, which clearly would not necessarily pertain to the particular cell in Fig. 1 a. Close to an exact match between this individual experiment and the simulation could be achieved if the receptor distributions in the model were permitted to vary within one SD from their means in the parameter space (data not shown). On the other hand, the columns, labeled uniform BKR, show the results of simulations where the BK receptor (BKR) density is set to a uniform distribution along the plasma membrane. The general features of the calcium wave remain unchanged, indicating that the surface receptor distribution is not a critical determining factor. Although an elegant indirect method has been described for monitoring intracellular InsP 3 , there is no available fluorescent indicator for [InsP 3 ]. So, the model can provide a unique view of the spatial and temporal distribution of this key metabolite. The calculated [InsP 3 ] cyt dynamics show a rapid buildup in the neurite to a peak of ∼10 μM, whereas [InsP 3 ] cyt in the soma increases more slowly, and to lower peak concentrations (∼3 μM). The production of InsP 3 is much faster than its diffusion throughout the intracellular volume and also outpaces the rate of degradation through putative cytosolic kinase and phosphatase based pathways. Therefore, because InsP 3 is produced from the plasma membrane, the cytosolic concentrations of InsP 3 will rise faster and with greater maximum amplitude in the neurite than in the soma. This is primarily because of the high surface to volume ratio of the neurite compared with the soma, with the higher surface density of BKR in the proximal neurite serving only to somewhat focus the site of initiation . When the [InsP 3 ] values obtained from our radioligand binding experiments are plotted against the simulated average [InsP 3 ] for the entire cell, an excellent match was obtained . Of course, because the soma contains the highest proportion of the cell volume, the average [InsP 3 ] at any time is only slightly higher than the [InsP 3 ] in the soma. Indeed, in addition to providing a picture of the spatiotemporal profile of [InsP 3 ] within the cell, our analysis allows us to extract rates for the stimulated flux of InsP 3 from the plasma membrane and degradation in the cytosol from the constraints imposed by the data of Fig. 2 d. We also modeled uniform InsP 3 uncaging throughout the intracellular volume. In agreement with the experiment , the simulation shows [Ca 2+ ] cyt to be significantly higher in the soma than in the neurite, and no calcium wave behavior is evident. The higher calcium levels in the soma result from its greater density of ER and InsP 3 R as established in our immunofluorescence analysis . This also explains why a higher level of InsP 3 is required in the neurite to produce a Ca 2+ signal comparable to that produced in the soma. Thus, the combination of experiment and modeling reveals the interplay of structural features which both produce and require a higher [InsP 3 ] cyt in the neurite after BK stimulation. Despite the higher amplitude of [InsP 3 ] cyt in the neurite, the amplitude of the calcium response remains relatively uniform throughout the cell because the calcium stores are biased toward a higher density in the soma. To further probe whether this amplified [InsP 3 ] in the neurite is necessary and sufficient for initiation and propagation of a calcium wave, we modeled the condition of a local BK stimulation in three distinct cell regions: soma, middle of the neurite, and growth cone (or distal neurite). The results of these simulations are shown in Fig. 4 a. When BK is added to only the soma, elevation of calcium levels occur after a long delay, no wave is generated, and peak [Ca 2+ ] cyt is low (∼500 nM). When BK is applied to the neurite, a calcium wave propagates in both directions; whereas [Ca 2+ ] cyt in the neurite is comparable to that produced by global BK application, [Ca 2+ ] cyt in the soma is lower (∼500 nM). Finally, when the application of BK is simulated for only the distal neurite, an immediate calcium increase is seen in that region. However, the wave fails to propagate far up the neurite, and [Ca 2+ ] cyt soon returns to baseline levels. To check these predictions, we performed a series of experiments in which BK was focally applied by pressure ejection to the cells . When BK was focally applied to the soma, a gradual increase of calcium was seen in the soma, which failed to propagate down the neurite as a wave (observed in 7/8 cells). When BK is focally applied to only the neurite, a calcium wave is typically initiated (9/13 cells), although [Ca 2+ ] cyt in the soma didn't reach the levels seen with global BK application. Finally, when BK is focally applied to only the most distal neurite (or growth cone), a local elevation of calcium was observed (7/11 cells), which failed to propagate as a calcium wave to the soma. Together, these experiments validate the predictions made by the simulations, and show that the morphologically enhanced InsP 3 signal in the neurite is necessary and sufficient for initiation and propagation of a calcium wave. This study illustrates how an interplay of structural, morphological, and geometric features can play a critical role in defining the spatiotemporal characteristics of intracellular signaling. Such considerations should be of even greater importance for InsP 3 -dependent signaling in primary neurons because of their even greater morphological complexity. For example, it has been previously reported that Purkinje neurons, cells with a particularly extensive dendritic arborization, require very high (>10 μM) concentrations of uncaged InsP 3 to elicit a calcium response . The very high surface to volume ratio that exists in the dendritic tree may underlie the ability of the Purkinje cell to deliver such high InsP 3 concentrations in response to metabotropic synaptic inputs. Of course, these ideas may be applicable beyond InsP 3 -dependent processes. For example, fine membranous structures, such as lamellipodia and filopodia, play a role in pathfinding and directed motility . The intensified signals that are possible in such confined intracellular spaces may represent a common mechanism for such specialized functions. Exploration of these ideas will be facilitated by studies that intimately combine quantitative image-based models with experiment. | Study | biomedical | en | 0.999997 |
10579715 | All insertion plasmids used in this study are derivatives of the previously described plasmid pINCO , which contains a DHFR-TS mutant, pyrimethamine-resistance gene, and a targeting sequence consisting of the distal part of TRAP lacking nucleotides (nt) 1–67 and 1.4 kb of downstream sequence. The 3′ end of TRAP bearing the T Δ L deletion was generated by PCR using 5′ primer P008 (5′ CGCGAAGCTTCTGAATGTTCTACTACATGTGACAATG 3′), which hybridizes from nt 736 of TRAP onwards, and 3′ primer P011 (5′ CGC TTAATTAA CGCTACTTCCTGCTATAAAATTATAACC 3′), which hybridizes at the 3′ end of TRAP and introduces a stop codon as well as a PacI restriction site (bolded). The resulting PCR product was then cloned into plasmid pCRScriptSK, yielding plasmid pΔL1. A linker encompassing the first 24 bp of the TRAP 3′ UTR, including the natural XbaI site located 18 bp from the stop codon, was then cloned downstream from the TRAP coding sequence in plasmid pΔL1 using PacI and EcoRI adaptors, creating plasmid pΔL2. Further 3′ UTR, borne by a XbaI–XmnI 1.4-kb fragment, was then cloned downstream from the linker in plasmid pΔL2 digested with XbaI and EcoRV, yielding plasmid pMutΔL. The HincII–AflII internal portion of plasmid pMutΔL, which extends from nt 1150 of TRAP to 0.6 kb 3′ to its stop codon, was then used to replace its wild-type counterpart in plasmid pINCO, giving rise to plasmid pTΔL. The 3′ end of TRAP bearing the T Δ S deletion was generated by PCR using 5′ primer P017 (5′ GAATGGAGTGAATGTTCTACTACATGTG 3′), which hybridized from nt 738 of TRAP onwards, and 3′ primer P012 (5′ CGC TTAATTAA CAACAATACCCTTTTCATCATCTGC 3′) that hybridizes at the 3′ end of TRAP and introduces a stop codon as well as a PacI restriction site (bolded). The resulting PCR product was then cloned into plasmid pCRScriptSK, yielding plasmid pΔS1. The 3′ UTR of TRAP , borne by the PacI-KpnI fragment of plasmid pMutΔL, was further cloned into plasmid pΔS1, yielding plasmid pMutΔS. The HincII-AflII internal portion of plasmid pMutΔS was then used to replace its wild-type counterpart in plasmid pINCO, giving rise to plasmid pTΔS. The exchanged fragments in plasmids pTΔS and pTΔL were sequenced and confirmed to differ from the corresponding wild-type fragment only by the desired mutation. The DNA encoding the cytoplasmic tail of MIC2 was amplified from the XhoI fragment of BAC G11-11 using 5′ primer P27 (5′ AAAA CTGCAG GATCCCCATCCGCGGAGATAG 3′) and 3′ primer P28 (5′ TGC TCTAGA TATATATGTTTATTAAAATTACTCCATCCACATATCACTATCG 3′), which contain a PstI and a XbaI site, respectively (bolded). The resulting PCR fragment was digested with PstI and XbaI and cloned into plasmid pMutΔS digested with the same enzymes, yielding plasmid pMut-MIC2. The AgeI-AflII internal portion of plasmid pMut-MIC2 was sequenced, confirmed to contain the expected sequence, and used to replace its wild-type counterpart in plasmid pINCO, giving rise to plasmid pTMIC. Both TRYP and ACID mutations were generated using 5′ primer P017 (5′ GAATGGAGTGAATGTTCTACTACATGTG 3′) and 3′ primer SK01 for the TRYP mutation (5′ TCA TCTAGA TATATATGTTTATTAAAATTA GCTAGC GTCATTATCTTCAGGTAATTTAAACT- GCTC 3′) or SK02 for the ACID mutation (5′ TCA TCTAGA TATATATGTTTATTAAAATTAGTTCCAGGCATT GCTAGC AGG- TAATTTAAACTGCTC 3′). Both 3′ primers contain an XbaI site, and a mutation-tagging NheI site (bolded). PCR fragments were cloned into plasmid pCRScript, excised as an AgeI-XbaI insert and cloned into plasmid pMutΔS digested with AgeI and XbaI. The AgeI-AflII fragments of the resulting plasmids that encompassed the mutations were sequenced, verified to differ from their wild-type counterpart only by the desired mutations, and used to replace the corresponding fragment in plasmid pINCO, yielding plasmids pTRYP and pACID. Parasite transformation and selection was performed as described . Anopheles stephensi mosquitoes were fed on infected young rats and sporozoites dissected out at days 14–18 postfeeding. Preparation of sporozoites from the various mosquito compartments was as described . For immunofluorescence assays, sporozoites were incubated in RPMI–3% BSA on ice for 3 h, pelleted, and resuspended in 0.5% BSA/PBS containing primary antibody at 1:50. After 30 min at 37°C, sporozoites were pelleted, washed three times with PBS, and air dried in wells on glass IFA slides. For permeabilized staining, some wells were incubated again with primary antibody after drying. Revelation was performed with anti–rabbit IgG-FITC (Kirkegaard & Perry Laboratories, Inc.) at 1:40 in 0.5% BSA/PBS for 30 min at 37°C, slides were washed three times with PBS and mounted. For cell invasion assays, ∼10 5 HepG2 cells were seeded in eight chamber slides and grown to semiconfluency. Sporozoites (∼15,000/well) were added, incubated 2 h at 37°C, and washed off. After 48 h, parasite exoerythrocytic forms (EEF) were revealed as described using primary antibody against parasite HSP70, which is expressed only in maturing liver stages. Samples were fixed for 30 min at 4°C with 1% formaldehyde, 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed samples were washed, dehydrated, and embedded in LR White resin (Polysciences Inc.) as described previously . Thin sections were blocked in PBS containing 5% wt/vol nonfat dry milk and 0.01% vol/vol Tween 20 (PBTM). Grids were then incubated with primary antibodies diluted 1:50 to 1:300 in PBTM for 2 h at room temperature. After washing, grids were incubated for 1 h in 15-nm gold-conjugated goat anti–rabbit IgG (Amersham Life Sciences), diluted in 1:20 in PBS containing 1% wt/vol bovine serum albumin and 0.01% vol/vol Tween 20 (PBTB), rinsed with PBTB, and fixed with glutaraldehyde to stabilize the gold particles. Samples were stained with uranyl acetate and lead citrate, and examined in an electron microscope (CEM902; Carl Zeiss, Inc.). Polyclonal antibodies against the TRAP repeats (antirepeats) were obtained using a recombinant polypeptide corresponding to residues 263–428 of TRAP, and polyclonal antibodies against the TRAP cytoplasmic tail (antitail) using the synthetic peptide corresponding to D 586 DE to DND 604 of the TRAP tail. We generated Plasmodium berghei sporozoites that produced TRAP proteins lacking the cytoplasmic tail. Sequence comparison of the cytoplasmic tails of the TRAP proteins sequenced so far reveals that the 14 carboxy-terminal residues are the most highly conserved . We thus created sporozoites whose TRAP lacked the 14 or 37 carboxy-terminal residues, named TΔS and TΔL, respectively. For this, modifications in the single-copy TRAP gene were introduced via a single recombination event promoted by targeting insertion plasmids . As shown in Fig. 1 B, homologous integration of these targeting plasmids generate two TRAP copies: the first is full-length, bears the mutation, and is flanked by expression sequences, whereas the second lacks the 5′ part of the gene and promoter sequences. Three such plasmids, in which the targeting sequence was wild type (WT) or contained the TΔS- or the TΔL-encoding mutations (plasmids pINCO, pTΔS, and pTΔL, respectively), were independently transformed into WT merozoites. One parasite clone from each transformation experiment, named INCO (integration control), TΔS, and TΔL, was selected in rats by limiting dilution. Southern hybridization demonstrated that parasites in each clone had a single copy of the corresponding plasmid integrated into chromosomal TRAP , as depicted in Fig. 1 B (data not shown). The first TRAP duplicate, which should contain the mutation present in the targeting plasmid, was specifically amplified by PCR using primers O1 and T7 and analyzed by restriction digestion. The PCR products obtained from TΔS and TΔL parasites contained the corresponding deletion tagged with a PacI site, as shown in Fig. 1 C. Mutant parasite clones, created at the red blood cell stage, were then transmitted to Anopheles stephensi mosquitoes. Sporozoites are formed inside oocysts in mosquito midguts and are released in the hemolymph, the fluid that bathes the mosquito body cavity. TRAP production was assessed in sporozoites collected from mosquito midguts; i.e., freshly released from, or still within, oocysts. Sporozoite extracts were subjected to Western blot using antibodies directed against the TRAP extracellular repeats (antirepeats) or the TRAP cytoplasmic tail (antitail). As shown in Fig. 2 A, wild-type and INCO sporozoites produced similar amounts of TRAP. TΔS and TΔL sporozoites expressed comparable amounts of their TRAP truncate that, as expected, did not react with antitail antibodies. The presence of TRAP and TRAP truncates on the sporozoite surface was then examined by immunofluorescence. Sporozoites collected from mosquito hemolymph were used because, as described below, TΔS and TΔL sporozoites did not invade the salivary glands. In the WT and INCO clone, virtually all sporozoites permeabilized before staining strongly reacted with antirepeats as well as antitail antibodies, in a typical patchy pattern . However, when staining was performed with live parasites, only ∼5% of sporozoites in both populations reacted with antirepeats, but not antitail, antibodies. Fluorescence patterns mostly resembled caps covering the sporozoite body to various extents, frequently over less than half its length. In addition, a ring-type pattern could be observed in a small number of WT sporozoites. In the TΔS and TΔL clones, ∼5% of the live sporozoites also strongly reacted with antirepeats antibodies and displayed the cap-type fluorescence pattern , indicating that the cytoplasmic tail of TRAP is dispensable for surface exposure of the protein. To determine the subcellular localization of TRAP, WT and TΔL sporozoites were examined by immunoelectron microscopy with antirepeats and antitail antibodies . Using antitail antibodies, WT, but not TΔL, sporozoites were labeled. However, micronemal localization was not unambiguous with these antibodies. Labeling with antirepeats antibodies showed association of TRAP with the micronemes in both TΔL and WT (data not shown) sporozoites. This subcellular localization was similar to that previously described for Plasmodium falciparum TRAP/SSP-2 , indicating that the TΔL truncate was correctly targeted to the parasite micronemes. We then examined the phenotype of recombinant sporozoites ( Table ). Similar numbers of sporozoites were found in the midguts of mosquitoes infected with the three parasite populations. However, dramatically fewer sporozoites were found associated with the salivary glands of mosquitoes infected with TΔS or TΔL parasites than with INCO or WT parasites. This indicated that the former, like TRAP(−) sporozoites , did not infect salivary glands. Sporozoite infectivity to the vertebrate host was estimated by injecting sporozoites intravenously into rats and measuring the prepatent period of erythrocytic infection. Whereas WT and INCO sporozoites induced erythrocytic infections with similar prepatent periods, TΔS and TΔL sporozoites were not infective. Microscopic examination of sporozoites deposited on glass slides revealed that WT and INCO sporozoites glided with similar speed (∼2 μm/s) and pattern, but that TΔS or TΔL sporozoites did not exhibit typical gliding (TΔS sporozoites displayed a limited locomotion described below). Sporozoite invasion into mammalian cells was examined by immunostaining of EEF of the parasite that developed within hepatoma HepG2 cells. Whereas WT and INCO sporozoites generated similar numbers of EEF, no EEF was detected in cells incubated with either TΔS or TΔL sporozoites. Therefore, the cytoplasmic tail of TRAP is dispensable for surface presentation of the protein, but is essential for sporozoite gliding motility and cell invasion. MIC2 is a TRAP-related protein expressed by tachyzoites of Toxoplasma gondii ( Table ). MIC2 is first secreted at the apical pole of the parasite upon attachment to the host cell, and is then translocated to the posterior pole during parasite penetration into the cell . We tested the hypothesis that TRAP and MIC2 have similar functional properties by generating a TRAP variant, named TMIC, in which the cytoplasmic tail was substituted by that of MIC2 starting at the TΔL deletion site . The insertion plasmid that contained the sequence encoding the tail switch, pTMIC, was transformed into WT merozoites, and one resistant clone, TMIC, was selected. Southern blot hybridization confirmed that a single copy of the plasmid was integrated at the TRAP locus in TMIC parasites (data not shown), and PCR amplification of the first TRAP duplicate confirmed the presence of the additional BamHI site tagging the exchange . To rule out the possibility that a discontinuous gene conversion event had preserved the BamHI site but corrected downstream heterologies, the sequence of two independent PCR products generated with primers O1 and T7 was determined. Both sequences contained the desired exchange. As shown in Table , TMIC sporozoites behaved similarly to WT or INCO sporozoites in all tests performed. Most strikingly, TMIC sporozoites glided at the same average speed and followed a similar circular pattern than WT or INCO sporozoites . In addition, TMIC sporozoites were as infectious to the rodent host as WT sporozoites. In all rodent infection experiments, Southern hybridization indicated that the blood stages of the parasite induced by TMIC sporozoites still contained the TMIC recombinant locus (data not shown), with no trace of WT TRAP that could have been recreated via plasmid excision. We conclude that the cytoplasmic tail of MIC2, despite little primary amino acid sequence similarity to the TRAP cytoplasmic tail, can function in its place during gliding locomotion and cell invasion by malaria sporozoites. The cytoplasmic tails of these proteins must then interact with homologous partners in the respective Apicomplexan host. Although their primary sequence is not conserved, the cytoplasmic tails of the TRAP-related proteins have two common features ( Table ). They are rich in acidic residues (18–30%) and contain a tryptophan as the penultimate or antepenultimate residue. To test the contribution of these residues, we created two additional TRAP mutants. One had the carboxy-terminal WN residues modified to AS, named TRYP, and one had the last three acidic residues ED[N]D modified to AS[N]A, named ACID . Parasite clones TRYP and ACID were selected after transformation with the targeting plasmids containing the corresponding mutation, pTRYP and pACID. Each clone was confirmed by Southern hybridization (data not shown) and PCR analysis to contain the expected TRAP recombinant locus, with a mutated (NheI-tagged) first TRAP duplicate. Surface expression of the TRYP and ACID variants was confirmed by IFA using antirepeat antibodies (data not shown). In both TRYP and ACID sporozoites, cap-like structures were seen that were similar to those in WT, TΔS, and TΔL sporozoites. Ring-type patterns, however, seemed less intense in TRYP and ACID sporozoites than in the WT. However, since the ring-type patterns showed some variability in WT sporozoites themselves , the significance of this difference remains questionable. As shown in Table , both TRYP and ACID sporozoites were not infective to the mosquito salivary glands or the rodent liver, nor did they invade HepG2 cells in vitro. Surprisingly, the gliding phenotype of TRYP and ACID sporozoites was not abolished but drastically modified, and identical to the gliding pattern of TΔS sporozoites. Whereas ∼10% of WT hemolymph sporozoites typically described circles at a constant speed, the same proportion of hemolymph sporozoites in the TRYP, ACID, and TΔS clones all displayed an identical phenotype of “pendulum” gliding. This new phenotype consisted in repeated cycles of (a) gliding over one third of a circle, (b) stopping for usually 1–2 s, and (c) moving back to the original position . This phenotype was not observed with TΔL or TRAP knock-out sporozoites, indicating that it depends on the cytoplasmic tail of TRAP. Importantly, it was not due to low-level expression (as verified with TΔS sporozoites) or mistargeting of the TRAP variant (since their surface presentation was not affected), and thus appeared to be a direct consequence of the modifications. In WT sporozoites, TRAP is released on the substrate during gliding locomotion . Labeling of TRAP on the substrate revealed a periodic intensity pattern reminiscent of the nonuniform expression of TRAP on the sporozoite surface. Also, TRAP released on the substrate is detected by antirepeats, but not antitail, antibodies (data not shown), suggesting that TRAP release occurs after cleavage of the protein. Therefore, the pendulum phenotype may be explained by the sporozoite capacity to translocate TRAP to its posterior tip, making it glide over one third of a circle (corresponding approximately to the sporozoite length), and incapacity to release TRAP accumulated at its posterior pole, making it stop. The TRAP cytoplasmic tail could therefore have a bifunctional character; the membrane proximal part would be required for protein translocation along the cortical microfilaments, whereas the distal part would contain a signal for TRAP release at the posterior pole, probably by proteolytic cleavage. Whatever the molecular basis for the pendulum phenotype, it demonstrates a direct role of TRAP as a transmembrane link in gliding motility (and cell invasion). The critical role of the conserved tryptophan and acidic residues also indicates a similar role for TRAP-related proteins. The results presented here show that TRAP and structurally related proteins constitute a family of functionally homologous bridging proteins involved in parasite gliding motility and cell penetration. The interchangeability of the TRAP and MIC2 cytoplasmic tails suggests the conservation of the molecular machinery that drives protein redistribution. The cytoplasmic tails of these proteins may interact with some component of the cortical microfilament system, possibly a motor protein. Recent evidence suggests that myosin colocalizes with actin underneath the parasite plasma membrane in a circumferential pattern and powers motility and cell invasion by Toxoplasma gondii tachyzoites . Favorable signals on the parasite surface may trigger interaction between the cytoplasmic tail of the TRAP-related protein with myosin, and their translocation along submembranous actin filaments. In addition, if our interpretation of the pendulum phenotype is correct (i.e., if the distal part of the TRAP cytoplasmic tail and the conserved tryptophan are necessary for protein release), then the product(s) necessary for releasing the bridging protein may also be conserved in these parasites. Members of the TRAP family of proteins have been found in the malaria sporozoite and ookinete, as well as in invasive stages of Toxoplasma , Cryptosporidium , and Eimeria . An invasion system dependent on the redistribution of TRAP-related proteins, which contain various associations of A-type and TSR domains, may be restricted to penetration of Apicomplexan parasites into epithelial cells. For example, the bridging protein of the capping machinery used by the malaria merozoite for invading a red blood cell, which remains unknown, probably requires specialized cell-binding modules. However, the cytoplasmic tail of such protein might carry the features shared by members of the TRAP protein family. Further unraveling of the mechanisms of gliding motility and cell invasion by Apicomplexa should reveal new features of cell adhesion associated with force transduction and may identify common targets for blocking their infectivity. | Study | biomedical | en | 0.999999 |
10579716 | Diagrams of the infectious clones used in this study are shown in Fig. 1 A (below). The plasmids pU3/12 and pU3/12 ΔM-RV were used to generate infectious transcripts of wild-type (wt) TMV and TMV that does not express a functional MP (ΔM), respectively. The plasmids pT-MfCP (MP:GFP-CP) and pTMV-M:GfusBr (MP:GFP-ΔC) contain the MP fused to GFP in which serine at amino acid position 65 is replaced by threonine . vRNA-MP:GFP-ΔC does not contain the CP open reading frame . The plasmid pTR447 was used as template to produce the nonradioactive (digoxigenin or fluorescein) labeled RNA probes used in Northern blot and in situ hybridization experiments. To create pTR447, the XbaI–HindIII fragment of the TMV replicase coding sequence was removed from pU3/12 ΔM-RV and subcloned into the plasmid vector Bluescript SK+ (Stratagene Inc.) previously digested with the same restriction endonucleases. Infectious RNAs were obtained by in vitro transcription of TMV clones using the MEGAscript T7 kit (Ambion Inc.). Protoplasts were transfected by electroporation as described by Watanabe et al. 1987 and collected by centrifugation at 4, 8, 14, 20, and 30 h post-infection (hpi). Total RNA was isolated from 5 × 10 4 protoplasts using TRIzol reagent following the recommendations of the manufacturer (GIBCO-BRL). RNA was recovered from the aqueous phase after centrifugation at 12,000 g for 15 min at 4°C, precipitated with isopropanol, and resuspended in an adequate volume of sterile water. To monitor plus- and minus-strand accumulation, the RNAs were denatured with glyoxal following the procedure described by McMaster and Carmichel 1977 . RNAs extracted from equal numbers of protoplasts were loaded onto the gel and electrophoresed in 20 mM Hepes, pH 7.00, 1 mM EDTA. After electrophoresis, the gels were soaked in 50 mM NaOH for 15 min and 50 mM sodium acetate/acetic acid for an additional 15 min. The nucleic acids were then transferred to nitrocellulose membranes in 10× SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) according to standard procedures . Before hybridization, the membranes were stained with methylene blue to verify that the levels of ribosomal RNA in each sample were similar. Prehybridization, hybridization, and colorimetric detection were performed as previously described by Más and Pallás 1996 . Single-stranded digoxigenin-RNA probes that recognized plus or minus strands of the genomic TMV RNA were obtained by in vitro transcription of the plasmid pTR447 clone previously linearized with XbaI or HindIII and using T7 or T3 RNA polymerases, respectively (Boehringer Mannheim Biochemicals). Protoplasts were fixed in paraformaldehyde and spun onto polylysine-coated slides (Sigma Chemical Co.) as described by Kahn et al. 1998 . For immunostaining, the samples were washed two times in PBS-TE buffer (140 mM NaCl, 2.5 mM KCl, 10 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 0.5% Tween 20, and 5 mM EGTA). The slides were then immersed in cold methanol for 10 min and washed again in PBS-TE before incubation for 2 h at room temperature with the primary antibody. Monoclonal mouse anti–α-tubulin and anti–actin antibodies (Amersham Corp.) were diluted 1:100 in PBS-TE. The antibody against the rabbit anti–ER lumenal binding protein (BiP), kindly provided by R.S. Boston (North Caroline State University, Raleigh, NC), was used at 1:100 dilution in PBS-TE. To immunolocalize TMV replicase, a polyclonal antibody against the 126/183-kD replicase protein was diluted 1:50 in PBS-TE. After incubation with the primary antibody, the samples were washed two times in PBS-TE and incubated for 2 h at room temperature with 1:100 dilution of the secondary antibody:TRITC-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) or TRITC-conjugated goat anti–rabbit IgG (Pierce Chemical Co.). The samples were then washed again and either mounted in Mowiol (Calbiochem Corp.) or processed by in situ hybridization to detect vRNA. Before hybridization, the samples were treated with proteinase K (1 μg/ml) for 5 min, washed, and refixed with paraformaldehyde at room temperature for 30 min. After fixation, the samples were washed again and immersed in 0.25% acetic anhydride, 100 mM triethanolamine-HCl, pH 8.0, for 10 min to prevent nonspecific binding of the probe to positively charged amino groups. After acetylation, the samples were dehydrated by 10 min washes in 70, 80, 90, 95, and 100% ethanol solutions. The samples were then hybridized overnight at 55°C in a moist chamber with the hybridization solution containing 50% (vol/vol) of deionized formamide, 4× SSC, 0.1% (wt/vol) SDS, 8% (wt/vol) dextran sulphate, and 10 ng/μl of fluorescein-RNA (fluor-RNA) probe. The fluor-RNA probes that recognized plus or minus strand of the genomic TMV RNA were obtained by in vitro transcription of pTR447 as described above. Probes were labeled with fluorescein-12-UTP (Boehringer Mannheim Biochemicals) following the recommendations of the manufacturer. After hybridization, the samples were washed two times in 2× SSC for 10 min at room temperature and two times in 0.1× SSC for 10 min at 50°C, air-dried, and mounted as described above. To disrupt microtubules, infected protoplasts were treated with 10 μM oryzalin (ChemService) for 2 h. To disrupt microfilaments, infected protoplasts were treated with 100 μM cytochalasin D (Calbiochem Corp.) for 2 h. Stock solutions of oryzalin and cytochalasin D were prepared in DMSO so that the final concentration of DMSO added to the protoplasts did not exceed 0.1% (vol/vol). After these treatments, the protoplasts were fixed and processed for in situ hybridization following the procedure described above. Confocal imaging was performed as previously described using a confocal laser scanning microscope (IX70; Olympus Corp.). Optical sections were made at 0.8-μm intervals and projections of serial optical sections were obtained using the software provided by the manufacturer. To examine fluorescein and GFP fluorescence, optical planes were scanned with the 488-nm argon laser using a 550-nm (FVX-BA550RIF) barrier filter and 60× 1.4 NA PlanApo or 100× 1.35 NA UPlanApo oil-immersion objectives. The TRITC signal was detected with the 568-nm krypton laser (FVX-BA585IF). In experiments of dual localization (with fluorescein and TRITC), both fluorophores were examined independently, or by attenuating the 488-nm line to 25% intensity to reduce the possibility of crossover between the channels. Furthermore, single immunodetection controls verified the absence of fluorescence crossover. Before in situ hybridization analysis, the specificity of the RNA probe and the accumulation of viral RNA of the different TMV constructs used in this study were analyzed by Northern blot hybridization . Total RNAs from equal numbers of protoplasts were extracted at different times after infection and subjected to Northern hybridization with a probe labeled with digoxigenin (dig-RNA). The RNA probe contains a fragment of the replicase sequence (see details in Materials and Methods). The probe hybridized with an RNA of ∼6.4 kb, representing the full-length, plus-strand genome of TMV . As anticipated, vRNA accumulated to approximately the same level in cells infected with wt TMV RNA and in cells infected with TMV RNA that lacked the MP (ΔM), confirming previous studies showing that the MP is not required for replication . RNAs from mock-inoculated protoplasts did not react with the probe . Time-course analysis showed that vRNA accumulated to detectable levels at 4 hpi, increased until 14 hpi, and then gradually decreased to a lower level through 30 hpi . The levels of accumulation of vRNA in mutants that lack the CP sequence and express the MP:GFP fusion protein were somewhat lower than in wt infection, especially at 30 hpi . Since MP:GFP-ΔC produced no CP, the lower levels of vRNA in these samples are presumed to reflect the lack of encapsidated vRNA. Protoplasts infected with RNAs derived from constructs containing the CP open reading frame and MP:GFP fusion protein (MP:GFP-CP) showed lower levels of vRNA than those observed in wt infection . The levels of ribosomal RNAs in each sample were similar, as visualized by staining with methylene blue before hybridization (not shown). When samples were hybridized with a probe to detect minus-strand RNA, the signal was observed only at early stages of infection (4–8 hpi) and in much lower amounts than plus strands (not shown). Similar results were described by Ishikawa et al. 1991 in TMV (L strain)-infected protoplasts. These studies show that the nonradioactive RNA probes were sufficiently sensitive and specific for use in situ hybridization procedures. They also indicate that vRNA produced by wt, ΔM, and MP:GFP virus infections accumulate to approximately the same degree throughout infection. The intracellular accumulation of TMV RNA during infection was visualized by in situ hybridization. Protoplasts isolated from BY-2 tobacco cells were transfected with wt RNA, collected at different times after infection, and hybridized with fluor-RNA probes. The fluorescent signals were visualized by confocal microscopy after collecting optical sections with a focal depth of 0.8 μm. Since infection of BY-2 protoplasts is apparently not synchronous , the accumulation of vRNA was considered in three stages: early, 6–12 hpi; mid, 12–22 hpi; and late, 22–36 hpi. Representative examples are shown in Fig. 2 . At early stages of infection , vRNA was primarily localized in faint fluorescent vesicle-like structures surrounding the nucleus . The fluorescent signal was also observed in small, discrete cytoplasmic patches of uniform size, dispersed randomly throughout the cell . In some cells, vRNA tended to accumulate in large amounts around the nucleus as shown in an optical section in Fig. 2 C. At midstages of infection , vRNA was associated with large irregularly shaped bodies . High magnification of the image revealed that these bodies were, in many cases, associated with a weakly fluorescent reticulated network that resembled the cortical ER. Short fluorescent strands of tubules, small fluorescent circles, and vesicles of variable size and shape were also observed . During the middle stage of infection, most of the large bodies were localized around the nucleus, although some were dispersed throughout the cytoplasm and in some cells occupied much of the cytoplasm . Clear localization of vRNA in filamentous structures was visible during this period ; most of the filaments appeared to be associated with the fluorescent bodies . At late stages of infection , vRNA was localized to small, intensely labeled spots around the nucleus, throughout the cytoplasm, and at the periphery of the cell . The number and intensity of these spots decreased over time. By 40 hpi, 15% of infected protoplasts showed vRNA localized to elongated structures that appeared to protrude through the plasma membrane . The protrusions resembled those previously described as binding the MP:GFP fusion protein . We conclude from these studies that vRNA accumulates in different subcellular compartments throughout infection. This compartmentalization most likely reflects the roles of vRNA in replication, translation, and assembly in viral particles. Mock-inoculated protoplasts identically processed and imaged did not show fluorescent signals . In addition, no signal was observed when either the fluor-RNA probe was omitted from the hybridization reaction or when the samples were treated with RNAse A before the hybridization reaction (not shown). Furthermore, 90–95% of the inoculated protoplasts showed hybridization signal, coinciding with the percentage of protoplasts that are infected after inoculation with wt vRNA. As anticipated from the Northern hybridization studies described above and those described by Ishikawa et al. 1991 , in situ hybridization experiments using a probe that bound minus-strand RNA revealed fluorescent signals only at early stages of infection (4–8 hpi). As illustrated in Fig. 3 , fluorescence accumulated primarily in perinuclear vesicles and in small cytoplasmic patches, similar to the accumulation of plus-strand vRNA . Interestingly, the surface of the nucleus was not uniformly covered by fluorescence in these reactions. In protoplasts visualized at 4–6 hpi, the fluorescent signal was clearly absent from a discrete region of most nuclei . Visualization at high magnification revealed circular regions free of RNA in protoplasts with weak or strong fluorescence around the nucleus. Taken together, these results indicate that the fluorescent signal obtained after in situ hybridization of U1-infected protoplasts reflects the intracellular distribution of TMV RNA. To examine the relationship between the sites of vRNA accumulation and the location of the viral replicase, protoplasts infected with wt TMV RNA were collected at early stages of infection, first immunostained with antibody raised against the TMV replicase protein , and subsequently processed for in situ hybridization with the fluor-RNA probe. The distribution of the replicase, visualized with TRITC-conjugated secondary antibody was similar to that of vRNA (green). Superimposition of coincident green and red signals are presented in yellow (merged image) and are most apparent in bodies and filaments in the cytoplasm. The antireplicase antibody also labeled structures that lacked vRNA (red in merged image). It is not known whether these differences reflect differences in distribution of replicase protein and vRNA or differences in intensity of each fluorophore. In double labeling experiments, the fluorophores were scanned independently to minimize crossover between the two channels. Similar patterns of distribution of replicase were observed when the samples were processed only by immunostaining with the replicase antibody, indicating that the results were not due to artifacts produced as a result of the in situ hybridization procedure. The patterns of vRNA accumulation over time resembled the pattern of synthesis, accumulation, and degradation of the MP during TMV infection . To investigate the spatial relationship between vRNA and MP, protoplasts infected with vRNA-MP:GFP-CP were hybridized with the digoxigenin-RNA probe and hybridization was visualized by immunostaining with a rhodamine antidigoxigenin antibody. The distribution of vRNA-MP:GFP-CP was similar to that of vRNA in wt infection, indicating that the presence of the MP:GFP fusion protein did not significantly alter the pattern of vRNA distribution. Similar patterns of fluorescence were also observed in protoplasts infected with vRNA-MP:GFP-ΔC, a mutant virus that expresses the MP:GFP fusion and lacks the CP sequence (not shown). The hybridization procedure was incompatible with simultaneous detection of MP:GFP due to bleaching of GFP fluorescence during the treatment of the samples. Therefore, to compare the accumulation of both the MP and vRNA in the same cell, protoplasts infected with vRNA-MP:GFP-ΔC were harvested at midstages of infection, fixed, and spun onto poly-lysine–coated slides (see Materials and Methods). Cells showing the characteristic fluorescence pattern of MP:GFP accumulation as previously described were scanned using the 488-nm argon laser of the confocal microscope and the samples were then processed for in situ hybridization. Before the hybridization reaction, the cells were imaged to verify the absence of fluorescence due to GFP. After hybridization with the fluor-RNA probe, the samples were examined to detect the products of hybridization (vRNA). As shown in Fig. 5 , there was a striking coincidence between the distribution of MP and the sites of vRNA accumulation. vRNA was also found in small cytoplasmic patches that lacked detectable MP (arrows). Similar patterns of distribution of replicase, MP, and vRNA distribution confirmed the hypothesis that the replication of vRNA and the accumulation of the MP occur at the same subcellular sites. As shown above, MP colocalizes with the main sites of vRNA accumulation during TMV infection. To clarify the role of the MP in establishing the sites of virus replication, protoplasts infected with vRNA-ΔM were processed for in situ hybridization. At early stages of infection, a similar pattern of fluorescence was observed in protoplasts infected with wt vRNA or vRNA-ΔM. The fluorescent signal was localized in vesicle-like structures surrounding the nucleus and in small cytoplasmic patches . In contrast to infection by wt TMV, vRNA-ΔM also accumulated in stacks of tubular structures near the nucleus . Higher magnification revealed vesicles of different sizes (2–5 μm diameter) around the nucleus as well as throughout the cytoplasm . At midstages of infection, the most striking feature that distinguished this pattern of vRNA-ΔM distribution from that of wt vRNA was the absence of large fluorescent bodies. Instead, smaller fluorescent spots were distributed throughout the cell, interconnected by fewer fluorescent reticulated strands than in wt infection . We often observed stacks of large lamellar cisternae that were up to 10 μm in length and 4–5 μm in depth . At late stages of infection, the fluorescent signal was observed only as small, intensely labeled spots around the nucleus and scattered throughout the cytoplasm . Cells infected with vRNA-ΔM did not exhibit fluorescent protrusions from the surface of the cells as in cells infected with wt vRNA . Similar distribution of vRNA was observed when protoplasts were infected with vRNA-ΔM-GFP-ΔC, a virus construct that lacks CP and MP and produces free GFP (not shown). As noted above, the reticulated pattern of fluorescence that represents the location of wt vRNA resembled the distribution of the ER. Furthermore, previous studies demonstrated colocalization of TMV MP and the replicase with ER on the fluorescent irregularly shaped bodies . To determine if vRNA is associated with the ER, in situ hybridization was performed on protoplasts collected at early stages of infection. The samples were also stained using an antibody to the ER BiP and a TRITC-conjugated secondary antibody. As shown in Fig. 7 , BiP was localized to perinuclear areas and small aggregates dispersed in the cytoplasm. vRNA was localized in structures surrounding the nucleus and scattered in the cytoplasm and in fluorescent bodies. After superimposing the images, we concluded that a portion of the vRNA detected is associated with the ER (yellow in merged image). BiP and vRNA were not fully coincident, indicating that a fraction of vRNA was independent of the ER. In protoplasts treated at early stages of infection with 50 μg/ml of Brefeldin A (BFA), a fungal metabolite that disrupts the endomembrane system in plant cells , the vRNA was dispersed throughout the cytoplasm in small fluorescent spots. No fluorescent vesicle-like structures were observed surrounding the nucleus in treated samples . These results corroborate the hypothesis that at early stages of infection vRNA is associated with ER. At early and midstages of infection, vRNA was associated with fluorescent cytoplasmic filaments . To determine if vRNA is associated with elements of the cytoskeleton, infected protoplasts that were immunostained with a monoclonal antibody to α-tubulin were processed for in situ hybridization. A variety of different experimental conditions was evaluated before selecting conditions that permitted visualization of both signals. The procedure that was used involved treating fixed protoplasts with 1 μg/ml of proteinase K for 5 min to partially digest cross-linked proteins and subsequent refixation to avoid disintegration of the cells. These procedures improved the accessibility of the probe while preserving the integrity of the microtubules. Fig. 9 shows confocal micrographs of tubulin (in red) and vRNA (in green) in an infected protoplast processed at midstage of infection. Merging the images clearly showed the coalignment of both signals (merged image). Most of the green fluorescent filaments corresponding to sites of vRNA accumulation coaligned with the cytoplasmic strands of microtubules . Furthermore, some of the bright fluorescent spots that contained vRNA accumulated in tracks that were aligned with microtubule filaments . Protoplasts infected with vRNA-ΔM that were identically processed and imaged did not show fluorescent signals associated with cytoskeleton (except in cytochalasin D–treated protoplasts, see below). The results indicate that colocalization of vRNA with microtubules was not due to artifactual aggregation that occurred during fixation procedures. These conclusions are based on replicated data, either in the number of experiments or in the number of protoplasts analyzed per experiment. In companion studies, we attempted to stain with antiactin antibodies and with the probe for vRNA. However, we did not resolve a typical pattern of filamentous actin distribution (not shown). Most of the cells showed very short fluorescent strands of actin dispersed throughout the cytoplasm. In some cells, we observed a coincidence between the short strands of TRITC-fluorescent signal and vRNA (not shown). It is possible that under the conditions of these experiments, the integrity of the microfilaments was not maintained. A relationship between actin filaments and vRNA can not be ruled out since disruption of microfilaments by treating infected protoplasts with cytochalasin D altered the pattern of vRNA distribution (see below). The results provide strong evidence for colocalization of vRNA with microtubules and suggest that cytoskeletal elements may be involved in distribution of vRNA in protoplasts. To clarify the role of the cytoskeleton in vRNA distribution, protoplasts infected with wt vRNA were treated with specific cytoskeletal inhibitors and vRNA accumulation was examined by in situ hybridization. Representative examples are shown in Fig. 10 . Treatment of protoplasts at early stages of infection with oryzalin, a plant microtubule depolymerizing agent , abolished the accumulation of vRNA around the nucleus and most vRNA was dispersed throughout the cytoplasm (not shown). When oryzalin was added at midstages of infection, nearly all of the fluorescence was associated with enlarged fluorescent bodies on or near the nuclear envelope . In nontreated protoplasts, most of the fluorescent bodies were dispersed throughout the cytoplasm at this stage of infection . When oryzalin was added late in infection, we did not observe changes in distribution of vRNA. In treated as well as nontreated protoplasts, vRNA was associated with the filament-like structures protruding from the surface of the cells . In protoplasts treated during early stages of infection with cytochalasin D, there was a clear delay in the formation of the cytoplasmic bodies compared with nontreated protoplasts. Interestingly, the pattern of vRNA accumulation in cytochalasin D–treated cells was similar to that observed in cells infected with vRNA-ΔM . At midstages of infection, at a time when the large cytoplasmic bodies were formed, the vRNA in cytochalasin D–treated protoplasts (not shown) was detected in enlarged bodies that appeared as more elongated structures than those observed in nontreated cells. In protoplasts infected with vRNA-ΔM and treated with cytochalasin D at early stages of infection, vRNA-ΔM was associated with filaments resembling microtubules . In these protoplasts, vRNA-ΔM also accumulated at the periphery of the cell, but no fluorescence was observed around the nucleus or in small bodies throughout the cytoplasm, as in the case of nontreated, vRNA-ΔM–infected protoplasts . The effects of cytochalasin D were more dramatic in infections with vRNA-ΔM than with wt vRNA, suggesting an influence of the MP in the localization of vRNA in cytochalasin D–treated protoplasts. The results of these studies demonstrate that disruption of the cytoskeleton produces changes in the pattern of vRNA localization and suggest an important role of microtubules and microfilaments in the distribution of vRNA during virus infection. The replication of many positive-strand RNA viruses occurs in close association with membranes . In this study, TMV vRNA was specifically localized with several types of cellular membranes. Early in infection, vRNA was associated with a perinuclear reticulated network and with strands of tubules and small vesicles. These structures closely resembled the cortical polygonal ER network of tubules and sheets with intervening lamellar segments connected to the nuclear envelope, previously described in plant cells . Colocalization of vRNA with BiP, a resident protein of the ER and the disruption of the fluorescent structures by BFA , support the hypothesis that vRNA is associated with ER. Our observations that viral replicase and the complementary minus-strand RNA (used as template for vRNA replication) were localized in these membranous structures strongly suggest that replication of TMV vRNA takes place in close association with the ER. Earlier reports described a relationship between TMV replication complexes and membranous extracts from infected cells . Minus-strand RNA was also localized to structures (presumed to be ER) that surround the nucleus, with the exception of a discrete region. The absence of minus-strand RNA from this region could reflect compartmentalization of the perinuclear ER, or it may indicate that the ER is divided into subdomains with specific morphological or functional properties . Ishikawa et al. 1986 suggested that the synthesis of plus and minus strands of vRNA requires different types of factors or molecular interactions, an observation that may have relevance to our studies. vRNA and viral replicase were colocalized in small patches that are distributed throughout the cytoplasm. As previously indicated in poliovirus infection , we suggest that patches that contain vRNA and replicase correspond to replication complexes in association with ER that compartmentalize the required components of replication to enhance virus production. At early stages of infection, vRNA was associated with fluorescent filaments that resembled elements of the cytoskeleton (not shown). Based on the effects of oryzalin and cytochalasin D on the distribution of vRNA in the cytoplasm, we suggest that there is association of vRNA with the cytoskeleton at a very early stage of infection. In this scenario, vRNA exploits the cytoskeleton for transport from the cytoplasm to perinuclear positions. This hypothesis is based in part on the observation that treating protoplasts with oryzalin at the time of inoculation prevented the localization of vRNA to the perinuclear region. A recent report described a mechanism in newt lung epithelial cells by which the ER membranes attached to microtubules are transported toward the cell center through actomyosin-based retrograde flow of microtubules with ER attached as cargo . Following this model, it is possible that vRNA-replicase complexes associated with ER are transported via microtubules to perinuclear positions using a similar mechanism. Throughout the infection, vRNA was localized in different subcellular compartments. We suggest that this reflects movement of vRNA to different compartments, but cannot eliminate the possibility that compartmentalization represents the synthesis and subsequent degradation of vRNA rather than movement of vRNA from one compartment to another. When protoplasts were infected with vRNA-ΔM, a mutant of TMV that does not produce MP, vRNA-ΔM was localized to vesicle-like structures around the nucleus and in small patches in the cytoplasm. These results indicate that at an early stage of infection, association with the ER is an intrinsic property of vRNA and/or the replicase and does not require MP. It is possible that vRNA contains sequences that target to the ER. Some cellular mRNAs are known to contain specific signals that direct them to the rough ER for translation . At midstages of infection, vRNA was localized in fluorescent, irregularly shaped bodies, some of which were vesicle-like in appearance. Furthermore, the replicase and MP colocalized with vRNA on these bodies. Since such structures were not observed in protoplasts infected with vRNA-ΔM, we conclude that MP is required for the formation and/or stabilization of the bodies. These structures may correspond to the previously described “viroplasms or amorphous inclusions” induced by TMV infection . Our observations support an earlier suggestion that replication complexes associated with rough ER function as mRNAs . During its synthesis, MP may remain associated with vRNA, acting as an anchoring protein and trapping vRNA on ER-derived structures. Accordingly, the large accumulation of MP is coincident with dramatic morphological changes that take place on the ER . Over time, the cytoplasmic bodies become elongated structures, often associated with fluorescent filaments, directed toward the periphery of the cell. Immunostaining with antitubulin antibody and in situ hybridization reactions provide clear evidence of colocalization of vRNA with microtubules. Treatment of protoplasts at midstage of infection with oryzalin prevented the dispersion of the bodies to the periphery of the cell, suggesting that microtubules play a role in intracellular distribution of vRNA. Two different microtubule-based mechanisms, membrane sliding and tip attachment complexes, participate in the movement of ER from the cell center to the periphery of newt lung epithelial cells . In TMV infection, the complexes that contain MP, vRNA, and replicase that are associated with ER may be transported towards the periphery of the cells using such mechanisms. Treatment of infected protoplasts with cytochalasin D clearly altered the pattern of vRNA distribution and caused a delay in the appearance of the large bodies, suggesting a role of microfilaments in their formation and/or stabilization. However, depolymerization of microtubules can also lead to disruption of microfilaments ; it is therefore difficult to define the specific role of each cytoskeletal component in the intracellular spread of vRNA. vRNA-ΔM was not associated with elements of the cytoskeleton unless the cells were treated at early stages of infection with cytochalasin D. Such treatment resulted in accumulation of vRNA-ΔM in fluorescent spots at the periphery of the cell, as well as on filaments that were similar in appearance to microtubules. These results suggest that vRNA was associated with microtubules when both MP and microfilaments were absent. The transport of mRNAs along cytoskeletal components has been described in a variety of biological systems, especially in relation to cell differentiation and development . In those cases, RNA transport mechanisms involved specific sequences in the 3′ untranslated region that are recognized by proteins that bind these sequences and mediate the interaction with the cytoskeleton . In our study, the change in the distribution pattern of vRNA that is induced by cytochalasin D was more evident in protoplasts infected with vRNA-ΔM vs. wt vRNA. These data suggest some influence of the MP in localization of vRNA in cytochalasin D–treated protoplasts. The implications of MP and microfilaments in the formation and anchoring of the cytoplasmic bodies are discussed below. Throughout infection, vRNA was localized around the nucleus, although at mid and late stages, vRNA was also dispersed throughout the cytoplasm and at the periphery of the cell. Accumulation of vRNA around the nucleus may facilitate association with host components that are required for virus infection. However, the accumulation of the much larger bodies that surround the nucleus may imply a different biological role in replication. Recently, a model was proposed by which misfolded proteins that escape the proteosome-mediated pathway of degradation can aggregate to form large structures, referred to as “aggresomes.” Aggresomes are transported on microtubules from peripheral sites to a ubiquitin-rich nuclear location at the microtubule-organizing center, where they are entangled with collapsed intermediate filaments . Following this model, during TMV infection, the bodies that contain MP, vRNA, and replicase may be transported on microtubules in order to be degraded. In other recent studies, we described the ubiquitination of MP and the role of proteosomes in degradation of MP (C. Reichel and R.N. Beachy, manuscript submitted for publication). At late stages of infection, vRNA was localized in structures that protrude from the surface of the cell. These protrusions were not disrupted by oryzalin or cytochalasin D. Furthermore, in other studies, we observed that the protrusions were stained with DiOC 6 (3) (3, 3′-dihexyloxacarbocyanine iodide), a vital fluorescent stain of ER (not shown). Other studies indicate that these ER-containing structures are not induced by virus infection per se, but may be stimulated by infection (P. Más and R.N. Beachy, manuscript in preparation). We propose that the protruding structures are related to desmotubules, the appressed ER that comprises the central component of plasmodesmata . Alternatively, they could be a consequence of cell damage at late stages of infection, which results in the extrusion of ER through the plasma membrane. Interestingly, vRNA-ΔM was not associated with the protrusions, indicating a role of the MP in localization of vRNA with these structures. Together, these results may imply that at least two different types of ER are involved in TMV infection. One type of ER is involved in vRNA replication and does not require the presence of the MP. A second type corresponds to the filamentous protrusions that may be involved in the intercellular spread of the virus and requires a functional MP. The data presented here and in previous publications are consistent with a model of TMV infection in which the replication of vRNA takes place in close association with membranes of the ER . In this model, cytoskeletal elements are involved in targeting vRNA/replicase complexes to the perinuclear ER, perhaps via a retrograde flow of microtubules with ER attached as cargo. The ER-associated nascent vRNAs in replication complexes function as mRNAs for the synthesis of MP . The MP remains associated with vRNA in the complex, resulting in the formation of large ER-derived structures . At this point, the distribution of vRNA would be determined by a balance between the formation and anchoring of the large structures and their spread towards the periphery of the cell. Our results are consistent with a model in which MP and microfilaments participate in the formation and anchoring of the ER-derived structures , while microtubules are involved in the transport to their final destinations; i.e., to the periphery for intercellular spread, or toward the nucleus for degradation . Several types of experimental evidence support this model. First, there is a dramatic effect on distribution of vRNA-ΔM in cytochalasin D–treated protoplasts. Not only were bodies not found in these protoplasts, in contrast to wt vRNA, but vRNA-ΔM was located on or near the cell periphery but not in the protrusions from the plasma membrane. Since in nontreated protoplasts vRNA-ΔM was not localized at the periphery in early stages of infection, the intracellular spread that normally occurs later in infection was apparently accelerated in the absence of microfilaments. Second, the clear association of vRNA-ΔM with microtubules would explain the role of microtubules in the intracellular spread of the virus towards the periphery of the cell. Third, at late stages of infection, the close relationship between the ER and microtubules explains the association of vRNA in the presence of the MP to a precursor of the plasmodesmata that would result in the cell-to-cell spread of the virus in leaf tissues. | Study | biomedical | en | 0.999998 |
10579717 | TOM holo complex and TOM core complex were isolated and purified from mitochondrial membranes of a Neurospora strain (GR-107) in which the wild-type Tom22 is replaced with a version of Tom22 encoding a protein with a hexahistidinyl tag at its COOH terminus. Growth of the Neurospora cells and preparation of mitochondria were performed as described previously . Mitochondria were solubilized for 30 min at 4°C in 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 1% (wt/vol) n-dodecyl β-D-maltoside (DDM; Anatrace Inc.) in the presence of 1 mM PMSF at a protein concentration of 10 mg ml −1 . Insoluble material was removed by centrifugation at 257,320 g for 30 min. The clarified extract was loaded onto a nickel-nitrilotriacetic acid-agarose (Ni-NTA; Quiagen) column using 4 ml resin per 1 g of total mitochondrial protein. The column was washed with 20 vol 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, 0.1% DDM, and 40 mM imidazole. Specifically bound material was eluted with 300 mM imidazole in the same buffer. For further purification, the TOM complex containing fractions were pooled and loaded onto a MonoQ (Pharmacia Biotech) anion-exchange column equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM. The TOM complex was then eluted by a linear 0–500 mM KCl gradient in the same buffer. Stock solutions of purified TOM complex were stored at a protein concentration of ∼5–10 mg ml −1 at 4°C. The identity of the individual Tom proteins was verified by immunodetection with antibodies specific for the Tom components. An average preparation of the TOM core complex started with ∼1.5 kg of Neurospora cells (wet wt) which yielded ∼7 g of mitochondrial protein. The final preparation contained ∼10–15 mg pure TOM core complex. For determination of the stoichiometry of Tom components, core complex was isolated from strain GR-107 grown in the presence of 35 S-sulfate. The purified radio-labeled TOM core complex was subjected to SDS-PAGE. For the detection and quantification of radio-labeled proteins, dried gels were analyzed by phosphorimaging analysis. Holo complex containing all established Tom components was purified from isolated Neurospora mitochondrial outer membrane vesicles in 0.5% (wt/vol) digitonin as previously described . For preparing of TOM core complex that lacks the hydrophilic receptor domains, core complex (900 μg) was incubated with 100 μg ml −1 trypsin in 100 μl 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM at 0°C for 30 min. Proteolysis was stopped with trypsin inhibitor (0.5 mg ml −1 ) and proteolytic cleavage of TOM complex was assessed by SDS-PAGE. A COOH terminally His-tagged fusion protein consisting of the presequence of subunit 9 of the F 0 -ATPase (residues 1–69) and dihydrofolate reductase (pSu9-DHFR) was expressed in Escherichia coli and purified by Ni-NTA chromatography. In brief, bacteria were grown overnight in 250 ml LB medium at 37°C. The overnight culture was diluted and grown to an OD 600 of 0.8–0.9. Expression of pSu9-DHFR was induced by adding isopropyl thiogalactoside to a final concentration of 2 mM. Cells were grown for 1 h and harvested by centrifugation. The bacterial pellet was resuspended in buffer containing 50 mM NaHPO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF, 10 μg ml −1 α-macroglobulin, 10 μg ml −1 leupeptin, and 10 μg ml −1 pepstatin, and then sonicated using a Branson 250 sonifier. Unbroken cells were removed by centrifugation and supernatants were loaded onto a Ni-NTA affinity column. The column was washed with 10 column vol phosphate buffer, and bound material was eluted by a linear 10–300 mM imidazole gradient in the same buffer. The peak fractions containing 3–4 mg ml −1 of purified pSu9-DHFR were stored at −80°C. Purified TOM complex (100 μg) was loaded onto a TosoHaas TSK G4000 PW XL size-exclusion column equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 10% glycerol, and 0.5% digitonin at room temperature using an Äkta chromatography system (Pharmacia Biotech, Inc.). Protein was eluted at a flow rate of 0.45 ml min −1 . The absorbance of the eluant was monitored at 280 nm. The molecular weights of TOM complexes were calculated using thyroglobulin (669 kD), apoferritin (443 kD), alcohol dehydrogenase (155 kD), and carboanhydrase (29 kD) as protein standards. TOM core complex used for EM was passed over a Superose 6 gel filtration column (Pharmacia Biotech), equilibrated with 50 mM potassium acetate, 10 mM MOPS, pH 7.0, 20% glycerol, and 0.1% DDM. Native PAGE was performed using a 4–15% acrylamide gradient (PhastGel; Pharmacia Biotech, Inc.). For blue native polyacrylamide electrophoresis , purified TOM complex (50 μg) was dissolved in 25 μl sample buffer, 0.5% (wt/vol) Coomassie brilliant blue G-250, 10 mM bis-Tris, pH 7.0, and 50 mM aminocaproic acid, and electrophoresed through 6–16.5% polyacrylamide gradient gels. Immunodecoration was performed by standard procedures and detection was achieved by the enhanced chemiluminescence method (Nycomed Amersham). SDS-PAGE was performed according to the procedure described by Laemmli 1970 , using 16% acrylamide and 0.6% bisacrylamide. To achieve a higher resolution of the smaller TOM components, high Tris/urea gels were used . Conductance measurements of TOM complex in planar black lipid membranes were carried out as previously described . Membranes were formed from a 1% (wt/vol) solution of dipytanoyl phosphatidyl choline (DPPC; Avanti Polar Lipids) in n-decane/butanol (9:1 vol/vol) across circular holes (surface area ∼0.1 mm 2 ) in the wall of a Teflon cell separating two aqueous compartments of 5 ml each. The aqueous solutions contained 1 M KCl, 5 mM Hepes-KOH, pH 7.0 (σ 0 = 96.7 mS cm −1 ). The channel size of native TOM complex and TOM subcomplexes was determined by analyzing the partitioning of differently sized PEGs into the TOM complex channel. The electrolyte contained 1 M KCl, 5 mM Hepes-KOH, pH 7.0, and 20% (wt/vol) polyethylene glycol (PEG) of various molecular weight (Fluka; Sigma Chemical Co.). The bulk electrolyte conductances, σ PEG , were the same for all PEG solutions (σ PEG = 58.1 ± 0.4 mS cm −1 , mean ± SEM). Membrane currents were measured at a membrane potential of +20 mV with a pair of Ag/AgCl electrodes (Metrohm) using a Keithley 428 current amplifier. Amplified signals were monitored with an analogue/digital storage oscilloscope (Hameg HM 407) and recorded with a strip chart recorder . The conductance of each buffer solution, σ 0 and σ PEG , was measured using a Greisinger GLM 200A conductance meter. Purified TOM complex preparations (0.1 mg protein ml −1 ) were adsorbed to glow-discharged carbon-coated specimen support grids (Cu, 600 mesh or 400 × 100 mesh) for 30 s. The grids were washed twice with deionized water, blotted with filter paper, and stained with 2% (wt/vol) aqueous uranyl acetate for 60 s. Projection images of isolated TOM complex were recorded at 0° tilt in a Philips CM12 transmission electron microscope operating at 120 kV at 43,800× and an underfocus of between 1 and 2.5 μm. Single particle images were processed as described . In brief, micrographs were digitized into 2048 × 2048 × 16 bit arrays using an Eikonix densitometer at a step size of 0.34 nm/pixel. Further image processing was carried out on Silicon graphics workstations using the EM software described by Hegerl 1996 . Images were low-pass filtered to the first order of the electron microscope contrast function, corresponding to cutoff frequencies between (0.18 nm) −1 and (0.3 nm) −1 . 1,598 particles were manually marked in the digitized images. After extraction of frames with 64 × 64 pixels, the images were iteratively aligned with respect to translation and rotation via cross-correlation to a synthetic double ring model with smooth borders as a first reference. Using multivariate statistical analysis , 30 eigenimages were calculated. Using the 10 most significant eigenimages, the data set was divided into 20 classes. Two sets of images were generated from classes representing particles with one and two pores. For the images showing two pores, two independent averages were calculated from images with odd and even numbers. From the Fourier-ring correlation between those averages, a resolution of (2.5 nm) −1 was determined. Electron tomography of TOM core complex stained with 2% (wt/vol) uranyl acetate was carried out using a Philips 200 FEG electron transmission microscope equipped with a VIPS-1000 computer (TVIPS) and a large-area CCD camera . The microscope was operated at 120 kV at an underfocus of 1.5 μm, an EM of 26,950× and a postmagnification factor of 2.05 on the CCD camera. This corresponded to a pixel size of 0.344 nm at the specimen. Data were collected within a tilt range of ±60°, with 6° angular increments. The mean dose per image was ∼1,600 e − nm −2 . The direction of the tilt axis was determined from an independent tilt series of a specimen with 10-nm gold clusters. Image processing of the tomographic data included the alignment of the projections of each tilt series to a common origin, the selection of single particles at 0° tilt, and the 3D reconstruction of individual particles by means of weighted backprojection. From the 3D maps of 321 particles, projection images were calculated, aligned, and subjected to MSA. Particles that did not show two pores were excluded from the data set. For calculation of the final 3D model, the largest homogenous class of particles ( n = 116) was subjected to refined 3D alignment with respect to all six alignment parameters (i.e., three Cartesian coordinates and three angles) using the 3D map of the previous cycle as a reference. To avoid biased 3D alignment and merging molecules with different up and down orientation, each individual particle was allowed to rotate by all three Euler angles in each refinement cycle. For the visualization of the 3D model of the TOM core complex, the AVS/Express 4.0 software package (Advanced Visual Systems Inc.) was used. The surface models were, based on a protein density of 1.3 g cm −3 , thresholded to a volume with an expected mass of 410 kD. The TOM core complex was prepared in two different ways: either by treatment of purified TOM holo complex with high concentrations of nonionic detergent and size-exclusion chromatography or by direct isolation from detergent-solubilized mitochondria in DDM. The TOM holo complex containing all the established Tom components was purified from isolated mitochondrial outer membrane vesicles prepared from a Neurospora strain bearing a hexahistidinyl-tagged form of Tom22 . Fig. 1 B shows the elution profile of TOM holo complex fractionated by size exclusion chromatography. The fractions were analyzed for polypeptide composition by PAGE. The peak fractions contained the established components, Tom70, Tom40, Tom22, Tom20, Tom7, and Tom6. According to the elution profile, the apparent molecular mass of the holo complex was estimated to be ∼490 kD, in agreement with earlier studies . Incubation of the holo complex with DDM at a concentration of 0.33% (wt/vol) at 37°C for 1 h led to dissociation of the import receptors, Tom70 and Tom20, and formation of a defined subcomplex. Size-exclusion chromatography of this material gave a profile similar to that of the holo complex, which was slightly shifted towards the low molecular weight range . The main fraction contained nearly all of Tom40, Tom22, and the smaller Tom components, Tom7 and Tom6. Tom40 and Tom22 were present at roughly the same proportion as in the holo complex, as indicated by quantification of Coomassie staining . Most of Tom70 and Tom20 eluted in fractions corresponding to lower molecular weight. Similar results were obtained when intact TOM complex was treated with the nonionic detergent Triton X-100 at concentrations above 0.33% (data not shown). Treatment of the TOM holo complex with SDS, in contrast, completely dissociated the complex into its individual components (data not shown). Thus, Tom40, Tom22, Tom7, and Tom6 form a defined, and rather stable subcomplex that we designated as the TOM core complex. According to the elution from the sizing column, the apparent molecular mass of this complex was estimated to be ∼410 kD. To isolate the TOM core complex directly from mitochondria in high amounts, mitochondria from a strain with a hexahistidinyl-tag on Tom22 were solubilized in DDM at a concentration of 1% (wt/vol). The extract was loaded onto a Ni-NTA affinity column and, after extensive washing, bound material was eluted with an imidazole gradient. Tom40, Tom22, and the smaller Tom components all coeluted within five major fractions that accounted for ∼0.2% of protein loaded onto the column . Further anion-exchange chromatography resulted in a virtually pure TOM core complex . The yield of purified core complex was ∼2 mg complex per 1 g isolated mitochondrial protein. Upon size exclusion chromatography, the isolated TOM core complex was recovered in a single peak that contained only Tom40, Tom22, and the smaller Tom components . Tom70 and Tom20 were not detected in this complex. Low amounts of Tom20 were present in the preparation eluted from the Ni-NTA column but, as determined by immunoblotting, these were completely removed after the gel filtration step . TOM core complex was purified from Neurospora cells that were grown in the presence of 35 S-sulfate. In the purified complex, Tom40, Tom22, Tom7, and Tom6 were present in molar ratios of ∼8:4:2:2 ( n = 2). Isolated core complex was incubated with low amounts of trypsin and analyzed by size-exclusion chromatography. Proteolytic cleavage left Tom40 intact and removed the hydrophilic domains of Tom22 and the small Tom components, yielding fragments <3–5 kD. Immunoblotting using specific antisera against the COOH and NH 2 terminus of Tom22 did not recognize a fragment of the protein. With antiserum against Tom6, no protein could be detected (data not shown). The trypsinized complex eluted in a defined peak corresponding to a high molecular mass complex of ∼410 kD. A similar observation was made when the TOM holo complex, isolated in digitonin, was treated with protease (data not shown). This result indicated that the hydrophilic domains of Tom22 and of the small Tom components are not important for the structural integrity of the core complex. To further confirm the tight association of Tom40, Tom22, and the smaller Tom proteins in a defined subcomplex, purified TOM holo complex and TOM core complex were examined by native PAGE. Single high molecular weight bands were observed upon staining with Coomassie brilliant blue . The different migration behavior of the complexes is due to the different detergents. The holo complex was solubilized in digitonin whereas the core complex has been purified in DDM. Immunoblotting of the holo complex with monospecific antisera confirmed the presence of Tom70, Tom40, Tom22, Tom20, Tom7, and Tom6 (data not shown). The band representing the core complex yielded a positive signal using antibodies against Tom40, Tom22, and Tom6. When the holo complex was treated with DDM (0.33%) or Triton X-100 (0.33%), the resulting core complex had the same electrophoretic mobility as the core complex isolated from mitochondria, and Tom70 migrated close to the running front (data not shown). Only Tom40, Tom22, and the smaller Tom components were detected in the band corresponding to the core complex. Examination of the holo and core complexes by blue native gel electrophoresis, a method in which the binding of Coomassie brilliant blue adds negative charges to the protein complexes, gave results similar to those obtained without inclusion of a dye . However, these conditions resulted in partial dissociation of the TOM holo complex since Western blotting and decoration of proteins with specific antibodies revealed that Tom70, and most of Tom20, no longer comigrated with Tom40 (data not shown). This partial disintegration of the TOM holo complex can be attributed to a destabilizing effect of the negatively charged dye used on the complex . To test whether the isolated core complex contains pores, we analyzed its channel forming activity after reconstitution into lipid membranes. Purified core complex was added to both sides of a black lipid membrane bilayer. Current recordings showed characteristic steps of conductance increase that reflect insertion of the core complex into the lipid bilayer. An average conductance of ∼2.3 nS in the presence of 1 M KCl was observed . The trypsin-treated TOM holo complex had an average conductance of 2.7 nS (data not shown). These average conductances were similar to that of the holo complex . The hydrophilic import receptor domains of Tom70, Tom22, and Tom20 apparently play only a minor role in the channel properties of the TOM complex. To probe the channel size of the isolated core complex, we studied its conductance in the presence of differently sized nonelectrolyte polymers. Low molecular weight polyethylene glycol, PEG 1000 , led to decreased channel conductances . High molecular weight polyethylene glycols, such as PEG 8000 , affected the channel conductance to a lesser degree . The results indicate that the TOM core complex channel can be blocked by molecules of up to ∼6 kD . Does the TOM core complex retain its ability to bind a chemically pure preprotein in the presence of detergent? To address this question, we incubated chemical amounts of pure preprotein (pSu9-DHFR) with mitochondrial outer membrane vesicles. Membranes were solubilized with DDM at a concentration identical to that used for the isolation of the core complex directly from mitochondria, and the lysate was subjected to size exclusion chromatography. All column fractions were analyzed by SDS-PAGE and immunoblotting. A large fraction of pSu9-DHFR coeluted with Tom22 and Tom40 (not shown) in a high molecular weight complex. Only background binding was observed when DHFR lacking a mitochondrial presequence was analyzed, excluding the possibility that formation of the TOM-pSu9-DHFR complex was the result of unspecific binding. Thus, pSu9-DHFR remained firmly bound to the TOM core complex in a signal-sequence dependent manner, even at high levels of nonionic detergent. Electron micrographs of negatively stained TOM core complex particles displayed predominantly two stain filled openings or pores, but particles representing a single ring were also present . The length of the two pore particles was ∼12 nm, with a width of ∼7 nm. For further image processing, a total of 1,598 particle images were extracted and aligned with respect to translation and rotation via cross-correlation . Using MSA , 30 eigenimages were calculated and the data set was broken up into 20 classes using the 10 most significant eigenimages. The class averages contained predominantly two or one pores . Preparations of the TOM holo complex contained roughly equal amounts of particles with either two or three rings . None of the three ring structures were observed in the core complex preparations. Group averages that showed one and two pores, respectively, were merged, yielding two main groups. Classification analysis was then used to eliminate remaining core complexes with poorly defined structures. This analysis resulted in projection maps of two core complex classes . Projection maps of the TOM core complex treated with trypsin were calculated. The class averages of the trypsinized complex predominantly showed particles with two pores ( n = 326) and one pore . Frequently, one of the channels appeared less distinct. This may be due to stain fluctuations, as can be seen in the original micrograph . The overall structure of the trypsinized core complex was similar to that of the intact core complex. Do the stain filled openings of the TOM core complex, as seen in the 2D projections, span the entire complex? A 3D map of the TOM core complex was constructed by means of electron tomography. A total of 321 core complex particles were individually reconstructed in three dimensions from 6,741 projections, and subjected to 3D alignment and classification. As mentioned in Materials and Methods, the orientation of each particle was checked before averaging. As a result, 19% of the particles had to be flipped from up to down orientation. We cannot exclude, however, that the final 3D average is slightly distorted, due to possible flattening of the molecules that would mimic a common orientation of actually differently oriented particles. An average of 116 reconstructions corresponding to the most prominent class of particles is shown in Fig. 8 . The resolution of this average was 1/2.4 nm −1 , based on the Fourier shell correlation function and 1/3.4 nm −1 following the stronger phase residual criterion . The top view of the 3D model shows a two-ring structure. The density of the contacts between the two rings is as strong as the density of the walls of the rings, thus excluding the possibility that the two-ring structure was due to association of two independent translocation pores. The diameters of the two channels measure ∼2.1 nm. Based on the final reconstruction, and taking into account possible flattening and incomplete staining effects occurring during specimen preparation, the height of the TOM complex is ∼7 nm. We have isolated and analyzed the TOM core complex, which lacks the receptors Tom70 and Tom20, but retains several of the essential properties of the TOM holo complex. Tom40 is present in the holo and the core complex in the same number and constitutes the main component of the protein conducting channel. Tom22, which appears to have both a receptor function and a role in translocation, is firmly associated with Tom40. Likewise, the small Tom components, Tom6 and Tom7, which are believed to be required for the stability of the TOM complex , are present in the core complex. An equivalent of the yeast Tom5 has not been identified in the Neurospora TOM complex. However, a band in the size range of yeast Tom5 was resolved upon SDS gel electrophoresis of the Neurospora complex. Both the core and the holo complex contain the same two pores likely representing protein conducting channels. On the other hand, the TOM core complex lacks the third density seen in the holo complex that could represent another protein mass with or without pore character. It is conceivable that the third density is due to the presence of the receptors Tom70 and Tom20. The Tom70 and Tom20 molecules seem to be attached to the periphery of the holo complex, as they are easily released in the presence of very mild detergents. The ion conductance properties, as defined by electrophysiology, are essentially the same with both types of complexes . The isolated TOM core complex, after binding preprotein to intact mitochondrial outer membrane vesicles, retains the preprotein. Furthermore, the purified TOM core complex is able to bind preproteins in a specific manner (our unpublished results). The hydrophilic domains of Tom70 and Tom20 may increase the rate and specificity of preprotein binding. We conclude that the TOM core complex encompasses the function of the protein conducting channel. Our data also allows us to define the minimal structural requirement of the translocation pore to Tom40, the transmembrane segments of Tom22, and the small Tom components. Whether the transmembrane segments are essential for the formation of the double pore structure remains an open question. Although the sequence homology between Tom40, mitochondrial and bacterial porins is limited, circular dichroism data suggest a common β-barrel-like structure . Renaturation and reconstitution of E . coli -expressed Tom40 into lipid vesicles yielded channels similar in conductance to that of the core complex . It is presently unclear whether single Tom40 molecules formed these pores, or whether renatured Tom40 assembled into a multisubunit structure to create pores. EM and image analysis of the isolated TOM core complex revealed mainly double ring structures, but a significant fraction of single ring particles were also observed (∼19%). This percentage of single ring particles is higher than observed with the holo complex . Analysis of the TOM core complex by both native gel electrophoresis and size exclusion chromatography yielded a homogenous population of molecules of ∼410 kD, which correspond to the double ring structures. Therefore, the TOM core complex may display a somewhat increased instability, or the placement on grids and negative staining of samples may promote disassembly. The height of the TOM core complex of ∼7 nm is ∼2 nm larger than the thickness of a lipid bilayer. In fact, the extra membrane loops of Tom40 and receptors, or intermembrane space domains of Tom22, and the small Tom components would not be able to form large masses on either side of the complex. When edge-on views of the core complex become available, it should be possible to resolve the cytosolic and intermembrane space domains of the TOM core complex in more detail. The 3D reconstruction of the TOM core complex shows several globular elements. Given that the TOM core complex is composed of about eight Tom40 molecules, these elements could represent dimers of Tom40. At present, the cytosolic and intermembrane space sides of the TOM core complex cannot be distinguished. Probing both surfaces of the molecule with tags or antibodies should help resolve this issue. As higher resolution images become available, it will also be possible to better resolve the surface boundaries and internal surfaces of the putative translocation channels. The size of the pores of the TOM core complex, as derived from single particle analysis, can be compared with that calculated from conductance measurements of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Low molecular weight polyethylene glycols that were able to partition into the pores of the core complex reduced the mean conductance of the complex. Intermediate and large size polyethylene glycols with molecular weights of >6,000 reduced the currents mediated by the TOM complex to a lesser extent. Apparently, molecules with hydrodynamic radii larger than that of PEG 6000 were not able to penetrate the core complex channel. Given a radius of ∼2.5 nm of PEG 6000 , the size of the core complex channel should not exceed 5 nm. This value is roughly two times larger than that indicated by EM (∼2.1 nm). High molecular weight PEG molecules might block the entrances of the import channels of the TOM complex. Conceivably, the two-ring structure is a dynamic assembly. A structural flexibility of the TOM complex in terms of alterations of subunit interactions during import of matrix-targeted preproteins has indeed been observed . Furthermore, it seems possible that the two rings undergo a rearrangement to form a structure with a single large pore, when preproteins are imported that appear to cross the TOM complex not in an extended state, but rather in a folded state, as suggested for the precursors of Tom40 and the ADP/ATP carrier . In the case of import of integral proteins of the outer membrane, the rings of the TOM complex may have to open to release the preprotein into the lipid phase. Furthermore, it remains to be determined whether the assembled TOM complex is in an equilibrium with its individual subunit constituents, e.g., with monomers or dimers of Tom40 . The TOM core complex shares a number of interesting characteristics with the Sec61p complex, which facilitates protein translocation in the ER . Although there are no structural relationships between the proteins constituting the two complexes, both complexes appear to form a functionally similar passive conduit for polypeptides. Both complexes are organized as oligomers, with a major component that spans the membrane several times, Tom40 and Sec61α, respectively, and contain additional small components that span the membrane only once. Further, both complexes form ring-like structures with the characteristics of hydrophilic pores . Even the size of the pore appears similar, and the putative protein conducting channels appear to be dynamic in terms of their sizes . Finally, the TOM core complex and the Sec61p complex are basic elements of larger, more complex assemblies, and they associate with auxiliary proteins, such as receptors for targeting signals, or, as shown in the case of the ER translocon, enzymes modifying the translocating chains. So, it appears that during evolution, protein conducting channels have been generated that have certain functional properties in common, but differ entirely in the origin of their constituents . | Study | biomedical | en | 0.999997 |
10579718 | Centrosomes were isolated from mouse L5178Y cells and treated with 1 M KCl . Xenopus egg extracts were prepared as described previously . Centrosomes (1 ml) were incubated with Xenopus egg extracts (5 ml) for 30 min at 20°C. After dilution with 15 ml of buffer A (10 mM Pipes, pH 7.2, 1 mM EGTA, 1 mM MgCl 2 , 0.9 M glycerol, 12.5 mM β-glycerophosphate, 1 mM DTT, 4 μg/ml cytochalasin B, and 7 μg/ml nocodazole), centrosomes were recovered by centrifugation through a 40% sucrose cushion at 50,000 g for 30 min at 2°C. The precipitate was suspended in 5 ml of buffer A and centrifuged again through the same sucrose cushion. The precipitate was then extracted with 200 μl of 1 M KCl in buffer B (20 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgCl 2 ) on ice for 30 min, followed by centrifugation at 22,000 g for 20 min at 2°C. The supernatant, containing pericentriolar material from the egg extracts, was dialyzed against PBS and used as an antigen for mAb production. Hybridomas were produced and screened as described previously . We obtained several independent clones producing mAbs including W8C3, which recognized centrosomes of A6 cells on immunofluorescence microscopy. A λZAP II cDNA expression library of Xenopus embryo (Stratagene) was screened using mAb W8C3, and several positive clones including clone n1 (2.3-kb cDNA fragment) were obtained. A λgt11 Xenopus oocyte cDNA library (Clontech Laboratories, Inc.) was then screened by hybridization with the clone n1 as a probe. Finally, the full-length Xenopus PCM-1 cDNA (XPCM-1; clone 23a) was obtained. The predicted open reading frame (ORF) contained 6,093 nucleotides encoding a protein of 2,031 amino acids (aa) with a calculated molecular mass of 228 kD, which showed 56.8% identity to human PCM-1 (hPCM-1) at the amino acid sequence level. A mouse cDNA library constructed from F9 cells was screened by hybridization with the coding region of XPCM-1 cDNA, and the full-length cDNA for mouse PCM-1 (mPCM-1; clone 16-111) was isolated. The predicted ORF contained 6,075 nucleotides encoding a protein of 2,025 aa with a calculated molecular mass of 229 kD, which showed 57.2% and 87.3% identity to XPCM-1 and hPCM-1 at the amino acid sequence level, respectively. SDS-PAGE (7.5%) was performed according to the method of Laemmli 1970 , and proteins were stained with Coomassie brilliant blue. For immunoblotting, proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Co.), which were subsequently incubated with the first antibodies. Bound antibodies were detected with biotinylated second antibodies and streptavidin-conjugated alkaline phosphatase (Nycomed Amersham Inc.). Nitroblue tetrazolium and bromochloroindolyl phosphate were used as substrates for detection of alkaline phosphatase. The cDNA encoding aa 1,346–2,031 of XPCM-1 (clone n1) or aa 1,299–2,025 of mPCM-1 was subcloned into pGEX-4T-1 or pGEX-5X-3 (Pharmacia Biotech Sverige), respectively, to produce fusion proteins with glutathione S-transferase (GST). These GST fusion proteins were expressed in E . coli , purified using glutathione Sepharose 4B columns , and used as antigens to generate polyclonal antibodies (pAbs) in rabbits. These pAbs were affinity-purified on PVDF membranes with the bands of respective fusion proteins. Full-length XPCM-1 and its middle portion were fused with GFP at their COOH termini (GFP-FX and GFP-MX, respectively). To construct the expression vector for GFP-FX, XbaI sites were introduced into both ends of the ORF of XPCM-1 cDNA by site-directed mutagenesis using a Transformer Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc.) with primers 5′-CTGCAAACCATGTCTAGAGGAGGAGGTCC-3′ and 5′-GCCATCCACCCTGCATCTAGAGAAACTGACAAACAAG-3′. The XbaI–XbaI fragment was subcloned into the NheI site of the pQB125 GFP expression vector (Quantum Biotechnologies, Inc.). To construct the expression vector for GFP-MX, MluI sites and KpnI sites were introduced into both ends of the regions encoding aa 1–744 and aa 1272–2031, respectively, in the GFP-FX expression vector by site-directed mutagenesis with primers 5′-CGCGCAAGAAATGACGCGTGGAGGAGGTCCAC-3′/5′-CCAAATAACACGCGTCCAATGTGTC-3′ and 5′-CTTTCAGCAGGTGGTACCTCAGCCAAGCTGG-3′/5′-GCCATCCACCCTGGTACCAGCAAAG-GAGAAGAAC-3′. This plasmid was digested with MluI, followed by self-ligation, and then digested with KpnI, followed by self-ligation, to generate the GFP-MX expression vector. All expression vectors were confirmed by sequencing. A6 cells were transfected with the GFP-FX or GFP-MX expression vector, and stable transfectants were obtained (GFX-A6 and GMX-A6 cells, respectively) as described previously . These transfectants were observed using a DeltaVision microscope (Applied Precision, Inc.) equipped with an Olympus IX70 microscope and a cooled charge-coupled device (CCD) system. Each image was acquired with 1-s exposure of the CCD camera. Centrosomes were isolated from A6 cells, and rhodamine-labeled tubulin was obtained from bovine brain as described previously . GMX-A6 cells were collected and homogenized in PEM35 buffer (35 mM Pipes, pH 7.1, 0.5 mM EGTA, 0.5 mM MgCl 2 ) containing 0.2 M sucrose, 1 mM DTT, 0.4 μg/ml nocodazole, 10 μg/ml pepstatin, 10 μg/ml leupeptin, and 1% aprotinin, and were then layered onto a 0.3–1.2 M linear sucrose density gradient of PEM35 buffer. After centrifugation at 20,000 g for 30 min, the fraction enriched in GFP-tagged centriolar satellites was collected and centrifuged at 100,000 g for 1 h. The pellet was resuspended in the supernatant (200,000 g for 10 min) of Xenopus egg extract, and 1/20 vol of isolated centrosomes and 1/80 vol of rhodamine-labeled purified porcine brain tubulin were added on ice. The mixture was spread onto glass coverslips, warmed to 22°C, and then observed with the DeltaVision microscope. In some experiments, AMP-PNP (Sigma Chemical Co.), sodium orthovanadate (Wako Pure Chemicals), or mouse antidynein intermediate chain mAb (m70.1; Sigma Chemical Co.) was added to the mixture on ice for 20 min, followed by further incubation for 10 min at 22°C before observation. Nasal respiratory epithelia of C57/B6 mice were irritated with 1% aqueous ZnSO 4 solution as described previously . The ZnSO 4 treatment induces necrosis of ciliated cells in the C57/B6 strain of mice. As a result, the entire layer of epithelium is sloughed off so that the cilia-bearing cells are reformed with concomitant centriologenesis and ciliogenesis . In brief, a drop of 1% ZnSO 4 solution was applied to the orifice of external nares to be inhaled by the mouse. This procedure was repeated until at least three drops had been aspirated into the nasal chambers. The mice were treated in this manner three times at intervals of 30 min. Control mice were exposed to distilled water. Mice were killed 4 d after treatment, and 1% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) was immediately injected into the nasal cavities. Nasal epithelial tissues were quickly removed from the mice, treated with the same fixative for 30 min on ice, washed with PBS three times, and processed for immunofluorescence and immunoelectron microscopy. Xenopus A6 cells cultured on glass coverslips were fixed with methanol for 5 min at −20°C and processed for immunofluorescence microscopy as described previously . Mouse anti–α-tubulin mAb (DM1A; Sigma Chemical Co.) and mouse anti–γ-tubulin mAb (GTU-88; Sigma Chemical Co.) were used as the primary antibodies, and Cy3-conjugated goat anti–rabbit IgG antibody and Cy2-conjugated donkey anti–mouse IgG antibody (Nycomed Amersham, Inc.) were used as secondary antibodies. Small pieces of mouse nasal respiratory epithelia fixed in 1% paraformaldehyde were infused with 2.3 M sucrose containing 20% polyvinylpyrrolidone at 4°C overnight, rapidly frozen in liquid nitrogen, then cut into cryosections ∼0.5-μm thick at −110°C. They were then mounted on poly- l -lysine coated glass coverslips, treated with 0.12% glycine in PBS for 30 min, and processed for immunofluorescence microscopy as described previously . Specimens were observed using a fluorescence microscope (Axiophot photomicroscope; Carl Zeiss, Inc.), or an MRC 1024 laser-scanning confocal microscope (Bio-Rad Laboratories) equipped with a Zeiss Axioplan 2 photomicroscope. Images gained by laser-scanning confocal microscopy were integrated from the optical sections recorded at intervals of 0.2 μm. Conventional EM for A6 cells and nasal respiratory epithelium of mouse was performed as described previously . For immunoelectron microscopy of A6 cells, cells cultured on glass coverslips were fixed with 0.25% glutaraldehyde in PEM80 buffer (80 mM Pipes, pH 6.8, 1 mM EGTA, and 1 mM MgCl 2 ) containing 1% Triton X-100 for 10 min at room temperature, and processed for immunoelectron microscopy according to the method described previously . For immunoelectron microscopy of in vitro reconstituted asters, samples were prepared as follows. The fraction enriched in centriolar satellites was prepared from nontransfected A6 cells as described above. This fraction was resuspended in the supernatant of Xenopus egg extract, and then 1/10 vol of isolated centrosomes was added. The mixture was incubated at room temperature for 20 min and processed for immunoelectron microscopy as described previously . For preembedding immunoelectron microscopy of mouse nasal epithelia, samples were treated with PEM80 buffer containing 0.5% Triton X-100 for 6 min, fixed with 0.25% glutaraldehyde in PEM80 buffer containing 1% Triton X-100 for 10 min, and processed as described previously . For postembedding immunoelectron microscopy of mouse nasal epithelia, small pieces fixed in 1% paraformaldehyde were infused with 2.3 M sucrose containing 20% polyvinylpyrrolidone at 4°C overnight and rapidly frozen in liquid nitrogen. Ultrathin cryosections were cut and processed for immunolabeling, according to the method developed by Tokuyasu 1980 ; Fujimoto et al. 1992 . Goat anti–rabbit IgG coupled to 10-nm gold (Nycomed Amersham Inc.) was used as a secondary antibody. Samples were examined with an electron microscope at an accelerating voltage of 100 kV. A movie file corresponding to Fig. 4 a is available online (http://www.jcb.org/cgi/content/147/5/969/F4/DC1). Time-lapse images collected using DeltaVision were processed with Adobe Photoshop software and converted to a QuickTime movie (JPEG compression) with Adobe Premiere Software. This movie contains the time-lapse images from the first to the last panel in the corresponding figures. Images were recorded for 3 min at 5–6 s intervals. To analyze the molecular components of centrosomes, we raised mAbs against pericentriolar material isolated from Xenopus egg extracts. Since one of these mAbs, W8C3, stained centrosomes of cultured Xenopus epithelial A6 cells, we isolated a full-length cDNA encoding its antigen by screening a Xenopus embryo λZAP II cDNA expression library. DNA sequencing revealed that its product encoded a protein of 2,031 aa with significant similarity to human PCM-1 (hPCM-1; 56.8% identity at the amino acid sequence level), indicating that W8C3 recognized a Xenopus homologue of PCM-1 . As this mAb showed some cross-reactivity with α-tubulin, we then raised a pAb against recombinant XPCM-1 produced in E . coli . As shown on immunoblots, this pAb specifically recognized a 230-kD band in A6 cell lysates, as well as Xenopus egg extracts . Judging from the molecular mass of human PCM-1 (∼230 kD) and from the reactivity of this pAb with recombinant Xenopus PCM-1, we concluded that this pAb specifically recognized XPCM-1. Next, to examine the subcellular localization of XPCM-1 by immunofluorescence microscopy, cultured A6 cells were doubly stained with the anti–XPCM-1 pAb and anti–γ-tubulin mAb . The γ-tubulin signal was exclusively detected in centrosomes, whereas XPCM-1 was concentrated on and/or around γ-tubulin–positive centrosomes in large amounts, and was also scattered in the cytoplasm in a punctate manner in small amounts. In metaphase, its concentration around centrosomes became obscure as previously described in HeLa cells . Interestingly, when these XPCM-1–positive granular structures were examined in Triton X-100–treated A6 cells by immunoelectron microscopy, anti–XPCM-1 pAb specifically labeled electron-dense spherical granules 80–90 nm in diameter located around centrosomes . Some of these granules appeared to be associated with MTs. Conventional EM of A6 cells also identified similar electron-dense granules gathering around centrosomes, which were not surrounded by membranes . Judging from their morphological characteristics, we concluded that these granules were identical to the previously described structures designated as centriolar satellites . As shown in Fig. 2d and Fig. e , pale granules with a similar diameter were also observed around centrioles, but these granules were not labeled with anti–XPCM-1 pAb. Occasional association of PCM-1–containing granules with MTs led us to examine whether MTs are required to determine their pericentriolar localization, i.e., whether their localization is affected by the MT-depolymerizing agent nocodazole. When A6 cells were treated with 0.4 μg/ml nocodazole for 2 h, followed by immunostaining doubly with anti–XPCM-1 pAb and anti–α-tubulin mAb, most of the centriolar satellites (XPCM-1–positive dots) were released from centrosomes and were scattered into the cytoplasm with concomitant destruction of MT networks (data not shown). When the nocodazole was washed out from these cells, within 9 min MTs began to elongate from centrosomes and concomitantly centriolar satellites increased in number around centrosomes. At 12 min after washing out of nocodazole, most of the centriolar satellites were reconcentrated around centrosomes, from which a well developed MT network was reorganized. In contrast, the actin-depolymerizing agent cytochalasin B (1–10 μg/ml) did not affect the localization of centriolar satellites (data not shown). These findings indicated that centriolar satellites are concentrated around centrosomes in a manner dependent on the MT network. To examine the interaction between centriolar satellites and MTs in more detail, we observed the behavior of centriolar satellites in live A6 cells. We constructed cDNAs encoding fusion proteins of GFP with the full-length or middle portion of XPCM-1 (designated as GFP-FX and GFP-MX, respectively), and introduced them into A6 cells to obtain stable transfectants (designated as GFX-A6 and GMX-A6 cells, respectively). In both transfectants, GFP-derived fluorescence was detected as small granules in large numbers around centrosomes and in small numbers scattered in the cytoplasm . Since the anti–XPCM-1 pAb described above was raised against the COOH-terminal region of XPCM-1 , this pAb recognized endogenous XPCM-1, but not GFP-MX. However, when GMX-A6 cells were stained with this pAb, the GFP fluorescence signal overlapped the region of staining with the anti–XPCM-1 pAb . These findings indicated that each centriolar satellite contains multiple XPCM-1 molecules, and that the middle portion of the XPCM-1 is sufficient for incorporation into centriolar satellites. Since GMX-A6 cells gave stronger GFP fluorescence signals than GFX-A6 cells, we used the former transfectants to examine the movement of centriolar satellites in live cells. As shown in Fig. 3 a, in these cells, individual granules were not resolved around centrosomes, but were readily detected in the cytoplasm. These single granules moved linearly at maximum rates of ∼0.7–0.8 μm/s. They frequently changed their velocity, as well as direction of movement, and repeatedly cycled through moving and stationary states. In cultured GMX-A6 cells, single centriolar satellites in the cytoplasm appeared to move not only toward centrosomes, but also toward the cell periphery . Nocodazole (0.4 μg/ml), but not cytochalasin B (1–10 μg/ml), affected these directional movements of centriolar satellites (data not shown). Observations in live cells suggested that centriolar satellites moved along MTs. Therefore, we next examined the interaction of MTs and centriolar satellites in vitro. GFP-tagged centriolar satellites were partially purified from GMX-A6 cells. Asters were reconstituted in vitro from centrosomes isolated from A6 cells, rhodamine-labeled tubulin purified from porcine brain, and 200,000 g supernatant of Xenopus egg extracts. Then, the isolated centriolar satellites were mixed with reconstituted asters in the presence of ATP. As shown in Fig. 4 a and the movie, at the beginning of observation by fluorescence microscopy, numerous GFP-tagged centriolar satellites were already gathered in the center of reconstituted asters, but close inspection revealed single granules moving along MTs. These granules moved toward the minus end of MTs, i.e., toward the centrosomes. No granules were observed moving toward the plus end of MTs. Similarly to the in vivo observations, these granules repeatedly alternated between the stationary and moving states. Their maximum velocity was 0.7 μm/s, which was compatible to that in vivo. As shown in Fig. 4 a, these granules had frequent changes of MTs. We then examined the centriolar satellites gathered around centrosomes in the in vitro reconstituted system by immunoelectron microscopy. Numerous electron-dense nonmembranous granules, ∼80–90 nm in diameter, were observed around centrosomes, and these were labeled with anti–XPCM-1 pAb . Of course, no granules were observed in asters that were reconstituted in the absence of isolated centriolar satellites (data not shown). At the periphery of asters, anti–XPCM-1 pAb-labeled electron-dense granules were occasionally seen to be associated with MTs, which may have been on the way to centrosomes (data not shown). To identify the motor protein responsible for this in vitro movement of GFP-tagged centriolar satellites, we examined the effects of some inhibitors of motor proteins . At 10 μM, vanadate abolished the accumulation of centriolar satellites around centrosomes. This finding suggested that dynein was involved, since dynein, but not kinesin, is inhibited by low concentrations of vanadate . AMP-PNP did not affect centriolar satellite accumulation at a concentration of 100 μM, whereas at higher concentrations, such as 2 mM, AMP-PNP showed complete suppression. This again favored the notion that dynein is responsible for the centriolar satellite movement, since 100 μM AMP-PNP inhibits kinesin, but not dynein . In good agreement with these observations, antidynein intermediate chain mAb (m70.1; 60 μg/ml) completely abolished the accumulation of GFP-tagged centriolar satellites around centrosomes, while control IgG had no effect. The pericentriolar localization of PCM-1–containing centriolar satellites and their disappearance in mitotic cells suggested some association of these granules with the replication cycle of centrioles. During experimentally induced ciliogenesis, numerous centrioles (ciliary basal bodies) were known to be replicated in a synchronized manner within individual cells, providing a good system to examine centriolar replication . We then examined the expression and behavior of PCM-1 in mouse nasal respiratory epithelium, since ciliogenesis can be induced simply by irritation with 1% aqueous ZnSO 4 . First, full-length cDNA encoding mouse PCM-1 (mPCM-1) was isolated. Its product encoded a protein of 2,025 aa with significant similarity to hPCM-1 and XPCM-1 . Then, using recombinant mPCM-1 produced in E . coli as an antigen, a pAb was generated. This pAb specifically recognized an ∼230-kD band in the total lysate of mouse Eph4 cells on immunoblots and exclusively labeled centriolar satellites of Eph4 cells at the electron microscopic level (data not shown). Interestingly, when cryosections of mouse nasal respiratory ciliated epithelium were immunofluorescently stained with this pAb, the mPCM-1 signal was specifically detected at their apical cytoplasm in a granular pattern . Four days after irritation of the nasal epithelia with 1% aqueous ZnSO 4 in situ, cilia were completely removed from their apical surface and, interestingly, the mPCM-1 signal was markedly elevated at the apical cytoplasm . Then, we examined the ZnSO 4 -induced morphological changes of these ciliated epithelia at the electron microscopic level. Conventional ultrathin EM revealed that in nontreated ciliated cells, electron-dense granules ∼100 nm in diameter were scattered beneath the layer of basal bodies of cilia . Curiously, these granules were morphologically indistinguishable from centriolar satellites. As shown in Fig. 7b and Fig. c , both preembedding and postembedding immunolabeling revealed that these granules were exclusively labeled with anti–mPCM-1 pAb. When cilia were removed from these respiratory epithelia by ZnSO 4 treatment, these granules appeared to increase in number and aggregated extensively . In previous reports, these granules were called fibrous granules and were thought to be absent in nonciliogenic cells , but this was not likely. This will be confirmed by the subsequent immunoelectron microscopy. In or close to the aggregation of these granules called fibrogranular area, so-called deuterosomes with multiple replicating procentrioles appeared . These morphological characteristics indicated that synchronized multiple centriolar replication and subsequent ciliogenesis were induced in these cells. Preembedding immunoelectron microscopy revealed that these aggregated fibrous granules, but not deuterosomes, were heavily labeled with anti–mPCM-1 pAb . Since deuterosomes were very large electron-dense structures, it was possible that antibodies cannot access the antigen within deuterosomes. However, postembedding immunolabeling did not detect mPCM-1 within deuterosomes, excluding this possibility . Taken together, we concluded that so-called fibrous granules, which had been intensively examined from the viewpoint of centriolar replication, may be identical to PCM-1–containing centriolar satellites. Various types of membranous and nonmembranous organelles have been described in eukaryotic cells, and their structures and functions have been analyzed in detail. However, there are likely to be many organelles that have not been identified or characterized. The centriolar satellite, electron-dense spherical granules ∼70–100 nm in diameter, occurring around centrioles in most types of cells, is one such uncharacterized type of nonmembranous organelle. In this study, we identified PCM-1 as the first component of the centriolar satellite in Xenopus A6 and mouse Eph4 cells. Transfection experiments of a truncated form of XPCM-1 showed that multiple XPCM-1 molecules were incorporated into each granule, and our preliminary experiments showed that these molecules bind directly to each other to form dimers or oligomers, suggesting that PCM-1 is a kind of scaffold protein constituting the centriolar satellites. Fibrous granules also constitute an uncharacterized type of nonmembranous organelle. These granules were thought to appear in ciliated cells only during ciliologenesis, but we found that they were also distributed close to ciliary basal bodies in nonciliogenic phase. These granules also had the appearance of electron-dense spherical granules ∼80–90 nm in diameter, and were indistinguishable morphologically from the centriolar satellites, although this resemblance has not been described previously. Interestingly, we found that these granules also contained PCM-1. Therefore, we propose here that centriolar satellites and fibrous granules can be regarded as the same novel nonmembranous organelles, defined by their specific component, PCM-1. One of the most characteristic features of centriolar satellites (so probably also fibrous granules) is their ability to move along MTs; they moved along MTs toward their minus ends, i.e., toward centrosomes, in reconstituted asters in vitro in the presence of ATP. The effects of AMP-PNP, vanadate, and antidynein mAb suggested that dynein, but not kinesin, was involved in their movement. In good agreement, Balczon et al. 1999 recently reported that PCM-1 was coprecipitated with MTs from CHO cell extracts, and that immunodepletion with antidynein antibody, not antikinesin antibody, from CHO extracts significantly decreased the amount of coprecipitated PCM-1. Furthermore, PCM-1 was shown to directly bind to Huntingtin-associated protein 1 (HAP1) by yeast two hybrid analyses . Since HAP1 bound to the p150 Glued subunit of dynactin complex , HAP1 may function as a cross-linker between PCM-1, i.e., the centriolar satellites (and also fibrous granules), and the dynein/dynactin complexes. The dynein-dependent, minus end-directed movement can explain the pericentriolar localization of centriolar satellites. In live A6 cells, GFP-tagged centriolar satellites moved not only toward centrosomes, but also toward the cell periphery. It is clear that the centripetal movement dominates as a whole, since centriolar satellites accumulated around centrosomes in live cells, but it remains unclear whether centriolar satellites also bear plus end-directed motors such as kinesins, or some fraction of MTs of A6 cells do not originate from centrosomes and these MTs are responsible for the centrifugal movement of centriolar satellites. The localization of fibrous granules close to ciliary basal bodies in ciliated epithelial cells in the nonciliogenic phase could also be explained in the same way. In simple epithelial cells, most of the minus ends of MTs are not anchored at the centrosome, but are scattered throughout their apical regions with MTs running parallel along the apico-basal axis . Therefore, if the fibrous granules are also associated with dynein, they would accumulate at the apical regions of epithelial cells. The physiological functions of the centriolar satellites remain unclear. As shown in this study, however, their resemblance to the fibrous granules not only morphologically, but also in their molecular composition, suggested that they may play some roles in centriolar replication. Fibrous granules were reported to be associated with the replication of basal bodies . In good agreement, the level of PCM-1 was markedly elevated when ciliogenesis was induced in nasal epithelial cells . In addition to fibrous granules, larger electron-dense spherical structures called deuterosomes also emerged during centriolar replication in ciliogenic cells, from which multiple procentrioles grew , and previous electron microscopic observations suggested that fibrous granules were fused to form deuterosomes . However, this was not likely since PCM-1 was detected in fibrous granules, but not in deuterosomes . The experimentally induced ciliogenesis examined in this study will be an advantageous system to further analyze the relationship between PCM-1–containing granules and centriolar replication in future studies. Previous studies on PCM-1 itself also suggested its possible association with centriolar replication. It is widely accepted that centriolar replication begins near the G1/S boundary, continues through S phase, and is completed during G2 phase . In good agreement, PCM-1 at centrosomes is released into the cytoplasm on the entry to M phase, and on the entry to interphase this molecule is reconcentrated at centrosomes . PCM-1 mRNA levels increase through G1 and S phases, and became undetectable during G2 and M phases in CHO cells . Interestingly, PCM-1 mRNA levels remained elevated during multiple rounds of centrosome replication in CHO cells arrested at the G1/S boundary by hydroxyurea with a concomitant increase in number of centriolar satellites . On the other hand, fibrous granules were also suggested to function as axonemal precursors . Recent studies using Chlamydomonas identified intraflagellar transport (IFT) particles as large preassembled precursors for various axonemal structures in cytoplasm that were concentrated around centrioles . However, it is not likely that fibrous granules are the counterparts of IFT particles; IFT particles are lollipop-shaped electron-dense granules, ∼14–19 nm in diameter , which is much smaller than fibrous granules. IFT particles were detected within flagella, while fibrous granules or PCM-1 was not observed within cilia. Furthermore, PCM-1 immunofluorescence was abundant in the apical cytoplasm of nonciliated epithelial cells, such as intestinal and gastric epithelial cells (Kubo, A., A. Yuba-Kubo, S. Tsukita, and N. Shiina, unpublished data). These findings are against the notion that fibrous granules function as axonemal precursors. Further identification of other components of fibrous granules/centriolar satellites will answer these questions more clearly. In this study, we identified pericentriolar satellites and fibrous granules as PCM-1–containing novel nonmembranous organelles, which were accumulated around centrosomes and ciliary basal bodies, respectively, through their minus end-directed movement along MTs. These findings then suggested the possible association of these PCM-1–containing organelles with centriolar replication. Further detailed analyses of these organelles, as well as PCM-1 molecules, will lead to a better understanding of the molecular mechanism of centriologenesis in general. | Study | biomedical | en | 0.999997 |
10579719 | Standard techniques were used . Rich yeast media, containing either 2% glucose (YPD) or 2% galactose (YPG), and synthetic yeast media were prepared as described . Strains were cured of URA3 -containing plasmids using 5-fluoroorotic acid . Cell cycle arrests were induced by incubating for 3 h with 10 μM α-factor, 0.1 M hydroxyurea, or 15 μg/ml nocodazole (Sigma Chemical Co.). The pRS315-derived plasmid p315-P GAL -HFSMT3, for expressing His6- and FLAG-tagged SUMO(G98) from P GAL10 , has been described . p315-CDC3-HA was pRS315-derived and contained ∼1,000 bp of the 5′ flanking sequence of CDC3 and the CDC3 coding region fused to a COOH-terminal influenza virus hemagglutinin epitope tag (HA) sequence encoding GYPYDVPDYAAFL . p315-CDC3-Δ94-HA lacked the coding region for the NH 2 -terminal 94 residues of Cdc3, so that the expressed protein began MSQING. p315-P GAL -CDC3-HA expressed full-length Cdc3-HA from P GAL10 . Plasmids expressing variants of Cdc3 with TEV protease cleavage sites or Lys to Arg mutations were produced by oligonucleotide-directed site-specific mutagenesis of p315-CDC3-HA using the Mutagene kit (BioRad) according to the manufacturer's instructions. Single stranded p315-CDC3-HA was produced using helper phage R408 (Promega Corp.). Partial protein sequences encoded by TEV site-containing Cdc3 constructs, including the altered segment are as follow: pCDC3-TEV-K2, RQH 21 ENLYFQGS D 26 VQI; pCDC3-TEV-K3, DGV 39 ENLYFQGS Q 48 NDD; pCDC3-TEV-K4, GLG 69 ENLYFQGS Q 76 SEK; pCDC3-TEV-K6, IRRQ 95 ENLYFQGS I 96 NGY. Introduced sequence is underlined. Superscripts indicate positions of amino acid residues in the unaltered sequence. TEV protease cleaves between Gln and Gly in the introduced sequence. Correctly mutagenized plasmids were identified by digesting with BamHI, whose cleavage site encodes the Gly-Ser in the introduced sequence. Plasmids containing Lys to Arg mutations in CDC3 were identified by cleaving with the following restriction enzymes: K4R, PstI; K11R, BamHI; K30R, AatII; K63R, BsiWI; K415R, BamHI; K443R, MscI. All oligonucleotide sequences and construction details are available on request. S . cerevisiae strains used are listed in Table . Strains in which the genomic copies of genes bear epitope tags, deletions, Lys to Arg mutations, or previously isolated mutant alleles, were produced by transforming yeast with the products of assembly PCR reactions, made as follows, which were integrated into the chromosome by homologous recombination. Three PCR products that overlapped by 17–20-bp were used as the templates in a second round PCR reaction using outside primers to produce a product containing the 5′ flanking sequence and/or the coding sequence of the gene of interest, with or without a tag or a mutation, followed by a selective marker and then the 3′ flanking sequence. Transformants were selected for the appropriate selective marker and were screened further by immunoblotting with an mAb against the HA epitope, by amplifying the gene of interest from chromosomal DNA and digesting with an appropriate restriction enzyme diagnostic for a point mutation, or by screening for the desired temperature sensitive (ts) phenotype. Strains containing multiple altered genes were derived from the single mutants by mating and tetrad dissection. Chromosomal DNA derived from MY254 (a generous gift of M. Yuste and F. Cross, Rockefeller University, NY, NY) and STX339-1C (Yeast Genetic Stock Center) was used as source of the cdc15-2 and cdc12-1 alleles, respectively. In constructing EJY309, the SHS1 coding sequence was amplified in two overlapping pieces, one containing the K426R mutation and the other containing the K437R mutation with a BstBI restriction site included in the overlapping region between the two mutations. Cdc12 with a COOH-terminal green fluorescent protein (GFP) tag was constructed by inserting the CDC12 coding sequence into pYX242-GFP . As the high level of Cdc12-GFP expression produced from this plasmid was toxic to EJY318, but not to wild-type cells, we integrated the GFP tag into one of the CDC12 loci in the diploids EJY318 and JD51, as described above, to produce EJY320 and EJY319, respectively. Cultures of EJY320 contained a greater proportion of abnormal cells than either the parental strain EJY318 or the wild-type control strain EJY319. All construction details and oligonucleotide sequences are available on request. A rabbit polyclonal antibody was raised against NH 2 terminally His 6 -tagged SUMO(G98) (Cocalico Biologicals) and was affinity purified on a His 6 -FLAG-SUMO(G98) affinity column as described . In a whole cell lysate from an arrested ubc9 mutant, the affinity-purified anti-SUMO antibody recognized only free unconjugated SUMO (data not shown), demonstrating its specificity. A rabbit polyclonal antibody against Cdc3, a generous gift of J. Pringle (University of North Carolina, Chapel Hill, NC), was affinity purified on a nitrocellulose filter containing Cdc3-lacZ as described . The unpurified anti-Cdc3 serum recognized primarily an ∼100-kD band unrelated to Cdc3. The affinity-purified antibody also contained a small amount of antibody against this other protein. Other antibodies used were the 16B12 mAb against the HA epitope , the 3F10 mAb against the HA epitope , the B5-1-2 mAb against tubulin (a generous gift of Mike Rout, Rockefeller University, NY, NY), and a rabbit polyclonal antibody against Cdc11 (Santa Cruz Biotechnology). Yeast whole cell lysates were prepared as described . Acetone-washed TCA precipitates were resuspended in 0.5 M Tris base, 6.5% SDS, 100 mM dithiothreitol (DTT), and 12% glycerol, heated at 65°C for 20 min and the debris removed by microcentrifugation. Lysates were subjected to immunoblotting, followed by chemiluminescent detection as described . For anti–HA-epitope immunoprecipitations, 45 μl of lysate (∼1 mg protein) was added to 1.5 ml RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing 2 μg/ml of each of the protease inhibitors antipain, aprotinin, chymostatin, leupeptin, and pepstatin, plus 50 mM N-ethylmaleimide (Sigma Chemical Co.). 10 μl of anti-HA–Sepharose (Berkeley Antibody Co.) was added, followed by incubation at 4°C for 2 h with rotation. Beads were washed three times with RIPA buffer plus 0.1% SDS, resuspended in Laemmli loading buffer lacking reducing agent, and incubated at 65°C for 20 min. The supernatant was removed, supplemented to 100 mM DTT, and analyzed by SDS-PAGE and immunoblotting as described. For the TEV protease cleavage experiment, washed HA–Sepharose beads bearing Cdc3-HA variants were washed once with 50 mM Tris, pH 8.0, 0.5 mM EDTA, and 1% Triton X-100, and incubated with 20 U TEV protease (Life Technologies) for 4 h at 25°C in 20 μl of the same buffer. HA-Sepharose beads were pelleted by centrifugation, and the supernatant “unbound” fraction was removed. One 20 μl wash of the beads with RIPA buffer and 0.1% SDS was added to the unbound fraction, and the beads were washed three times with 1 ml RIPA buffer and 0.1% SDS and prepared for SDS-PAGE as described above to yield the “bound” fraction. EJY251-11b containing p315-P GAL -HFSMT3 was grown at 30°C in 4 liter of YP containing 2% raffinose and 1% galactose to an A 600 of 2. Cells were harvested by centrifugation and lysed 10 min on ice in 200 ml cold 1.85 NaOH, 7.5% β-mercaptoethanol. Protein was precipitated by addition of 200 ml 50% TCA, collected by centrifugation, and the pellet was washed with 200 ml ice-cold acetone. The pellet was resuspended in 400 ml Buffer A (6 M guanidine HCl, 100 mM sodium phosphate, 10 mM Tris/HCl, pH 8.0) and incubated at 25°C for 1 h with rotating. Lysates were clarified by centrifugation at 27,000 g max , adjusted to pH ∼7.0 (measured using pH paper) with 1 M Tris base, supplemented to 20 mM imidazole, and bound in batch for 2 h to 2 ml of Ni-nitriloacetic acid (NTA) agarose (Qiagen). The Ni-NTA agarose was loaded into a column, washed with 20 ml of Buffer A, and then with 100 ml of Buffer B (8 M urea, 100 mM sodium phosphate, 10 mM Tris, pH 6.3), and then eluted with 200 mM imidazole in Buffer B. 4 ml of the eluate was added to 46 ml RIPA buffer containing 0.1% SDS, 1 mM PMSF, and 1 mM β-mercaptoethanol, and bound in batch overnight at 4°C to 0.4 ml anti-FLAG sepharose (IBI/Kodak). The anti-FLAG Sepharose was loaded into a column, washed with 50 ml of RIPA buffer with 0.1% SDS, and then eluted with 100 mM glycine, pH 2.2, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS. The eluate was fractionated on a 5–20% acrylamide gel. Protein bands detected by staining with Coomassie brilliant blue were excised and identified by direct analysis of a Lys-C endoproteinase digest by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry at the Rockefeller University Protein/DNA Technology Center (NY, NY) as described . Two different bands yielded fragments of 1032.6, 1226.6, 1274.8, 1356.2, 1607.9, 1844.6, 1995.86, 2268.5, and 3574.5 D, which match the masses predicted by a theoretical digest of Cdc3. Some of the other bands identified contained other sumoylated proteins, but many contained proteins that bound nonspecifically to the affinity columns. Yeast cells were prepared for indirect immunofluorescence microscopy essentially as described . Cells were fixed in 3.7% formaldehyde for 10 min. Proteins were visualized by incubating with the primary antibodies described above, followed by Cy3-conjugated donkey anti–rabbit IgG or fluorescein (DTAF)-conjugated donkey anti–mouse IgG (Jackson Laboratories). Cells were mounted in 4′,6-diamidino-2-phenylindole (DAPI)-containing medium to visualize DNA. GFP fluorescence and Calcofluor staining of bud scars were visualized in live yeast stained with Fluorescent Brightener 28 (Calcofluor white; Sigma Chemical Co.) as described . All micrographs were taken using a 63× oil objective on a Axiophot microscope (Carl Zeiss). Images were transferred directly to Adobe Photoshop 3.05 using a Sony DKC-5000 digital photo camera. Overlaying of Cy3 and fluorescein signals in double-label immunofluorescence microscopy experiments was done in Adobe Photoshop. To identify substrates of the SUMO pathway, we constructed a strain that expressed His6- and FLAG-tagged SUMO as its only copy of the SUMO-encoding SMT3 gene. When proteins bearing both tags were purified by affinity chromatography, the resulting fraction contained a smear of high molecular weight bands , similar to the previously observed pattern of SUMO conjugates in yeast . Two of these bands were identified by direct mass spectrometric analysis of a protease digestion mixture (see Materials and Methods) as containing the product of the S . cerevisiae CDC3 gene. To confirm that Cdc3 could be modified by SUMO and to ask whether any of the other four yeast septins expressed during vegetative growth were sumoylated, we tagged the genomic copies of the CDC3 , CDC10 , CDC11 , CDC12 , and SHS1 genes with the HA epitope tag . The HA-tagged versions of all five septins were functional and were incorporated normally into the septin ring at the bud neck (not shown). When these strains were treated with nocodazole (see below), significant amounts of higher molecular weight forms of Cdc3, Cdc11, and Shs1 were observed, whereas Cdc10 and Cdc12 were present only as single species of the predicted molecular weight . Immunoprecipitation of the HA-tagged septins and immunoblotting with a polyclonal antibody against SUMO confirmed that all of the high molecular weight species of Cdc3, Cdc11, and Shs1 contained SUMO . To test whether septin sumoylation is cell cycle dependent, cells expressing Cdc3-HA were arrested in G 1 with the mating pheromone α-factor, in the S phase checkpoint with hydroxyurea, in the G 2 /M spindle checkpoint with nocodazole, or in late anaphase with the ts cdc15-2 allele. In all cases, >90% of cells were arrested at the appropriate point in the cell cycle (data not shown). Both Cdc3-HA and Cdc11 were most heavily modified in nocodazole-arrested cells and somewhat less so in cdc15 -arrested cells . Very little, if any, of either septin was modified in α-factor- or hydroxyurea-arrested cells, although the results were partially obscured by cross-reacting bands. To define the stages when septins are sumoylated in more detail, SUMO was visualized by double label immunofluorescence microscopy. The protein detected in addition to SUMO was either Cdc3-HA or tubulin , whose localization was used to determine the stage of the cell cycle occupied by individual cells. SUMO localized to the nucleus at all points in the cell cycle , but more strikingly it localized to a ring at the bud neck only during mitosis. Specifically, a ring of SUMO appeared at the bud neck of large budded cells just before initiation of anaphase spindle elongation and nuclear division . This SUMO ring persisted through anaphase and it disappeared abruptly at cytokinesis, virtually simultaneously with septin ring separation and the beginning of spindle breakdown . Surprisingly, the SUMO ring did not completely colocalize with the septins. SUMO coincided only with the mother cell side of the septin “double ring,” appearing on the side next to the undivided nucleus or on the side of the larger cell in mitotic cells . Thus, SUMO conjugation to the septins was asymmetrical with respect to the mother–bud axis. To determine the function of septin sumoylation, we wanted to generate yeast mutants in which septins would not be modified. The most direct way to eliminate septin sumoylation specifically was to identify the Lys residues that serve as SUMO attachment sites and to replace them with Arg residues, which cannot be modified, thereby eliminating septin sumoylation without disturbing conjugation to other substrates. Our initial approach to identifying SUMO attachment sites on Cdc3 was to affinity-purify SUMO-Cdc3 conjugates from strains expressing Cdc3-HA and His6- and FLAG-tagged SUMO(G98) or SUMO(A98). These preparations varied only in the identity of the COOH-terminal residue of SUMO, which is the residue involved in the isopeptide bond with the substrate. This material was digested with trypsin and with endoproteinase Lys C and analyzed by MALDI-TOF mass spectrometry with the goal of identifying species varying by the 14 mass unit difference between the COOH-terminal Gly and Ala in the two preparations. One such species was identified, with a molecular weight of 2,327 D, which was consistent with SUMO attachment at Lys11 of Cdc3 (data not shown). Examination of the sequence surrounding Lys11 revealed that three other Lys residues in the NH 2 -terminal domain of Cdc3 were embedded in similar sequence motifs having the consensus (IVL)KXE, which appeared to be a potential sumoylation site consensus sequence. Since there are multiple SUMO-Cdc3 conjugate bands , such a consensus sequence might be used in either of two different ways. SUMO might be attached at one or the other of these Lys residues as a chain containing SUMO–SUMO linkages, analogous to the multi-Ub chain. Alternatively, multiple single copies of SUMO might be attached with one SUMO moiety per Lys residue. To distinguish between these possibilities, we designed a series of constructs containing cleavage sites for the TEV protease at different sites along the NH 2 -terminal domain of Cdc3, either after the first two Lys residues (TEV-K2), after the third (TEV-K3), after the fourth (TEV-K4), or after the sixth . These constructs also contained a COOH-terminal HA tag, which was used to immunoprecipitate the Cdc3 variants from lysates of nocodazole-arrested yeast. Immunoprecipitated proteins were cleaved with TEV protease while still bound to the beads and separated into an unbound fraction containing the fragment NH 2 -terminal to the cleavage site, and a bound fraction containing the COOH-terminal cleavage product. This experiment showed that for each additional Lys residue included NH 2 -terminal to the TEV site, an additional sumoylated species appeared on the NH 2 -terminal product and disappeared from the COOH-terminal product. There was one major NH 2 -terminal SUMO-containing species from TEV-K2, two from TEV-K3, and three from TEV-K4 and TEV-K6 , and there were two major SUMO-containing COOH-terminal cleavage products from TEV-K2, one from TEV-K3, and none from TEV-K4 or TEV-K6 . These results were generally consistent with a model where single copies of SUMO are attached to Cdc3 at three to four major sites, at Lys-4 and/or Lys-11, at Lys-30, and at Lys-63. A separate experiment in which these Lys residues were altered by site-directed mutagenesis showed that both Lys-4 and Lys-11 can serve as sumoylation sites (data not shown). However, this model does not completely explain the pattern of high molecular weight SUMO conjugates on Cdc3, which consisted of seven bands between ∼95 and ∼200 kD . For several reasons, it is more likely that the additional bands reflect the presence of another mobility-altering factor, rather than attachment of multiple copies of SUMO at the same Lys residue. One is that cleavage at the TEV-K2 site produced only two detectable SUMO-containing COOH-terminal fragments . This result can be explained most easily if a single mobility-altering factor lies NH 2 -terminal to the TEV site at position 27. Once the NH 2 terminus is cleaved off, the number of bands associated with the COOH-terminal fragment would reflect the number of attached SUMO moieties, whereas NH 2 -terminal fragments would still be present in two forms for every SUMO moiety. This explanation is also consistent with the pattern of NH 2 -terminal cleavage products, where there is a minor band above each of the three major SUMO-containing bands from TEV-K4 and TEV-K6 . A second reason is that several of the SUMO-Cdc3 bands and corresponding NH 2 -terminal TEV cleavage product bands appear to be too close together to differ by a whole SUMO moiety. Free SUMO runs at ∼20 kD on SDS-PAGE, but two of the pairs of SUMO-Cdc3 bands appeared to differ by significantly less than 20 kD, and the first two sets of major and minor NH 2 -terminal cleavage products appeared to differ by 10 kD or less . A third reason is that the TEV-K4 variant significantly reduced the amount of the third SUMO-containing conjugate with a proportionate increase in the second band, but there was no reduction in intensity or change in position of the fourth or sixth SUMO-containing bands . This result is most easily explained if the TEV-K4 mutation reduced the other mobility-altering factor without affecting SUMO conjugation. We do not know what this other factor is. It might be another posttranslational modification, but it is also possible that conjugates bearing the same number of SUMO moieties could have different mobilities resulting from SUMO attachment at different sites. Inspection of the Cdc11 and Shs1 sequences for sumoylation site consensus sequences revealed that Cdc11 contained one such sequence near its COOH terminus and that Shs1 contained two such sequences in its COOH-terminal coiled-coil domain . To determine the effects of eliminating these Lys residues on septin sumoylation and on cellular function, we made yeast strains in which the genomic copies of septins were replaced with mutant versions lacking these Lys residues. Deleting the coding sequence for the 94 NH 2 -terminal residues of Cdc3 eliminated the vast majority of the SUMO-Cdc3 conjugates in nocodazole-arrested cells, confirming the conclusion that most SUMO is attached to this NH 2 -terminal domain . However, Cdc3-Δ94 was still sumoylated at a very low level by one copy of SUMO . This result was confirmed by immunoprecipitating with the anti-HA antibody and immunoblotting with an antibody against SUMO (data not shown). Mutating two more Lys residues in CDC3 -Δ94, Lys-415 in the sequence IKQD, and Lys-443 in the sequence AKLE, had no effect on this residual sumoylation (data not shown). The only other sequence in Cdc3 that resembles the sumoylation site consensus sequence is the AKSD containing Lys-287, which is in the septin homology domain. This Lys residue is conserved in all members of the septin family in all organisms, except for Shs1 and one hypothetical open reading frame in S . pombe . Mutant cdc3 containing a Lys to Arg mutation in this position was unable to complement the lethality of the cdc3 Δ strain, even when all the NH 2 -terminal sumoylation site Lys residues were present (data not shown). It is likely that this result reflects a requirement for this Lys residue in some septin function other than SUMO conjugation. When we mutated Lys-412 of Cdc11, and Lys-426 and Lys-437 of Shs1 to Arg, similar results were observed. In both cases, the vast majority of SUMO conjugates were eliminated, but a small amount of residual conjugation remained, reflecting low levels of SUMO conjugation at other sites . As all of the strains with sumoylation site mutations in only one of the septins still contained substantial SUMO rings at the bud neck, we proceeded to construct a triple mutant in which all of Cdc3, Cdc11, and Shs1 lacked the major sumoylation sites. Analysis of a series of strains in which progressively fewer septins contained sumoylation sites demonstrated that the majority of the total SUMO in nocodazole-arrested cells was attached to Cdc3 and that the most of the remainder was attached to Cdc11 or Shs1 . Thus, septins are by far the most abundant SUMO conjugates at this point in the cell cycle. However, a longer exposure of this blot revealed a large number of lower abundance substrates in the triple mutant strain (data not shown). To minimize any non-SUMO–related effects from deleting the entire Cdc3 NH 2 -terminal domain, we constructed a different cdc3 sumoylation site mutant in which all four attachment site Lys residues were replaced with Arg. This mutant's effects on Cdc3 sumoylation were indistinguishable from those of the deletion mutant (data not shown), and subsequent experiments were done using this cdc3 allele. We analyzed the sumoylation site triple mutant ( cdc3-R4,11,30,63 cdc11-R412 shs1-R426,437 ) for phenotypes and found that in most respects it was very similar to the parental wild-type strain. Its growth rate was virtually indistinguishable from that of the wild-type (data not shown), with a doubling time of 95 min, versus 94 min for wild-type, a statistically insignificant difference. This strain was not hypersensitive to either high or low temperatures, to 1 M sorbitol, to DNA damaging agents (UV light or methyl methane sulfonate), to the microtubule depolymerizing drug benomyl, or to the cell wall perturbing agent Calcofluor white (data not shown). It mated efficiently and sporulated with the same efficiency as wild-type to produce viable segregants (data not shown). Haploid cells correctly positioned their bud sites axially, and most diploid cells positioned their bud sites bipolarly, although both this strain and the parental wild-type diploid strain had a significant frequency of cells with random bud sites (data not shown). The actin cytoskeleton, as visualized by rhodamine-phalloidin staining, also appeared normal in the triple mutant (data not shown). We also analyzed the triple mutant by double-label immunofluorescence microscopy with antibodies against SUMO and against the HA tag on the septins. The triple mutant displayed a dramatic reduction in SUMO staining at the bud neck , although a faint ring could be seen in a few cells (data not shown), and other cells contained a bulge of SUMO staining at the bud neck near the nuclear envelope that did not appear to colocalize completely with the mother cell half of the septin ring . This slight SUMO localization to the bud neck probably resulted from the residual sumoylation of the septins, although some other bud neck protein may also be sumoylated at a very low level. The striking difference that we noted between the triple mutant strain and wild-type was that in the mutant, at least one extra septin ring could be observed in virtually all budded cells . This is in contrast to the wild-type strain, in which budded cells never contained septin rings other than at the neck of the growing bud . The presence of these extra septin rings was a synthetic phenotype of the triple mutant, as none of the single mutants or the cdc11-R412 shs1-R426,437 double mutant had this property (data not shown). To test the hypothesis that the extra septin rings in the triple mutant were remnants of septin rings from previous cells divisions, we compared the localization of the extra septin rings with that of the bud scars. Every time an S . cerevisiae cell divides, the division site on the mother cell is marked by a chitin-containing bud scar, stainable with the fluorescent dye Calcofluor white, which persists through the lifetime of the cell. Although they sometimes had many bud scars, wild-type budded cells never contained more than the one septin ring at the base of the growing bud . In the mutant, however, a septin ring colocalized with virtually every bud scar , indicating that the extra septin rings are likely to be undisassembled septin rings from previous bud sites. Unlike mother cells, daughter cells do not have Calcofluor-staining structures marking the division site. Examination of budded cells lacking bud scars revealed that they always contained exactly one extra septin ring, indicating that daughter cells are also defective in septin ring disassembly . There are two simple reasons why the triple sumoylation site mutant might be defective in septin ring disassembly. One is that attachment of SUMO to the septin ring promotes disassembly. The other is that the sumoylation-site Lys residues also have some other function, and that this other function promotes septin ring disassembly. For example, the purpose of these Lys residues could be to serve as ubiquitination sites, or perhaps acetylation or methylation sites. One way to distinguish between these possibilities might be to determine whether mutants in the SUMO conjugation pathway also have septin ring disassembly defects. Examination of Cdc11 localization in ubc9 and uba2 (data not shown) ts mutants showed that no extra septin rings were visible in cells from either strain, either when grown at the permissive temperature or after transfer to the restrictive temperature. This result may suggest that SUMO conjugation, per se, does not promote septin ring disassembly. Alternatively, it may indicate that, although SUMO conjugation may promote septin ring disassembly, as long as these ts mutants retain enough SUMO conjugating activity to divide, they also retain sufficient SUMO conjugation to disassemble their septin rings. In contrast to a previously published report , the levels of Cdc3 and Cdc11 did not decrease noticeably when the ubc9 ts mutant was incubated at the nonpermissive temperature for 4 h , and Cdc11 was still present at the bud neck in similar amounts as in wild-type . It is likely that the other septins were also present, as septins assemble cooperatively. However, the septin rings in arrested ubc9 and uba2 mutants were unlike those in wild-type preanaphase cells in that they appeared to be discontinuous in the middle , almost as in cells undergoing cytokinesis. This result may be a secondary effect of the prolonged G 2 /M arrest, as we sometimes saw a similar effect in nocodazole-arrested cells, or, this observation may suggest that SUMO conjugation prevents septins from undergoing a reorganization usually associated with cytokinesis prematurely. To test whether septins are ubiquitinated or degraded during cytokinesis, we synchronized a culture of cdc15-2 cells by incubating them at 37°C, which arrests them in late anaphase, and releasing them at 25°C to allow them to complete cytokinesis . Between 30 and 50 min after release, all the SUMO-conjugated forms of Cdc3 and Cdc11 disappeared from these cells. However, there was no significant reduction in the steady-state level of either Cdc3 or Cdc11 at this point, which is consistent with the observation by immunofluorescence microscopy that septin rings do not disappear suddenly at cytokinesis. Also, in several different experiments, we never observed high molecular weight ubiquitinated Cdc3 species. However, we did sometimes observe minor bands that might be degradation products of Cdc11 and of Cdc3-HA (data not shown). We conclude that, at most, a small fraction of Cdc3 and Cdc11 is degraded immediately at cytokinesis. We also tested whether the sumoylation site triple mutant interacted genetically with either SUMO conjugation pathway mutants or septin mutants. A quadruple mutant also containing a chromosomally integrated version of the uba2 ts10 allele grew at a range of rates similar to those seen with the uba2 ts10 strain alone (data not shown), which grew very poorly and was heterogeneous. Thus, the attachment site mutations did not strongly exacerbate or suppress the phenotypes of the uba2 ts10 mutant. However, when we crossed the sumoylation site triple mutant to a cdc12-1 mutant, we obtained the surprising result that the cdc3-R4,11,30,63 mutation alone was synthetically lethal with the cdc12-1 mutation. cdc12-1 single mutants grow well with near normal morphology at 25°C, but lose septin rings and develop the distinctive phenotypes of septin mutants at 37°C (see introduction). At 25°C, cdc3-R4,11,30,63 cdc12-1 cells overexpressing wild-type CDC3-HA exhibited near normal morphology . When CDC3-HA expression was repressed, the double mutant developed the phenotypes of septin mutants, even at 25°C, producing branched, elongated cells and failing to undergo cytokinesis . This effect was not general for all septin mutants, as the sumoylation site mutations did not exacerbate the phenotypes of the cdc10-1 mutant (data not shown). We have identified the septins Cdc3, Cdc11, and Shs1 as the three SUMO conjugation pathway substrates that account for the most abundant sumoylated species during yeast mitosis. Septin sumoylation was highly regulated, occurring only during mitosis and only on the mother cell side of the bud neck. We identified the major SUMO attachment site Lys residues in these septins and found that they are surrounded by a short consensus sequence that has also been observed around the mammalian SUMO attachment sites . Multiple copies of SUMO could be linked to a single Cdc3 polypeptide by attachment of single SUMO moieties to several different Lys residues. We could see no evidence of SUMO chain formation analogous to the chains formed by Ub. A mutant lacking these sumoylation sites on septins grew well and was not hypersensitive to the stress conditions tested, but was deficient in disassembly of the septin rings after cytokinesis. The cdc3 sumoylation site mutation also showed synthetic lethality with the cdc12-1 mutation. These results raise many interesting issues regarding the regulation of SUMO conjugation and the structural and functional alterations of the septin ring that take place during and after cytokinesis. While this manuscript was in preparation, another paper was published reporting that Cdc3 is a SUMO substrate , but there are many contradictions between the two reports. For example, they fail to find that Cdc11 is modified and they observe that SUMO is found primarily as the unconjugated form, with small amounts of low molecular weight conjugates, rather than primarily in high molecular weight conjugates, as observed by others . These results can be explained by noting that their protocol for preparing yeast lysates does not, in our experience, inhibit the isopeptidases that cleave SUMO off its substrates virtually instantly when cells are lysed under native conditions. Other groups working in this field lyse cells under denaturing conditions to prevent this problem . Despite this problem, Takahashi et al. 1999 do detect a SUMO conjugate of a Cdc3-GFP fusion protein. Using this reporter, they make the following observations that contradict our results: that there is only a single SUMO-Cdc3-GFP conjugate band; that 50–100% of the total Cdc3-GFP is sumoylated ; that Cdc3-GFP is sumoylated at this same high level in α-factor-, hydroxyurea- and nocodazole-arrested cells; that the SUMO-Cdc3-GFP conjugate persists through cell lysis under native conditions (in our hands, Cdc3-HA is rapidly desumoylated under these conditions; data not shown); and that Cdc3-GFP disappears from the bud neck upon transfer of ubc9 ts cells to the nonpermissive temperature. The first four of these results also contradict other published reports about Cdc3, where Cdc3 runs on SDS-PAGE as a single band of the expected size for the unmodified protein . It is likely that the Cdc3-GFP reporter protein is responsible for these discrepancies. There is no evidence to support the statement that this protein is functional, and all of these experiments have been performed in strains also containing the wild-type untagged genomic copy of CDC3 . The findings that the protein level of Cdc3-GFP decreases during cytokinesis and in ubc9 ts cells at the nonpermissive temperature contradict our results and appear to involve technical problems with their Western blots. The most interesting discrepancy is that they observe SUMO localizing to both sides of the bud neck, whereas we see it only on the mother cell side. This observation could result from a strain difference, but it seems more likely that their result is a phenotype of HA-SUMO overexpression. Our results indicate that the G 2 /M cell cycle arrest phenotypes of the SUMO conjugation pathway mutants probably do not result from a reduction in septin sumoylation. The sumoylation site triple mutant, which eliminated the vast majority of SUMO conjugation to the septins, grew at the same rate as wild-type and did not exhibit any cell cycle arrest phenotype. Furthermore, a uba2 ts mutation did not exacerbate the phenotype of the triple mutant, indicating that SUMO conjugation to other sites on the septins or to some other substrate is not compensating for the loss of the major septin sumoylation sites. It is still possible that the minor septin sumoylation sites play a distinct essential role for which the major sites cannot substitute. However, we believe it is more likely that the SUMO substrates involved in the essential and cell cycle-related roles of the SUMO pathway participate in an entirely different cellular function, possibly chromosome segregation, as SMT3 , the SUMO-encoding gene, was identified as a high copy suppressor of a mutant impaired in chromosome segregation . Septin sumoylation might be controlled by the cell cycle machinery through regulation of SUMO attachment, SUMO removal, or both. It is difficult to determine the fate of sumoylated septins at cytokinesis because of the small fraction modified, but it is most likely that the septins are desumoylated by a SUMO-specific isopeptidase such as Ulp1 or the similar protein Ulp2/Smt4 . In fact, one possibility is that the level of SUMO–septin conjugates at all stages in the cell cycle reflects a dynamic equilibrium between sumoylation and desumoylation. We found that SUMO–septin conjugates disappeared rapidly from nocodazole-arrested ubc9 ts cells, but not from wild-type cells, upon shift to the nonpermissive temperature (data not shown), suggesting that SUMO may have to be continually reattached to maintain a constant level of septin sumoylation. Thus, septins may be constitutively desumoylated, which would allow the level of SUMO conjugates to be tightly regulated solely by controlling the rate of the conjugation reaction. Several issues depend on the structure of the septin-containing neck ring, which has not been described in detail. Purified septins form linear filaments in vitro, and the present model for the structure of the ring holds that it consists of parallel septin filaments running through the bud neck parallel to the mother-bud axis . These filaments are linked laterally to form the ring by an unknown mechanism involving the Nim1-related kinases Gin4, Hsl1, and Kcc4 . Other proteins associate with the septin ring in four distinct arrangements: throughout the septin ring, on the mother cell side, on the bud side, and in the middle between the mother cell and bud. One model for how such regions might be established derives from the fact that the mother and bud sides of the neck ring are assembled at different points in the cell cycle . Thus, different septin-associated proteins could be incorporated into the different sides, establishing stable zones within the ring. Other proteins, such as SUMO conjugation pathway enzymes, could specifically bind the asymmetrically localized proteins initially incorporated into the different regions. A second model relies on the potential inherent polarity of the septin filaments running through the bud neck. Associated proteins might move along the filaments in one direction or the other until they reach the mother or bud side of the ring. The data on septin-SUMO conjugation is less consistent with the model where various regions are preestablished, because it is possible for the daughter side to be sumoylated. Takahashi et al. 1999 observed SUMO on both sides of the bud neck, probably as a result of HA-SUMO overexpression, and we also observed SUMO on both sides of some septin rings in nocodazole-arrested cultures (data not shown). Thus, septin sumoylation appears to be dosage dependent, such that the mother cell side is modified first, with the daughter cell side modified when the system is overexpressed or abnormally induced. Another question is why such a small fraction of each of the septins is modified. If the septin polypeptides that are sumoylated are functionally and structurally equivalent to the ones that are not, it would be less likely that SUMO is blocking Lys residues against attachment of some other modification, since these Lys residues would still be available on the vast majority of potential substrates. However, the various septin polypeptides may not be equivalent, either because all positions in the septin lattice may not be equivalent structurally or because of the presence of other posttranslational modifications. We did see variant forms of SUMO-Cdc3 conjugates, which may bear another posttranslational modification. There did not appear to be proportional amounts of a corresponding variant of unsumoylated Cdc3, suggesting that the other modification may target the same subpopulation or that one modification is a prerequisite for the other. The triple sumoylation site mutant is the first mutant isolated with a defect in septin ring disassembly. We have mentioned two models for how the triple mutant might affect disassembly of the septin ring. One is that SUMO itself promotes septin ring disassembly, and the other is that the sumoylation-site Lys residues play some other role in disassembly, possibly by serving as ubiquitination sites. In the second model, SUMO might play a regulatory role by preventing the Lys residues from taking part in the other function. Alternatively, the other function might be completely independent from SUMO. Any model for SUMO involvement in septin ring disassembly has to explain two sets of seemingly contradictory results. One is that SUMO is attached only to the mother cell side of the bud neck, but the septin rings fail to be disassembled in both the mother cell and in the daughter cell . The other is that SUMO–septin conjugates disappear suddenly at cytokinesis, but disassembly of the septin rings does not take place until G 1 of the next cycle. The simplest way to explain the symmetry of the phenotype is if the SUMO-related event takes place in the center of the septin ring. During cytokinesis, an actomyosin contractile ring forms in the center of the septin ring and colocalizes with other proteins required for contractile ring function . Contraction of the ring is triggered by an unknown mechanism close to the same time the SUMO ring disappears. A SUMO–septin-related event could take place in this same region that would be required for the complete disassembly of both the mother and daughter cell sides of the ring. It is worth noting that the old septin rings in the sumoylation site mutant contained much less septin than the ring at the base of a growing bud, indicating that some disassembly did take place. Perhaps the remaining structure is “capped” in such a way that a posttranslational modification at the SUMO attachment sites is required to remove it. Either model can also explain the timing of SUMO removal and septin ring disassembly. If SUMO itself promotes disassembly, SUMO might be involved in an initial event, either the SUMO-dependent association with the septin ring of another protein that is later involved in disassembly, or direct SUMO participation in a preliminary disassembly event, whose effects only become apparent later in the cell cycle. Using this model, it is easier to explain the fact that only a small fraction of each of the septins is modified, because a small amount of modified septin might be sufficient to attract some other protein to the bud neck. On the other hand, the model where SUMO prevents disassembly by inhibiting some other process is more consistent with the timing of desumoylation, as septin ring disassembly actually starts after SUMO is removed. Removal of SUMO might serve as an initiation signal, allowing access to other proteins that trigger the septin rearrangements that take place during and after cytokinesis. However, as mentioned above, the main problem with this model is the extremely low percentage of septin polypeptides that are actually modified. This seems like an inefficient way to block an interaction or modification, since >95% of all sites still would be available. Another key question related to this model is whether any fraction of the septins is ubiquitinated and degraded at this point in the cell cycle. We were unable to detect high molecular weight Ub conjugates or any dramatic reductions in the steady-state levels of Cdc3 or Cdc11, but it is possible that a small fraction of the septins may be degraded during cytokinesis. Another important question is whether septin sumoylation plays other roles in initiation of anaphase, during anaphase, or in cytokinesis. It is still possible that septin sumoylation participates in a process carried out by two or more partially redundant pathways, or that it is part of a checkpoint monitoring a process that we have not perturbed in any of our experiments. Further analysis of genes that interact with the septin sumoylation site mutant should lead to a clearer picture of the role of septin sumoylation in yeast growth. | Study | biomedical | en | 0.999996 |
10579720 | The following primary antibodies were used: affinity-purified rabbit anti-ankyrin-B (COOH terminus specific) polyclonal antibody ; monoclonal antibody against the dihydropyridine receptor, α2 subunit, (Affinity Bioreagents); SERCA, type 1 and 2 (Affinity Bioreagents); ryanodine receptor type 1 and 2 (Affinity Bioreagents); triadin (Affinity Bioreagents); IP3R (type 1, 2, and 3; Accurate Biochemicals); and sarcomere α-actinin (Sigma). Secondary antibodies used were: rhodamine- or fluorescein-conjugated goat anti–mouse IgG (Pierce and Jackson ImmunoResearch Laboratories) at 5 μg/ml and Cy-5- or rhodamine-conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories) at 5 μg/ml. F-actin was visualized using biotin-phalloidin (5 U/ml; Molecular Probes) and Cy5-labeled streptavidin (Jackson ImmunoResearch Laboratories). Mouse genotypes were established by Southern blot analysis , or by PCR. Tissue samples and cells were processed for SDS-PAGE/immunoblots and immunofluorescence and by laser scanning confocal microscopy as described in previous publications . Hearts were dissected from neonatal (1–3-d-old) mice under sterile conditions by bathing the animal in 70% alcohol. Each heart was then placed in a 12-well flat-bottom tissue culture plate containing 2 ml of Ham's F10 nutrient media. The atrium and major blood vessels were removed and the ventricle was rinsed by squeezing with forceps to wash away the blood. The ventricle was minced finely in 1.5 ml of 0.05% trypsin, 0.2 mM NaEDTA, and placed in a humidified incubator (37°C, 95% air-5% CO2) for 15 min. Ventricles were then resuspended several times in a 1-ml Eppendorf pipette and incubated for another 15 min. After incubation, 0.2 ml of 2-mg/ml soybean trypsin inhibitor was added, followed by the addition of 0.2 ml of 0.2-mg/ml collagenase (type VII; Sigma). The homogenate was resuspended several times and incubated further for 30–50 min until dissociation of cells was complete. Then 2 ml of complete media (DME/Ham's F10, 10% FBS, and 10% HS) was added, and cells were pelleted by centrifugation at 500 g for 5–10 min. Cells were resuspended in 2 ml complete media and transferred to a 35-mm petri dish to remove fibroblasts by differential adherence (2 h at 37°C). Cardiomyocytes in the supernatant were collected by centrifugation at 500 g for 5–10 min, and resuspended in 1 ml complete media. 0.25 ml of cell suspension were plated on a fibronectin-coated plate at a density of 1 × 10 6 /ml and washed 24 h later with complete media to remove dead cells and debris. To minimize growth of nonmuscle cells, complete media was replaced with defined growth medium (DMEM/F10) with additions of insulin (1 μg/ml), transferrin (5 μg/ml), LiCl (1 nM), NaSeO 4 (1 nM), ascorbic acid (25 μg/ml), thyroxine (1 nm), or with serum-free medium (DMEM/F10). Cardiomyocytes were transfected using Effectene (Qiagen) with 0.1 μg of cDNA encoding 220-kD ankyrin-B or 190-kD ankyrin-G , both with EGFP positioned at the COOH termini of ankyrins in a pEGFP vector (Clontech). Quadriceps muscles from 7-d-old littermates were placed directly in Karnovsky glutaraldehyde/formaldehyde/cacodylate fixative plus tannic acid (pH 7.4) and sliced into smaller pieces . After fixing for 3 h (room temperature) or overnight (4°C), pieces were thoroughly rinsed in MOPS buffered mammalian Ringer and further fixed in 0.2% tannic acid, 3% glutaraldehyde in MOPS buffered Ringer (pH 7.0) for 30 min. After rinsing in MOPS buffered Ringer, followed by 100 mM phosphate buffer, pH 6.1, tissue was post-fixed for 1 h in ice-cold 1% OsO4 in 100 mM potassium phosphate buffer, pH 6.1, rinsed in water, block stained in 2% aqueous uranyl acetate for 1 h, rinsed in water, dehydrated in a graded ethanol series, and embedded in Araldite 506 . Ultrathin sections were photographed at magnifications ranging from 3,800 to 18,500 on a Philips EM420 microscope. Cytosolic Ca 2+ levels were viewed in spontaneously contracting neonatal cardiomyocytes loaded with 10 μM fluo-3/AM (Molecular Probes) by ratioing fluo-3 images (excitation 488 nm, emission 510 nm) against a reference image acquired at rest (I/I 0 ) as described . A 40× 1.2 NA water immersion objective was used and images were recorded at 6–12 frames/s. Data were analyzed using the LSM 410 data analysis software. Creatine kinase activity in mouse serum was measured using a Sigma Diagnostics Creatine Kinase kit. Fresh rat skeletal muscle samples were homogenized in a buffer (1 g/15 ml) containing 0.25 M sucrose, 100 mM Tris, pH 7.4, 5 mM sodium EDTA, 5 mM sodium EGTA, PMSF (200 μg/ml), leupeptin (10 μg/ml), AEBSF (0.5 mM), and pepstatin (10 μg/ml), centrifuged at 1,300 g for 10 min, rehomogenized and spun again. The supernatant was spun at 9,000 g for 10 min. The second supernatant was spun at 190,000 g for 1 h. Pellets were resuspended in 3 ml buffer and loaded on a 20–50% sucrose gradient, and then centrifuged at 150,000 g for 16 h. Samples were treated as described above. Ankyrin-B (−/−) mice display abnormal posture with kyphosis and winged scapulae . Creatine kinase activity also is elevated about fourfold in sera from ankyrin-B (−/−) mice compared with normal littermates, based on determinations of seven litters at ages ranging from postnatal day 1 to 13 . Measurements of neonatal heterozygotes revealed some mice (8 out of 26) with a two- to threefold increase and other mice with normal levels of serum creatine kinase activity. For comparison, patients with congenital myopathies or mild muscular dystrophy frequently have normal levels or small elevation in levels of creatine kinase activity. Transmission electron micrographs of ankyrin-B (−/−) heart (not shown) and skeletal muscle demonstrate overall normal sarcomere organization. However, occasional localized sites of severely disorganized sarcomeres and loss of striations encompassing 3–6 sarcomeres were observed in ankyrin-B (−/−) skeletal muscle . Disorganized sarcomeres were not observed in skeletal muscle from normal littermates, and also were not evident in heart sections of either wild-type or ankyrin-B (−/−) mice (not shown). Sites of disorganized sarcomeres, combined with elevation of serum creatine kinase activity and reduced myofibril size (not shown), support the conclusion that ankyrin-B (−/−) mice suffer from a neonatal myopathy and suggest a physiological role of ankyrin-B in muscle. Ankyrin-B polypeptides of 220 and 150 kD are expressed in skeletal muscle and heart of normal mice, and are not detectable in ankyrin-B (−/−) mice . Ankyrin-B (−/−) mice are born in Mendelian ratios, but suffer 70–80% mortality on postnatal day 1, and 100% by postnatal day 21. Causes of neonatal death are likely to include compromised function of striated muscle and the immune system (see below), in addition to previously described defects in the nervous system . Previous reports that ankyrin associates with IP3 receptors and ryanodine receptors suggested the possibility that Ca 2+ homeostasis might be abnormal in ankyrin-B (−/−) mice. Dynamic patterns of intracellular Ca 2+ release and uptake were measured in cardiomyocytes cultured from 1-d-old ankyrin-B (+/+) and (−/−) mice and maintained for 96 h, using Fluo-3 as an indicator . Ankyrin-B (+/+) and (−/−) cardiomyocytes in these cultures spontaneously contract, and have assembled sarcomeres and T-tubules, based on patterns of fluorescent labeling of α-actinin , F-actin , and voltage-gated calcium channels . Normal cardiomyocytes exhibit a regular sinusoidal oscillation of cytosolic Ca 2+ levels with a frequency of about 1 Hz and an increase in Ca 2+ elevation over the basal level of ∼2.5-fold . Ankyrin-B (−/−) cardiomyocytes exhibit an irregular pattern of Ca 2+ release with periods of prolonged elevation (average 2.5 times longer than normal) combined with a threefold reduction in frequency . The spatial pattern of Ca 2+ release also was abnormal in ankyrin-B (−/−) cells . Wild-type cardiomyocytes exhibited one or two well resolved sites of elevated Ca 2+ which then propagated along the cell , while mutant cells exhibited multiple simultaneous foci of elevated calcium . The distribution of SR proteins involved in Ca 2+ release and uptake was examined in ankyrin–4-B (+/+) and (−/−) cardiomyocytes maintained in culture for 4–6 d and in sections of skeletal muscle . SERCA 2 is distributed in normal cardiomyocytes in a longitudinal and cross-striated pattern coinciding with the network SR and sarcomeres, which are identified by labeling of α-actinin, a component of the Z-line . In striking contrast, SERCA 2 of ankyrin-B (−/−) cardiomyocytes is restricted to a perinuclear distribution and is completely absent from striations associated with sarcomeres . The absence of SERCA 2 from contractile units of ankyrin-B (−/−) cardiomyocytes could contribute to the prolonged time of decay of Ca 2+ transients observed in these cells . SERCA 1 also is missing from sarcomeres in sections of skeletal muscle of 7-d-old ankyrin-B (−/−) mice . Labeling for SERCA does occur along muscle fibers, but this may represent nonspecific interactions of antibodies with components of connective tissue . Ryanodine receptors are abnormally localized in ankyrin-B (−/−) cardiomyocytes , and skeletal muscle . Ryanodine receptor type 2 of normal cardiomyocytes is present in a single periodic pattern that overlays the Z-line (not shown) and corresponds to the site of T-tubules in heart cells. Ryanodine receptor type 1 in normal skeletal muscle is distributed in a periodic pattern as a double row , as expected from a distribution flanking T-tubules in these cells. Ryanodine receptor type 2 in ankyrin-B (−/−) cardiomyocytes is localized in 0.5–1-μm structures dispersed throughout the cell and in a pattern distinct from striations observed in normal cells . Ryanodine receptor type 1 in ankyrin-B (−/−) skeletal muscle exhibits only occasional sites of label within muscle fibers . Fluorescent label for the ryanodine receptor type 1 is localized adjacent to the plasma membrane in ankyrin-B (−/−) fibers , but this may represent nonspecific interactions of antibodies as noted above with SERCA. Transfection of ankyrin-B (−/−) cardiomyocytes with cDNA encoding 220-kD ankyrin-B with a COOH-terminal green fluorescent protein (GFP) tag restored normal striated patterns of distribution of SERCA 2 as well as ryanodine receptor type 2 . 220-kD ankyrin-B–GFP in rescued cardiomyocytes was visualized with antibody against GFP, and exhibited a striated distribution similar to that of native ankyrin-B . The presence of sarcomeres in these cardiomyocytes was established by labeling F-actin with biotin-phalloidin/Cy5-streptavidin . Ability to restore normal organization of both SERCA 2 and ryanodine receptor type 2 with 220-kD ankyrin-B demonstrates that other ankyrin-B spliceoforms, such as the 150-kD polypeptide, are not required for this activity. Transfection with cDNA encoding 190-kD ankyrin-G–GFP did not restore SERCA 2 or ryanodine receptor type 2 patterns . Moreover, 190-kD ankyrin-G–GFP was not localized in a striated pattern but instead was distributed more or less evenly throughout the cytoplasm of transfected cardiomyocytes . 190-kD ankyrin-G–GFP polypeptide contains the same domains as 220-kD ankyrin-B, but has a shorter COOH-terminal domain . Ankyrin-G and ankyrin-B thus have distinct and nonoverlapping localizations and functions in striated muscle. The abnormal localization of ryanodine receptors and SERCA in cardiomyocytes and skeletal muscle could be due either to defects in targeting of these protein to otherwise normal SR and junctional SR sites, or to abnormal localization of the entire SR and junctional SR. Immunofluorescence using antibodies against established components of SR/T-tubule junctions and electron microscopy were used to distinguish between these possibilities. The pattern of labeling for triadin, a SR protein associated with ryanodine receptors at SR/T-tubule junctions , is normal in ankyrin-B (−/−) skeletal muscle . Moreover, labeling for the dihydropyridine receptor also is normal in skeletal muscle as well as cardiomyocytes . These results suggest that T-tubules and the SR are present in the correct location and are linked by junctions. Direct evidence for apparently normal SR/T-tubule junctions and SR in ankyrin-B (−/−) skeletal muscle is provided by electron micrographs . Wild-type and ankyrin-B null skeletal muscles both have small T-tubules (small arrows) positioned about midway between the Z-bands (Z) and the M-lines of well-ordered sarcomeres . Where the section plane is favorably oriented, the network of membranes of the longitudinal sarcoplasmic reticulum (SR) can be visualized as well-organized around the myofibrils and sarcomeres in both normal and ankyrin (−/−) fibers . Junction (triads) between the T-tubule (lumen marked by t) and the SR membranes (arrows) also appear similarly well-organized overall, with the density between the SR and T-tubule membranes of the triads (arrows) equally evident in normal and ankyrin-B (−/−) fibers. A gallery of skeletal muscle triads from normal (A–D) and ankyrin-B (−/−) (E–H) animals visualized at higher magnification are presented in Fig. 7 . In both wild-type and ankyrin-B (−/−) skeletal muscle, the junctional SR (JSR) membrane forms a sac (arrowhead) abutting the T-tubule, where the JSR is seen en face. The JSR membrane is usually marked by a row of particles parallel to the junction and is frequently associated with filamentous densities extending away from the junction at right angles. The particles evident in ankyrin-B (−/−) muscle presumably are comprised of the voltage-gated calcium channel, triadin, and perhaps other SR and/or T-tubule proteins, but not ryanodine receptor 1, which is absent based on immunofluorescence . Measurements across 9 examples of well-oriented T-tubule–JSR membrane junctions in normal and 9 in ankyrin-B (−/−) skeletal fibers visualized at the same magnification indicated a ten percent reduction in thickness of the ankyrin-B (−/−) junctions. (The range in normal fibers was 0.9–1.1 mm [average 1.01 mm]; the range in mutant was 0.8–1.0 mm [average 0.92 mm].) Complete absence of ryanodine receptor type 1 in the RYR1 (−/−) mouse results in loss of particles in SR–T-tubule junctions and a reduction in thickness of ∼40% . Retention of structures in SR–T-tubule junctions in our sections of ankyrin-B (−/−) skeletal muscle suggests that ryanodine receptors are not completely absent. One possible explanation is that ryanodine receptors are reduced in amount such that they are not evident by immunofluorescence, especially at high contrast, but are present in sufficient quantities to detect by electron microscopy. The combined observations of normal immunofluorescent patterns of labeling of triadin and DHPR and electron microscopy strongly support the interpretation that the SR and SR–T-tubule junctions are normal overall in localization in ankyrin-B (−/−) skeletal muscle. The reduced amount of ryanodine receptors and absence of SERCA from a normal striated pattern in ankyrin-B (−/−) skeletal muscle therefore reflects a defect in targeting of these proteins to their cellular sites. A similar conclusion also is true for heart based on electron microscopy (not shown), and the normal appearance of DHPR in cardiomyocytes . Ankyrin-B in skeletal muscle is located in two sites that can be resolved in longitudinal and in transverse sections of muscle fibers. One site, visualized in an optical section along the surface of the plasma membrane, is in a costamere pattern at the sarcolemma, and is aligned with the Z-lines that are labeled by α-actinin . The other location, visualized with an optical section through the interior of the fiber, is in intracellular punctate structures aligned with the A-band . Transverse sections also reveal ankyrin-B staining at the sarcolemma, and in a punctate intracellular pattern . The pattern of ankyrin-B labeling in costameres and in intracellular sites over the A-band is distinct from the localization of ankyrin noted at T-tubules . However, ankyrin-B localization does closely resemble the labeling obtained by Nelson and Lazarides 1984 with antibody raised against chicken erythrocyte ankyrin. Double immunofluorescence labeling of transverse sections of skeletal muscle reveals that ankyrin-B is localized at sites distinct from both SERCA 1 and from ryanodine receptor type 1 . The majority of SERCA1 and ryanodine receptor type 1 are clearly not in close contact with ankyrin-B . Therefore, ankyrin-B cannot participate as a common structural component of the SR–T-tubule junction. Possible mechanisms involving a catalytic role of ankyrin-B in localization of ryanodine receptors and SERCA are discussed below. Subcellular fractionation of skeletal muscle supports the idea that the punctate intracellular staining of ankyrin-B represents vesicles . The majority of 220-kD ankyrin-B pelleted after centrifugation for 10 min at 2,000 g , and presumably is associated with myofibrils (data not shown). However, some 220-kD ankyrin-B sedimented only at high speeds (at least 30 min at 100,000 g ). Particulate 220-kD ankyrin-B sediments with a similar density to SERCA and ryanodine receptors after sedimentation to equilibrium in sucrose density gradients, and therefore most likely is associated with membranes . Visualization of membrane-associated ankyrin-B by immunofluorescence microscopy revealed small structures <1 μm in diameter that presumably represent small vesicles and are distinct from vesicles labeled for SERCA (not shown). IP3 receptors are widely expressed channels responsible for intracellular Ca 2+ release regulated by IP3 and Ca 2+ , and are coexpressed with ryanodine receptors in striated muscle as well as other tissues. IP3 receptors visualized by immunofluorescence are mis-sorted in ankyrin-B neonatal cardiomyocytes . IP3 receptors in normal cardiomyocytes are distributed in a striated pattern, while in mutant cells IP3 receptors are in an irregular punctate distribution . IP3 receptors in normal skeletal muscle are distributed in a SR pattern distinct from the pattern of ryanodine receptors, while IP3 receptors in ankyrin-B (−/−) skeletal muscle are distributed throughout the cytoplasm (not shown). Levels of accumulation of IP3 receptors are reduced by at least 50% in ankyrin-B (−/−) heart tissue . The neonatal thymus is an active site of T cell differentiation, a process that is regulated by intracellular calcium transients mediated in part by IP3 receptors and ryanodine receptors . 220- and 150-kD ankyrin-B polypeptides are expressed in normal thymus . The 220-kD polypeptide is not detectable and the 150-kD polypeptide is reduced >90% in ankyrin-B ( − / − ) mice . IP3 receptors are abundantly expressed in normal thymus and are localized in a punctate perinuclear pattern in thymic lymphocytes . IP3 receptors in ankyrin-B ( − / − ) thymus are reduced in immunoblots by ∼50% . IP3 receptors in ankyrin-B ( − / − ) lymphocytes also exhibit an altered pattern of localization . IP3 receptors in these lymphocytes are confined adjacent to the plasma membrane, and are generally not present in the perinuclear pattern observed in normal cells . Reduced accumulation and abnormal localization of IP3 receptors in ankyrin-B (−/−) thymus would be anticipated to interrupt normal calcium signaling and differentiation of T cells. Consistent with such a loss of signaling, Toluidine blue–stained sections of ankyrin-B (−/−) neonatal thymus reveal cell death of a major fraction of T cells . Epithelial cells are present in equivalent numbers and organization, while the majority of T cells are missing or exhibit pyknotic nuclei. In contrast, sections of normal thymus are densely populated with T cells containing normal nuclei. This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (−/−) mice, as well as dramatic alterations in intracellular localization of key components of the Ca 2+ homeostasis machinery in ankyrin-B (−/−) striated muscle and thymus. The SR and SR–T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (−/−) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and DHPR. The abnormal localization of SERCA and ryanodine receptors therefore represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (−/−) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (−/−) cardiomyocytes is rescued by expression of the 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence for a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Evidence in support of some form of direct interactions of ankyrin-B with ryanodine receptors and IP3 receptors is provided by in vitro binding assays . In addition, our laboratory has isolated IP3 receptors from cerebellum with an ankyrin-B membrane-binding domain affinity column, and determined a relatively modest K D of 0.2 μM for ankyrin-B–IP3 receptor binding (data not shown). Such an affinity would be consistent with weak or short-lived interactions. SERCA has not been examined for ankyrin-B binding activity. However, SERCA shares overall sequence similarity with the Na/K ATPase, which does associate with ankyrin . Direct but transient interaction of Ca 2+ homeostasis proteins with ankyrin-B could occur during transit of these proteins between the ER and the SR. Exchange of proteins between ER and Golgi is well known to involve a machinery for recruitment of specific cargo proteins into vesicles, and transfer of these vesicles between organelle compartments. A comparable system may also mediate transfer of functionally defined proteins from the ER to specialized sites within the SR. In this case, the basic defect in ankyrin-B (−/−) cells underlying mis-sorting of multiple Ca 2+ homeostasis proteins could be a failure in some step required for segregation of these proteins and/or their transport between the ER and SR. According to this hypothesis, ankyrin-B would function as a SR-specific guide or escort using the touring metaphor for protein sorting . Ankyrin-B is a multifunctional protein that could participate in ER protein sorting at several levels. The multivalent ankyrin membrane-binding domain could bind to and laterally segregate selected proteins within the plane of the ER membrane . Ankyrin-B also could participate in coupling vesicles to transport systems through interactions of the spectrin-binding domain or the microtubule-association site . Coordinated assembly and spatial organization of Ca 2+ release channels and SERCA within the ER is essential in cells of the immune system as well as in striated muscle. Lymphocytes encode information in the amplitude and frequency of intracellular waves of Ca 2+ . Unlike the highly organized SR of striated muscle, lymphocytes are likely to have variable states of organization of ryanodine receptors, IP3 receptors, and SERCA that depend on their differentiation state. As a consequence of such plasticity, signals that elevate calcium can have opposite effects in naive and differentiated cells . Our findings suggest that ankyrin-B is a candidate to participate in targeting IP3 receptors and possibly other Ca 2+ homeostasis proteins to their physiological sites in T-lymphocytes. The fact that IP3 receptor accumulation is reduced in the absence of ankyrin-B also implies regulation by ankyrin-B at some level of IP3 receptor biosynthesis in addition to spatial targeting. Ankyrin-B deficiency apparently has profound consequences for survival of neonatal thymic lymphocytes , as expected if signals promoting differentiation are interrupted. The specialized neuronal ER involved in calcium homeostasis is located in dendritic spines and growth cones of axons and resembles the SR found in muscle as visualized by electron microscopy . 220-kD ankyrin-B is expressed in neuron cell bodies and dendrites in the postnatal brain in a time frame that approximates development of the dendritic spine apparatus of hippocampal and Purkinje neurons , while 440-kD ankyrin-B is targeted to axons early in development . Although ankyrin-B (−/−) mice did not survive long enough to obtain sufficient numbers for a complete study, in two examples accumulation of IP3 receptors was significantly reduced in dendrites and cell bodies of Purkinje neurons (data not shown). These preliminary results suggest that 220-kD ankyrin-B is a good candidate to participate in targeting ER proteins dedicated to calcium homeostasis to their physiological sites in dendrites of neurons, while 440-kD ankyrin-B may have a similar function in axons. Complete ankyrin-B deficiency in humans would be anticipated to involve defective development of the nervous system, as well as severe dysfunction of striated muscle and immune system that would not be compatible with prolonged postnatal life. However, individuals with weak alleles or partial deficiency of ankyrin-B may be viable. Ankyrin-B (+/−) mice have reduced expression of ankyrin-B and survive to adulthood, although with musculoskeletal defects that remain to be characterized (not shown). These animals may provide models for certain autosomal dominant human channelopathies involving Ca 2+ release channels and SERCA, with loss of function due to mis-sorting rather than direct mutations in these proteins. For example, malignant hyperthermia, a leading cause of anesthesia-associated mortality, can be caused by mutations in the ryanodine receptor . However, the genetic basis for this disorder is heterogeneous, suggesting that proteins in addition to the ryanodine receptor also are involved . Another candidate disease involving ankyrin-B is long QT interval type 4, an autosomal dominant cardiac arrhythmia associated with sudden death, that maps to the same chromosomal region of 4q25-27 as the ANK2 gene encoding ankyrin-B . Ankyrin-G and ankyrin-R polypeptides are still expressed in the ankyrin-B (−/−) cardiomyocytes (not shown), and 190-kD ankyrin-G cannot rescue ankyrin-B (−/−) cardiomyocytes . These data indicate that ankyrins, unlike many other multigene families, do not compensate for each other, and presumably have gene-specific functions. The lack of complementation between ankyrins suggests the presence of binding partners specific for each ankyrin, which have not yet been identified. Therefore, the activities proposed above for ankyrin-B in ER protein sorting are likely to involve protein interactions unique for ankyrin-B and not accessible to ankyrin-G. It will be of interest in future experiments to use chimeric ankyrins to define key domains involved in rescue of SERCA and ryanodine receptor sorting as well as cellular targeting of ankyrin-B. Ankyrin-G and ankyrin-B both have been implicated in segregating diverse proteins within functionally defined membrane domains. However, ankyrin-G–dependent proteins are associated with specialized regions of the plasma membrane , and ankyrin-B–dependent proteins are shown here to be localized in the Ca 2+ homeostasis compartment of the ER. These parallels suggest the possibility that related mechanisms involving ankyrins but with distinct interacting proteins are responsible for restriction of proteins within certain specialized regions of plasma membrane and within the ER. A common feature of both pathways may be recognition of target proteins by ankyrin membrane-binding domains and their constituent ANK repeats. Since proteins lacking any obvious sequence homology interact with multiple distinct sites on the ankyrin membrane-binding domain , these protein interactions are likely to result from an evolved fit mechanism rather than from targeting through a conserved sequence motif. A consequence is that evolution of ankyrin-based recognition could be driven by functional considerations of increased physiological efficiency. Other aspects of the pathways for segregation of ankyrin-dependent plasma membrane and ER proteins are also likely to be conserved, but are currently undefined. A working hypothesis for future work is that both plasma membrane and ER segregation pathways will involve formation and transport of vesicle/tubule intermediates similar to currently established communication between the ER and Golgi. | Study | biomedical | en | 0.999997 |
10579721 | NIH3T3 fibroblast cells were cultured in DME supplemented with 10% newborn calf serum (Gibco-BRL Life Technologies). For serum starvation, cells were cultured for 24–36 h in DME supplemented with 0.2% newborn calf serum. Stable cell lines expressing the hemagglutinin (HA) epitope–tagged proteins C1199Tiam1 and C580Tiam1 (encoding the 1,199– and 580–COOH-terminal amino acids of Tiam1) or the Myc epitope–tagged GTPases were generated by retroviral transduction and selected with neomycin or zeocin. HA-tagged C1199Tiam1, C580Tiam1, and Myc-tagged V12Rac have been described and were cloned into an LZRS-IRES-Neo retroviral vector, a modified LZRS vector , conferring neomycin resistance. Myc-tagged RhoV14 and N19Rho were cloned as an EcoRI fragment in LZRS-IRES-Zeo that confers zeocin resistance. Myc-tagged L61Rac, L61RacA37, and L61RacC40 were cloned from pRK5 as a ClaI-NotI fragment into the SfuI and NotI sites from LZRS-IRES-Neo. The Myc-tagged fast cycling F28LCdc42 mutant was generated by a standard PCR procedure and cloned into the EcoRI site of LZRS-IRES-Neo. Activated Pak1 E423 was provided by G. Bokoch (The Scripps Research Institute, La Jolla, CA) and J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA) and cloned as a SalI-NotI fragment into LZRS-IRES-Neo. To produce retroviruses, Phoenix packaging cells were transfected with retroviral constructs encoding Tiam1 or GTPase proteins as described . Transductants were selected in DME containing neomycin (0.8 mg/ml), zeocin (0.2 mg/ml), or a combination of both drugs in case of doubly transduced cell lines. Expression of proteins in the pool of transduced cells was analyzed by Western blot, as described by Stam et al. 1997 . Dissociation assays were performed as described . The cells were scraped in culture medium and suspended by repeated pipetting. To demonstrate calcium sensitivity of cell–cell adhesion, calcium in the medium was chelated by the addition of 4 mM EGTA for 3 h before the dissociation assay. The number of particles (cell clusters) was counted and divided by the number of total cells (Np/Nc). For association assays, cells were scraped off the dish and suspended in medium by repeated pipetting to single cells. Dissociated cells were allowed to associate for 1 h in culture medium while rotating (37°C, 5% CO 2 ). The number of particles was counted and divided by the number of total cells (Np/Nc). Cells grown on glass coverslips were proceeded as described Stam et al. 1998 . C1199Tiam1 and C580Tiam1 were stained with anti–DH antibody or mAbs against the HA tag (12CA5; Boehringer Mannheim), N- and P-cadherins with the monoclonal Pan-cadherin antibody (Sigma Chemical Co.), vinculin with the monoclonal hVinc-1 (Sigma Chemical Co.), and α-, β-, and γ-catenin and p120 CAS were stained with respective mAbs from Transduction Laboratories. For the scratch assay, cells were nearly grown to confluency before 12 h serum starvation. The cell monolayer was wounded with a plastic pipet tip, generating a wound spanning ∼15–20 cells. Migration of cells was judged by monitoring the closure of the wound over a period of 12 h in medium containing 0.2% newborn calf serum. Cell migration assays were performed using transwell migration chambers (diam, 6.5 mm; pore size, 8 μm; Costar Corp.) coated on both sides of the membrane with fibronectin (10 μg/ml; Sigma Chemical Co.) in PBS for 12 h at 4°C. The coated filters were rinsed once with PBS and placed into the lower chamber containing medium supplemented with 20 ng/ml PDGF (Calbiochem-Novabiochem). Trypsinization of cells was kept to a minimum time to preserve the presence of cell surface adhesion molecules as much as possible. 70,000 cells were added to the upper compartment of the transwell chamber and allowed to migrate to the underside of the top chamber for 2–3 h. Nonmigrated cells on the upper membrane were removed with a cotton swab, and migrated cells attached to the bottom surface of the membrane were fixed for 10 min in methanol, stained with Giemsa, and counted. Cloning of the GST-PAK-CD fusion protein (encompassing amino acids 56–141 from PAK1B), containing the Rac and Cdc42 binding region from human PAK1B, has been described . GST-C21 has been described by Reid et al. 1996 , Reid et al. 1999 and contains the NH 2 -terminal 90 amino acids, representing the Rho binding domain, from the Rho effector protein Rhotekin. Escherichia coli BL21 cells transformed with the GST-PAK-CD construct were grown at 37°C, cells transformed with the GST-C21 construct were grown at 30°C to OD 600 0.3. Expression and purification of recombinant proteins has been described . GTPase activity assays were performed as described . In brief, lysates of NIH3T3 cells were prepared and incubated with bacterially produced GST-PAK-CD or GST-C21 fusion proteins, bound to glutathione-coupled Sepharose beads. The beads and proteins bound to the fusion protein were washed in an excess of lysis buffer, eluted in Laemmli sample buffer, and analyzed for bound Cdc42, Rac1, or RhoA molecules by Western blot using antibodies against Cdc42 (rabbit polyclonal antibody from Santa Cruz Biotechnology), Rac1 (mAb from Transduction Laboratories or when indicated Upstate Biotechnology Inc.) or RhoA (mAb from Santa Cruz Biotechnology). To study the molecular basis of the morphological transformation of NIH3T3 fibroblasts upon expression of Tiam1 , we generated NIH3T3 cell lines stably expressing C1199- and C580Tiam1 cDNAs by retroviral transduction . Stable pools of cells expressing C1199Tiam1 grew in small groups of flat, spread cells and displayed an epithelial-like morphology. The C1199Tiam1 protein was predominantly localized at the plasma membrane and induced extensive membrane ruffling. Moreover, C1199Tiam1 was also enriched at the sites of cell–cell contact, which was accompanied by increased actin polymerization . We previously showed that Tiam1-induced Rac activation promotes E-cadherin–mediated cell–cell adhesion in epithelial cells . Therefore, we studied whether the Tiam1-induced epithelial-like morphology in NIH3T3 fibroblasts was due to the establishment of cadherin-based cell–cell adhesions. NIH3T3 fibroblasts do not express E-cadherin, but instead express the family members N- and P-cadherin . In the C1199Tiam1-expressing cells, N-cadherin and P-cadherin as well as the cadherin-associated catenins, such as α-, β-, and γ-catenin (not shown) and p120 CAS localized to sites of cell–cell contact. The adhesion-related proteins were to some extent also detected in Tiam1-induced membrane ruffles . In contrast, the phenotype of cells expressing the C580Tiam1 protein was not different from untransfected control cells . C580Tiam1, lacking the NH 2 -terminal PH domain as a membrane localization signal , was predominantly cytoplasmic and did not result in phenotypic changes such as ruffling or increased actin polymerization between neighboring cells . Cells expressing the nonfunctional C580Tiam1 protein displayed no relocalization of components of cadherin adhesion complexes , indicating that membrane localization of Tiam1 is required to induce the recruitment of cadherins and members of the cadherin complex to sites of cell–cell contact. To demonstrate a functional increase in the strength of cell–cell adhesion induced by Tiam1, we used dissociation and association assays. For dissociation studies, subconfluently grown control NIH3T3 cells or cells expressing C1199- or C580Tiam1 were scraped off the dish, suspended by repeated pipetting, and the number of cell aggregates was counted. Expression of C1199Tiam1 increased the number and size of aggregates considerably when compared with C580Tiam1, indicative for increased cell–cell adhesion. Formation of aggregates by expression of C1199Tiam1 was calcium-dependent, supporting a role for cadherin-based cell–cell adhesions . In the reverse experiment, NIH3T3 cells expressing either Tiam1 protein were suspended by pipetting to single cells and subsequently rotated in cell culture medium to allow the formation of cell–cell contacts. Quantification of the cell aggregates that reformed showed that C1199Tiam1 but not C580Tiam1 induced cell aggregation, indicating that C1199Tiam1 promotes adhesion between cells . Taken together, these data indicate that C1199Tiam1-induced Rac activation leads to recruitment of cadherin-based adhesion complexes to sites of cell–cell contact that results in a functional increase of cell–cell adhesion and an epithelial-like morphology of NIH3T3 fibroblasts. We could not detect changes in protein expression of cadherins or associated complex members in the Tiam1-expressing cell lines compared with wild-type fibroblasts (data not shown). Most likely, the Tiam1/Rac–mediated increase in cell–cell adhesions is due to increased actin polymerization at cell–cell contacts, which facilitates the formation of functional cadherin-based adhesions, similarly as found in epithelial cells . C580Tiam1 still contains the catalytic DH domain, but does not localize at the cell membrane and does not induce an epithelial-like morphology, including membrane ruffling. To study whether C580Tiam1 is able to activate Rac, we compared the activation of endogenous Rac in cells expressing C1199- or C580Tiam1 . Cell lysates expressing either one of the Tiam1 proteins were incubated with a fusion protein of glutathione-S-transferase (GST) and the CRIB domain of the Rac/Cdc42 effector molecule Pak (GST-PAK-CD). Activated, i.e., GTP-loaded Rac bound to GST-PAK-CD, was analyzed by Western blot with an antibody directed against Rac1. C1199Tiam1 strongly activated endogenous Rac, compared with control NIH3T3 cells . Expression of the cytoplasmic C580Tiam1 containing the catalytic DH domain only weakly stimulated Rac activity . This indicates that efficient activation of endogenous Rac by Tiam1 requires membrane localization of the GEF. In addition to Rac, Cdc42 also has been implicated in the formation of cell–cell adhesions in epithelial cells . To determine whether the observed Tiam1-induced cell–cell adhesion in fibroblasts could involve activation of Cdc42, GST-PAK-CD precipitates were incubated with an antibody directed against Cdc42. Cdc42 activity was not increased by either Tiam1 protein . These data demonstrate that in NIH3T3 fibroblasts, Tiam1 functions as a specific activator of Rac and not Cdc42. We conclude that activation of Rac and not Cdc42 is responsible for the morphological transformation and increased cell–cell adhesion of Tiam1-expressing NIH3T3 cells. In addition to promoting cadherin-mediated cell–cell adhesion in fibroblasts, C1199Tiam1 also induced increased spreading of fibroblasts when compared with control cells . Enhanced cell spreading was found on different substrates including vitronectin, fibronectin, collagen I, and laminin 1 (data not shown). This is consistent with findings in neuronal and lymphoid cells, where Rac was shown to promote integrin-mediated cell spreading and adhesion . To study the effect of Tiam1-mediated Rac activation on migration, we quantified the migratory behavior of control and C1199Tiam1-expressing fibroblast cells using a transwell invasion assay. Expression of C1199Tiam1 resulted in inhibition of fibroblast migration through filters coated with fibronectin as compared with the control cells . Fibroblasts expressing C580Tiam1, which does not activate Rac , showed a migration rate similar to control cells (data not shown). This indicates that the migratory behavior of fibroblasts is inhibited by Tiam1-mediated Rac activation, similarly as found in epithelial cells . Upon serum starvation, C1199Tiam1-expressing NIH3T3 cells lost their epithelial-like morphology. The cadherin-mediated cell–cell contacts were largely disassembled and many cells acquired a polarized, sickle-shaped phenotype . As determined in scratch assays, these serum-deprived cells did not migrate (data not shown). Addition of serum partly restored the epithelial-like morphology within 3–5 h, the cells appeared more round again and began to re-establish cell–cell contacts . Growth factors such as LPA, PDGF, insulin, and bradykinin were tested for their capability to substitute for serum to restore the epithelial-like phenotype of the serum-starved C1199Tiam1-expressing NIH3T3 cells. From all growth factors tested, only LPA was capable of partly restoring the epithelial-like morphology, similar to serum . The C1199Tiam1 protein was localized to membrane ruffles that were also restored in the presence of LPA. Serum or LPA had to be present for a few hours before restoration of the epithelial-like phenotype was observed. LPA is a major component in serum and a potent activator of Rho . To substantiate the role of Rho in the maintenance of the epithelial-like phenotype, Rho was inactivated by treating C1199Tiam1-expressing NIH3T3 cells grown in the presence of serum with C3 transferase toxin. Similar to serum-starved cells , these cells became sickle-shaped and lost their epithelial-like morphology including most membrane ruffles . From these data we conclude that Rho activity is required for both cadherin-mediated cell–cell adhesion and membrane ruffles. To assess activation of endogenous Rho protein by LPA, we used the Rho binding domain (GST-C21) of the Rho effector Rhotekin fused to GST . As expected, control fibroblasts showed a significant downregulation of Rho activity upon 24 h serum starvation . LPA (lane 3) or serum (not shown) treatment of these cells led to complete restoration of the Rho activity. Similarly, LPA led to the activation of endogenous Rho (lanes 5 and 4) but not Rac protein (lanes 6 and 7) in serum-starved C1199Tiam1-expressing fibroblasts. These biochemical data are consistent with the established link between LPA-mediated receptor stimulation and subsequent activation of Rho . However, comparing control and Tiam1-expressing fibroblasts, we found that in serum-starved C1199Tiam1-expressing cells almost no Rho activity was detectable (compare lanes 5 and 2), whereas C1199Tiam1-expressing cells grown in the presence of serum still showed some Rho activity . Similarly, Rho activation after stimulation with LPA was much lower in C1199Tiam1-expressing cells than the activation of Rho by LPA in control cells . Thus, expression of C1199Tiam1 resulted in decreased Rho activity and a desensitized stimulation by LPA. This suggests, that sustained activation of Rac by Tiam1 resulted in downmodulation of Rho activity. Complete inactivation of Rho, either by the absence of serum or C3 transferase treatment , gave rise to sickle-shaped C1199Tiam1-expressing cells. The phenotypic switch because of complete inactivation of Rho could be restored by LPA- or serum-mediated activation of Rho, indicating that the maintenance of the Rac-induced epithelial-like morphology of NIH3T3 cells requires at least a basal Rho activity. The different levels of Rho activities in serum-starved control and C1199Tiam1-expressing cells suggested that the phenotype of C1199Tiam1-expressing cells, characterized by an epithelial-like morphology and inhibited migration, might not only be determined by activation of Rac, but also by downregulation of Rho signaling. Therefore, we determined in more detail the effect of Tiam1-mediated Rac activation on the activity state of endogenous Rho protein. In the presence of serum, expression of C1199Tiam1 caused a strong decrease of Rho activity, compared with control cells . Expression of C580Tiam1 resulted in a minor decrease of Rho activity, consistent with the little activation of Rac found to be induced by this protein . Similar to C1199Tiam1, also constitutively active V12Rac resulted in downregulation of endogenous Rho activity , indicating that inhibition of Rho activity occurred downstream of Rac. It could be argued that downregulation of Rho is a consequence of Rac-induced cellular spreading, which is accompanied by cytoskeletal changes rather than by Rac-mediated signaling events. To address this issue, we measured Rho activity of attached and suspended cells in relation to Rac activation. V12Rac-expressing cells showed a similar degree of Rac activity, independent from attached or suspended conditions . Irrespective of cell adhesion, these cells also showed a strong downregulation of Rho activity when compared with the control cells . This argues that the downregulation of Rho activity is not due to cytoskeletal rearrangements caused by Rac-induced cell spreading, but rather the result of signaling events downstream of Rac. Although wild-type and Tiam1-expressing fibroblasts display different cell shapes and are, therefore, difficult to compare, we tried to address the corresponding cytoskeletal changes after LPA stimulation with respect to typical Rho-dependent cytoskeletal organization. We monitored the distribution of vinculin-containing adhesion complexes and actin stress fibers. For this, wild-type and C1199Tiam1-expressing cells were mixed and grown on coverslips. After serum starvation, the cells were subsequently stimulated with LPA or serum and analyzed by immunocytochemistry. After LPA or serum stimulation, wild-type fibroblasts are characterized by relatively high Rho activity, whereas Tiam1-expressing cells show decreased levels of Rho activity, as a result of Tiam1-mediated Rac activation . In wild-type NIH3T3 cells, we observed the typical Rho-induced pattern of pronounced punctuate spots of focal contacts containing vinculin at the end of thick, heavy bundles of stress fibers upon LPA or serum (data not shown) stimulation. In C1199Tiam1-expressing cells (characterized by their roundish shape, see arrows), the actin fibers appeared finer and less bundled compared with wild-type cells . Vinculin-containing adhesion complexes were present at the cell periphery as well as small fine spots throughout the basal side of the cell . The larger spots at the cell periphery may correspond to the previously described Rac-induced focal complexes , and mostly did not overlap with the endpoints of stress fibers. The distribution of the small vinculin-containing complexes throughout the basal side of the cell followed the distribution of thin stress fibers and, therefore, most likely represent true Rho-induced focal contacts. The Rho-dependent cytoskeletal organization is, therefore, consistent with the low Rho activity observed after LPA or serum stimulation in Tiam1-expressing cells. Compared with wild-type fibroblasts, Tiam1-expressing cells exhibit less bundled and very thin stress fibers, which are accompanied by small focal contacts. To get insight into the signaling events downstream of Rac responsible for downregulation of Rho activity, we generated NIH3T3 cells stably expressing the Rac effector mutants L61Rac, L61RacA37, and L61RacC40. Consistent with the findings in Swiss 3T3 fibroblasts , activated L61Rac induced similar phenotypic changes as activated V12Rac, including membrane ruffling and lamellae formation in NIH3T3 fibroblasts . L61RacA37, which has been described to bind Pak1 and to activate the Jun kinase pathway, did not cause cytoskeletal rearrangements, whereas L61RacC40, which cannot bind Pak1 or activate the Jun kinase pathway, induces cytoskeletal reorganization similar to L61Rac. By assaying binding of the Rac effector mutants to GST-PAK-CD, we confirmed that L61Rac and L61RacA37 efficiently bound to GST-PAK-CD, whereas L61RacC40 did not . To gain insight into the possible Rac effectors mediating the downregulation of Rho, we determined Rho activities in fibroblast lines expressing the Rac effector mutants . Downregulation of Rho by L61Rac was comparable to V12Rac . Expression of both L61RacA37 and L61RacC40 resulted also in strong downregulation of Rho activity , although L61RacA37 mediated this response somewhat less effectively than L61Rac and L61RacC40. A stable cell line expressing an activated mutant of Pak1, Pak E423 , did not show downregulation of Rho activity (data not shown). This confirms the findings with the L61RacC40 effector mutant, and suggests that the Rac-mediated downregulation of Rho is not dependent on Pak1. Furthermore, on the bases of the L61RacA37 mutant, activation of the Jun kinase pathway seems not to be involved in signaling towards Rho. Similarly, Rac signaling pathways leading to reorganization of the cytoskeleton, do not play a role in inactivation of Rho, substantiating the conclusions drawn from the experiments with adherent and suspended cells. To address whether Rac and Rho regulate their activities in a reciprocal manner or whether downregulation of Rho activity is specifically mediated by Rac, we established an NIH3T3 cell line stably expressing Myc-tagged V14Rho . Rac activity in V14Rho-expressing cells was not significantly changed compared with control cells , consistent with the equal Rac activities found in serum-starved Tiam1-expressing cells and cells stimulated with LPA . Similarly, blocking Rho activity by dominant negative N19Rho or treatment of cells with the Rho-kinase inhibitor Y27632 (not shown) did not affect endogenous Rac activity. From these data, we conclude that V14Rho or LPA-mediated Rho activation does not result in downmodulation of Rac activity, and neither does dominant negative N19Rho result in upregulation of Rac activity. Apparently, modulation of Rho activity itself does not affect cellular Rac activity in NIH3T3 cells. To investigate the role of Cdc42 in the regulation of Rho activity, we established an NIH3T3 cell line expressing a Myc-tagged fast cycling Cdc42 mutant F28ACdc42 that has been previously described by Lin et al. 1997 . We used this fast cycling Cdc42 mutant because we were unable to obtain stable cell lines expressing V12Cdc42. F28ACdc42 downregulated Rho activity, similar to V12Rac , indicating that both Rac and Cdc42 are capable of modulating Rho activity. F28ACdc42 clearly functioned as an activated mutant of Cdc42 and also activated endogenous Rac to some extent in NIH3T3 cells , consistent with previous findings based on cytoskeletal changes in Swiss 3T3 fibroblasts . Therefore, downregulation of Rho activity by Cdc42 may either involve activation of Rac or occur independently of Rac, possibly by a downstream signaling pathway shared by Cdc42 and Rac. To address whether inactivation of Rho is a phenomenon restricted to sustained activation of Rac in established cell lines or whether growth factor–induced transient activation of Rac leads to downregulation of Rho, we stimulated serum-starved control NIH3T3 cells with PDGF. In agreement with the established link between PDGF-mediated receptor stimulation and Rac activation, PDGF activated Rac and not Cdc42 (data not shown) after 3 and 7 min of stimulation. PDGF-mediated activation of Rac was found to be a transient effect and was no longer detected 1 h after PDGF stimulation . Simultaneously with activation of Rac, we found a time-dependent inactivation of Rho in response to PDGF stimulation . Similarly to the activation of Rac, Rho activity returned to basal levels 1 h after PDGF stimulation. These data suggest that PDGF-mediated Rac activation also results in downregulation of Rho activity, as the kinetics of Rac activation correspond with inactivation of Rho. Expression of Tiam1 or activated Rac in NIH3T3 fibroblasts resulted in immotile cells with established cell–cell contacts . This phenotype is characterized by high Rac activity and downregulated Rho activity. Therefore, raising Rho activity exogenously in Tiam1-expressing cells should change the phenotype again towards fibroblastoid, motile cells. We generated an NIH3T3 cell line stably expressing C1199Tiam1 as well as V14Rho to test this hypothesis. Expression of C1199Tiam1 was not changed upon introduction of V14Rho (data not shown). Whereas C1199Tiam1-expressing cells grew in small epithelial-like islands of immotile cells, cells expressing Tiam1 as well as titrated amounts of V14Rho did not establish cell–cell contacts and exhibited a more motile, contractile phenotype . Thus, restoration of Rho activity by V14Rho in Tiam1-expressing cells induces transition from an epithelioid to a more fibroblastoid phenotype. These data suggest that the balance of Rac and Rho activities determines the cellular phenotype and migratory behavior in NIH3T3 fibroblasts. Stable expression of the GEF Tiam1 in NIH3T3 fibroblasts increases cell–substrate interactions and induces an epithelial-like morphology that is characterized by increased N- and P-cadherin–mediated cell–cell adhesion and inhibition of cell migration. This phenotypic transformation of NIH3T3 fibroblasts is due to Tiam1-mediated activation of Rac and Rac-mediated downregulation of Rho activity. Both sustained Cdc42 and Rac activation result in downregulation of endogenous Rho activity, demonstrating that Cdc42 and Rac antagonize Rho by regulating its GTP level. This crosstalk between Rac and Rho signaling pathways determines morphology and migratory behavior of NIH3T3 fibroblasts. In epithelial cells, both Rac and Cdc42 GTPases promote the formation of E-cadherin–mediated cell–cell adhesions . We found that Tiam1-induced formation of cadherin-based cell–cell contacts in NIH3T3 cells is due to activation of Rac and not Cdc42, confirming that Tiam1 functions as a specific GEF for Rac in fibroblasts. In MDCK cells, expression of Tiam1 resulted in inhibition of cell migration by increasing E-cadherin–mediated adhesion . In fibroblasts, Tiam1-mediated Rac activation also resulted in strong inhibition of migration, by increasing cell–cell and cell–matrix adhesions. The formation of Rac-induced cell–cell contacts does not exclude a requirement for Rac in migratory responses, as was shown previously by us and others . In epithelial cells, we showed earlier that Rac may influence cell migration in a positive or negative fashion, predominantly because of its effect on cell–cell adhesion . V12Rac has been shown to increase haptotaxis towards an immobilized collagen gradient in NIH3T3 cells . Besides using an inducible expressing system, these variant results might originate from similar effects on cell–cell adhesion, which is largely determined by cell density applied in the migration assay. As established in epithelial MDCK cells, high cell densities favor cell–cell adhesion and inhibit migration, whereas increased Rac-mediated migration is only seen at low cell densities . In addition, preservation of cell surface adhesion proteins such as cadherins during the trypsinization procedures may account for differences in the formation of cell–cell adhesions and, therefore, affect migratory behavior. The morphology and migratory behavior of Tiam1-expressing fibroblasts seems to be determined by the crosstalk of Rac and Rho proteins. V12Rac or Tiam1-mediated Rac activation resulted in a substantial reduction of Rho activity, leading to an epithelial-like morphology. However, complete inactivation of Rho activity by serum starvation or treatment with C3 transferase toxin results in disruption of cell–cell contacts in Tiam1-expressing fibroblasts. Most likely, this reflects a requirement for a basal Rho activity in the maintenance of Rac-induced cell–cell contacts. Similarly, both Rac and Rho activities have been shown to be required for cell–cell adhesion in epithelial cells . LPA partly restored the epithelial-like morphology in serum-starved fibroblasts. We have ruled out that LPA exerts its effect by activation of Rac, and confirmed that LPA functions as a potent activator of endogenous Rho protein. However, LPA-mediated activation of Rho occurred within minutes after stimulation and preceded the restoration of the epithelioid phenotype by hours. In addition to Rho activation, LPA may stimulate other signaling pathways required for phenotypic restoration. Rac-induced inactivation of Rho occurred downstream of Rac and was found in attached as well as suspended cells. Moreover, L61RacA37, which is unable of inducing the typical Rac-mediated cytoskeletal changes, still causes downregulation of Rho. This makes it unlikely that inactivation of Rho is a secondary consequence of Rac-induced spreading and cytoskeletal rearrangements, but rather reflects a signaling event by Rac. Inactivation of Rho activity was mediated by both Rac effector mutants, L61RacA37 and L61RacC40. Moreover, an activated mutant of the Rac downstream effector Pak1 was not effective in downregulation of Rho activity. This suggests that Pak1 and signaling pathways that stimulate Jun kinase activity or cytoskeletal changes are not involved in the regulation of Rho activity by Rac. However, this does not exclude the possibility that other Pak-like kinases participate in downregulating Rho activity. In Swiss 3T3 fibroblasts, activated V12Rac or PDGF-mediated Rac activation was shown to induce the accumulation of actin in membrane ruffles followed by the delayed Rho-dependent formation of stress fibers, suggesting activation of Rho by Rac . However, our data show inactivation of Rho activity by Rac in NIH3T3 fibroblasts. We found a similar inactivation of Rho by Rac in epithelial MDCK cells (Zondag, G.C.M., E.E. Evers, J.P. ten Klooster, L. Janssen, and J.G. Collard, manuscript submitted for publication) and in Cos-7 cells . Our findings are in agreement with a recent study in Swiss 3T3 cells, where the authors concluded that Rac and Rho activities are mutually antagonistic by monitoring the formation of Rac and Rho-dependent adhesion structures. After inhibition of the p160 Rho kinase, Rottner et al. 1999 observed a shift from focal contacts to focal complexes, which is accompanied by enhanced membrane ruffling. The authors suggested that inhibition downstream of Rho leads to activation of Rac. However, by direct measuring of the GTPase activities, our results suggest a unidirectional signaling from Rac towards Rho. Up- or downregulation of Rho activity, using either V14Rho, LPA, N19Rho, or the p160 Rho kinase inhibitor Y27632, did not affect GTP loading of Rac. Therefore, it is likely that the cytoskeletal changes that occur after inhibition of a Rho downstream effector originate from a shifted balance in the Rac and Rho pathways rather than from a direct, Rho-mediated modulation of Rac activity. In addition to the direct regulation of GTPases shown here, crosstalk between Rac and Rho downstream effector pathways can occur, as was shown for Pak1 regulating myosin light chain phosphorylation . We found that downregulation of Rho by Rac or Cdc42 was not restricted to stable cell lines showing sustained activation of Cdc42 or Rac. A transient increase in Rac activation induced by PDGF, similarly resulted in a transient decrease in Rho activity, suggesting a coupling of both Rac and Rho activities upon PDGF receptor stimulation. However, we cannot exclude that Rac and Rho activities are regulated by separate pathways initiated by stimulation of the PDGF receptor. Stimulation of other receptors for growth factors results in different activation patterns of Rho and Rac proteins (Sander, E.E., J.P. ten Klooster, S. van Delft, A. van der Kammen, and J.G. Collard, unpublished results), thereby mediating different cellular responses depended on the type of receptor stimulated. Similar mechanisms of opposing Rac and Rho activities as seen in fibroblasts seem to operate in other cell types as well, as deduced from cytoskeletal changes specific for Rac and Rho. Stimulation of C3H10T1/2 murine fibroblasts with EGF results in the formation of membrane ruffles, indicative of Rac activation, and at the same time in the dissolution of F-actin stress fibers, indicative of Rho inactivation . In neuronal N1E-115 cells, both Rac and Rho GTPases have been proposed to counteract each other's activities . Rac activation promotes a loss of contractility associated with increased cell spreading, whereas activation of Rho by LPA promotes contraction and cell rounding . These phenotypic changes have been attributed to Rac-induced myosin heavy chain phosphorylation . Moreover, Rac has been suggested to negatively influence Rho activity since Rac-expressing neuronal cells are refractile to LPA stimulation, and expression of V14Rho is able to overcome this defect . This is consistent with our biochemical data, demonstrating that Rac mediates downregulation of Rho activity and that activation of Rac inhibits LPA-mediated stimulation of Rho, indicating the desensitization of receptor-mediated Rho activation by Rac . In fibroblasts, this Rac-mediated downregulation of Rho activity in Tiam1-expressing cells is reflected in a fine network of thin stress fibers, which are less bundled and correspond to small focal contacts, when compared with wild-type cells . Upon integrin-mediated cell substrate adhesion, Rac and Rho activities might similarly have opposing roles. Rho activity in fibroblasts plated on fibronectin is low within approximately the first 15 min of cell spreading, followed by an increase of Rho activity . The initial low Rho activity could be the result of Rac activation, as Rac is activated early upon integrin-mediated adhesion . Rac-mediated downregulation of Rho occurred downstream of Rac. This signal regulates Rho activity upstream of Rho since low levels of GTP-bound Rho were found. The biochemical analysis of the actual GTPase activities in NIH3T3 fibroblasts supports a model that Rac and Rho signaling antagonize each other to control the cellular phenotype and migratory behavior. Activation of Rac induces cell spreading, resulting in an epithelioid phenotype. Activation of Rho promotes a more fibroblastoid, motile phenotype because of enhanced contractility by the formation of stress fibers. Note that for an epithelioid as well as for a migratory phenotype, the activities of both Rac and Rho GTPases are required, but the balance of their reciprocal activities determines the cellular morphology and the migratory behavior. At the molecular level, this is controlled by the negative regulation of Rho activity involving Cdc42 and Rac signaling. Activity of the distinct GTPases might be regulated locally in cells in response to extracellular stimuli, allowing cellular migration by coordinated activation/inactivation of Rho-like proteins. Our current research is focusing on the signaling pathways downstream of Cdc42 and Rac involved in downregulation of Rho. Complex formation and/or activity of regulators of Rho, including GAP, GEF, and GDI proteins, might be altered. p190RhoGAP may be a good candidate involved in Rac-mediated downregulation of Rho, as p190 becomes phosphorylated on tyrosine after EGF stimulation and β1 integrin signaling . A PKC-mediated pathway could also play a role, since in Swiss 3T3 fibroblast, LPA-induced stress fiber formation is inhibited by short pretreatment with PDGF or the phorbol ester PMA. Stress fibers induced by microinjected V14Rho are not sensitive to PMA treatment . In light of the present data, Rac might activate a PMA-sensitive isoform of PKC, thereby negatively regulating Rho activity. As phosphorylation of the myosin light chain by Rho contributes to increased contractility , a consequence of Rac-induced downregulation of Rho might be the inhibition of myosin light chain phosphorylation leading to loss of cellular contraction and allowing cell spreading. Taken together, we conclude that the molecular crosstalk of different Rho family GTPases, specifically Rac-mediated downregulation of Rho activity, determines cellular morphology and migratory behavior of NIH3T3 fibroblasts. | Study | biomedical | en | 0.999997 |
10579722 | The expression plasmid of Botulinum C3 ADP-ribosyltransferase (pGEX-C3) was kindly provided by Dr. A. Hall (University College London, London, UK). The MDCK cells and the cDNA-encoding mouse moesin (1–577 amino acids [aa]) were gifts from Dr. S. Tsukita (Kyoto University, Kyoto, Japan). Monoclonal mouse anti-MBS Ab (anti-mMBS Ab; antigen: 371–511 aa of M130) was kindly provided by Dr. D.J. Hartshorne . HA1077 was kindly provided by Asahi Chemical Industry (Shizuoka, Japan). Y-32885 was synthesized as described . Human recombinant hepatocyte growth factor (HGF) was produced and purified as described . TM71 , anti-pp2b Ab , anti-pT558 Ab , anti-pT445 Ab , and polyclonal rabbit anti-MBS antibodies were generated. A rabbit polyclonal antibody against ERM (ezrin/radixin/moesin) family proteins (anti-ERM Ab) was generated as follows. Glutathione- S -transferase-mouse moesin (GST-mouse moesin; 357–577 aa) was produced and purified from Escherichia coli as an antigen. The obtained antiserum was then affinity-purified against mouse moesin (357–577 aa). Anti-ERM Ab specifically recognized ERM family proteins (data not shown). Protein kinase C (PKC) was prepared from rat brain as described . Phosphatidyl serine, bisbenzimide Hoechst, anti-MLC Ab, nocodazole, and N 6 ,2′- O -dibutyryladenosine 3′:5′-cyclic monophosphate (dibutyryl cAMP) were purchased from Sigma Chemical Co. γ-[ 32 P]ATP was purchased from Amersham Corp. Tetradecanoylphorbol-13-acetate (TPA) and calyculin A were purchased from Wako Pure Chemical Industries, Ltd. All materials used in the nucleic acid study were purchased from Takara Shuzo Co. Other materials and chemicals were obtained from commercial sources. GST-catalytic domain of Rho-kinase (GST-CAT; 6–553 aa) and full-length Rat3 MBS were produced in Spodoptera frugiperda cells in a baculovirus system and purified as described . Maltose-binding protein-RB/PH(TT) [MBP-RB/PH(TT); 941–1388 aa], GST-MBS-NH 2 -terminal domain (GST-MBS-NT; 1–763 aa), GST-MBS-COOH-terminal domain , GST-MBS-CT S854A, T855A (GST-MBS-CT AA), GST-RhoA I41 and GST-C3 were produced and purified from E . coli . For microinjection, GST-C3 and GST-RhoA I41 were cleaved with thrombin, and purified to remove the GST. MBP-RB/PH(TT), C3 and RhoA I41 were concentrated, and during the concentration the buffer was replaced by microinjection buffer (20 mM Tris-HCl at pH 7.4, 20 mM NaCl, 1 mM MgCl 2 , 0.1 mM EDTA, and 5 mM 2-mercaptoethanol). For incubation, the buffer of C3 was replaced by PBS. The guanosine 5′-(3- O -thio)triphosphate (GTPγS) bound form of RhoA I41 was prepared as described . The kinase reaction for Rho-kinase was carried out in 50 μl of kinase buffer A (50 mM Tris-HCl at pH 7.5, 5 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT) containing 100 μM γ-[ 32 P]ATP (1–20 GBq/mmol), 5 pmol of GST-CAT and 20 pmol of GST-MBS-NT, GST-MBS-CT, GST-MBS-CT AA, or full-length MBS. The kinase reaction for PKC was carried out in 50 μl of kinase buffer B (25 mM Tris-HCl at pH 7.5, 4 mM MgCl 2 , 400 nM CaCl 2 , 150 nM TPA, 10 μg/ml phosphatidyl serine, and 1 mM DTT) containing 100 μM γ-[ 32 P]ATP (1–20 GBq/mmol), 5 pmol of PKC, and 20 pmol of full-length MBS. After an incubation for 15 min at 30°C, the reaction products were boiled in SDS sample buffer and aliquots of the reaction products were subjected to SDS-PAGE. The radiolabeled bands were visualized and estimated by an image analyzer . To determine the sites of phosphorylation of MBS, the recombinant full-length MBS (1 nmol of protein) was phosphorylated with GST-CAT (200 pmol of protein) in 1 ml of kinase buffer A containing 100 μM γ-[ 32 P]ATP for 1 h at 30°C, and the reaction product was digested with Achromobacter protease I at 37°C for 20 h. The obtained peptides were applied onto a C18 reverse phase column (SG120; 4.6 × 250 mm; Shiseido) and eluted with a linear gradient of 0–48% acetonitrile for 100 min at a flow rate of 1.0 ml/min by high-performance liquid chromatography (System Gold; Beckman). The radioactive peptides were separated and phosphoamino acid sequencing was carried out with a peptide sequencer (PPSQ-10; Shimazu). The fractions obtained from each Edoman degradation cycle were measured for 32 P in a Beckman liquid scintillation counter. A rabbit polyclonal antibody against MBS phosphorylated at Ser-854 (anti-pS854 Ab) was prepared as described . The phosphopeptide Cys-Arg 849 -Glu-Lys-Arg-Arg-phosphoSer 854 -Thr-Gly-Val-Ser-Phe 859 was chemically synthesized as an antigen and bound to the carrier protein, keyhole limpet hemocyanin at the NH 2 -terminal cysteine residue, by Peptide Institute Inc. The obtained antiserum was then affinity-purified against the phosphopeptide. MDCK cells were grown in DME containing 10% calf serum, penicillin and streptomycin in an air-5% CO 2 atmosphere at constant humidity. REF52 cells were grown in DME containing 10% fetal bovine serum, penicillin, and streptomycin in an air-5% CO 2 atmosphere at constant humidity. We employed the conditions for C3 treatment as described with slight modifications. MDCK cells were seeded at a density of 4.0 × 10 5 cells in 6-cm dishes and incubated for 24 h. Then, the cells were deprived of serum for 24 h. For the C3 treatment, the seeded cells were incubated first for 8 h, and then in the presence of various doses of C3 for 16 h. Next, the cells were deprived of serum for 24 h in the presence of C3. For some experiments, serum-deprived cells were treated with various doses of HA1077 or Y-32885 for 30 min. The cells were then incubated in DME containing 200 nM TPA or 50 pM HGF at 37°C for various periods. The TPA- or HGF-treated cells were treated with 10% (wt/vol) trichloroacetic acid. The resulting precipitates were subjected to immunoblotting with anti-pS854 Ab and anti-pnMBS Ab. MDCK cells were seeded at a density of 1.2 × 10 6 cells in 10-cm dishes. Non- and TPA-stimulated MDCK (5 × 10 7 ) cells were fractionated as described with slight modifications. In brief, cells were washed and homogenized in buffer H (20 mM Hepes at pH 7.4, 5 mM KCl, 1 mM MgCl 2 , 50 mM NaF, 30 mM sodium pyrophosphate, 20 μg/ml leupeptin, 50 μg/ml PMSF, 1 mM DTT, and 0.1 μM calyculin A) with dounce homogenizer. The homogenates were loaded onto 1 M sucrose in buffer H, and centrifuged at 1,600 g for 10 min. The precipitates contained nuclei. The supernatant fluids were further centrifuged at 100,000 g for 30 min. The resulting supernatants were used as cytoplasmic fraction. The resulting precipitates were used as membrane fraction. The precipitates containing nuclei were resuspended with 1 M sucrose in buffer H, and centrifuged at 1,600 g for 5 min. The resulting precipitates were used as nuclear fraction. We confirmed by phase contrast microscopy that nuclear fraction contained nearly the pure nuclei, and by immunoblot analysis of nuclear (CREB), cytoplasmic (Rho GDI), and membrane (E-cadherin) marker proteins that subcellular fractionation was successful . MDCK cells in interphase, early mitotic (metaphase), and later stages of cell division cells were prepared as described with slight modifications. MDCK cells were seeded at a density of 1.2 × 10 6 cells in a 10-cm dish, and cultured for 24 h. Nocodazole was added directly to the medium at 0.33 μM, and then cells were cultured for additional 11 h. Mitotic cells were collected with PBS containing 4 mM EGTA and 0.33 μM nocodazole by mechanical shake off, and adherent cells were used as interphase cells. Mitotic cells were rinsed twice and suspended with nocodazole free medium, and plated onto the dishes. Cells at 0, 30, 60, or 90 min after removal of nocodazole were used as early mitotic and later stages of cell division cells. These cells were treated with 10% (wt/vol) trichloroacetic acid as described above. MDCK cells were fixed with 3.0% formaldehyde in PBS for 20 min and treated with PBS containing 0.2% Triton X-100 for 10 min on ice or acetone for 10 min at −20°C (for anti-pp2b Ab). REF52 cells were fixed with 3.0% formaldehyde in PBS for 10 min and treated with PBS containing 0.2% Triton X-100 for 10 min at room temperature. After being washed with PBS three times, the cells were incubated with anti-pS854 Ab, anti-pnMBS Ab, anti-pT558 Ab, anti-ERM Ab, anti-pp2b Ab, or anti-MLC Ab at 4°C overnight, TM71 for 2 h at room temperature, or anti-mMBS Ab for 1 h at room temperature. The cells were washed with PBS three times, then incubated with fluorescein isothiocyanate (FITC)-conjugated anti–rabbit or –mouse Ig Ab, Texas red–conjugated anti–rabbit, –mouse, or –rat Ig Ab for 1 h at room temperature or tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin for 30 min at room temperature. DNAs were stained with 1 μg/ml of bisbenzimide Hoechst for 3 min at room temperature. Fluorescently labeled cells were examined with a Zeiss LSM510 (Carl Zeiss). In some experiments, fluorescent images were taken with PXL cooled CCD camera (Photometrics) with DeltaVision processing software (Applied Precision Inc.). Exposure time was adjusted to obtain FITC and Texas red or rhodamine images with roughly equal intensities in nonstimulated MDCK cells . Under the same condition, the images of TPA- or HGF-stimulated MDCK cells were taken. The images of Fig. 4 A and 5 A, or Fig. 4 B and 5 B were treated with same establishment of contrast and brightness, respectively. Grayscale FITC and Texas red images were converted into green and red images, respectively, and then merged to synthesize RGB color images. A ratio image was created using an Image-Pro image processing system (Media Cybernetics). A grayscale image of anti-pS854 Ab was divided by a corresponding grayscale image of anti-mMBS Ab, and the resultant image was multiplied by 50. REF52 cells were seeded at a density of 3 × 10 3 cells per 13-mm cover glass in 6-cm dishes and incubated for 48 h. MBP (5 mg/ml), C3 (0.1 mg/ml), C3 (0.1 mg/ml) plus GTPγS·RhoA I41 (0.4 mg/ml), or MBP-RB/PH(TT) (5 mg/ml) was microinjected along with a marker protein (rabbit or mouse IgG at 1 mg/ml) into the cytoplasm of cells. After injection, the cells were incubated at 37°C for 30 min, and fixed as described above. We first determined the sites of phosphorylation of MBS by Rho-kinase in vitro as follows. Full-length MBS was expressed in Spodoptera frugiperda cells in a baculovirus system and purified. Purified MBS was then incubated with the GST-catalytic domain of Rho-kinase (GST-CAT; 6–553 aa). CAT has been previously shown to be constitutively active in vitro and in vivo . Phosphorylated MBS was digested with Achromobacter protease I and subjected to high-performance liquid chromatography. Two major (AP-3 and -4) and 6 minor (AP-1, -2 and -5–8) radioactive peptides were obtained . As shown in Table , we identified 14 phosphorylation sites of MBS by Rho-kinase. Thr-697 and Ser-854 turned out to be the major phosphorylation site in AP-3 and AP-4, respectively. When GST-MBS-NT (1–763 aa) or CT was used as a substrate instead of full-length MBS, the essentially similar results were obtained (data not shown). Taking our previous studies and this result into consideration, the consensus sequence of Rho-kinase phosphorylation site is RXXS/T or RXS/T . Rho-kinase seems to require the basic amino acid such as Arg close to its phosphorylation site. It has been previously reported that MBS is phosphorylated by protein kinase A (PKA), protein kinase C (PKC), and the endogenous kinase that is copurified with MBS from chicken gizzard . MBS is phosphorylated at Thr-850 of Chicken M133, which corresponds to Thr-855 (one of the phosphorylation sites in AP-4) of Rat3 MBS, by PKA . The sites of phosphorylation of MBS by PKC are unknown. It should be noted that Thr-697 (a major phosphorylation site in AP-3) corresponds to Thr-695 of Chicken M133, has been shown to be phosphorylated by the endogenous kinase . The phosphorylation of chicken MBS by the endogenous kinase results in inhibition of myosin phosphatase . Thus, the phosphorylation of MBS at Thr-697 by Rho-kinase may result in inhibition of myosin phosphatase. The endogenous kinase has not been identified. However, the endogenous kinase appears to be distinct from Rho-kinase because some characters of the endogenous kinase are different from those of Rho-kinase . To investigate how the phosphorylation of MBS by Rho-kinase is regulated in vivo, we prepared the site- and phosphorylation state-specific antibody for MBS. As shown in Table , Rho-kinase phosphorylated multiple sites of MBS in vitro. Thr-697 was one of the major sites of phosphorylation of MBS by Rho-kinase in vitro. However, we can not distinguish the phosphorylation of Thr-697 by Rho-kinase and the endogenous kinase in vivo as described above. Ser-854, which was also one of the major sites of phosphorylation of MBS by Rho-kinase in vitro , is the phosphorylation site specific to Rho-kinase among known MBS kinases in vitro (We confirmed that PKC did not phosphorylate MBS at Ser-854: see below). The phosphorylation at Ser-854 can serve as a pertinent indicator to study MBS phosphorylation by Rho-kinase in vivo. Thus, we prepared the rabbit polyclonal antibody (anti-pS854 Ab), raised against the synthetic phosphopeptide (CEKRRphoshoS 854 TGVSF). The specificity of anti-pS854 Ab was examined by immunoblot analysis. Equal amounts of GST-MBS-CT with various ratios between phosphorylated and unphosphorylated forms were loaded on the gel. GST-MBS-CT phosphorylated by GST-CAT in vitro was specifically detected by anti-pS854 Ab in a dose-dependent manner . The binding was neutralized by preincubation of the antibody with the antigen phosphopeptide. We also confirmed that anti-pS854 Ab recognized neither GST-MBS-CT S854A, T855A (substitution of residues by Ala), GST-MBS-NT phosphorylated by GST-CAT nor full-length MBS phosphorylated by PKC . These results indicate that anti-pS854 Ab specifically recognizes MBS phosphorylated at Ser-854 by Rho-kinase. Rho/Rho-kinase are implicated in HGF- and phorbol ester–induced membrane ruffling in MDCK and KB epithelial cells . We examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway in vivo. When total cell lysate of nonstimulated MDCK cells was immunoblotted with anti-pS854 Ab, MBS phosphorylated at Ser-854 was weakly detected. The addition of TPA enhanced the phosphorylation of MBS at Ser-854 . Similar results were obtained when the cells were stimulated with HGF. A minor band with apparent relative molecular mass of 90 kD was also detected. This minor band may be a degradation product of MBS, because the immunoreactive band detected by anti-MBS Ab was found at the same position (data not shown). After TPA stimulation, the phosphorylation level of MBS at Ser-854 elevated within 3 min, reached maximum at ∼30 min, and was sustained for at least 2 h . The maximal phosphorylation level of MBS at Ser-854 was about fivefold the basal level. The stoichiometries of phosphorylation at Ser-854 were ∼0.04 at the basal level and ∼0.20 at the maximal level, respectively. Similar results were obtained when the cells were stimulated with HGF, although the level of MBS phosphorylation induced by HGF was slightly lower than that induced by TPA . We also confirmed that the addition of dibutyryl cAMP did not induce the phosphorylation of MBS at Ser-854 , whereas it induced the phosphorylation of cAMP-response element binding protein (CREB) at Ser-133 (data not shown). These results indicate that MBS is phosphorylated during the action of TPA and HGF in MDCK cells. We furthermore examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway during the action of TPA and HGF in MDCK cells . Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with endogenous Rho functions, inhibited the focal adhesion formation in MDCK cells (∼50% inhibition by incubation of 100 μg/ml of C3; data not shown). Under the conditions, the TPA-induced MBS phosphorylation was inhibited to a similar extent . A similar inhibition was observed when the cells were pretreated with HA1077 or Y-32885, both of which are inhibitors of Rho-kinase . We investigated the subcellular distribution of MBS phosphorylated at Ser-854 in MDCK cells. In the nonstimulated MDCK cells, the immunoreactivity of phosphorylated MBS was strong in the nucleus and diffuse in the cytoplasm, but not detected in cell–cell contact sites and free end of plasma membrane . The TPA-induced membrane ruffling was detectable within 5 min and reached maximum at 15 min of exposure to TPA in the outer cell edge colonies . Under the conditions, the addition of TPA enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm, and the immunoreactivity of phosphorylated MBS was weakly detected in the membrane ruffling area , where F-actin , MBS and phosphorylated α-adducin accumulated . Similar results were obtained when the cells were stimulated with HGF instead of TPA . The immunoreactivity of phosphorylated MBS in the nucleus, cytoplasm and membrane ruffling area was abolished by preincubation of the antibody with the antigen phosphopeptide . Next, we compared the distribution of phosphorylated and total MBS. In nonstimulated MDCK cells, the immunoreactivity of total MBS was strong in the cytoplasm . In TPA-stimulated MDCK cells, the immunoreactivity of total MBS was detected in the cytoplasm and the TPA-induced membrane ruffling area . The merged image of anti-pS854 Ab (green) and anti-MBS Ab (red) immunofluorescence enabled us to roughly estimate the phosphorylation state of MBS. Green, yellow, and red images indicate high, intermediate, and low levels of phosphorylation of MBS at Ser-854, respectively. The merged image of phosphorylated and total MBS of the cytoplasm and free end of plasma membrane in the TPA-stimulated cells was more greenish than that of the cytoplasm in the nonstimulated cells . Consistently, the ratio (phosphorylated MBS/MBS) was high in cytoplasm and membrane ruffling area of TPA-stimulated MDCK cells as compared with that of nonstimulated MDCK cells . The ratio in nucleus was also increased by TPA stimulation. We found that phosphorylated MBS was more enriched in the membrane ruffling area as compared with Rho GDI, which is one of the cytoplasmic proteins . Similar results were obtained when the cells were stimulated with HGF instead of TPA . To further determine the subcellular distribution of phosphorylated MBS, we carried out the subcellular fractionation analysis . Total MBS was detected mainly in cytoplasmic fractions in non- and TPA-stimulated MDCK cells. The amount of total MBS in each fractions of subcellular fractionation is consistent with the result of immunofluorescence study . In nonstimulated MDCK cells, phosphorylated MBS was weakly detected in nuclear and cytoplasmic fractions. The addition of TPA increased phosphorylated MBS mainly in the cytoplasmic fraction and modestly in the nuclear fraction. In addition, phosphorylated MBS was weakly detected in membrane fraction. It should be also noted that although the immunoreactivity of phosphorylated MBS in immunofluorescence study was strong in the nucleus in the presence or absence of TPA , the phosphorylation level of MBS in the nuclear fraction in the cells stimulated with TPA was lower than that in the cytoplasmic fraction in subcellular fractionation analysis . This might be due to the rapid dephosphorylation of MBS during nuclear isolation. Severe dephosphorylation of MBS was not prevented during the subcellular fractionation under the present conditions. Therefore, the amount of phosphorylated MBS after subcellular fractionation may be less than that of phosphorylated MBS in intact cells. We found the additional band above phosphorylated MBS in nuclear fraction. We do not know exactly whether the additional band is an isoform of MBS or a nonspecific band. In fact, Shimizu et al. identified two isoforms of MBS (M130 and M133) from chicken gizzard . The similar isoforms may exist in MDCK cells. However, we could not rule out the possibility that pS854 Ab cross-reacted with other insoluble nuclear component in immunofluorescence study. We compared the subcellular distribution of phosphorylated MBS with that of F-actin and phosphorylated MLC at Ser-19 in migrating MDCK cells. Between 2 and 16 h after the addition of TPA, the cells dissociated from each other and migrated, with polarized morphology and membrane ruffling in the leading edge. In the TPA-induced migrating cells, phosphorylated MBS was localized in the leading edge, where F-actin accumulated, and the posterior region containing the nucleus . Similar results were obtained as to the distribution of phosphorylated MLC in migrating MDCK cells as described . The merged image of phosphorylated and total MBS in the leading edge and the posterior region containing the nucleus in migrating MDCK cells was more greenish than that in the cytoplasm in the nonstimulated MDCK cells . Consistently, the ratio (phosphorylated MBS/MBS) was high in the leading edge and the posterior region containing the nucleus in migrating MDCK cells as compared with that in nonstimulated MDCK cells . We further investigated the subcellular distribution of phosphorylated MBS, F-actin, and phosphorylated MLC in REF52 fibroblasts. REF52 cells grown in 10% fetal bovine serum have thick actin stress fibers and vinculin-containing focal adhesions, and show a filamentous periodical pattern of myosin on stress fibers. We have previously shown that total MBS is localized on stress fibers . Phosphorylated MBS was localized on stress fibers, cortical actin filaments, and in the nucleus in REF52 cells . The double-immunostaining by TRITC-phalloidin and anti-pS854 Ab or anti-pp2b Ab demonstrated coexistence of phosphorylated MBS, F-actin and phosphorylated MLC on stress fibers and cortical actin filaments. The intracellular localization of phosphorylated MBS is consistent with in vitro binding of MBS and myosin . There was the difference in localization of phosphorylated MBS between REF52 and MDCK cells. REF52 cells highly developed thick stress fibers , whereas serum-starved MDCK cells had a few thin stress fibers . Thus, phosphorylated MBS might not be detected in stress fibers in MDCK cells. Next, we compared the distribution of phosphorylated and total MBS. The distribution of phosphorylated MBS was similar to that of total MBS . The ratio (phosphorylated MBS/MBS) image showed that the phosphorylation level of MBS was high on stress fiber and in nucleus . We examined the effects of C3 and dominant negative Rho-kinase [RB/PH(TT)] on the localization of phosphorylated MBS in REF52 cells . C3 ADP-ribosylates Rho at Asn-41 and inactivates it, whereas RhoA I41 is not ADP-ribosylated by C3 and is insensitive to C3. RB/PH(TT) is composed of Rho-binding (RB) and pleckstrin-homology (PH) domains of Rho-kinase . RB/PH(TT), which has point mutations in the RB domain and does not bind to Rho, interacts with the kinase domain of Rho-kinase and thereby inhibits the Rho-kinase activity without titrating out Rho in vitro . RB/PH(TT) functions as the dominant negative form of Rho-kinase in vivo . The microinjection of C3 into REF52 cells disrupted the stress fibers and decreased the phosphorylated MLC staining as described . The filamentous pattern of phosphorylated MBS was also perturbed by the microinjection of C3 . Under these conditions, total MBS and MLC were scattered in the cytoplasm . The coinjection of GTPγS·RhoA I41 with C3 reversed the effects of C3 . The microinjection of RB/PH(TT) also disrupted the stress fibers and decreased the staining of phosphorylated MBS and MLC in REF52 cells . Similar results were obtained when cells were treated with Rho-kinase inhibitors (data not shown). It should be noted that the microinjection of C3 into REF52 decreased the staining of phalloidin in most cells, whereas that of RB/PH(TT) induced the disorganization of actin filaments . C3 may induce depolymerization of F-actin through the inhibition of other Rho-targets such as p140mDia. It has been shown that ERM family proteins and MLC phosphorylated at Ser-19 highly accumulate at the cleavage furrow during cytokinesis . In a recent study, we have found that Rho-kinase also highly and circumferentially accumulates at the cleavage furrow in various cell lines , and that dominant negative Rho-kinase inhibits the progress of cytokinesis . Here we examined the distribution of phosphorylated MBS during the different mitotic stages of MDCK cells. Phosphorylated MBS was enriched at the mid zone between the daughter chromosomes in late anaphase and at the cleavage furrow in telophase . Phosphorylated MBS persisted at the mid body until the end of cytokinesis . Next, we compared the distribution of phosphorylated MLC and ERM family proteins to that of phosphorylated MBS during different mitotic stages of MDCK cells. The staining patterns of phosphorylated MLC were spatially and temporally similar to that of phosphorylated MBS in dividing cells . Phosphorylated ERM family proteins accumulated in the microvilli-like structures in the cell body at all stages as described , and highly and circumferentially accumulated around the mid zone in late anaphase, and the cleavage furrow in telophase. The staining patterns of phosphorylated ERM family proteins were also similar to that of phosphorylated MBS, but phosphorylated ERM family proteins did not persist at the mid body until the end of cytokinesis . Vimentin is the most widely expressed intermediate filament protein, which is phosphorylated by Rho-kinase at Ser-71 . Using TM71, which recognizes the phosphorylation of vimentin at Ser-71, vimentin is shown to be specifically phosphorylated at the cleavage furrow whereas total vimentin is diffusely localized throughout the cytoplasm . Although phosphorylated vimentin, MBS and ERM family proteins accumulated around the cleavage furrow, they were not completely colocalized . Phosphorylated adducin, which is one of the Rho-kinase substrates, was diffusely localized throughout the cytoplasm . It should be noted that total MBS was diffusely localized throughout the cytoplasm, but not accumulated at the cleavage furrow . In contrast, phosphorylated MBS strongly accumulated at the cleavage furrow , indicating that MBS was phosphorylated specifically at the cleavage furrow. Total ERM family proteins was diffusely localized throughout the cytoplasm, and at the microvilli and cleavage furrow , whereas phosphorylated ERM family proteins accumulated at the microvilli and cleavage furrow preferentially . To further confirm that the phosphorylation level of MBS at Ser-854 elevates during the cytokinesis, immunoblot analysis of synchronized MDCK cells lysates was carried out . By release from mitotic arrest by nocodazole, early mitotic cells synchronistically entered into later stages of cell division, and at 30 min after release of mitotic arrest, ∼40% of the cells were in late anaphase, telophase or cytokinesis. At later phases, the cells were in post mitotic spreading (60–180 min) as described . The immunoreactivity of anti-pS854 Ab was increased at 30 min after release of mitotic arrest as compared with that in interphase and early mitotic cells . This increment of phosphorylation level of MBS was reversed at 60 min. Here we identified Ser-854 as one of the major sites of phosphorylation of MBS by Rho-kinase in vitro , and prepared a rabbit polyclonal antibody (anti-pS854 Ab), raised against the synthetic phosphopeptide. Anti-pS854 Ab specifically recognized MBS phosphorylated by Rho-kinase, but not by PKC in vitro. Since Ser-854 in MBS is the phosphorylation site specific to Rho-kinase among known MBS kinases, the phosphorylation of MBS at Ser-854 appears to be a useful indicator of the Rho/Rho-kinase activation in vivo. We found that Thr-697 was also one of the major sites of phosphorylation of MBS by Rho-kinase in vitro. We have previously reported that the phosphatase activity toward MLC is inhibited when MBS is phosphorylated by Rho-kinase . The endogenous kinase that is copurified with MBS from chicken gizzard , phosphorylates MBS at Thr-695 (M133), which corresponds to Thr-697 of Rat3 MBS, and thereby inactivates the phosphate activity . The phosphorylation of MBS at Thr-697 by Rho-kinase may result in inhibition of myosin phosphatase. The endogenous kinase appears to be distinct from Rho-kinase because the endogenous kinase is not inhibited by H7, which is one of the PKC inhibitors, whereas Rho-kinase is inhibited by H7 (unpublished data), and because the endogenous kinase but not Rho-kinase phosphorylates MLC at PKC sites . Since several sites of MBS including Thr-697 and Ser-854 were phosphorylated by Rho-kinase in vitro , further studies are necessary to determine which sites are the major sites of phosphorylation of MBS by Rho-kinase in vivo, and which phosphorylation sites are responsible for the inhibition of the phosphatase activity by Rho-kinase in vitro and vivo. We have previously shown that expression of dominant active Rho in NIH 3T3 cells results in an increment of MBS phosphorylation . MBS is phosphorylated and the myosin phosphatase activity is inactivated during the action of thromboxane A 2 in platelets, and both reactions are inhibited by a prior treatment of platelets with C3 . Similar observations are obtained in endothelial cells during the action of thrombin . Here we found by use of anti-pS854 Ab that the stimulation of MDCK cells with TPA or HGF induced the phosphorylation of MBS at Ser-854, and that pretreatment of the cells with C3 or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. It is possible that TPA induced the phosphorylation of MBS at Ser-854 through direct phosphorylation by PKC. However, this possibility is unlikely because anti-pS854 Ab did not recognize MBS phosphorylated by PKC in vitro. Phosphorylated MBS accumulated on stress fibers in REF52 cells. The microinjection of C3 or dominant negative Rho-kinase into REF52 cells weakened the accumulation of phosphorylated MBS. These results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in non-muscle cells. Myosin phosphatase binds to phosphorylated Rho-kinase substrates such as MLC via MBS and dephosphorylates them. Rho-kinase phosphorylates MBS, which leads to the inactivation of myosin phosphatase in vitro . Taken together, these observations suggest that the phosphorylation of MBS by Rho-kinase is involved in regulating the phosphorylation level of Rho-kinase substrates in non-muscle cells. Evidence is accumulating that Rho regulates the phosphorylation level of MLC through Rho-kinase and myosin phosphatase in smooth muscle cells . Because contraction of smooth muscle cells determines the size of lumen in blood vessels, airways, the gastrointestinal tract, uterus, and bladder, abnormal contraction can cause diseases such as hypertension and asthma . It has recently been shown that Y-27632 (one of the specific inhibitors for Rho-kinase) selectively inhibits smooth muscle contraction and corrects blood pressure in several hypertensive rat models . Thus, the Rho-kinase–mediated pathway appears to be involved in the pathogenesis of hypertension. Phosphorylation of MBS by Rho-kinase may play an important role in generating a certain types of abnormal contraction of smooth muscle. In this regard, we have recently found that Rho-kinase phosphorylates MBS at Ser-854 during porcine coronary artery spasm . Membrane ruffling is observed in the leading edges of motile cells and is thought to be essential for cell motility . A force arising from actin polymerization appears to drive lamellipodial protrusion , which is thought to be regulated by the small GTPase Rac . Actin in the membrane ruffling area is thought to be continuously depolymerized and then repolymerized during cell movement . A force derived from myosin II driven by MLC phosphorylation, which is thought to be regulated by Rho in the membrane ruffling area and posterior region of motile cells may also contribute to cell movement . Indeed, injection of anti–MLC-kinase Ab diminishes the cell motility of macrophages . Moreover, Matsumura et al. 1998 have recently shown that the phosphorylation level of MLC is high in the leading edge and posterior region containing the nucleus during the cell migration. It has been previously reported that the addition of TPA decreases force that the whole cell applies to the substrate in certain migrating fibroblasts . The cycling between phosphorylated and nonphosphorylated states of MLC may be necessary for cell migration. In this regard, we have recently reported that microinjection of either dominant negative or constitutively active Rho-kinase inhibits cell migration of NRK cells . We have recently found that the microinjection of dominant negative Rho-kinase inhibits the TPA- or HGF-induced membrane ruffling in MDCK cells, indicating that Rho-kinase is necessary for the cell motility . Here we found that MBS phosphorylated at Ser-854 as well as MLC phosphorylated at Ser-19 were localized in the leading edge and posterior region in migrating MDCK cells. We have recently found that phosphorylated α-adducin accumulates in the leading edge in migrating MDCK cells . Myosin phosphatase interacts with both MLC and adducin through MBS, and dephosphorylates the phosphorylated MLC and α-adducin . Taken together, the above observations suggest that myosin phosphatase and Rho-kinase cooperatively regulate the MLC phosphorylation in the leading edge and posterior region in migrating MDCK cells, and the α-adducin phosphorylation in the leading edge. Rho-kinase is thought to regulate the formation of actin stress fibers . We have recently found that the expression of mutant MLC T18D, S19D (substitution of residues by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chain in vitro , also enhances the formation of stress fiber . Thus, it is likely that the Rho/Rho-kinase pathway plays a critical role in the formation of stress fiber through myosin II activation. Here we found that phosphorylated MBS was localized on stress fibers in REF52 cells. The microinjection of C3 or dominant negative Rho-kinase into REF52 cells disrupted stress fibers and weakened the accumulation of phosphorylated MBS. These observations suggest that myosin phosphatase and Rho-kinase cooperatively regulate the MLC phosphorylation in fibroblasts, which in turn induce the formation of stress fiber. Rho, Rho-kinase, and ERM family proteins accumulate at the cleavage furrow , where MLC phosphorylation occurs . The expression of C3 or dominant negative Rho-kinase inhibits cytokinesis, resulting in multiple nuclei . Thus, MLC phosphorylation by the Rho/Rho-kinase pathway appears to provide contractility to the contractile ring and to play a critical role in cytokinesis. Rho-kinase also phosphorylates intermediate filament proteins such as glial fibrillary acidic protein (GFAP) and vimentin, exclusively at the cleavage furrow during cytokinesis . The expression of GFAP mutated at Rho-kinase phosphorylation sites induces impaired segregation of glial filament into postmitotic daughter cells . Thus, Rho-kinase appears to be essential not only for cytokinesis but also for the segregation of GFAP filaments into daughter cells which in turn ensures efficient cellular separation. Here we found that phosphorylated MBS as well as phosphorylated ERM family proteins accumulated at the cleavage furrow, where phosphorylated MLC and vimentin accumulated. Indeed, the phosphorylation level of MBS elevated during cytokinesis. Taken together, these results suggest that myosin phosphatase spatiotemporally regulates the phosphorylation state of certain substrates including MLC and ERM family proteins during cytokinesis in cooperation with Rho-kinase under the control of Rho. Recently, it has been shown that citron kinase, another Rho-binding kinase with structural similarity in the kinase domain to Rho-kinase, accumulates at the cleavage furrow and may play an important role in the contractile process during cytokinesis . Further analysis of different and redundant functions of both Rho-binding kinases will help us to elucidate the molecular mechanism underlying cytokinesis downstream of Rho. | Study | biomedical | en | 0.999996 |
10579723 | Dictyostelium myosin II–null cells (HS1), transformed with pBigMyD to create wild-type myosin II cells and with pBIG-ASP to create 3×Asp myosin II cells, were grown as described . The HL-5 growth medium was supplemented with 60 μg/ml streptomycin, 60 U/ml penicillin, and 5 μg/ml G418 (Geneticin; Life Technologies, Inc.). Bacterial lawns were prepared by spreading 2 ml of an overnight Klebsiella aerogenes culture on SM/5 plates . Transformations were performed by electroporation and transformed cells were selected by 5 μg/ml G418. Suppressors of 3×Asp myosin II cells were generated by treatment with 4-nitroquinoline- N -oxide (NQNO) or UV irradiation. Conditions for treatment of 3×Asp myosin II cells using chemical mutagen NQNO were as described by Patterson and Spudich 1995 . After treatment with NQNO, cells were shaken in suspension for 30 min, washed twice with HL-5, and 1.2 × 10 6 cells were plated onto bacterial lawns. For suppressors generated by UV irradiation, 1.2 × 10 6 3×Asp myosin II cells were spotted onto plates containing bacterial lawn and irradiated with UV (0.8–6.4 mJ/cm 2 ). Plates from either treatment were placed at 22°C and the developmental phenotype was observed 5 d later to screen for suppressors, which recovered the ability to sporulate. Clonal isolation of suppressors was performed as follows: a single sorus from each plaque was picked by a sterile pipet tip, resuspended in 5 ml HL-5, and spread on a new bacterial lawn to obtain single plaques. The tail region of each suppressor was sequenced using standard methods to locate each mutation. The mutations were then introduced into the 3×Asp myosin II sequence by PCR overlap extension mutagenesis , and then subcloned into pLittleMyo . These constructs were subsequently transformed into myosin II–null cells, and the developmental phenotypes were examined. Whole-cell lysates were electrophoresed on SDS/7.5% polyacrylamide gels and transferred to nitrocellulose paper. The paper was probed with anti- Dictyostelium myosin II mAbs, My6 or mAb 55 (kindly provided by Dr. Guenther Gerisch, Max Planck Institut für Biochemie, Martinsried, Germany) , and then incubated with a HRP-coupled secondary antibody (Bio-Rad). Signals were visualized with an enhanced chemiluminescence system (DuPont). Wild-type and 3×Asp myosin IIs were purified from cells grown and harvested as described , after a modified purification protocol that does not require filament assembly . After being washed with 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, the cell pellets were resuspended in 2 vol/g cells 25 mM Hepes, 50 mM NaCl, 2 mM EDTA, 1 mM DTT, pH 7.4, containing a mixture of protease inhibitors (buffer A) . The cell suspension was frozen by dripping into liquid nitrogen and stored at −80°C. Immediately before preparation, frozen pellets were thawed and lysed in 7 vol/g cells buffer A at 0°C. After sedimentation at 36,000 g for 30 min, the pellets were suspended in 4 vol/g cells 10 mM Hepes, 130 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4. The cell suspension was centrifuged at 36,000 g for 15 min. Extraction of myosin II was achieved by resuspending the pellets in 1.5 vol/g cells 10 mM Hepes, 150 mM NaCl, 5 mM MgCl 2 , 4 mM ATP, and 1 mM DTT, pH 7.4. After centrifugation at 200,000 g for 30 min, RNase A (5 μg/ml) and PMSF (0.1 mM) were added to the supernatant. The solution was dialyzed against a 12% Aquacide III (Calbiochem) solution in 10 mM Hepes, 200 mM NaCl, 1 mM DTT, pH 7.4, to concentrate the volume ∼20-fold, and further dialyzed in 10 mM Hepes, 200 mM NaCl, 1 mM DTT, pH 7.4 (column buffer). After adding 0.6 M potassium iodide, 20 mM sodium pyrophosphate, and 2 mM ATP, the concentrated dialysate was loaded onto a Biogel A15 gel (Bio-Rad) filtration column (80 ml total volume), which had been equilibrated previously with column buffer and preloaded with 10 ml 0.6 M KI, 20 mM NaPPi. Fractions containing myosin II were pooled and concentrated as mentioned before. Typical final concentrations for myosin IIs were 1–3 mg/ml, as determined by the Bradford method , using rabbit skeletal muscle myosin II as the standard. Samples were diluted to 30–50 μg/ml myosin II in 50–70% glycerol, 10 mM Hepes, 200 mM KCl, 1 mM DTT, pH 7.4, and sprayed immediately onto freshly cleaved mica and viewed as described . The locations of bends along the tail of myosin II monomers were measured on prints. We used a random mutagenesis approach to identify suppressors responsible for recovery of myosin II function for 3×Asp myosin II cells. 3×Asp myosin II cells are phenotypically identical to myosin II–null cells . Both fail to complete the Dictyostelium developmental cycle. They arrest at the mound stage . After treatment of cells with the chemical mutagen NQNO or UV irradiation, 3×Asp myosin II cells were spread on bacterial lawns. Any colony that developed past the mound stage was scored as a suppressor. Depending on the extent of suppression, the suppressors were sorted into three groups: limited, medium, and full . Mutagenesis was performed on a strain of Dictyostelium that had its endogenous mhcA gene deleted (HS1) and contains an extrachromosomal plasmid expressing mhcA –3×Asp myosin II (pBIG-ASP). To check whether the suppressor mutations were intragenic or extragenic, the plasmid from each suppressor was rescued and retransformed into unmutagenized myosin II–null cells, and the transformed cells were spread on bacterial lawns. The phenotypes of all the suppressors were reproduced, verifying that all 28 suppressor mutations were intragenic. The characteristics of the suppressors are shown in Table . Typically the expression level of myosins from the suppressors was similar to that from the wild-type and 3×Asp myosin II cells, but the size of the myosins varied . 7 of 28 of the suppressors were full-length myosin II, 9 were small internal deletions of 1–7 residues, and 12 were truncations from the COOH terminus. There was no correlation between the means of mutagenesis (NQNO or UV) and the sizes of myosin IIs expressed from the suppressors. The sizes of the 12 ΔCOOH terminus suppressor myosin IIs were the same or larger than a previously studied mutant myosin II called ΔC34. ΔC34–myosin II, a truncated Dictyostelium myosin II lacking the 34-kD COOH terminus of the tail , constitutively assembles into thick filaments, and ΔC34–myosin cells are able to complete the Dictyostelium developmental cycle and form fruiting bodies . Our ΔCOOH terminus myosin II suppressors are likely to be longer variants of the ΔC34–myosin II constitutive assembly phenotype, and we therefore focused on the remaining 16 suppressors. Sequencing results for all of the 16 full-length or near full-length suppressors revealed mutations that strikingly all lie in a 21-kD region (∼182 amino acids) towards the COOH terminus of the tail . All of the seven suppressors with single residue mutations resulted from changes of a single nucleotide base pair. The strongest suppressor was strain D1823Y, which had an aspartate to tyrosine mutation at position 1823 (denoted YDD). This position is one of the three targets for myosin II heavy chain kinase . This residue corresponds to position d in the heptad repeat of the myosin II tail. The rest of the suppressors with single residue changes resulted in the introduction of a proline, which does not exist in the tail of wild-type Dictyostelium myosin II. Interestingly, three independent suppressors recovered from our screen affected Arg 1880, and two affected Arg 1926. Such multiple hits imply that these positions may play critical roles in regulating filament assembly. The nine small internal deletion group of suppressors had deletions of one to seven amino acids in this region. As shown in Fig. 3 B, the locations of the deletion and the single-residue mutation groups did not mix. The deletions all mapped beyond position 1926. To be certain that the mapped mutations were responsible for the suppression phenotype and not mutations elsewhere in the myosin II that we may have missed, these mutations were recreated using PCR overlap extension mutagenesis of a 3×Asp myosin II gene contained within an extrachromosomal Dictyostelium expression vector. The plasmids were transformed into myosin II–null cells and the development ability on bacterial lawns was tested. The sporulation phenotypes were identical to the original suppressor strains . Dictyostelium 3×Asp myosin II molecules were monomeric at high ionic strength. Rotary shadowed 3×Asp myosin II exhibited primarily two conformations under this condition: straight and bent monomers . Various forms of the bent monomers were observed. In 20% of the bent monomer images, the COOH terminus of the tail folded back tightly and resulted in an apparently shorter tail . 77% of the 3×Asp myosin II molecules were found to be in the bent conformation ( n = 400). On the other hand, only 23% of the wild-type myosin II molecules were found to be bent ( n = 280). The percentage of the bent wild-type molecules is consistent with the previous finding that freshly purified wild-type myosin IIs are 20–30% phosphorylated in the heavy chain . The majority of the 3×Asp myosin II molecules bend at ∼1,200 Å, located at approximately two-thirds the length of the tail from the head–neck junction . This bent position is similar to that measured for wild-type myosin II monomers . However, we also observed a previously unfound, minor population of bends at ∼1,000 Å . These values are interesting, in that they fit well with the structural motifs described below for the myosin II tail. The relative proportion of bends at 1,000 and 1,200 Å appeared to be the same in 3×Asp and wild-type myosins (∼35%). The tail domain of Dictyostelium myosin II consists of 1,298 residues, and has no proline interruptions. The coiled–coil prediction algorithm Coils predicts small, distinct regions in the Dictyostelium myosin II tail that have low probabilities to form a coiled–coil structure . The two most unfavorable regions for coiled–coil structure locate at ∼1,000 and 1,200 Å from the head–neck junction. Consistent with this prediction, to optimize the pattern of charged and uncharged residues for the periodicity of stable coiled–coil structure in the tail, two skips of two amino acids each were necessary to be inserted into the tail sequence lineup at these two regions . Similar correlation between the bends observed from EM versus skips in the tail has been shown in smooth and skeletal muscle myosins , and in Acanthamoeba myosin . These results indicate that these two positions at ∼1,000 and 1,200 Å from the head–neck junction are hinge regions in the Dictyostelium tail domain, as seen in other myosin IIs . In another region of the tail, closer to the myosin head (∼400 Å from the head–neck junction), there is a small area that has a somewhat lower probability to be in a coiled–coil than the majority of the tail . In C . elegans , a similar domain has been described as the prehinge . A similar region (∼440 Å from the head–neck junction) predicted from muscle myosin IIs has been proposed to serve as a hinge, which allows the head domain to swing away from the thick filament, thus allowing the myosin heads greater freedom to interact with the actin filaments . An analysis of the Dictyostelium myosin II tail sequence revealed two regions (denoted A and B) in the tail that are unusually rich in alanine residues at the core (a or d) positions of the heptad repeats . Region A spans 18 heptad repeats . Region B contains 23 heptad repeats . More than 75% of all of the alanines at core a and d positions along the tail are found in regions A and B, although these regions account for only 22% of the total tail. The 300–amino acid gap between regions A and B contains the previously identified assembly domain , which contains only two alanines. The midpoint of the gap is at ∼1,200 Å from the head–neck junction. Core a and d positions rich in alanine residues have been shown to be important for the proper packing of four α-helices into an antiparallel α-helical coiled–coil structure . Moreover, X-ray crystal structure determination indicates that the ColE1 Rop protein forms a highly regular four stranded α-helix bundle with alanine residues populated in the hydrophobic core . The results presented here, together with earlier results , suggest the following structural model of phosphorylation control of myosin II thick filament assembly . Phosphorylation by myosin II heavy chain kinase produces charges on the outside of the coiled–coil tail that help stabilize the bent form of the myosin. Bent myosin II molecules cannot associate with other molecules to form parallel dimers, and therefore no antiparallel tetramers appear for the next phase of filament formation. Myosin II heavy chain phosphatase removes phosphates from the bent monomers, and the molecules return to their straight conformation. We propose that the threonine pair 1823/1833 could act as a nucleation site, which when phosphorylated, initiates the bent monomer conformation by orienting the two strands of dimeric coiled–coils. Once nucleated, regions A and B may zip up into an antiparallel four-stranded structure, possibly similar to that observed for the ColE1 Rop protein . Formation of such a structure results in a major bend at ∼1,200 Å from the head–neck junction. Moreover, the previously identified assembly domain is sequestered as a loop between regions A and B. This conformation of the assembly domain prevents intermolecular interactions that lead to formation of thick filaments. The equilibrium between the bent versus straight conformations is delicately poised, and can be easily disturbed by mutations at multiple sites in the tail . Position 1823 appears to be a particularly critical position for switching between the bent and open conformations. YDD is the only suppressor with a full-length myosin II that completely restores wild-type development. With the aspartate to tyrosine mutation at position 1823, YDD revives myosin II functions possibly by removing the negatively charged aspartate at position 1823 in 3×Asp myosin II that participates in an electrostatic interaction needed for the formation of the bent monomer. Unlike the other two myosin heavy chain kinase sites, residue 1823 corresponds to position d of the heptad repeat of the myosin tail. Position d is at the core of the coiled–coil, which most commonly consists of hydrophobic residues. To tolerate a negatively charged residue at this position in the bent conformation, it is conceivable that a positively charged residue(s) from region A contributes in the core of the antiparallel tetrameric coiled–coil structure. Two possible candidates are lysine 1481 or 1526, at positions a and d, respectively. An interruption of the heptad repeat by polar residues has been observed in the SNARE complex, which is also a highly regulated tetrameric coiled–coil structure . The relative importance of the three threonine targets for myosin heavy chain kinase and phosphatase have been explored by constructing all possible combinations of threonines and aspartates and examining the myosin II functions (Nock, S., W. Liang, H. Warrick, and J.A. Spudich, unpublished data). Consistent with the current report, threonine 1823 appears to be the most critical phosphorylation site for myosin II functions. Simply replacing aspartate at position 1823 with a threonine in 3×Asp myosin II (denoted TDD) is enough to reverse the null phenotypes into wild-type. On the other hand, DTD partially recovers myosin II function, which indicates that position 1833 does play some role. Position 2029 does not appear to be required, because DDT is identical to myosin-null cells. These arguments assume, of course, that an aspartate fully mimics a phosphorylated threonine. Because TDD has a wild-type phenotype, it is interesting that we did not get a simple reversion from aspartate to threonine in our suppressor screen. This could be due to the fact that this mutation would require two nucleotide changes, which is expected to occur in lower probability. It has been reported that kinases that specifically phosphorylate the three threonines also accept serines . The mutation from an aspartate to a serine would also require two nucleotide changes. The interaction within the alanine-rich core regions A and B appears to be highly sensitive to even single amino acid changes in the tail. All of the suppressors with single point mutations other than YDD, are located in region B, and except for YDD, they all involve the replacement of an amino acid residue with proline. That a single proline at any of three positions appears to be sufficient to destabilize the antiparallel tetrameric coiled–coil domain is possibly explained by its well-known disruptive effect on α-helical structure. The locations of suppressors with proline substitutions and deletion mutations are distinctly clustered. This suggests that the interactions that lead to suppression are different in the two sections of domain B. In one section, prolines may destabilize the bent monomer by disrupting the tetrameric coiled–coil structure. In the other section, deletions may shift the alignment of the coiled–coil regions in the bent monomer, resulting in disruption of critical contacts such as salt bridges important for maintaining the bent monomer. It is important to note that the model described in Fig. 7 only deals with the first step of filament assembly. It is possible that the interactions identified by the suppressors could be between different molecules in higher ordered structures. These structures would be the subsequent steps of the filament assembly pathway. Thus, the proline mutations may locally disrupt the thick filament substructure such that the aspartate residues could be better accommodated. This could shift the equilibrium to favor filament formation. Similarly, the shift of alignment by the deletion mutations could disrupt critical contacts important for the thick filament structure. It is interesting that no suppressors were found in region A. One possibility is that any mutation in this region affects another step in the pathway for thick filament assembly, which is therefore unable to survive our screening process. In fact, the second half of region A has been strongly implicated in formation of parallel dimers , which are the building blocks for thick filaments. Several myosin II tail mutants have been constructed with COOH-terminal and internal deletions. COOH-terminal deletion mutants are functional to different extents as long as the assembly domain is intact, allowing filaments to form . Mutants that remove parts of the proposed tetrameric coiled–coil structure give rise to intermediate in vivo phenotypes, indicating that regulation is impaired . Deletions that remove fragments between the head and region A appear to be functional, but if part of region A is removed an intermediate phenotype is observed . These observations are consistent with the proposal that the tetrameric coiled–coil structure is important for efficient regulation. Phosphorylation of myosin II heavy chain has been found to occur in a variety of nonmuscle cells as well as in the catch muscle of mollusks, and may be a general mechanism of regulating myosin II function . It is possible that the hinges in the tail domains of these other myosins are designed for regulation of myosin IIs through a mechanism similar to that proposed here. The bending position of Acanthamoeba myosin II locates at a proportionally similar position as the Dictyostelium region . Furthermore, the Acanthamoeba myosin II is phosphorylated at three serines located at the end of the tail . However, it is controversial whether phosphorylation regulates assembly of the Acanthamoeba myosin II . Myosin II from a molluscan catch muscle is phosphorylated at two serines in the tail domain . After phosphorylation, myosin II solubility is enhanced and the molecule folds , reminiscent of the behavior of Dictyostelium myosin II. Recently, heavy chain phosphorylation of vertebrate nonmuscle myosin IIs and even of smooth muscle myosin II has been reported . It remains to be determined whether the model proposed in this study is universal to myosin IIs that are regulated by heavy chain phosphorylation. Smooth muscle myosin II has two hinge regions located at approximately one-third and two-thirds the length of the tail from the head–neck junction domain . Although little is known regarding the effects of heavy chain phosphorylation for smooth muscle myosin, in vitro regulation of conformational changes in this myosin, and control of assembly has been reported to be mainly by light chain phosphorylation . However, there is controversy about the state or the extent of light chain phosphorylation change in the filamentous state of smooth muscle myosin II in vivo . Cells have evolved intricate mechanisms for the control of macromolecular assemblies. The myosin II thick filament is just one example where a delicately balanced equilibrium between monomer and filament forms is used to control cellular function. The model suggested here proposes that phosphorylation on a single threonine residue on a >200-kD protein results in stabilization of a bent monomer form of the molecule. This structural modification shifts the equilibrium to make filament assembly less favorable. Specific kinases and phosphatases can be activated, repressed, or spatially distributed to provide the appropriate regulation signals. A related model has been proposed for the regulation of the activity of heat shock factor in Drosophilia . There, coiled–coil regions of the protein interact within the protein as a monomer or between proteins as the active trimer form. The regulation of macromolecular assemblies via coiled–coil interactions is likely a widely applicable, highly dynamic, important cellular mechanism. | Study | biomedical | en | 0.999998 |
10579724 | Recombinant human APO-1/Fas–Fc IgG (Fas-Fc), soluble Fas-ligand (sFasL), and enhancer antibodies used for aggregating tagged sFasL were from Alexis Corp. Rabbit polyclonal antibodies against Fas (M-20) and against FasL (N-20) and the related control peptides were from Santa Cruz Biotechnology, Inc. Polyclonal rabbit antibodies to mouse FLICE-inhibitory protein (FLIP) were from R & D Systems, Inc., and mouse mAbs to neurofilament 68-kD subunit (NF-68) were from Sigma Chemical Co. mAb against FasL (H11) was purchased from Alexis Corp. Rat anti–mouse Fas mAbs (RMF6) were from Chemicon International, Inc. Control rat IgG 2a were obtained from PharMingen International and control rabbit Ig from Jackson ImmunoResearch Laboratories, Inc. DEVD-fmk (Asp-Glu-Val-Asp-fluoro methyl ketone) was purchased from Enzyme Systems Products and IETD-fmk (Ile-Glu-Thr-Asp-fluoro methyl ketone) from Clontech Laboratories, Inc. All mutant mice and control strains were purchased from Jackson Laboratories, Inc. Total RNA was isolated from different tissues and cells using the guanidine isothiocyanate-phenol chloroform method . Reverse transcription (RT) steps were performed using Expand™ reverse transcriptase (Roche Diagnostics) and random hexamer primers. Normalization of cDNA amounts was performed by PCR on a Perkin-Elmer Thermal Cycler, using primers for β-actin (sense primer TT GTA ACC AAC TGG GAC GAT ATG G and antisense primer GAT CTT GAT CTT CAT GGT GCT AGG). PCR reactions were performed for a total of 24 cycles. Fas and FasL cDNA were amplified using primers for rat Fas and rat FasL . The reaction mixture contained 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 0.2 mM each dNTP, 1.5 mM MgCl 2 , 1.5 U of Taq platinum polymerase (Roche Diagnostics), 20 pmol of each primer and a normalized amount of cDNA template. The PCR conditions for both sets of primers were 30 s at 94°C, 30 s at 60°C, 1 min at 72°C for a total of 35 cycles. Samples were then run on a 1.2% agarose gel, treated with 0.4 N NaOH and transferred to Hybond N + nylon membranes (Amersham Pharmacia Biotech). Blots were hybridized with 32 P-labeled probes corresponding to nucleotides 174–196, CCG ACA ACA ACT GCT CAG AAG G for rat Fas , and nucleotides 347–368, ACA TTC CTA ACC CCA TTC CAA C for FasL , at 42°C for 2 h. Membranes were then washed in decreasing concentrations of saline sodium citrate buffer (SSC), and exposed for autoradiography. For RT-PCR from cultures, a total of 50,000 motoneurons were seeded in five 35-mm dishes for each culture condition and rinsed with diethyl pyrocarbonate (DEPC)-treated PBS. Total RNA was isolated using TRIzol (Life Technologies, Inc.) reagent, and cDNA normalization was performed as above. Primers used were as follows: mouse Fas , nucleotides 273–292, CAC CAA CCT GTG CCC CAT GC, and nucleotides 999–1019, GTC CTT CAT TTT CAT TTC CAG ; mouse FLIP , nucleotides 226–247, TGA TGA AGA CGA GAA GGA GAT, and nucleotides 795–815, AAT CTT GGC TCT TTA CTT CGC . The PCR conditions were 30 s at 94°C, 30 s at 58°C for Fas or 54°C for FLIP , and 45 s at 72°C for a total of 35 cycles, using the same reaction conditions as described above. PCR products were separated on a 1.2% agarose gel and visualized by ethidium bromide staining. The relative intensity of ethidium bromide–stained bands was determined using an AGFA densitometer, and data were analyzed using the NIH Image 1.62 software. Rabbit polyclonal antibodies against Fas antigen (M-20) were immunoblot-purified using a procedure described previously . In brief, 5 mg of mouse thymus protein extract was submitted to preparative PAGE electrophoresis and Western blotting. The horizontal strip corresponding to Fas (45 kD) was cut out of the blot and incubated with 1 μg/ml M-20, then washed. Subsequently, bound antibody was eluted using a glycine buffer, pH 2.8, and rapidly buffered to pH 7.5. This step greatly decreased nonspecific staining observed using the initial preparation (data not shown). An identical immunopurification was performed using blots of E12.5 mouse ventral spinal cord extracts (see text). Tissues were collected in cold PBS, centrifuged to remove excess PBS and placed in lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 100 mM DTT, 1 mM PMSF, 2% SDS, 500 mM NaCl and Complete™ EDTA-free, a protease inhibitor mix purchased from Roche Diagnostics). After mechanical dissociation followed by sonication, lysates were centrifuged (12,000 rpm for 5 min) and the supernatant was heated at 95°C for 2 min in loading buffer (10% [vol/vol] glycerol, 2% [wt/vol] 2-mercaptoethanol, 250 mM Tris, pH 6.8, 0.005% bromophenol blue). Loading was 20 μg of proteins for adult tissue extracts and 40 μg for all embryonic tissues and cells, as determined using a modified Bradford reaction (Bio-Rad Laboratories). Proteins were separated by SDS-PAGE (12% acrylamide gel) and transferred to Immobilon membranes (Millipore). Membranes were blocked with 10% nonfat dry milk in PBS 0.1% Tween 20 (PBT) for 2 h at room temperature, then incubated with primary antibodies diluted in PBT containing 4% milk (100 ng/ml for FasL N-20 and Fas M-20 antibodies, 450 ng/ml for caspase-8 antibodies, 1 μg/ml for c-FLIP antibodies, and 50 ng/ml for NF-68 antibodies) overnight at 4°C. After washing with PBT, membranes were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) diluted in PBT containing 4% milk, for 1 h, washed with PBT and then developed using the ECL system (Amersham Pharmacia Biotech). Scanning was performed as for RT-PCR. E14 rat and E12.5 mouse spinal motoneurons were purified as described previously . In brief, cells were dissociated from ventral spinal cord after trypsin treatment. The largest cells were isolated by centrifugation on a 6.5% metrizamide density gradient. The immunoaffinity purification step performed previously by immunopanning was replaced by a cell-sorting step using magnetic microbeads . For rat, cells in the metrizamide fraction were incubated with a mouse mAb against rat p75 NTR (Ig192); for mouse, with a rat mAb against mouse p75 NTR (Chemicon). Subsequently, motoneurons were incubated with magnetic microbeads conjugated to either mouse anti–rat or rat anti–mouse secondary antibodies, thus allowing the purification of motoneurons on separating columns (Miltenyi Biotec, Inc.). After a final centrifugation through a BSA cushion, motoneurons were resuspended in either L15 or neurobasal medium and plated in 4-well dishes (Nunc) previously treated with polyornithine/laminin as described . Neurobasal medium (Life Technologies, Inc.) was supplemented with 2% (vol/vol) horse serum, 25 μM l -glutamate, 25 μM β-mercaptoethanol, 0.5 mM l -glutamine, and 2% (vol/vol) B-27 supplement (Life Technologies, Inc.). L15 (Leibovitz) medium (Life Technologies, Inc.) was supplemented with 2% (vol/vol) horse serum, 3.6 mg/ml glucose, 100 U/ml penicillin-streptomycin, 20 nM progesterone, 5 μg/ml insulin, 0.1 mM putrescine, 0.1 mg/ml conalbumin, 30 nM sodium selenite. For rat motoneurons, the different neurotrophic factors were used at the optimal concentrations as follows: human brain-derived neurotrophic factor (BDNF) (R & D Systems, Inc.) at 1 ng/ml, rat glial cell line–derived neurotrophic factor (GDNF) (Sigma Chemical Co.) at 100 pg/ml, mouse cardiotrophin-1 (CT-1) (Genentech, Inc.) at 10 ng/ml. Mouse motoneurons were cultured in the presence of a cocktail of trophic factors (NTFs) consisting of ciliary neurotrophic factor (CNTF; 10 ng/ml). All factors were added at the time of cell seeding. Rat or mouse motoneurons were seeded in 4-well plates and cultured in the indicated medium at the density of 1,000–2,000 cells per 16-mm well. Motoneurons were grown for 24 h at 37°C before being incubated with Fas activators at the indicated concentrations diluted in neurobasal medium. Treatments with sFasL were always performed in the presence of 1 μg/ml enhancer antibody. The activity of the different death-inducing agents was determined after different periods of treatment by direct counting of living cells using a phase-contrast microscope. Fas-Fc or IETD-fmk were added at the time of seeding, and the surviving motoneurons were counted after 24 h at 37°C in the indicated culture medium. Counts were performed using a phase-contrast microscope: ∼100 motoneurons in 2 diameters of each well were evaluated as described previously . All assays were performed in duplicate or triplicate wells in each of two to four independent experiments. Approximately 3,000 purified motoneurons were seeded per polyornithine/laminin–coated 12-mm diameter glass coverslip, and grown for 1 or 3 d at 37°C in neurobasal medium with trophic factors. Cultures were fixed on ice using freshly made paraformaldehyde, first for 10 min with 2% paraformaldehyde in PBS-neurobasal medium (1:1), then for 10 min with 4% paraformaldehyde in PBS. Cultures were washed three times with PBS, permeabilized with 50 mM l -Lysine, 0.1% Triton X-100 in PBS for 10 min, and incubated overnight at 4°C in PBS containing 4% BSA, 2% goat or donkey serum (species depending on the secondary antibodies). The primary antibodies were diluted in PBS containing 4% BSA, 2% donkey and goat serum at the following concentrations: 0.2 μg/ml for rabbit polyclonal antibodies against FasL (N-20) and Fas (M-20), 20 μg/ml for anti-Fas (RMF-6), 0.9 μg/ml for caspase-8 rabbit polyclonal antibodies, and matching concentrations for each control antibody. Cells were incubated with primary antibodies overnight at 4°C, then washed with PBS containing 0.1% Triton and incubated for 30–45 min at room temperature with fluorophore-conjugated anti–rabbit, anti–mouse, or anti–rat secondary antibodies (Jackson ImmunoResearch, Inc.) diluted in PBS 10% horse serum. After washing with PBS containing 10% horse serum and PBS, cultures were rinsed with double-distilled water before being mounted on glass slides using DABCO mounting solution and examined by fluorescence microscopy. Immunostaining using the polyclonal antibody CM1 to detect the activated form of caspase-3 was performed as described . To label all nuclei, immunostained preparations were incubated with a solution of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 200 ng/ml in PBS for 15 min in the dark before washing and mounting. We first asked whether Fas and FasL are expressed at the stage at which motoneuron PCD is about to begin. By RT-PCR followed by Southern blotting using specific oligonucleotide probes, we showed that, as reported , Fas and FasL were both expressed in a variety of adult tissues , and that FasL was expressed by ventral spinal cord of E14 rat embryos . In addition, we showed that Fas was expressed at significant levels by freshly isolated E14 ventral spinal cord . Because levels of mRNA were not high enough to be reproducibly detected by in situ hybridization (data not shown), we used an alternative approach. To determine whether motoneurons themselves expressed Fas and FasL , we purified E14 rat motoneurons to near-homogeneity using the metrizamide-immunoaffinity method (see Materials and Methods). All purification steps were performed below 20°C to reduce de novo synthesis. mRNAs for both Fas and FasL were clearly present in freshly isolated motoneurons , demonstrating that motoneurons about to undergo PCD in vivo potentially express the elements of the Fas–FasL system. Next, we determined whether motoneurons express significant levels of Fas and FasL protein. By Western blotting using a polyclonal antibody to FasL, we detected a band of the appropriate molecular mass (38 kD) in adult thymus but not in heart, as expected from previous reports and our RNA data . The same antibody clearly detected FasL in ventral spinal cord from both E14 rat and E12.5 mouse embryos and in purified rat motoneurons of the same age . Several commercial antibodies to Fas we tested reacted with multiple bands on Western blots (data not shown). Therefore, we affinity-purified anti-Fas antibodies from polyclonal rabbit Ig (M-20) raised against the intracellular domain of Fas (see Materials and Methods). On Western blots of thymus from P17 mice, this antibody recognized a band that migrated with an apparent molecular mass of 45 kD, the expected molecular mass for Fas . The intensity of this band was greatly reduced in thymus from P17 lpr/lpr mice , in which levels of Fas have been reported to be at least 10-fold lower than normal . Also as expected , Fas protein was detected in adult heart, but not eye . In the same conditions, the immunopurified antibody recognized a band of identical molecular mass in extracts of ventral spinal cord from E14 rat and E12.5 mouse, and in motoneurons freshly purified from E14 rat spinal cord . When the antibody was preincubated with the immunogenic peptide, no staining was seen (data not shown). As a final demonstration that the polypeptide present in spinal cord and motoneurons was indeed Fas, we immunopurified the Fas antibodies by incubation with the 45-kD band cut out from Western blots of E12.5 mouse spinal cord. The eluted antibodies recognized a 45-kD band in thymus that disappeared in lpr/lpr tissue (data not shown). Thus, Fas protein is expressed at significant levels by spinal motoneurons (and perhaps other spinal neurons) at the onset of motoneuron PCD. However, we were not able to immunolocalize Fas reliably on embryonic tissue sections (data not shown). We performed immunolabeling studies on purified mouse and rat (data not shown) motoneurons cultured for 1 d with neurotrophic factors. For Fas, using the affinity-purified Ig described above, granular membrane labeling was observed in nearly all cultured motoneurons, although some were more intensely labeled than others . Similar results (data not shown) were obtained using rat mAb RMF6 raised against the extracellular domain of Fas. The rabbit antibodies also reproducibly stained the nuclei of cultured motoneurons; both this and the membrane immunoreactivity were absent when the antibody was preincubated with the peptide used for immunization . Antibodies to FasL also stained nearly all motoneurons , and double-labeling experiments confirmed that most individual neurons coexpressed Fas and FasL . Since motoneurons coexpressed the death receptor Fas together with its ligand, we asked whether Fas might play a role in motoneuron cell death. When purified rat motoneurons were grown in basal medium at low density (1,000 per 16-mm well) for 24 h, about half of them died . Their death was completely prevented by BDNF (1 ng/ml). Fas-Fc, which contains the extracellular domain of Fas fused to an Ig Fc domain, is an antagonist known to block activation of Fas by FasL. Fas-Fc inhibited ∼75% of the death of trophically deprived motoneurons in a dose-dependent manner . Higher doses of Fas-Fc were slightly less efficient (data not shown), but this may have been due to toxicity of the reagent. This suggested that endogenous FasL was activating Fas in these neurons, thereby triggering the cell death process. Fas-triggered cell death in nonneuronal systems involves activation of caspase-8, which then activates downstream caspases such as caspase-3 . Using a specific antibody to caspase-8 that recognizes both the inactive procaspase and the active forms , we found that nearly all cultured motoneurons showed a characteristic pattern of cytoplasmic labeling . We then tested the hypothesis that caspase-8 might be an essential relay in the death of motoneurons induced by trophic deprivation. Prior application of the caspase-8–selective inhibitor peptide IETD-fmk , which alone did not affect survival in BDNF, inhibited 75% of the motoneuron loss observed in the absence of trophic support , consistent with a requirement for caspase-8 in this process. To determine whether caspase-8 is present in motoneurons in vivo, we performed Western blots on different tissue extracts . As expected, nonapoptotic HeLa and Jurkat cells showed a principal doublet around 53–55 kD, which corresponds to the forms referred to as caspase-8/a and caspase-8/b . Spinal cord extracts and freshly purified motoneurons showed a single major caspase-8–immunoreactive band at 43 kD . It is not possible to determine from these data whether this form is identical to p43, a known intermediate in the caspase-8 activation process in nonneuronal cell types , and it is possible that it results from other posttranslational modifications than those observed in cell lines. However, we do not believe that the 43-kD form in nervous tissue is proteolytically active, since we detected it at similar levels in normal adult spinal cord, where little or no PCD occurs (data not shown). Thus, caspase-8, a central component of the Fas signaling pathway, is present in normal spinal cord during the period of naturally occurring motoneuron death. In view of the finding that blockade of the Fas system saves motoneurons deprived of trophic support , we next asked whether Fas activation could trigger motoneuron death even in the presence of trophic factors. To do this, we took advantage of reagents that have been shown to cluster Fas at the cell membrane in other systems and thereby exogenously trigger Fas-dependent cell death. Motoneurons purified from E12.5 mouse embryos were cultured in the presence of a cocktail of trophic factors (BDNF, CNTF, and GDNF) at concentrations determined previously to ensure optimal survival . After 24 h to allow attachment and initial neurite outgrowth, antibody to the extracellular domain of mouse Fas was added in the continued presence of trophic factors to induce clustering of Fas. 2 d later (i.e., after 3 d in vitro [DIV]), anti-Fas antibody had caused dose-dependent motoneuron loss, up to a maximum reduction of 45% using 10 ng/ml antibody . The half-maximal dose of antibody required to induce death of motoneurons (0.7 ng/ml) is close to that reported for lymphoid cells . Control antibodies including the enhancer antibody (see below) had no effect on survival (data not shown). To confirm the generality of this observation, we performed an analogous experiment on cultured rat motoneurons. Since rat Fas is not recognized by the clustering antibody, we used a recombinant form of the extracellular domain of FasL (sFasL) that is epitope-tagged. Addition of sFasL, followed by an antibody to this tag (enhancer antibody), clusters sFasL and thereby Fas, leading to Fas activation. Rat motoneurons were cultured for 1 d in the presence of optimal concentrations of BDNF (1 ng/ml). They were then treated with enhancer, either alone or in the presence of increasing concentrations of sFasL. Enhancer antibody alone (1 μg/ml) did not affect survival, but in the presence of sFasL, 40% of motoneurons were lost 2 d later in a dose-dependent fashion, with an EC 50 of 1 ng/ml . This is close to the half-maximal dose necessary to trigger death of A20 B lymphoma cells . Strikingly, in this model too, motoneuron death was not enhanced by further increasing the concentrations of sFasL. We compared the percentage of motoneurons induced to die by Fas activation in the presence of different factors. Rat motoneurons were cultured as described above, except that BDNF (1 ng/ml) was replaced by CT-1 (10 ng/ml), or GDNF (0.1 ng/ml), or CNTF (10 ng/ml; data not shown). The reduction in motoneuron number after 2 d was expressed as a percentage of the number surviving in the same conditions without sFasL . In each case, sFasL triggered death of ∼30% of motoneurons, and similar results were obtained using FasL in basal medium alone (data not shown). To demonstrate that the motoneurons lost after Fas activation had activated a cell death program, we studied the involvement of caspases. We first focused on caspase-3, a downstream caspase known to be required for PCD of many neurons , and of motoneurons in particular . Using an antibody (CM1) that specifically recognizes the activated form of caspase-3 , we showed that many cultured motoneurons exposed to Fas activators expressed activated caspase-3, and that this reactivity was especially strong in the motoneurons with apoptotic nuclei that had not yet detached from the culture dish . We quantified CM1-positive motoneurons 30 h after treatment (or not) with anti-Fas antibodies . At this stage, there was not yet a significant difference in total motoneuron numbers in the presence or absence of anti-Fas ( P > 0.5; data not shown). Without Fas activator, 8.0 ± 0.9% (mean ± SEM; n = 5) of motoneurons expressed intense CM1 immunoreactivity and showed fragmented chromatin using Hoechst staining. Using anti-Fas antibodies, this value was significantly increased (19.0 ± 1.6%; P = 0.001 by t test). To test the functional significance of caspase-3 activation, we used the active-site peptide DEVD, a potent cell-permeable caspase inhibitor that is selective for caspase-3 , but can also inhibit caspase-8 . Increasing concentrations of DEVD-fmk were added to rat motoneurons cultured in BDNF, 2 h before addition of sFasL (10 ng/ml) and enhancer antibody. Motoneuron loss was inhibited in a dose-dependent fashion , and the concentration of DEVD required to completely prevent Fas-dependent motoneuron death (10 μM) was similar to or lower than those reported to prevent PCD in other cell types . DEVD was also able to completely inhibit the cell death of mouse motoneurons induced by sFasL or anti-Fas antibodies . Thus, Fas most likely triggers PCD of motoneurons by a mechanism involving caspase-3. In similar experiments, the caspase-8 inhibitor IETD-fmk was able to completely prevent the motoneuron loss induced by sFasL , consistent with a requirement for caspase-8 in this process. It was striking that ∼50% of motoneurons were resistant to the effects of exogenous Fas activation in the presence of trophic factors. We tested the survival of mouse motoneurons treated at different stages with Fas activators . In a first experiment analogous to those in Fig. 5 , mouse motoneurons were seeded in a cocktail of neurotrophic factors, treated with either sFasL or anti-Fas at 1 DIV, and their survival was counted 4 d later, at 5 DIV . Some motoneurons (∼10%) died over the 5-d culture period even in the presence of trophic factors, and so values were expressed as a percentage of the value at 5 DIV with trophic factors alone. In these conditions, Fas activation led to loss of 50% of motoneurons. To rule out the possibility that incomplete motoneuron loss reflected instability of reagents in the culture dish, we then added Fas activators at both 1 and 3 DIV, and counted survival at 5 DIV . No further motoneuron loss was observed, demonstrating that some motoneurons are truly resistant to Fas activation. We then asked whether this resistance might be regulated in vitro, by first culturing motoneurons for 3 DIV in the presence of trophic factors, then adding Fas activators, and counting survival at 5 DIV. Surprisingly, no motoneuron death at all was triggered in these conditions . We asked whether all motoneurons might at some stage be susceptible to Fas activation. Because it was not technically possible to purify motoneurons from younger embryos, we added sFasL to E14 rat motoneurons at 0 h of culture. As at 24 h, it was not possible to trigger the death of more than half of them (data not shown). By extension, it seems likely that half the motoneurons in situ are already resistant to Fas activation as cell death begins. To exclude the possibility that the resistant neurons were intrinsically incapable of responding to Fas activation, we performed the experiment in the presence of cycloheximide (10 μM). As with many other cell types, this increased the number of motoneurons induced to die by Fas activators to 80% (two independent experiments; data not shown). Our results suggested: (a) that in the absence of trophic support, Fas becomes sufficiently activated by FasL to induce motoneuron cell death ; and (b) that in the presence of trophic factors, motoneurons become resistant to even exogenous activation of Fas . We used semiquantitative RT-PCR to follow the regulation of the expression of critical molecules in cultured motoneurons. Purified motoneurons were cultured in the absence and presence of neurotrophic factors. Cells were harvested after 1 and 3 DIV and levels of indicated mRNAs were determined by semiquantitative RT-PCR. First we asked whether trophic deprivation led to upregulation of components of the Fas system. Levels of Fas , normalized to actin , were relatively unaffected by the presence or absence of trophic factors . In contrast, whereas levels of FasL steadily decreased in motoneurons cultured with trophic factors, they were strongly upregulated in trophically deprived motoneurons . Thus, regulation of levels of FasL may underlie the dependence of motoneurons on external trophic support. Next, we searched for molecular correlates for the state in which motoneuron death can no longer be triggered by exogenous Fas activators. As stated above, after a 3-d exposure to BDNF, motoneurons still expressed Fas mRNA . We confirmed this by demonstrating immunoreactivity for Fas at their surface , and further showed that caspase-8 immunoreactivity was also still present in treated motoneurons . Surprisingly, therefore, all major components of the Fas pathway for cell death were still present in Fas-resistant motoneurons. This suggested that trophic factor–treated motoneurons became resistant at a level downstream from Fas. Normally, Fas activates caspase-8 through the binding of the adaptor molecule FADD to the cytoplasmic domain of Fas. FADD can then recruit procaspase-8 and lead it to self-activate. FLIP is an endogenous cytoplasmic decoy that can competitively inhibit the binding of procaspase-8 to FADD, and thereby prevent caspase-8 activation . Using semiquantitative RT-PCR, we showed that Flip mRNA was strongly upregulated after 3 DIV with neurotrophic support . Scanning of the gels showed that the intensity of the Flip band was 6.8-fold greater at 3 DIV with NTFs than at 1 DIV with NTFs, when both were expressed relative to the intensity of the band for actin . To confirm that the change in mRNA levels was reflected in an increase of FLIP protein, we performed Western blots on cultures of motoneurons in the same conditions using a specific antibody to mouse FLIP . As reported in other cell types , two major molecular species were labeled by this antibody in motoneuron extracts, with apparent molecular masses of 52 and 55 kD. Levels of both were markedly increased at 3 DIV with NTFs. Scanning of the blots showed that there was a mean 6.3-fold increase relative to neurofilament over the 2-d period in culture. Thus, motoneurons that survive in the presence of neurotrophic factors not only downregulate the death-inducer FasL but also upregulate the death inhibitor FLIP. PCD of motoneurons during development of the spinal cord has long been a classical paradigm, and its study was at the origin of the discovery of neurotrophic factors . However, we still know very little about exactly what drives the death of embryonic motoneurons. We have shown here that the death receptor Fas and its ligand FasL, which are coexpressed by motoneurons as they enter the phase of naturally occurring cell death, may potentially play an important role. Activation of Fas in cultured embryonic motoneurons is both necessary for PCD in the absence of neurotrophic factors, and sufficient to trigger death of a significant proportion of motoneurons in their presence. Levels of endogenous Fas activators and inhibitors are regulated by neurotrophic factors, suggesting that the Fas–FasL system may be a central pathway through which motoneuron numbers are controlled. Further experiments will be required to determine the role of Fas in naturally occurring and/or pathological motoneuron cell death in vivo. The presence of Fas on developing motoneurons (and perhaps other spinal neurons) was unexpected, although Fas expression has recently been reported in both normal and lesioned adult central nervous system , and a recent paper reported Fas in cerebral cortex at earlier stages . Therefore, we substantiated this observation by the use of different techniques (RT-PCR, Western blotting, and immunocytochemistry using several independent antibodies) wher-ever possible in two different species (mouse and rat). Our results unequivocally demonstrate the presence of both Fas and FasL in ventral spinal cord, and freshly purified motoneurons at the stage at which motoneuron PCD is about to begin. It will be of interest to determine the pattern of expression of Fas in vivo on a cell-by-cell basis. Our preliminary data using in situ hybridization and immunohistochemistry on rodent spinal cord were not included here, since Fas and FasL signals are too close to the threshold of detection. This may also explain apparent discrepancies between our findings and those of French et al. 1996 , who, although they described the presence of FasL in embryonic spinal cord, reported Fas to be absent from the embryonic nervous system. To better understand the potential roles of Fas and FasL, we studied two potential modes of Fas activation in vitro: endogenous and exogenous. Motoneurons coexpress FasL and Fas, and both Fas-Fc and IETD-fmk can prevent their spontaneous PCD in the absence of trophic factors, demonstrating that the binding of endogenous FasL to Fas is a motor for PCD in these conditions. The fact that these results were obtained in low-density cultures suggests that this was probably an autocrine effect, as already demonstrated for T lymphocytes . Similar events may occur in cerebellar granule neurons and PC12 cells which, like motoneurons, upregulate FasL in the absence of trophic support . Our results raise the possibility that some motoneurons die in vivo by a cell-autonomous Fas-related mechanism. Activation of Fas by trophic deprivation is by definition blocked by neurotrophic factors. In contrast, exogenous Fas activation inhibited the survival-promoting effects of all trophic factors we tested. The loss of motoneurons observed in these conditions was very similar in both extent (50%) and time-course (3–4 d) to motoneuron PCD in the embryo, although we cannot be sure that the 50% of motoneurons that die in these conditions are the same as the 50% that die from trophic deprivation. This raises the possibility that some motoneurons in vivo may die not because they have insufficient trophic support, but because they have been induced to do so by FasL presented in a paracrine fashion by nearby cells. Both the autocrine and paracrine hypotheses will need to be tested during development in vivo. Mice bearing either hypomorphic or null mutations for Fas, or a point mutation in FasL are known . We have obtained these mice and are currently analyzing the detailed pattern of motoneuron PCD and lesion-induced cell death in the absence of normal Fas signaling. It is not certain that Fas plays the same role in all motoneurons. It is striking that Fas-Fc and IETD-fmk both protected at most 75% of the motoneurons that would normally have died as a result of trophic deprivation. It is possible that higher concentrations of these reagents were simply toxic, but this suggests that other Fas-independent pathways may also be involved in triggering death of some motoneurons. Another suggestion of heterogeneity comes from the observation that 50% of motoneurons were resistant to Fas activators in all experimental conditions, even when treated immediately after purification. Strikingly, the resistance to Fas activation becomes complete after incubation for 3 d with trophic factors, and this is correlated with the upregulation of FLIP, an endogenous inhibitor of caspase-8 activation . Upregulation of FLIP over time in culture may reflect either general maturation or a specific effect of neurotrophic factors. It is not currently possible to purify rodent motoneurons at different developmental stages, but it will be interesting in the future to keep motoneurons alive by mechanisms apparently distinct from the mode of action of trophic factors, e.g., in high cAMP concentrations , and then test their resistance to Fas activation at different stages. Which are the caspases that act downstream of the Fas–FasL system in inducing motoneuron death? Our results implicate both caspase-3 and caspase-8. The evidence for activation of the downstream protease caspase-3 comes both from inhibitor studies and from immunolabeling using the CM1 antibody; our results are consistent with the implication of caspase-3 in many apoptotic phenomena in neurons . In other dying neurons, activation of the caspase-3 zymogen has generally been considered to occur through the actions of caspase-9, and null mutants for this gene indeed show reduced neuronal death . Our results using the IETD peptide, which inhibits caspase-8 much more strongly than caspase-3 , suggest that Fas acts through caspase-8 in this system as in others . Our conclusions are not in agreement with those of a previous report , whose authors found that caspase-8–like activity is present in chick motoneurons but is not upregulated after removal of trophic support, and that motoneurons were not saved by IETD-CHO. This may reflect a species difference and/or the different form of the peptide inhibitor used. However, it is clear that our results and those of Li et al. 1998 need to be substantiated by methods other than those based on active-site peptides, and we are currently developing other methods to study activation of procaspase-8 in the low numbers of motoneurons available. Understanding the intracellular mechanisms that regulate sensitivity or resistance to the effects of Fas will be one of the keys to evaluating the role of Fas during normal motoneuron development, and in pathological systems. Fig. 8 E presents a possible synthesis of our current knowledge. Fas activation may occur by either endogenous or exogenous mechanisms. The fact that exogenous (but not endogenous) activation triggers PCD even in the presence of neurotrophic factors may simply reflect more intense activation of caspase-8. This could mean that the signaling through the Bcl-2 family in the mitochondrion triggered by NTFs is not sufficient to prevent subsequent release of cytochrome c and activation of caspase-3 in these conditions . However, in the medium term (1–3 d), neurotrophic factors can prevent PCD induced by either endogenous or exogenous Fas activation. This seems to involve transcriptional events such as downregulation of FasL or upregulation of FLIP. Unraveling the interplay between these pathways will be important for understanding the control of motoneuron survival. The Fas-dependent mechanism of PCD we have described for motoneurons may well be relevant to the study of other developing neuronal populations, most of which undergo a quantitatively significant phase of naturally occurring cell death. If our data on spinal motoneurons can be extrapolated, it may be that sensitivity to Fas activation will not be apparent at all stages. For instance, in paradigms involving culture of neurons with neurotrophic factors for a certain period before withdrawal , the neurons under study may become resistant to Fas activation before the stage at which their cell death is normally studied. An interesting possibility is that, just as different cell populations seem to depend for cell death on different members of the Bcl-2 family or on different caspases , different members of the tumor necrosis factor (TNF) receptor family such as p75 and Fas may be involved in the programmed elimination of different groups of neurons. It will be important to determine whether triggering of motoneuron death by Fas might underlie the specific loss of motoneurons that is observed in some circumstances at later stages of development or in the adult. These include experimental or traumatic lesions, and the neurodegenerative diseases that affect motoneurons, such as the pmn or wobbler mutations in mice, and spinal muscular atrophies (SMA) or amyotrophic lateral sclerosis (ALS) in humans . If Fas expression is observed in these patients, the pathways downstream of Fas may provide a novel and specific site for therapeutic intervention in these still incurable diseases. | Other | biomedical | en | 0.999997 |
10579725 | The RKO colon carcinoma cell line was obtained from M. Brattain (University of Texas, San Antonio, TX), and MDA-MB-435 breast carcinoma cells were obtained from the Lombardi Breast Cancer Depository (Georgetown University). The cloning of the human β4 cDNA, the construction of the β4 cytoplasmic domain deletion mutant (β4-Δcyt), and their insertions into the pRc/CMV (β4) and pcDNA3 (β4-Δcyt) eukaryotic expression vectors, respectively, have been described . RKO/β4Δcyt clone 3E1, RKO/β4 clone D4 (RKO/β4 clone 1), RKO/β4 clone A7 (RKO/β4 clone 2), MDA-MB-435/β4-Δcyt clone 3C12, MDA-MB-435/β4 clone 5B3 (MDA-MB-435/β4 clone 1), and MDA-MB-435/β4 clone 3A7 (MDA/β4 clone 2) were selected for analysis based on their expression of similar surface levels of α6β4 and α6β4-Δcyt, as we have previously demonstrated . Dominant negative p53-expressing RKO/β4-Δcyt and RKO/β4 subclones were obtained by cotransfecting RKO/β4-Δcyt clone 3E1 and RKO/β4 clone D4 with plasmids expressing the puromycin resistance gene and a dominant negative p53 (dnp53) construct (provided by M. Oren, Weizmann Institute for Science, Israel) that encodes for a carboxy-terminal domain of p53 that can heterodimerize with endogenous p53 and inhibit its transcriptional activity. Dnp53-expressing subclones were obtained and those subclones expressing high levels of dnp53 were selected by FACS using the Pab122 mAb (Boehringer Mannhein), which recognizes a conserved, denaturation stable epitope in dnp53. In addition, RKO/β4 and RKO/β4-Δcyt cells were transfected with the puromycin resistance gene plasmid alone to obtain puromycin-resistant mock transfectants. All assays were performed using cell maintained below passage 10. Stable transfectants of MDA/β4 clone 3A7 that expressed temperature-sensitive p53 were obtained by cotransfecting this cell line with plasmids expressing the puromycin resistance gene (1 mg) and a plasmid expressing a temperature-sensitive mutant of human p53 (tsp53; 4 μg) that assumes a functional conformation at 32°C, but not at 37°C using the Lipofectamine reagent (GIBCO BRL). After growing these transfectants in complete medium for 2 d, stable transfectants were selected by culturing these cells in puromycin-containing medium (2 μg/ml) for an additional 18 d. These bulk transfectants were expanded and tsp53 expression was confirmed by showing increased p53 levels in tsp53 transfectants relative to mock transfectants by immunoblotting with a goat anti-human p53, followed by HRP-conjugated donkey anti–goat IgG. All assays were performed on cells maintained below passage 5. Dominant negative AKT (dnAKT)/PKB–expressing MDA-MB-435/mock and MDA-MB-435/β4 transient transfectants were generated by cotransfecting these cell lines using the Lipofectamine reagent (GIBCO BRL) with a plasmid encoding for green fluorescent protein (pEGFP-1; CLONTECH Laboratories; 1 μg) and a dnAKT/PKB construct that contains inactivating mutations in the catalytic domain of AKT/PKB (4 μg) . The following antibodies were used: 439-9B, a rat mAb specific for the β4 integrin subunit , control rat IgG (Sigma Chemical Co.); Pab122, a polyclonal rabbit serum specific for p53 (Boehringer Mannheim); goat anti-human p53; rabbit polyclonal anti-AKT/PKB raised against a peptide corresponding to mouse AKT/PKB residues 466–479 (New England Biolabs); rabbit polyclonal anti-AKT/PKB phosphoserine 473 (New England Biolabs); rabbit anti-actin (Sigma Chemical Co.); and mouse anti-hemagglutinin (Boehringer Mannheim). Goat anti–mouse IgG and goat anti–rat IgG secondary antibodies, as well as HRP conjugates of these antibodies, were obtained from Jackson ImmunoResearch Laboratories, Inc. HRP-conjugated donkey anti–goat IgG was obtained from BioSource International. To induce apoptosis in the RKO and MDA-MB-435 transfectants, the cells were plated in complete medium for 8 h in tissue culture wells (12-well plate; 2.5 × 10 5 cells/well) that had been coated overnight at 4°C with poly- l -lysine (Sigma Chemical Co.; 2 ml of 25 μg/ml stock) and blocked with 1% BSA. After 8 h, this medium was replaced with serum-free culture medium containing 1% BSA. After 15 h at 37°C, adherent and suspension cells were harvested, combined, and the level of apoptosis in these cells was assessed as described below. For annexin V stains, cells were washed once with serum-containing medium, once with PBS, once with annexin V-FITC buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ), and incubated for 15 min at room temperature with annexin V-FITC (Bender MedSystems) at a final concentration of 2.5 μg/ml in annexin V buffer. After washing once with annexin V buffer, the samples were resuspended in the same buffer and analyzed by flow cytometry. Immediately before analysis, propidium iodide was added to a final concentration of 5 μg/ml to distinguish apoptotic from necrotic cells, and 5,000 cells were analyzed for each sample. For ApopTag reactions, cells were harvested as described above, fixed in 1% paraformaldehyde for 15 min on ice, and washed twice with PBS. The samples were resuspended in 1 ml ice-cold 70% ethanol and stored at −20°C overnight. After centrifugation at 2,500 rpm for 15 min, cells were washed two times in PBS before performing ApopTag reactions (Oncor) according to the manufacturer's recommendations. These samples were analyzed by flow cytometry. For in situ analysis of apoptosis in cells transfected transiently with the green fluorescent protein (GFP)–expressing vector pEGFP-1 (CLONTECH Laboratories) and dnAKT/PKB, the transfected cells were stained with annexin V-PE (PharMingen) according to the manufacturer's directions, and plated on coverslips. The percentage of GFP-positive cells that was annexin V-PE–positive was determined by fluorescence microscopy. A total of at least 80 GFP-positive cells from at least 10 microscopic fields were analyzed for each data point. To assess the expression of endogenous AKT/PKB protein, cells were incubated with either rat Ig or 439-9B as described above in the presence of either DMSO (1:500), a caspase 3 inhibitor (Z-DEVD-FMK; Calbiochem-Novabiochem; 4 μg/ml), or a caspase 8 inhibitor (Z-IETD-FMK; Calbiochem-Novabiochem; 4 μg/ml). After washing with PBS, the cells were plated in serum-free medium containing 1% BSA in wells of a 12-well plate that had been coated with anti–rat Ig (13.5 μg/ml) and blocked for 1 h at 37°C with 1% BSA-containing medium. After a 1-h stimulation, adherent and suspension cells were harvested and extracted with AKT/PKB lysis buffer (20 mM Tris, pH 7.4, 0.14 M NaCl, 1% NP-40, 10% glycerol, 2 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, 50 μg/ml leupeptin, 1 mM sodium orthovanadate). After removing cellular debris by centrifugation at 12,000 g for 10 min, equivalent amounts of total cell protein from these extracts were resolved by SDS-PAGE (8%) and transferred to nitrocellulose. The blots were probed with a rabbit anti-AKT/PKB antiserum, followed by HRP-conjugated goat anti–rabbit Ig, and the immunoreactive bands were visualized by enhanced chemiluminescence. These blots were also probed with a rabbit antiserum specific for actin to confirm the loading of equivalent amounts of protein. Relative AKT/PKB and actin expression levels were assessed by densitometry using IP Lab Spectrum software (Scanalytics). To determine the level of serine 473–phosphorylated AKT/PKB, cells were transfected transiently using the Lipofectamine reagent (GIBCO BRL) with an HA-tagged AKT/PKB cDNA (provided by A. Toker, Boston Biomedical Research Institute, Boston, MA). 20 h after transfection, these cells were harvested by trypsinization and subjected to antibody-mediated integrin clustering. Specifically, cells were incubated on ice for 30 min with either control rat IgG or 439-9B at a concentration of 10 μg/ml. After washing with PBS, the cells were plated in serum-free medium containing 1% BSA onto wells of a 60-mM tissue culture dish that had been coated at 4°C with anti–rat Ig (13.5 μg/ml) and blocked for 1 h at 37°C in 1% BSA-containing medium. After 1 h, adherent and suspension cells were harvested and washed twice with PBS. Proteins from these cells were extracted with AKT/PKB lysis buffer (see above). After removing cellular debris by centrifugation at 12,000 g for 10 min at 4°C, equivalent amounts of total cellular protein were precleared with a 1:1 mixture of protein A and protein G–Sepharose for 1 h at 4°C. Immunoprecipitations were performed for 1 h on these precleared lysates using an HA-specific mAb (1 μg; Boehringer Mannheim) and protein A/protein G–Sepharose beads. Proteins from these immunoprecipitates were subjected to reducing SDS-PAGE (8%), transferred to nitrocellulose, and probed with an AKT/PKB phosphoserine 473–specific rabbit antiserum (New England Biolabs) followed by HRP-conjugated goat anti–rabbit IgG. Phospho-AKT/PKB was detected on these blots by chemiluminescence (Pierce Chemical Co.). These samples were also probed with rabbit anti-AKT/PKB. The relative intensity of phosphoserine AKT/PKB and AKT/PKB bands was assessed by densitometry, as described above. Baculovirus-expressed AKT/PKB (0.5 μg; provided by A. Toker) was incubated with either active recombinant caspase 8 (2 mg; Calbiochem-Novabiochem) or active recombinant caspase 3 (2 μg; Calbiochem-Novabiochem) at 37°C for 1 h in a final volume of 10 μl. Subsequently, the reaction mixtures were divided into two aliquots and resolved by SDS-PAGE (8%). The gels were silver stained using the GelCode SilverSNAP Stain Kit (Pierce Chemical Co.) or transferred to nitrocellulose and probed with a rabbit AKT/PKB antiserum as described above. For our initial experiments, we used stable β4 transfectants of two α6β4-deficient carcinoma cell lines that differ in their p53 status: RKO colon carcinoma cells, which express wild-type p53 ; and MDA-MB-435 breast carcinoma cells, which express a mutant, inactive form of p53 . We also used RKO and MDA-MB-435 cells that express a cytoplasmic domain deletion mutant of α6β4 (RKO/β4-Δcyt; MDA/b4-Δcyt) that is signaling deficient. The characterization of these cells has been described previously . To explore the potential influence of α6β4 expression on the survival of serum-starved carcinoma cells deprived of matrix attachment, the α6β4 and α6β4-Δcyt–expressing RKO and MDA-MB-435 subclones were plated on poly- l -lysine in serum-free medium. The level of apoptosis in these populations was determined either by staining with annexin V-FITC to detect cells in the early stages of apoptosis or by performing terminal deoxynucleotidyl transferase end labeling reactions (Apoptag) to detect DNA fragmentation . In addition, we assessed the viability of these serum-deprived cells by measuring the cellular uptake of propidium iodide ( Table ). The ability of α6β4 to promote the survival of these cells was determined by subtracting the percent apoptotic α6β4-expressing cells from the percent apoptotic α6β4-Δcyt–expressing cells. The expression of α6β4 in MDA-MB-435 cells significantly increased the survival of these cells relative to MDA-MB-435 cells expressing α6β4-Δcyt, as assessed by annexin V-FITC staining , ApopTag staining , and propidium iodide uptake ( Table ). In contrast, the expression of α6β4 in RKO cells did not increase the survival of these cells relative to either the mock ( Table ) or RKO/β4-Δcyt transfectants . In fact, we observed a higher level of apoptosis and cell death in serum-starved RKO/β4 as compared with RKO/β4-Δcyt cells, in agreement with our previous demonstration that α6β4 can promote apoptosis in wild-type p53 carcinoma cells . Based on the fact that RKO and MDA-MB-435 cells differ in their p53 status, we reasoned that the ability of α6β4 to promote cell survival may be inhibited by p53. This hypothesis was examined by investigating the effect of α6β4 expression on the survival of RKO cells in which p53 activity had been inhibited by the expression of a dnp53 construct. Indeed, α6β4 expression promoted the survival of serum-starved, dnp53-expressing RKO cells as determined by ApopTag and annexin V-FITC staining . These results demonstrate that p53 can suppress the survival signaling mediated by α6β4 in serum-starved carcinoma cells. Given the importance of the AKT/PKB kinase in numerous survival signaling pathways , we investigated whether the survival function of α6β4 in serum-starved, p53-deficient carcinoma cells was AKT/PKB–dependent. The MDA-MB-435/β4–transfected clones, as well as the parental cells, were cotransfected with plasmids encoding for GFP and an HA-tagged, kinase-deficient AKT/PKB mutant that acts as a dominant negative construct (dnAKT/PKB) . Expression of this dnAKT/PKB construct was confirmed by immunoblotting extracts from these transfected cells with an HA-specific mAb (data not shown). After 15 h of serum starvation, the level of apoptosis in GFP-positive cells was assessed by annexin V-PE staining. As shown in Fig. 2 , MDA-MB-435/β4 clones demonstrated significantly less apoptosis than parental MDA-MB-435 cells in agreement with the data shown in Table . Importantly, dnAKT/PKB expression inhibited this α6β4 survival function in each of the two MDA-MB-435/β4 clones examined, but it did not alter the level of apoptosis in parental MDA-MB-435 cells. To understand the mechanism by which p53 inhibits α6β4-mediated survival, we investigated the possibility that p53 alters the ability of this integrin to activate AKT/PKB. Initially, we examined whether the antibody-mediated clustering of α6β4 in MDA-MB-435 cells resulted in the phosphorylation of AKT/PKB on serine 473, an event that has been shown to correlate with AKT/PKB activation . MDA-MB-435/β4 subclones were transfected with an HA-tagged AKT/PKB construct. These cells were incubated with either a control rat IgG or the β4-specific antibody 439-9B and plated in the absence of serum on secondary antibody–coated tissue culture wells for 1 h. HA immunoprecipitations were performed on extracts from these cells, and the levels of serine-phosphorylated AKT/PKB were assessed by blotting these immunoprecipitates with an antiserum specific for AKT/PKB molecules phosphorylated on serine residue 473. As shown in Fig. 3 A, the antibody-mediated clustering of α6β4 stimulated an increase in the level of serine-phosphorylated AKT/PKB in each of the two MDA-MB-435/β4 subclones relative to control cells (2.1-fold increase, β4 clone 1; 5.5-fold increase, β4 clone 2). This α6β4-induced increase in AKT/PKB serine phosphorylation was dependent on α6β4 signaling based on the inability of α6β4-Δcyt clustering to increase the level of the serine 473–phosphorylated AKT/PKB in MDA-MB-435/β4-Δcyt subclones (data not shown). To investigate the influence of p53 on the activation of AKT/PKB by α6β4, we explored whether α6β4 clustering induced the phosphorylation of AKT/PKB on serine residue 473 in MDA-MB-435/β4 that had been reconstituted with functional p53. Specifically, MDA-MB-435/β4 cells were transfected with a temperature-sensitive mutant of human p53 (tsp53) that assumes a functional conformation at 32°C but not at 37°C . This construct has been used extensively to study the influence of p53 on signaling pathways involved in cell growth and apoptosis . Stable transfectants of these cells were selected, and tsp53 expression was confirmed by immunoblotting (data not shown). Tsp53 and mock-transfected cells were transfected transiently with HA-AKT/PKB. After incubating these cells with either rat IgG or 439-9B, they were plated on secondary antibody–coated wells and subjected to a 32°C incubation to stimulate p53 activity, followed by a 37°C incubation to activate AKT/PKB. HA immunoprecipitations were performed on extracts from these cells, and these immunoprecipitates were subjected to immunoblotting with phosphoserine 473 AKT/PKB–specific rabbit antiserum. As shown in Fig. 3 B, the clustering of α6β4 significantly increased the level of phosphoserine 473-AKT/PKB in mock-transfected MDA/β4 cells (7.9-fold increase), but not in tsp53-expressing MDA/β4 cells (1.2-fold increase). The importance of p53 in the inhibition of the α6β4-associated activation of AKT/PKB was indicated by the finding that α6β4 clustering increased the level of phosphoserine 473 AKT/PKB in MDA/β4 + tsp53 transfectants that had been incubated at 37°C, the nonpermissive temperature for this tsp53 construct (data not shown). The ability of p53 to suppress the α6β4-mediated activation of AKT/PKB was explored further in RKO carcinoma cells, which express wild-type p53. In agreement with the results obtained in MDA/β4 cells that had been reconstituted with functional p53, the clustering of α6β4 in two independent RKO/β4 subclones did not result in increased amounts of serine phosphorylated AKT/PKB . Importantly, the expression of dnp53 in RKO/β4 cells restored the ability of α6β4 to activate AKT/PKB, as evidenced by an increase in phosphoserine 473-AKT/PKB immunoreactivity in RKO/β4 + dnp53 cells that had been subjected to antibody-mediated α6β4 clustering (8.6-fold increase), as described above . The ability of α6β4 to stimulate AKT/PKB activity in RKO/β4 + dnp53 cells but not in RKO/β4 cells was confirmed by performing in vitro kinase assays using histone H2B as a substrate (data not shown). As a control for specificity, we also demonstrated that the clustering of α6β4 on dnp53-expressing RKO/β4-Δcyt cells did not stimulate AKT/PKB activity (data not shown). To define the mechanism by which p53 inhibits the ability of α6β4 to activate AKT/PKB, we investigated whether p53 alters AKT/PKB expression levels in response to α6β4 clustering. RKO/β4 and RKO/β4 + dnp53-expressing cells were incubated with either rat Ig or 439-9B and stimulated on secondary antibody–coated wells for 1 h. The amount of total AKT/PKB in equivalent amounts of total protein from these lysates was assessed by immunoblotting. Importantly, the antibody-mediated clustering of the α6β4 integrin on each of two RKO/β4 subclones resulted in a significant reduction in the total level of AKT/PKB in these cells . In contrast, AKT/PKB levels were not reduced in dnp53-expressing RKO/β4 cells or in MDA-MB-435/β4 subclones (data not shown) after the antibody-mediated clustering of α6β4. We also observed decreased levels of HA-AKT/PKB protein in HA-AKT/PKB–transfected RKO/β4 cells, but not in HA-AKT/PKB–transfected RKO/β4 + dnp53 cells upon the antibody-mediated clustering of α6β4 (data not shown). Based on the reported ability of caspases to cleave signaling molecules that promote cell survival , we hypothesized that α6β4 may promote the caspase-dependent cleavage of AKT/PKB in wild-type p53-expressing carcinoma cells. Initially, we explored the importance of caspase 3 activity, which has been shown to play a crucial role in p53-dependent apoptotic pathways , in the α6β4-associated reduction of AKT/PKB expression levels. In agreement with the data shown in Fig. 4 , the clustering of α6β4 in control RKO/β4 cells significantly reduced the level of AKT/PKB in these carcinoma cells . However, RKO/β4 cells that had been pretreated with Z-DEVD-FMK, a cell permeable caspase 3 inhibitor, did not exhibit decreased levels of AKT/PKB in response to α6β4 clustering . In contrast, we detected a decreased amount of AKT/PKB after the clustering of α6β4 in RKO/β4 cells that had been pretreated with Z-IETD-FMK, a cell permeable caspase 8 inhibitor . Importantly, no effect of these inhibitors on AKT/PKB levels was observed upon the clustering of α6β4 on RKO/α6β4-Δcyt cells (data not shown). The ability of the caspase 3 inhibitor to restore normal AKT/PKB levels suggested that AKT/PKB is cleaved by caspase 3 upon the clustering of α6β4 in carcinoma cells expressing wild-type p53. To establish the caspase 3–mediated cleavage of AKT/PKB more rigorously, we investigated whether a recombinant form of this cysteine protease could cleave baculovirus-expressed AKT/PKB in vitro. Proteins in these reactions were resolved by SDS-PAGE and detected by silver staining. The results obtained revealed that the incubation of baculovirus-expressed AKT/PKB ( M r , 60 kD) with recombinant caspase 3 resulted in the formation of an AKT/PKB cleavage product ( M r , 49 kD) . In contrast, we did not detect an AKT/PKB cleavage product after the incubation of baculovirus AKT/PKB with recombinant caspase 8 . The caspase 3–generated AKT/PKB cleavage product was also detected by immunoblotting with an antiserum specific for the carboxy terminus of AKT/PKB, suggesting that caspase 3 cleaves AKT/PKB at its amino terminus (data not shown). Finally, to demonstrate that the caspase 3–dependent cleavage of AKT/PKB was responsible for the p53 inhibition of AKT/PKB activity in RKO/β4 cells, we explored the effects of a caspase 3 inhibitor on the ability of α6β4 to activate AKT/PKB. HA-AKT/PKB–transfected RKO/β4 cells were subjected to antibody-mediated α6β4 clustering in the presence of either DMSO or the caspase 3 inhibitor Z-DEVD-FMK. HA immunoprecipitates from extracts from these cells were subjected to immunoblotting with the phosphoserine 473 AKT/PKB–specific rabbit antiserum. As shown in Fig. 7 , the pretreatment of RKO/β4 cells with Z-DEVD-FMK restored the ability of α6β4 to stimulate the phosphorylation of AKT/PKB in these cells. These results demonstrate that α6β4 stimulates the caspase 3–dependent cleavage and inactivation of AKT/PKB in p53 wild-type, but not in p53-deficient carcinoma cells. The binding of extracellular matrix proteins to integrins initiates survival signals that inhibit anoikis, a form of apoptosis induced upon the detachment of cells from extracellular matrix . In the current studies, we show that the α6β4 integrin suppresses anoikis exclusively in carcinoma cells that lack functional p53. Furthermore, we demonstrate that this α6β4-associated survival function depends on the ability of this integrin to activate the serine/threonine kinase AKT/PKB in p53-deficient cells. Finally, we provide evidence that p53 inhibits the α6β4-mediated activation of AKT/PKB by promoting the caspase 3–dependent cleavage of this kinase. Collectively, our findings establish that p53 can inhibit an integrin-associated survival function, a phenomenon that has important implications for tumor cell growth. Our results suggest that the α6β4 integrin can enhance the survival of carcinoma cells in an AKT/PKB–dependent manner. Although previous studies have shown that cell attachment to matrix proteins promotes the survival of primary epithelial cells , α6β4 is the first specific integrin to be implicated in the delivery of AKT/PKB–dependent survival signals to carcinoma cells. The importance of AKT/PKB in α6β4 survival signaling was indicated in our studies by the ability of a dnAKT/PKB construct containing inactivating mutations in the catalytic domain to inhibit the survival effect of α6β4 in serum-starved MDA-MB-435 cells. Although this dnAKT/PKB has been used extensively to implicate AKT/PKB in survival pathways, it is possible that it associates with phosphoinositide-dependent kinases and inhibits their activity. However, our observation that the expression of a constitutively active AKT/PKB in MDA-MBA-435 enhances their survival (data not shown) strongly suggests that α6β4 expression promotes the survival of these cells by activating AKT/PKB. Our demonstration that p53 can inhibit AKT/PKB kinase activity is of interest in light of the recent finding that the PTEN tumor suppressor can also inhibit cell growth by inhibiting AKT/PKB in a manner that is dependent on its lipid phosphatase activity . Together, our current findings on p53 and the previously described activities of PTEN highlight the impact of tumor suppressors on integrin-mediated functions. Moreover, our demonstration that p53 inhibits α6β4 survival signaling by promoting the caspase-dependent cleavage of AKT/PKB provides a mechanistic link between tumor suppressor function and the regulation of integrin signaling, similar to the phosphatase activities of PTEN. Although previous studies have demonstrated that caspases can be activated by p53 in both cell-free systems as well as in response to DNA damage , our findings suggest that caspases can also be activated by an integrin in a p53-dependent manner. Indeed, it will be informative to determine if other activators of p53 such as DNA damage can promote the caspase-dependent cleavage of AKT/PKB. The finding that AKT/PKB activity can be regulated by caspase 3 substantiates the hypothesis that caspases play an important role in many forms of apoptosis based on their ability to cleave signaling molecules that influence cell survival. For example, caspases have been shown to cleave and inactivate an inhibitor of caspase-activated deoxyribonuclease (CAD). Importantly, the cleavage of this inhibitor results in the activation of CAD, which is the enzyme responsible for the DNA fragmentation that is characteristic of apoptosis . Caspase 3 has also been shown to cleave bcl-2, resulting in an inhibition of its anti-apoptotic function . While AKT/PKB has been suggested to be a target of caspase activity based on the reduced levels of this kinase observed in T cells in response to fas stimulation , our results extend this finding by establishing definitively that AKT/PKB is cleaved by caspase 3. More importantly, we provide evidence that this cleavage event results in the inhibition of AKT/PKB kinase activity, and implicate this event in the inhibition of α6β4 integrin survival function. It is important to consider the mechanism by which the α6β4-induced, caspase-dependent cleavage of AKT/PKB inhibits its kinase activity. We detected an AKT/PKB fragment ( M r , 49 kD) after the in vitro incubation of AKT/PKB with recombinant caspase 3. This fragment was recognized by a rabbit antiserum raised against a peptide corresponding to the extreme carboxy-terminal amino acids of the molecule, suggesting that caspase 3 cleaves AKT/PKB at its amino terminus. Interestingly, the pleckstrin homology domain, which resides in the amino terminus of AKT/PKB, is important in both the translocation of this kinase to the membrane and its subsequent activation . It is possible that the caspase 3–dependent cleavage of AKT/PKB prevents the membrane translocation of this kinase, thus, preventing its activation. However, we were unable to identify an AKT/PKB fragment in vivo after the clustering of α6β4, despite our detection of reduced AKT/PKB levels under these conditions. This result suggests that after the initial cleavage of AKT/PKB by caspase 3, this kinase is subjected to further cleavage by other caspases, as has been shown for ICAD . Moreover, our inability to detect AKT/PKB fragments in vivo after the clustering of α6β4 suggests that AKT/PKB cannot be detected by immunoblotting after its cleavage by multiple caspases. The ability of a caspase 3 inhibitor to restore both normal AKT/PKB levels as well as the α6β4-mediated activation of AKT/PKB suggests that the degradation of AKT/PKB observed in vivo is dependent on the initial cleavage of this kinase by caspase 3. In contrast to our finding that p53-dependent, caspase 3 activity inhibits AKT/PKB, other studies have concluded that constitutively active AKT/PKB can delay p53-dependent apoptosis , inhibit caspases , and block caspase-dependent forms of apoptosis . The demonstrated ability of AKT/PKB to inhibit p53 and caspase activity in these studies may relate to the kinetics of AKT/PKB activation. Specifically, the rapid stimulation of AKT/PKB may impede p53 or caspase activation. In contrast, the ability of α6β4 clustering to promote the caspase 3–dependent inactivation of AKT/PKB in p53 wild-type carcinoma cells may relate to the fact that α6β4 signaling stimulates caspase activity before AKT/PKB activity in these cells. Alternatively, it is possible that the ability of caspase 3 to cleave AKT/PKB was not observed in previous studies because insufficient amounts of endogenous caspase activity were present to inhibit the activity of exogenously introduced, active AKT/PKB. Nonetheless, these results suggest that an intimate crosstalk exists between AKT/PKB and caspases that contributes to the regulation of cell survival. We have previously demonstrated that the α6β4 integrin activates p53 function . The current studies describe an important consequence of this α6β4 activity, namely the inhibition of AKT/PKB activity and its associated cell survival function. Similar to previous results from our laboratory and others , the current studies demonstrate that the survival function of α6β4 is ligand-independent in β4-transfected, p53-deficient carcinoma cells. This ligand-independent survival function may be attributable to the ability of the β4 cytoplasmic domain to self-associate . In addition to demonstrating that p53 inhibits α6β4-mediated survival, we observed that α6β4 increases the level of apoptosis observed in serum-starved p53 wild-type carcinoma cells. This result suggests that the apoptotic signaling pathway activated by α6β4 can augment the apoptotic signaling initiated by serum deprivation. Although p53 has been implicated in the apoptosis induced in endothelial cells upon their detachment from matrix , others have reported that epithelial cell anoikis is p53-independent . In agreement with the results of the latter study, we observed apoptosis in p53-deficient cells, including MDA-MB-435 cells and dnp53-expressing RKO cells, upon their detachment from matrix. These results indicate that carcinoma cells are subject to a p53-independent form of anoikis. In combination with our previous observation that α6β4 apoptotic signaling requires p53 activity , our findings suggest that the p53-independent apoptosis of carcinoma cells that occurs in response to matrix detachment can be enhanced by p53-dependent, α6β4 apoptotic signaling. The current studies may explain why the α6β4 integrin has been implicated in the apoptosis of some cells and the survival of others. Specifically, α6β4 has been shown to induce growth arrest and apoptosis in several carcinoma cell lines as well as in endothelial cells . However, this integrin has also been shown to promote the proliferation and survival of keratinocytes. These apparently contradictory functions of α6β4 may relate to the fact that the functions of α6β4 are cell type–specific. The current studies establish that the p53 tumor suppressor is one critical signaling molecule that may influence α6β4 function in different cell types because this integrin promotes apoptosis only in wild-type p53-expressing cells and survival only in p53-deficient cells. Interestingly, the reported ability of α6β4 to promote keratinocyte survival may relate to the reported deficiency of p53 activity in these cells . One implication of our findings is that the α6β4 integrin is similar to a number of oncogenes that promote cell proliferation in some settings and cell death in others. The recent observation that oncogenes can deliver such death signals has led to their seemingly contradictory categorization as tumor suppressors in select environments. For example, although the stimulation of c-myc and E2F normally promotes cell proliferation, the activation of these oncogenes induces apoptosis in the presence of secondary stress signals such as p53 expression, serum starvation or hypoxia . The ability of these stress signals to stimulate oncogene-dependent apoptosis is thought to be important in eliminating tumor cells that escape normal proliferation checkpoints as a result of oncogene expression. Similarly, the α6β4 integrin, which promotes the survival of p53-deficient cells, could also be classified loosely as a tumor suppressor based on its apoptotic function in carcinoma cells that express wild-type p53. The current studies demonstrate that, similar to the activity of oncogenes, integrin function and signaling can be profoundly influenced by physiological stimuli that activate other signaling pathways in a cell. In summary, we have described the ability of the α6β4 integrin to promote the survival of the p53 mutant, but not p53 wild-type carcinoma cells. This ability of p53 to influence integrin-mediated functions so markedly derives from its ability to activate the caspase 3–dependent cleavage of AKT/PKB. The fact that AKT/PKB overexpression has been suggested to contribute to the transformed phenotype of tumor cells suggests that the introduction of the α6β4 integrin into p53 wild-type tumors may inhibit their growth by inducing the cleavage of this transforming protein. The ability of α6β4 to induce the p53-dependent cleavage of AKT/PKB also suggests that the acquisition of inactivating mutations in either p53 or caspase 3 will provide a selective growth advantage for carcinoma cells by stimulating α6β4-mediated AKT/PKB–dependent survival signaling. Moreover, given our previous demonstration that α6β4 promotes carcinoma cell migration and invasion , we suggest that carcinoma cells that express α6β4 and mutant forms of p53 or caspase 3 will have a distinct advantage in their ability to disseminate and survive as metastatic lesions. | Other | biomedical | en | 0.999998 |
10579726 | RNA display was performed using an RNAmap Kit (GenHunter) according to the manufacturer's instructions. The resulting PCR products were cloned into plasmid pBluescript for sequencing analysis. To screen the rat PC12 cDNA λgt11 library (Clontech) for full-length theta-associated protein (TAP) 20, both the 5′ and 3′ insert screening amplimers of λgt11 as the forward primers and a TAP20 3′ end sequence ACCATAAGAATGCAGACAAGA as the reverse primer were used for PCR reaction with pfu DNA polymerase (Stratagene). The PCR products were cloned into the pBluescript plasmid (Stratagene) and sequenced with T7 and T3 primers. To explore the function of TAP20 in mammalian cells, we constructed a TAP20 + green fluorescent protein (GFP) expression plasmid. The EGFP (enhanced GFP) gene from plasmid pEGFP-C1 (Clontech) including its 5′ end cytomegalovirus (CMV) promoter to 3′ end polyA signal region, was inserted into the EcoRI-KpnI restriction sites (non-multicloning sites) to create a GFP/pRc plasmid. TAP20 cDNA was then cloned into the HindIII-XbaI restriction sites in the plasmid GFP/pRc. Thus, in this vector the TAP20 and EGFP genes are controlled by separate CMV promoters. To localize TAP20 protein in the cell, we cloned the TAP20 cDNA into the BglII-SmaI restriction sites in plasmid pEGFP-C1 for expression of a GFP-TAP20 fusion protein in mammalian cells. ECV304 cells (a human bladder carcinoma cell line, distributed by American Type Culture Collection) were cultured in M199 medium supplemented with 10% FBS and antibiotics (GIBCO BRL), MV3 cells were cultured in DMEM medium with 10% FBS and antibiotics, and human umbilical vein ECs (HUVEC) were cultured in M199 with 20% newborn calf serum, and 5% human serum, at 37°C in a humidified 5% CO 2 atmosphere. Monolayers of cells were transfected with either control vector or TAP20 construct using lipofectin (GIBCO BRL) or GenePorter (Gene Therapy System) for HUVEC. 1 d after transfection of GFP-containing plasmids, cells were harvested and suspended in Hanks' buffer (137 mM NaCl, 5.4 mM KCl, 5.6 mM dextrose, 4.2 mM NaHCO 3 , 0.42 mM Na 2 HPO 4 , 0.44 mM KH 2 PO 4 , pH 7.4), and were sorted by GFP fluorescence using a FACScan flow cytometer (Becton Dickinson). The sorted cells were recultured in the complete medium. Expression of TAP20 was confirmed by Western analysis using the anti-TAP20 antibody. The sorted cells were tested in the cell adhesion, migration, and tube formation experiments. Total RNA was prepared using Trizol (GIBCO BRL). For Northern transfer analysis, 20 μg of total RNA was subjected to electrophoresis on a 1.5% formaldehyde-agarose gel and transferred to Gene-Screen Plus membrane according to the manufacturer's recommendations. The blot was hybridized with random-primed TAP20 cDNA probes at 65°C for 3 h in Quik-Hyb solution (Stratagene), and was washed under high stringency conditions before autoradiography. Immunoblotting was performed as follows: cell lysates were prepared by addition of 1 ml of lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 30 ml/ml aprotinin) per 10 7 cells. Samples were run on a 15 or 10% SDS polyacrylamide gel and electrophoretically transferred to Immobilon-P membranes (Millipore). The membranes were then hybridized with indicated antibodies (anti-αv polyclonal antibody; Santa Cruz Biotechnology), PKCθ, β1, and β3 mAb (Transduction Laboratories), and anti-β5 polyclonal antibody (Chemicon), in PBS containing 5% dry milk and detected via ECL (Amersham Pharmacia Biotech). The affinity-purified rabbit polyclonal anti-TAP20 antibody was produced by QCB Inc. Cell adhesion was performed as described previously with minor modifications. In brief, 96-well polystyrene plates (Costar) were coated with 10 μg/ml laminin (LN), fibronectin (FN), or VN in PBS. Cells were harvested, suspended in medium M199 (GIBCO BRL) with 2% BSA at a concentration of 100,000 cells/0.1 ml, and 50,000 cells were added to each well, with antibody as indicated. After incubation at 37°C for 1 h, nonadherent cells were removed with gentle washing. The cells in wells that were not washed were used as the standard (100%). Quantitation of cell attachment was determined by staining the cells with 0.5% crystal violet in 20% methanol, washing cells, and then eluting the dye with 0.1 M sodium citrate, pH 4.2, and measuring the absorbance at 596 nm. The integrin-blocking antibodies were purchased from Chemicon. The cell monolayer was briefly treated with trypsin to lift the cells, which were pelleted and washed once with PBS and resuspended in M199 medium containing 0.1% BSA to a final concentration of 500,000 cells/ml. Cells were added to 3 μm FALCON Cell Culture FluoroBlok Inserts (Becton Dickinson) at a density of 50,000 cells/insert. M199 (0.8 ml) with 10% FBS and VEGF (10 ng/ml) was used as a chemoattractant in the lower wells. The inserts were incubated for 4 h at 37°C. The membranes of the inserts were then mounted on glass slides and coverslips. Cells migrated through the FluoroBlok Inserts were quantitated by counting the fluorescent cells with a Nikon Optiphot-2 fluorescence microscope with a COHU video camera (COHU Electronics) and NIH Image software for Macintosh computer. Matrix gel (MATRIGEL Basement Membrane matrix; Becton Dickinson) plates were prepared in 12-well plates following the manufacturer's instructions. After transfecting and sorting, HUVEC (∼80% confluent) were treated with trypsin, and 5 × 10 4 cells were seeded on the top of plates with complete M199 medium. The photographs were taken under a 100× light microscope, monitoring with a COHU video camera, and captured with a Scion 7 video card (Scion Image) in a PowerMac computer. 24 h after transfection with GFP alone, TAP20 + GFP, or GFP-TAP20 plasmids, human MV3 cells were plated on 4-well glass chamber slides (Nalge Nunc) coated with VN (10 μg/ml) (Sigma Chemical Co.). Cells were incubated for the indicated times in DMEM containing 10% BSA, fixed with 4% paraformaldehyde and 0.1% Triton X-100 in PBS for 5 min, and then incubated with 4% paraformaldehyde in PBS for an additional 20 min. Focal adhesions were visualized by incubating first with mouse mAb against vinculin (1 μg/ml; Chemicon), paxillin (0.5 μg/ml; Transduction Laboratories), or FAK (2 μg/ml, Transduction Laboratories), and then with Cy-3–conjugated goat antibody to mouse IgG (dilution 1/600, Jackson ImmunoResearch Laboratories). Cells were viewed on a Nikon Optiphot-2 fluorescence microscope and COHU video camera as described above. The TAP20 cDNA was inserted into BamHI and XhoI cloning sites in pGEX4T1 (Amersham Pharmacia Biotech). Cytoplasmic domains of integrin β3 (amino acid sequence: KLLITIHDRKEFAKFEEERARAKWFTANNPLYKEATSTFTNITYRGT) or β5 (amino acid sequence: KLLVTIHDRREFAKFQSERSRARYEMASNPLYRKPISTHTVDFTFNKFNKSYNGTVD) were synthesized by the PCR method using the pfu enzyme and inserted into the BamHI and XhoI cloning sites. Glutathione S -transferase (GST) or GST fusion proteins were expressed in Escherichia coli BL21 cells and immobilized on glutathione Sepharose beads (Amersham Pharmacia Biotech) following the manufacturer's instructions. To precipitate protein from EC lysate, the GST-TAP20 coated beads were incubated with the cell lysate in PBS for 20 h at 4°C. To precipitate TAP20 with GST-integrin tail fusion protein, the GST-TAP20 beads were first incubated in thrombin containing buffer for 16 h at room temperature to release the TAP20 protein. The TAP20-containing solution was then incubated with GST-integrin tail fusion protein beads for 20 h at 4°C. After three washes with PBS, the proteins bound to the beads were eluted in 10 mM glutathione buffer and analyzed by Western blotting. The antibodies used included: GST polyclonal antibody (Amersham Pharmacia Biotech), αv polyclonal antibody (Santa Cruz Biotechnology), β1 mAb and β3 mAb (Transduction Laboratories), and β5 polyclonal antibody (Chemicon). We investigated the expression of genes regulated by PKCθ with the mRNA display method using mRNAs from clonal populations of rat capillary endothelial cells (RCE), in which either the kinase-negative PKCθ (PKCθ-kn, a dominant negative inhibitor) or a constitutively active form of PKCθ (PKCθ-ca) were overexpressed . An RNA of ∼0.8 kb whose expression depended upon the presence of active PKCθ was identified. Northern transfer analysis showed that this 0.8-kb mRNA is highly expressed in PKCθ-ca RCE but is dramatically suppressed in PKCθ-kn RCE . The partial sequence of this gene obtained from differential display was used to screen a rat PC12 cDNA library by the PCR method. The cDNA generated from the PCR was cloned, and the nucleotide sequence was determined . The open reading frame encodes a novel protein of 175 residues with a calculated molecular mass of ∼20 kD. An antibody raised against the COOH terminus of this putative protein recognized a protein of 20 kD , as assessed by immunoblotting of an RCE cell lysate, which confirmed that the expression of this protein depended upon functional PKCθ, as suggested by the Northern transfer analysis. In the PKCθ kinase-negative RCE, TAP20 expression was significantly decreased at the protein level. This anti-TAP20 antibody also recognized a 20-kD band in TAP20 transfected human cells , suggesting that TAP20 can be expressed as a 20-kD, full-length protein in the cells. Accordingly, this novel PKCθ-associated protein was designated TAP20. By searching the GeneBank database, we found that a partial 3′ end sequence of TAP20 had been reported previously , and the first 110 amino acid residues of TAP20 share 55% homology with human β3-endonexin , a 111-residue polypeptide that interacts with the β3 integrin subunit . TAP20 also has an additional 66-residue COOH terminus. Thus, although the differences suggest that TAP20 may have functional properties that differ from those of β3-endonexin, we hypothesized that TAP20 is involved in integrin-mediated cell functions that are regulated by PKCθ. The immunoblotting with the anti-TAP20 antibody that recognizes an epitope at the COOH terminus of the protein suggests that TAP20 is expressed as a full-length protein in RCE. To explore the function of TAP20 in human cells, TAP20 cDNA was cloned into the pRc/CMV vector and a cDNA encoding GFP was inserted into a separate region of the same plasmid; thus, these two cDNAs were controlled by separate CMV promoters . 1 d after transfection, human ECV304 or HUVEC cells were sorted by GFP fluorescence using FACS ® . Cells with GFP expression were recultured for 1–2 d before experiments. Cells were then harvested for cell adhesion, migration, and tube formation experiments, and also for the protein expression test . Blotting with anti-TAP20 antibody showed that the antibody does not detect antigen in the 20-kD range in wild-type (wt) or GFP transfected cells. The expression of TAP20 in unsorted TAP20 + GFP cells was 30–70% of the sorted cells, depending on the transfection efficiency. Furthermore, the expression level of PKCθ, integrin αv, β3, and β5 was not changed in the TAP20 + GFP expressed ECV cells compared with the untransfected or GFP transfected cells . To investigate the TAP20 effect on focal adhesion and to further characterize TAP20 distribution in the cells, either the TAP20 + GFP plasmid or the fusion protein GFP-TAP20 plasmid was transfected into MV3 cells, a β3 integrin–deficient cell line. The transfection efficiency varied from 20–40% in different experiments. 2 d after transfection, the cells were harvested and tested for TAP20 expression. Similar to ECV cells, the anti-TAP20 antibody did not detect antigen in the 20-kD range in nontransfected or GFP transfected MV3 cells, whereas a 20-kD band in the TAP20 + GFP transfected cells and an ∼45-kD band in the GFP-TAP20 transfected cells were recognized, which indicated the expression of TAP20 and GFP-TAP20 fusion protein, respectively . Transfected ECV cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 1–2 d. Adhesion of TAP20 + GFP expressing cells to VN, but not to FN and LN, was significantly attenuated compared with that of either group of control cells, i.e., the wt cells or the cells expressing GFP only (a mean of 33.7 ± 5.1% of the added TAP20 + GFP cells adhered to VN, in contrast to 73.5 ± 7.5% [wt] and 75.0 ± 6.3% [GFP] of the control cells, P < 0.01). This result indicates that TAP20 affects cell adhesion by modulating the VN receptors, i.e., the αvβ3 and/or αvβ5 integrins. To further investigate the TAP20 effect on VN receptors, antiintegrin antibodies were added to cells on VN-coated plates to block function of the VN receptors. As a control to rule out nonspecific effects of integrin ligation, an anti–α2β1 integrin (LN receptor) antibody, BHA2.1 was tested. The results obtained with the anti–VN receptor antibodies were compared with those with anti-α2β1 antibody . The anti–α2β1 integrin antibody BHA2.1 did not significantly affect cell adhesion on VN . Furthermore, adhesion of TAP20 + GFP cells on VN was reduced in the presence of BHA2.1 (31.6 ± 7.7% of added cells) to an extent similar to that with cells on VN plates without BHA2.1 antibody . In the experiments with the anti–VN receptor antibodies, the effects of the anti-αvβ3 antibody LM609 were compared with those of the control antibody BHA2.1. The degree of adhesion in the BHA2.1 group was used as basal (100%) for comparison with that of the LM609 and P1F6 groups in Fig. 3 B. LM609 caused a 50.3% reduction of adhesion of TAP20 + GFP cells, and also caused a 39.4% reduction in the wt cells and a 43.4% reduction in GFP cells. The difference in the magnitudes of the reductions between BHA2.1 and LM609 was not statistically significant among the three groups ( P > 0.5). In contrast, the anti-αvβ5 antibody P1F6 compared with the control antibody BHA2.1 caused only a 7.4% reduction of adhesion of the TAP20 + GFP cells, but caused significant reduction in both groups of the control cells (38.0 [wt] and 45.6% [GFP]). The magnitude of the reductions between BHA2.1 and P1F6 was significantly less in the TAP20 + GFP cells than that seen in either group of the control cells ( P < 0.01). These observations suggest that in the TAP20 transfectants, the αvβ3 integrin remains functional as a VN receptor, which can be blocked by the anti-αvβ3 antibody LM609. On the other hand, the VN receptor function of αvβ5 integrin was attenuated by TAP20 overexpression. Thus, blockage of αvβ5 in TAP20 transfectants by anti-αvβ5 antibody P1F6 did not further reduce cell adhesion on VN significantly, whereas the adhesion of the control cells with normal αvβ5 function was dramatically decreased by this anti-αvβ5 antibody. The results of adhesion with the integrin-blocking antibodies suggested that TAP20 affects cell function through the αvβ5 integrin. To further clarify the role of TAP20 on αvβ5 integrin, we next examined the effect of TAP20 on focal adhesion formation in MV3 cells , which are αvβ3-negative human melanoma cells and therefore have the αvβ5 integrin as the principal surface receptor for VN. Thus, when the cells are cultured on VN-coated plates, the focal adhesion formation is caused chiefly by the interaction of αvβ5 integrin with VN. We first investigated the cellular localization of TAP20. After 16 h of adhesion to VN, MV3 cells expressing either GFP alone or TAP20 + GFP were stained with anti-TAP20 antibody that was visualized with a Cy-3–conjugated secondary antibody . The strong staining in Fig. 4 A, panel d, suggests that TAP20 exists in transfected cells as a full-length protein. The TAP20 fluorescence was observed diffusely in the cytoplasm and the nucleus, which was also demonstrated by GFP fluorescence of the cells expressing the GFP-TAP20 fusion protein . When these cells were stained with antifocal adhesion component antibodies to visualize the focal adhesions, no strong colocalization of GFP-TAP20 with focal adhesions was observed, indicating a possibility that TAP20 might dissociate from or interrupt focal adhesion during the cell adhesion process. To characterize the effect of TAP20 on focal adhesions, MV3 cells were transfected with either control GFP plasmid or TAP + GFP plasmid and plated on VN-coated chamber slides. GFP transfectants formed numerous focal adhesions, as demonstrated by antibodies against vinculin, paxillin, or FAK . In TAP20 + GFP transfected cells, the number of focal adhesions was significantly reduced by 60–70% , compared with those in wt or GFP alone transfected MV3 cells. Next, we monitored the effects of TAP20 on cell migration with a modified Boyden chamber assay . Transfected HUVEC cells were sorted by GFP fluorescence using a FACScan flow cytometer and recultured for 1–2 d. 50,000 cells were seeded on each chamber. There was no significant difference in migration on an FN-coated membrane between the untransfected cells, GFP sorted GFP-expressing cells, and TAP20 + GFP expressing cells. Migration of cells on a VN-coated plate was markedly enhanced by expression of TAP20 (640.4 ± 78.8 [TAP20] versus 395.2 ± 36.2 [wt] and 377.2 ± 65.6 [GFP]). This enhancement (TAP20 versus the controls) could not be blocked by the control anti-α2β1 antibody, BHA2.1, or by the anti-αvβ3 antibody, LM609, but it could be blocked by the anti-αvβ3 antibody, P1F6 . Since TAP20 modulated cell adhesion and migration, we asked whether overexpressing TAP20 would alter the ability of cells to form tubes on matrix gel. When the GFP-sorted HUVEC cells were cultured in matrix gel, a three-dimensional matrix, tube formation by TAP20 + GFP transfectants was significantly enhanced . By 6 h, tube structures were observed in the TAP20 + GFP transfectants, but not in the control GFP cells. The number of tubes formed by TAP20 + GFP transfectants appeared to be dramatically increased at all timepoints compared with that by the control cells. The experimental evidence above suggested that the effects of TAP20 on cells were mediated by the αvβ5 integrin VN receptor. Immunoblotting with anti–β5 integrin antibody showed that the expression of the β5 integrin in TAP20 transfectants was similar to that in the control cells . Therefore, we asked whether the effects of TAP20 could result from direct interaction with the αvβ5 integrin. We used a GST-TAP20 fusion protein purified with glutathione Sepharose beads to precipitate proteins from cell lysate, and probed proteins bound to the complexes with the antibodies against αv, β1, β3, and β5 integrin subunits. GST alone did not precipitate any protein recognized by these antibodies. Only the β5 integrin subunit was precipitated by the GST-TAP20 fusion protein . To further confirm the TAP20–β5 integrin interaction, we used GST fusion proteins containing the cytoplasmic tail of either the β3 or β5 integrin subunit to precipitate TAP20 protein from lysate of ECV cells transfected with TAP20 + GFP. Immunoblotting with anti-TAP20 antibody demonstrated that TAP20 protein could coprecipitate with the GST–β5 tail fusion protein . To rule out an indirect interaction between the GST-TAP20 protein and the β5 integrin complex, or between the GST–β5 tail and the TAP20 protein complex, we then used the GST-integrin tail fusion proteins to precipitate purified TAP20 protein. The GST-TAP20 fusion protein was first purified with glutathione Sepharose beads, and then was digested with thrombin. After incubation of the GST–integrin tail–coated glutathione Sepharose beads with TAP20 released by thrombin, GST–β5 tail beads were able to precipitate TAP20, as shown in immunoblotting with TAP20 antibody . In this study, we identified a cDNA that encodes a novel 20-kD, 176–amino acid protein in RCE. Several observations in this study suggest that TAP20 is involved in integrin function in intact cells: (a) expression of TAP20 is dependent on PKCθ activity, which is required by RCE for migration and proliferation ; (b) overexpression of TAP20 in human ECV cells resulted in an attenuation of cell attachment to VN and a decrease of focal adhesion formation on VN-coated plates; (c) expression of TAP20 in HUVEC cells resulted in an increase of cell migration on VN-coated plates, which can be blocked by an antiintegrin β5 antibody, P1F6; (d) expression of TAP20 in HUVEC cells enhances tube formation on matrix gel; and (e) the GST fusion protein coprecipitation study demonstrated that TAP20 binds to the cytoplasmic domain of the β5 integrin subunit. β3-endonexin is the only gene closely related to TAP20. The overall amino acid sequence of TAP20 and the long form of β3-endonexin share 56% homology, and thus, these two proteins are encoded by two different genes. Because of different mRNA splicing, there are two forms of β3-endonexin–related messages in cells, but only the short form peptide has β3 integrin binding activity . An anti-TAP20 polyclonal antibody raised against a portion of the COOH terminus sequence of TAP20 recognizes a 20-kD band that is identical to calculated molecular weight of TAP20, indicating that a full-length form of TAP20 exists in cells. Overexpression of TAP20 in cells and the TAP20 binding experiments provide evidence that the full-length TAP20 protein can bind to the β5 integrin. Our data show that overexpression of TAP20 in ECV cells affects cell adhesion and migration on VN, but not on other ECM proteins such as FN and LN, indicating that TAP20 regulates the function of the αv integrin family. Furthermore, TAP20 effects were inhibited by a specific αvβ5 integrin–blocking antibody P1F6, not by an anti-αvβ3 antibody LM609, indicating that TAP20 may specifically interact with the αvβ5 integrin. A GST-TAP20 fusion protein coprecipitated specifically with detergent-solubilized β5 integrin from ECV cells, and thus the binding of TAP20 to β5 integrin does not depend on the existence of αv. A GST–β5 tail fusion protein coprecipitated specifically with TAP20 protein, indicating that the TAP20 binding site is in the cytoplasmic domain of β5 integrin. Unlike β3-endonexin, which binds the β3 integrin and increases the α IIb β3 receptor affinity when overexpressed in CHO cells , TAP20 interacts with the β5 integrin and negatively regulates αvβ5-based adhesion and focal adhesion formation. Therefore, the effect of TAP20 is to decrease the affinity of its associated integrin receptor αvβ5 for its ligand VN. These effects also differ from those of the αvβ5 blocking antibody P1F6, which can prevent cell migration and angiogenesis initiated by VEGF or PKC activation , presumably by blocking the outside-in component of the integrin bidirectional signaling pathway . This discrepancy suggests that TAP20 regulates EC functions by changing the inside-out component of αvβ5 signaling and thus modulating the functions of the αvβ5 integrin, indicating a different integrin regulating mechanism. Formation of focal adhesions requires ECM-integrin ligation and integrin clustering. The decrease in focal adhesions in the TAP20-overexpressing cells could result from the attenuation of ECM–β5 integrin ligation by TAP20. On the other hand, TAP20 may interfere with the interaction between β5 integrin and the cytoskeleton, which is required for focal adhesion formation. Perhaps as a result of decreased binding to VN, TAP20 enhances EC migration and in vitro tube formation. It is also possible that, despite negative modulation of β5 integrin function, TAP20 may upregulate recruitment of other molecules needed for migration, for instance. This study demonstrates that TAP20 requires enzymatically active PKCθ for its transcription. It is very likely that TAP20 expression is PKC isoenzyme specific. Direct interaction of TAP20 with the cytoplasmic tail of the β5 integrin subunit is at least one of the pathways by which PKCθ might modulate EC migration and tube formation. Therefore, controlling TAP20 expression presents a mechanism, alternative to direct protein phosphorylation , by which PKCs can regulate integrin function, and thus angiogenesis. | Study | biomedical | en | 0.999998 |
10579727 | An anti-peptide antibody, anti-IbαC, recognizing the COOH-terminal domain of GPIbα has been described previously . The monoclonal antibodies WM23 and AK2 against GPIbα, and purification of vWF and botrocetin were as described previously . The monoclonal antibody against GPIbα, P3221, was kindly provided by Dr. Zaverio Ruggeri (The Scripps Research Institute, La Jolla, CA). mAbs D57 and 15 against integrin α IIb β 3 were kindly provided by Dr. Mark Ginsberg (The Scripps Research Institute, La Jolla, CA). Monoclonal antibody, 4F10, against integrin α IIb β 3 complex was kindly provided by Dr. Virgil Woods (University of California at San Diego, CA). Monoclonal antibody against GPIbα, SZ2, monoclonal antibody against human β 3 , SZ21, and monoclonal antibody against vWF, SZ29, were generous gifts from Dr. Changgeng Ruan ; cDNA clones encoding α IIb and β 3 in CDM8 vector were kindly provided by Dr. Mark Ginsberg. In some experiments, botrocetin was also purchased from Centerchem. Ristocetin was purchased from Sigma Chemical Co. DNA encoding wild-type and mutant 14-3-3ζ was described previously . The wild-type and mutant 14-3-3ζ were subcloned into pEGFP-C2 vector (Clonetech) between EcoRI and XbaI sites. The constructs encode a wild-type or a mutant 14-3-3ζ fused to the COOH terminus of green fluorescent protein (GFP). Transfections of cDNA into CHO cells were performed according to the previously described methods using Lipofectamine . Selection markers (CDneo and CDhygro; Invitrogen) were cotransfected with desired DNA at a 1:10 ratio. Stably transfected cell lines were selected in selection media containing 0.5 mg/ml G418 and/or 0.2 mg/ml hygromycin, and further selected by mass cell sorting using antibodies recognizing GPIbα and/or integrin α IIb β 3 (D57). The following cell lines were established: cells expressing GPIb-IX complex (1b9) ; cells expressing integrin α IIb β 3 (2b3a); cells coexpressing GPIb-IX and α IIb β 3 (123); and cells coexpressing integrin α IIb β 3 and GPIb-IX mutants with truncated GPIbα cytoplasmic domains at residues 591 (Δ591/2b3a) and 559 (Δ559/2b3a) . Cells expressing comparable levels of integrins or/and GPIb-IX were further selected by cell sorting and monitored by flow cytometry. Microtiter wells were coated with 10 μg/ml vWF or fibrinogen in PBS at 4°C overnight. Cells in Tyrode's buffer in the presence of 5 μg/ml botrocetin were incubated in ligand-coated microtiter wells for 30 min at 37°C in a CO 2 incubator. As adhesion of the GPIb-IX and integrin-transfected CHO cells to vWF does not require botrocetin, botrocetin was omitted in some experiments. After three washes, cell spreading was examined under an inverted microscope (20× objective lens). In quantitative assays, 50 μl of 0.3% p -nitrophenyl phosphate in 1% Triton X-100, 50 mM sodium acetate, pH 5.0, was added to microtiter wells and incubated at 37°C for 1 h. The reaction was stopped by adding 50 μl of 1 M NaOH. Results were determined by reading OD at 405 nm wave length. A standard curve of acid phosphatase reaction was established by adding the acid phosphatase substrate to various known numbers of the same cells in parallel wells. Acid phosphatase assay of the standards confirmed that the OD value was proportional to cell number. The rate of cell adhesion was estimated from the ratio of the numbers of adherent cells to that of total cells. Cells were allowed to adhere and spread on vWF- or fibrinogen-coated glass chamber slides (Nunc). After three washes, cells were fixed by adding 4% paraformaldehyde in PBS. In experiments that required cell permeabilization, cells were permeabilized by adding 0.1 M Tris, 0.01 M EGTA, 0.15 M NaCl, 5 mM MgCl 2 , pH 7.4, containing 0.1% Triton X-100, 0.5 mM leupeptin, 1 mM PMSF, and 0.1 mM E64. The cells were then incubated with 20 μg/ml of various antibodies at 22°C for 1 h. After three washes, cells were further incubated with fluorescein- or rhodamine-labeled secondary antibodies at 22°C for 30 min. To stain the actin filaments, rhodamine-labeled phalloidin (Sigma Chemical Co.) was also added. After additional washes, cells were photographed under a fluorescence microscope. In some experiments, the data were collected by a cooled CCD camera and surface area quantitated using Image-Pro Plus (Media Cybernetics). Fluorescein-labeling of fibrinogen was prepared as described previously . Cells expressing recombinant proteins were harvested and suspended in modified Tyrode's buffer . Cells (∼1 × 10 7 /ml) were incubated for 30 min with 15 μg/ml fluorescein-labeled fibrinogen in the presence of 20 μg/ml vWF and 1 mg/ml ristocetin. As a negative control, cells were also incubated with fluorescein-labeled fibrinogen in the presence of 1 mg/ml ristocectin but in the absence of vWF. RGDS peptide (1 mM) was added in parallel assays for estimation of specific fibrinogen binding to the integrin. We showed previously that 1 mM RGDS completely abolished fibrinogen binding to integrin α IIb β 3 while 1 mM RGES had no effect . Fibrinogen binding was analyzed by flow cytometry. For vWF binding, the cells in Tyrode's buffer were incubated for 30 min at 22°C with vWF in the presence 1 mg/ml ristocetin. After washing, the cells were further incubated for 30 min with a monoclonal antibody against vWF, SZ29, and then analyzed by flow cytometry. CHO cells coexpressing integrin α IIb β 3 with wild-type GPIb-IX (123 cells) or GPIb-IX mutant Δ591 were solubilized as previously described . Cell lysates were incubated with 10 μg of WM23 against GPIbα or mouse IgG (Sigma Chemical Co.) at 4°C for 1 h and further incubated for 1 h after addition of protein G–conjugated Sepharose beads (Sigma Chemical Co.). After three washes, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting with a rabbit anti-GPIbα antibody or a rabbit antibody against 14-3-3ζ . Reaction of the antibodies was visualized using an enhanced chemiluminescence kit (Amersham-Pharmacia). To analyze the roles of GPIb-IX and integrin α IIb β 3 in vWF-mediated platelet adhesion and activation, stable CHO cell lines were established that express one of the two platelet receptors for vWF: GPIb-IX (1b9 cells) or integrin α IIb β 3 (2b3a cells). A stable cell line was also established that expressed both GPIb-IX and integrin α IIb β 3 at levels comparable to 1b9 and 2b3a cells, respectively . These cells were incubated in vWF-coated microtiter wells for 30 min in the presence of botrocetin, which binds to vWF and mimics the effects of subendothelial matrix to induce vWF binding to GPIb-IX . As a positive control, these cells were also incubated in fibrinogen-coated microtiter wells. Adherent cells were quantitated with an acid-phosphatase assay. As shown in Fig. 1 , <10% of the 2b3a cells (expressing only α IIb β 3 ) adhered to the vWF-coated surface compared with ∼55% adhesion to fibrinogen, suggesting only a background level of vWF-integrin interaction. This result is consistent with previous work showing a low affinity state of α IIb β 3 expressed in CHO cells , and is also consistent with results obtained in platelets showing that integrin α IIb β 3 interacts poorly with vWF without prior activation . The possibility of defective integrin function and expression in 2b3a cells can be excluded, as both the 2b3a cells and 123 cells but not 1b9 cells adhered to immobilized fibrinogen which is known to interact with integrin α IIb β 3 without prior activation . Thus, vWF is a poor ligand for unactivated integrin α IIb β 3 . In contrast to cells expressing α IIb β 3 (2b3a cells), those expressing GPIb-IX (1b9 cells) or those expressing both GPIb-IX and integrin α IIb β 3 (123 cells), adhered to vWF-coated wells in the presence of botrocetin . As platelet adhesion to immobilized vWF occurs in the absence of vWF modulators , we further examined the adhesion of 123 cells to immobilized vWF without botrocetin treatment. Fig. 1 C shows that 123 cell adhesion to vWF does not require botrocetin, indicating that adhesion of CHO cells expressing GPIb-IX and α IIb β 3 to vWF is similar to platelet adhesion. Furthermore, adhesion of 123 cells to vWF was inhibited by monoclonal antibodies against vWF-binding site of GPIbα . These results suggest that, as in platelets, GPIb-IX is required for cell adhesion to vWF in this CHO cell expression model. Under a microscope, most adherent 1b9 cells (expressing GPIb-IX only) on vWF showed a rounded morphology similar to nonadherent cells . In contrast, 123 cells (coexpressing GPIb-IX and α IIb β 3 ) spread on the vWF-coated surface . Spreading of 123 cells was abolished by RGDS peptide , indicating that spreading was mediated by integrins. Spreading of 123 cells was also inhibited by the monoclonal antibody 4F10, against human α IIb β 3 complex, and by anti-human β 3 antibody SZ21 . These data indicated that spreading was mainly mediated by integrin α IIb β 3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with α IIb β 3 in the 123 cell line resulted in constitutively active integrin α IIb β 3 , as 123 cells did not bind to soluble fibrinogen without prior activation . Thus, vWF binding to GPIb-IX induces integrin-vWF interaction and integrin-mediated cell spreading. To examine whether GPIb-IX–mediated signaling pathway in CHO cells mimics that in platelets, we examined the effects of platelet activation inhibitors. We found that the PGE1, which elevates intracellular cAMP, wortmannin, and calphostin C, which inhibit PI-3 kinase and PKC, respectively, also inhibited GPIb-IX and integrin-dependent CHO cell spreading on vWF. Thus, GPIb-IX expressed in CHO cells induced integrin interaction with vWF in a manner similar to that in platelets. To further exclude the possibility that integrin function in 123 cells may differ from that in the 2b3a cell line, we also examined integrin-mediated cell spreading on immobilized fibrinogen. Both 2b3a cells and 123 cells fully spread on immobilized fibrinogen , suggesting that α IIb β 3 expressed in both cell lines functioned in a similar manner. As shown above, only a small percentage of 2b3a cells adhere to vWF. Some of these adherent cells, however, also spread on vWF, suggesting that the background level GPIb-IX–independent interaction of α IIb β 3 with vWF in a small percentage of 2b3a cells can also mediate cell spreading. To examine the morphological changes in more detail, the adherent cells were stained with fluorescently labeled phalloidin, and examined by fluorescence microscopy under high magnification. Only 5% of 1b9 cells spread on vWF . Most 1b9 cells did not spread or only poorly spread on vWF. However, 58% of these poorly spread cells showed limited filopodium- or lamellipodium-like structures extending to the vWF-coated surface which was inhibited by RGDS peptide. This indicates a low level interaction between vWF and an endogenous integrin, which is consistent with the results obtained by Cunningham et al. 1996 . In contrast to 1b9 cells, ∼70% of 123 cells (expressing both GPIb-IX and integrin α IIb β 3 ) fully spread, which was inhibited by RGDS peptide . These results show that GPIb-IX induces integrin α IIb β 3 interaction with vWF which is responsible for 123 cell spreading on vWF. Two possible mechanisms could explain GPIb-IX–induced integrin-vWF interaction in the CHO cell expression model: (a) GPIb-IX may induce a cellular signal that increases the affinity of integrin for vWF (activation); or (b) the GPIb-IX binding to vWF may allow access of integrin to vWF, e.g., by changing the conformation of vWF. To differentiate between these two possibilities, we examined whether vWF activated integrin binding to another ligand of α IIb β 3 , soluble fibrinogen, in 123 cells. It is known that integrin α IIb β 3 binds soluble fibrinogen only after the integrin is activated . FITC-labeled fibrinogen was incubated with 123 cells in the presence of ristocetin which is known to induce soluble vWF binding to GPIb-IX and vWF-dependent platelet aggregation but not to induce fibrinogen-dependent platelet aggregation in the absence of vWF . As expected, there was no specific fibrinogen binding to 123 cells exposed only to ristocetin, indicating that ristocetin alone does not induce specific fibrinogen binding to integrin α IIb β 3 . When both vWF and ristocetin were present, however, there was significant binding of fibrinogen. vWF-induced fibrinogen binding was inhibited by RGDS peptide , and was also inhibited by an anti-GPIbα monoclonal antibody, AK2, known to inhibit ristocetin-induced vWF binding to GPIb-IX . Furthermore, vWF did not induce specific fibrinogen binding to 2b3a cells , suggesting that vWF-induced fibrinogen binding to integrin α IIb β 3 requires vWF interaction with GPIb-IX. Thus, vWF interaction with GPIb-IX not only stimulates vWF-α IIb β 3 interaction, but also induces the integrin to bind soluble fibrinogen. These data indicate that ristocetin-dependent vWF binding to GPIb-IX induces a cellular signal that activates the ligand-binding function of α IIb β 3 . We showed previously that the intracellular signaling molecule 14-3-3ζ binds to a site in the COOH-terminal 15 residues (residues 595–610) of the cytoplasmic domain of GPIbα. To investigate the role of 14-3-3 in GPIb-IX–mediated activation of α IIb β 3 in the CHO cell model, we established a CHO cell line (Δ591/2b3a cells) that coexpresses α IIb β 3 and a mutant GPIb-IX, Δ591, that lacks the 14-3-3–binding site (18 residues) at the COOH terminus of GPIbα, but retains the functional filamin-binding domain in GPIbα . As shown in Fig. 4 , wild-type GPIb-IX expressed in CHO cells (123 cells) coimmunoprecipitates with an endogenous CHO cell 14-3-3 protein reactive with anti–14-3-3ζ antibodies . The mutant GPIb-IX (Δ591), however, failed to coimmunoprecipitate endogenous CHO cell 14-3-3 . As a control, we also immunoblotted the same immunoprecipitates with an anti-GPIbα antibody, and observed that similar amounts of GPIbα were immunoprecipitated from both the Δ591/2b3a cells and 123 cells . Thus, the Δ591 mutant GPIb-IX is defective in binding to endogenous 14-3-3. To determine whether deletion of the 14-3-3–binding site in GPIbα affects GPIb-IX–mediated activation of the integrin α IIb β 3 , we examined whether ristocetin-induced vWF binding to the mutant GPIb-IX stimulates the binding of FITC-labeled fibrinogen to Δ591/2b3a cells. Fig. 5 A shows that vWF induces soluble fibrinogen binding to 123 cells which is inhibited by RGDS peptide. In contrast, vWF-induced fibrinogen binding to Δ591/2b3a cells is absent. It is unlikely that the defect in fibrinogen binding to Δ591/2b3a cells results from naturally occurring mutations developed in the CHO cells during selection as the Δ591/2b3a cells are established by mass sorting of cells reactive with both antibodies against α IIb β 3 and GPIb-IX and not by single cell cloning. It is also unlikely that the inhibition of integrin activation results from defective binding of vWF as vWF binding to 591/2b3a cells is not negatively affected . As Δ591/2b3a cells adhered and spread on fibrinogen , the possibility of a defective integrin function can be further excluded. Thus, our data indicate that the 14-3-3–binding site of GPIbα plays an important role in GPIb-IX–mediated integrin activation. We also examined vWF-induced fibrinogen binding to a CHO cell line (Δ559/2b3a), expressing integrin α IIb β 3 and a truncation mutant GPIb-IX lacking both the 14-3-3–binding domain and filamin-binding domain of GPIbα. No specific fibrinogen binding was detected in this cell line suggesting that inhibition of α IIb β 3 activation by deleting the 14-3-3–binding site of GPIbα was not reversed by further deletion of the filamin-binding site of GPIbα . To investigate whether 14-3-3 binding plays a role in GPIb-IX–induced integrin-vWF interaction and integrin-dependent cell spreading on vWF, the 123 cells and Δ591/2b3a cells were allowed to adhere to vWF-coated microtiter wells. As examined under the microscope, ∼70% of the 123 cells were spread on both vWF- and fibrinogen-coated microtiter wells. In contrast, only a small percentage (∼30%) of Δ591/2b3a cells appeared spreading on vWF, indicating that GPIb-IX–induced integrin-vWF interaction was inhibited . To quantitate the cell spreading objectively, cells adherent to vWF were permeabilized and stained with rhodamine-labeled phalloidin. Fluorescently stained cells in randomly selected fields were quantitated for cell surface area using Image-Pro Plus software (Media Cybernetics). As shown in Fig. 8 , the average surface area of Δ591/2b3a cells were about half of that of 123 cells, indicating that the spreading of the mutant cell line was significantly reduced but not totally abolished. Since Δ591/2b3a cells adhered and spread on fibrinogen in a manner similar to 123 cells, the ligand-binding function of α IIb β 3 and the integrin-mediated spreading process was not impaired in the Δ591/2b3a cell line. Thus, inhibition of GPIb-IX– and integrin-dependent spreading on vWF in this cell line is unlikely to be caused by a defect in ligand-binding function of α IIb β 3 or in the integrin's post-ligand occupancy events. These data suggest that 14-3-3ζ binding to the COOH-terminal region of GPIbα plays an important role in GPIb-IX–mediated activation of integrin α IIb β 3 . Spreading of a small percentage of mutant cells reflects a background level of α IIb β 3 -vWF interaction or the interaction of vWF with the endogenous CHO cell integrin . It has been shown previously that truncation of GPIb-IX at residue 559 (Δ559) of GPIbα abolishes filamin and 14-3-3 binding to GPIb-IX, and induces GPIb-IX–dependent cell spreading in the absence of integrin α IIb β 3 . To investigate functional effects of this truncation mutation on GPIb-IX–dependent vWF interaction with different integrins, we coexpressed Δ559 with integrin α IIb β 3 (Δ559/2b3a). Not only did the Δ559/2b3a cells exhibit no defect in spreading, but they actually showed enhanced spreading on vWF compared with 123 cells . The spreading of Δ559/2b3a cells was significantly inhibited by RGDS peptide but poorly inhibited by anti-α IIb β 3 antibody 4F10 and anti-β3 antibody SZ21 , suggesting that an endogenous integrin plays a significant role. This result is consistent with previous studies showing that CHO cells expressing the same mutant of GPIb-IX spread on vWF in the absence of integrin α IIb β 3 . However, when incubated with soluble fibrinogen in the presence of vWF and ristocetin, no specific binding of fibrinogen to Δ559/2b3a cells was detected . These results indicate that deletion of both 14-3-3 and filamin-binding sites of GPIbα inhibited GPIb-IX–mediated activation of fibrinogen binding to α IIb β 3 , but enhanced the interaction of vWF with an endogenous integrin (which only plays a very limited role in wild-type GPIb-IX–mediated cell spreading . Thus, it appears that two different mechanisms may be involved in the vWF interaction with integrins: a GPIb-IX–mediated 14-3-3-dependent mechanism that induces an activation signal leading to the activation of integrin α IIb β 3 , and an alternative mechanism that allows the interaction of vWF with an unidentified integrin. The latter mechanism becomes significant only when the association of GPIb-IX with the membrane skeleton structure is disrupted. We have recently shown that GPIbα binds to a site in the helix I region of 14-3-3ζ, distinct from the sites required for 14-3-3ζ binding to RSXpSXP-motif containing ligands such as c-Raf . To verify that 14-3-3ζ plays a role in GPIb-IX signaling, we constructed cDNAs encoding fusion proteins of green fluorescent protein (GFP) with wild-type 14-3-3ζ as well as a small fragment of 14-3-3ζ containing the GPIbα-binding site . Transient expression of the wild-type and the mutant 14-3-3 was indicated by the emission of green fluorescence . After transfection of pEGFP vector alone, ∼70% of 123 cells adhere and spread on vWF. Cells expressing GFP-1433 fusion protein showed an increase in the percentage of spreading (85%), suggesting that overexpression of 14-3-3 enhanced cell spreading on vWF-coated surface . In contrast, 90% of the cells expressing GFP-1433T12 fusion protein are rounded , and the rest (10% cells) only partially spread on vWF (not shown). These results suggest that the small fragment of 14-3-3ζ inhibited the function of endogenous 14-3-3 in a dominant negative fashion. In this study, we show that GPIb-IX binding to vWF induces signals that activate the ligand-binding function of integrin α IIb β 3 and integrin-dependent cell spreading using a reconstituted CHO cell expression model. We show that vWF-induced GPIb-IX signaling is inhibited by deletion of the 14-3-3–binding sites in the cytoplasmic domain of GPIbα . Thus, our study indicates that interaction between GPIb-IX and 14-3-3 plays an important role in GPIb-IX–mediated signaling leading to activation of integrin α IIb β 3 . Understanding the intracellular signaling mechanism induced by ligand binding to the platelet vWF receptor, GPIb-IX, as well as platelet signaling in general, has been hampered by the lack of specific means to interfere with platelet signaling intracellularly at a molecular level. Studies on the GPIb-IX–induced platelet activation by biochemical approaches have shown that ligand binding to GPIb-IX induces a series of intracellular biochemical changes such as generation of thromboxane A 2 , production of phosphatidic acid , activation of PI-3 kinase , increase in the cytoplasmic calcium level , and activation of protein kinases such as protein kinase C and tyrosine kinases . The consequence of these intracellular signaling events is the activation of integrin α IIb β 3 . However, specific molecular pathways leading to these signaling events are unclear. In many cell types, advances in understanding the molecular mechanisms of intracellular signaling are often achieved with the use of recombinant DNA transfection techniques to express specific intracellular signaling molecules or to specifically alter the function of such a molecule. As platelets do not have nuclei and cannot be maintained in culture, it is difficult to use recombinant DNA approach directly. Thus, we have developed a model system in a CHO cell line expressing both the human integrin α IIb β 3 and GPIb-IX. In our CHO cell expression model, GPIb-IX mediates signaling leading to the activation of integrin α IIb β 3 in a manner similar to that observed in platelets: (a) vWF binding to GPIb-IX in our CHO cell model not only induces integrin-vWF interaction but also induces soluble fibrinogen binding to the integrin α IIb β 3 , suggesting that GPIb-IX is unlikely to be simply presenting α IIb β 3 to vWF or inducing changes in vWF, but is inducing a cellular signal that activates the ligand-binding function of the integrin . This is consistent with previous findings in platelets showing vWF binding to GPIb-IX initiates signaling leading to integrin α IIb β 3 activation . (b) We show that GPIb-IX–induced integrin-dependent cell spreading on vWF was inhibited by prostaglandin E 1 (PGE 1 ), and the protein kinase C inhibitor calphostin C . These inhibitors also inhibit vWF-induced integrin activation in platelets . Reconstitution of the platelet GPIb-IX–mediated activation of α IIb β 3 in CHO cells is thus significant to further understanding the GPIb-IX–mediated signaling using specific molecular biological approaches. In our CHO cell expression model, the vWF modulator, ristocetin, was used to induce binding of soluble vWF to GPIb-IX expressed in CHO cells. It is known that platelets do not bind to soluble vWF under physiological conditions. At the site of vascular injury, vWF binds to exposed subendothelial matrix proteins such as collagen. Collagen binding causes the exposure of the GPIb-IX–binding site in vWF probably by inducing a conformational change . Shear stress may play a role in the conformational change of vWF induced by the subendothelial matrix . In vitro, the effect of subendothelial matrix proteins on vWF can be mimicked by desialation of vWF , natural occurring mutations in vWF or binding of artificial vWF modulators such as botrocetin and ristocetin . Although there is a report that ristocetin may flocculate fibrinogen and thus may increase nonspecific binding of fibrinogen, ristocetin-induced platelet aggregation in platelet-rich plasma is dependent on vWF binding to GPIb-IX, indicating that ristocetin cannot directly induce fibrinogen binding to integrin α IIb β 3 . Binding of vWF to platelets induced by ristocetin and other in vitro methods is similar to vWF binding induced by subendothelial matrix under flow conditions. In both cases vWF binds to essentially the same ligand-binding pocket on GPIb-IX in the NH 2 -terminal region of GPIbα, and can be inhibited by the same monoclonal antibodies (e.g., AK2) directed against the NH 2 -terminal region of GPIb-IX . Furthermore, vWF binding to GPIb-IX induced by vWF modulators, desialation or mutations initiate similar platelet responses to that observed when platelets adhere to matrix-bound vWF. These responses include activation of PKC, elevation of intracellular calcium, release of thromboxane A 2 , release of granule contents, and activation of the integrin α IIb β 3 . For these reasons, ristocetin as a modulator of vWF binding to GPIb-IX has been commonly used in clinical and research laboratories. Since all available evidence indicates that GPIb-IX signaling is initiated by vWF binding to the NH 2 terminus of GPIbα , and the experiments are controlled such that the effects of ristocetin alone can be excluded , differences in the methods of induction of vWF binding (desialation, mutation, modulators, or shear stress) are unlikely to be a significant factor causing GPIb-IX signaling to diverge into dramatically different pathways. Thus, data obtained using vWF modulators such as ristocetin are relevant to understanding GPIb-IX signaling during platelet adhesion to the subendothelial matrix in vivo. We have shown previously that 14-3-3, an intracellular signaling molecule, bound to the cytoplasmic domain of GPIb-IX, and that its binding was dependent upon the COOH-terminal region of GPIbα and the helix I region of 14-3-3 . In this study, we have investigated the role of 14-3-3 in GPIb-IX signaling in the CHO cell expression model and show that deletion of the 14-3-3–binding site in the COOH terminus of GPIbα inhibits GPIb-IX–mediated α IIb β 3 activation. As deletion of the COOH-terminal domain of GPIbα did not negatively affect vWF binding to GPIb-IX , it is unlikely that the inhibition in integrin activation resulted from a loss of vWF binding function of the mutant GPIb-IX. Since this GPIb-IX mutant still interacts with filamin at a nearby site , it is also unlikely that a gross disturbance of the tertiary structure of the GPIbα cytoplasmic domain or loss of the interaction with the filamin-membrane skeleton caused the inhibition in signaling. Consistent with the importance of 14-3-3 in vWF-induced signaling, the small dominant negative fragment of 14-3-3 that contains the GPIbα-binding site also inhibited GPIb-IX–mediated integrin activation . Thus, we conclude that 14-3-3 binding to GPIb-IX plays an important role in wild-type GPIb-IX–mediated signaling leading to integrin activation. This is the first identified early link between GPIb-IX and the integrin activation pathway. Filamin binding to the central region of the GPIbα cytoplasmic domain links GPIb-IX to the membrane skeleton structure (cross-linked short actin filaments) underlining the membrane . The association of GPIb-IX with this structure is important for platelets to maintain a discoid shape . A membrane skeleton-like structure can also be seen in the CHO cells expressing GPIb-IX complex (Du, X., unpublished data). It has been shown previously that truncation of GPIbα at residue 559 abolished association of GPIb-IX with the filamin-membrane skeleton. Cells expressing this truncated mutant GPIb-IX spread on vWF without coexpression of α IIb β 3 , a process that is inhibited by RGDS peptides (Du, X., unpublished data). Consistent with this, we found that Δ559/2b3a cells expressing this mutant GPIb-IX and integrin α IIb β 3 showed an enhanced spreading on vWF which was poorly inhibited by anti-α IIb β 3 antibodies that blocked ligand-binding sites but was significantly inhibited by RGDS peptide , suggesting that an RGDS-dependent endogenous integrin is responsible. In contrast to the truncation mutant, cells expressing wild-type GPIb-IX spread poorly on vWF in the absence of α IIb β 3 . This suggests that the function of this endogenous CHO cell integrin to mediate cell spreading on vWF is restrained by the membrane skeleton association with GPIb-IX and enhanced by disruption of this association. When coexpressed with integrin α IIb β 3 , however, wild-type GPIb-IX is able to induce cell spreading on vWF and soluble fibrinogen binding to integrin α IIb β 3 . Thus, GPIb-IX–mediated activation of α IIb β 3 is not restrained by the association of GPIb-IX with the membrane skeleton. This suggests that the functions of α IIb β 3 and the endogenous integrin are regulated by GPIb-IX via different mechanisms. Indeed, we showed that GPIb-IX–mediated activation of integrin α IIb β 3 involves the binding of 14-3-3 to the COOH terminus of GPIbα. In contrast, the Δ559/2b3a cells expressing the mutant GPIb-IX lacking both filamin and 14-3-3–binding sites were defective in vWF-induced fibrinogen binding . It remains unclear what types of endogenous CHO cell integrin are responsible for cell spreading on vWF in the absence of α IIb β 3 , and what mechanisms are involved in the upregulation of their function when GPIb-IX is dissociated from the membrane skeleton. CHO cells express an endogenous vitronectin receptor (αv complexed with β 1 or possibly β 5 ) and α 5 β 1 , both of which are inhibited by RGDS peptides . As these CHO cell integrins are known to interact with immobilized ligands without prior activation, one possibility is that binding of vWF to GPIb-IX may change the conformation of vWF or bring vWF to the vicinity of the endogenous integrin and thus allow their interaction via a localized signaling mechanism. Disruption of GPIb-IX association with the membrane skeleton may allow free lateral movement of GPIb-IX to the vicinity of these integrins without the restraint by the membrane skeleton structure, thus enhancing cell spreading on vWF. In contrast, the function of α IIb β 3 to interact with vWF and soluble fibrinogen is known to require prior activation via intracellular signaling . Thus, vWF-induced activation of αIIbβ3 is not restrained by the membrane skeleton structure, but requires a 14-3-3–dependent signaling mechanism. | Study | biomedical | en | 0.999997 |
10579728 | Immature and hypertrophic chondrocytes were isolated from the caudal one-third portion and the cephalic core portion of day 17–18 chick embryo sterna, respectively . Tissue fragments were pretreated for 2 h at 37°C with 0.25% trypsin to eliminate perichondrial and blood cells, and were then treated with a fresh 0.25% trypsin/0.1% collagenase enzyme mixture for an additional 2–3 h. The resulting cell populations were filtered, recovered by centrifugation, and plated at a density of 1.3 × 10 6 cells/60-mm tissue culture dish or 2 × 10 5 cells/22-mm well (12 well plates). Cells were grown continuously, without subculturing, for ∼2 wk in monolayer. During the first 2 d, cultures received 4 U/ml of testicular hyaluronidase to minimize cell detachment . Cultures were fed every other day with Dulbecco's modified high glucose Eagle's medium containing 10% fetal calf serum, 2 mM l -glutamine, and 50 U/ml penicillin and streptomycin (complete medium) . To allow cells to deposit mineral, confluent cultures received complete medium supplemented with 5 mM β‴glycerophosphate to serve as a phosphate source and with 25 μg/ml ascorbic acid or 35 nM all-trans-retinoic acid to serve as mineralization cofactors . Complete medium supplemented with ascorbate and β-glycerophosphate was termed medium A, and complete medium supplemented with retinoic acid and β-glycerophosphate was termed medium B. Mineral was revealed by staining with alizarin red . When indicated, cultures were treated with 3–10 μM vitamin K, warfarin, or a combination of both biochemicals. Stock solutions (1,000×) of warfarin and vitamin K3 were made in saline, whereas vitamin K1 and K2 stock solutions were made in 95% ethanol. Untreated cultures received equal volumes of vehicle. During treatment, the medium was changed daily. The APase activity of the cell layer was measured using p-nitrophenyl phosphate (pNP) as a substrate . Cells were scraped from the dish, recovered by centrifugation, and resuspended in ice-cold 0.9% NaCl in 3 mM Tris-HCl, pH 7.4. One half of each cell suspension was centrifuged for 1 min in a microfuge (10,000 g ) and the resulting cell pellet was solubilized in 0.9% NaCl and 0.2% Triton X-100. Samples were clarified by centrifugation for 5 min and the supernatants were mixed with one volume of 1 M Tris-HCl, pH 9.0, containing 1 mM pNP and 1 mM MgCl 2 . The reaction was stopped by addition of 0.25 vol of 1 N NaOH, and hydrolysis of pNP was monitored as a change in absorbance at 410 nm. The remaining half of each cell suspension was used to determine DNA content . For calcium determination, cultures in multiwell plates were rinsed three times with 0.9% NaCl in 10 mM Tris-HCl, pH 7.4, and extracted with 0.5 ml/well of 1 N HCl for 30 min at room temperature. The calcium concentration in the extract was determined by atomic absorption spectrophotometry in the presence of 0.1% LaCl 3 . Total RNAs were isolated from chondrocyte cultures as described . For Northern blot analysis, 10 μg of total RNA denatured by glyoxalation was fractionated on 1% agarose gels and transferred to Hybond-N membrane. Blots were stained with 0.04% methylene blue to verify that each sample had been transferred efficiently. Blots were hybridized in 6× SSC, 5× Denhardt's solution, 100 μg/ml sheared salmon sperm DNA, 2% SDS, and 50% formamide at 45°C overnight with 32 P-labeled cDNA MGP probe. A 306-bp full-length chick MGP cDNA clone was prepared in the pTA vector by reverse transcriptase PCR (RT-PCR), using primers designed on published sequence . The EcoRI-BamHI insert was excised and labeled with [ 32 P]CTP by random priming. Blots were exposed to Kodak x-ray Bio-Max films at −70°C for different lengths of time to insure linearity of exposure. This procedure was carried out as described . Cultures were rinsed three times with 0.9% NaCl in 10 mM Tris-HCl, pH 7.4, freeze-dried at −60°C, and stored dessicated. Samples were analyzed by FT-IR spectroscopy using a Nicolet Magna–IR spectrometer operated in the diffuse reflectance mode. Each sample was gently milled in an agate mortar and layered on KBr at a KBr:sample ratio of 300:1 (wt/wt). Routinely, 300 interferograms were collected at 4 cm −1 resolution; the background was subtracted using a nonmineralized chondrocyte preparation. Spectra were co-added and the resulting interferograms were fourier transformed; second derivative spectra (1,200–500 cm −1 ) were obtained using a software package (Omnic). Resolution factors are presented in the legend of each figure. Chondrocyte monolayer cultures were processed for these analyses as described previously . In brief, cultures were rinsed with PBS, fixed with 1:1 dilution of Karnovsky's fixative for 30 min, postfixed with full strength Karnovsky for 1 h, and then dehydrated by incubation in 10, 30, 50, 70, 80, 90, 95, and 100% ethanol (15 min each); incubation with 100% ethanol was repeated twice. After removal of the last ethanol, the cells were covered with Freon 113; the dishes were sealed with parafilm in which a few holes were punctured with a needle, and were allowed to dry slowly under a hood overnight. A circle ∼2 cm in diameter was cut from each dish and mounted by colloidal silver paint onto an aluminum stub and cells were carbon sputter–coated. Samples were viewed using a JEOL T330A scanning electron microscope and were simultaneously examined by energy dispersive x-ray microanalysis using Kevex hardware (Kevex Corp.) with Quantex 5 software. The entire coding sequence of chick MGP was amplified by RT-PCR (see below) and subcloned into the RCAS(A) retroviral vector . The resulting plasmid (RCAS-MGP) was resequenced to verify sequence fidelity. Recombinant plasmid DNA was transfected into chick embryo fibroblasts by a FuGENE6 transfection reagent (Boehringer Mannheim). Recombinant viral particles released into the medium were concentrated using a centrifugation concentrator and used to infect freshly isolated chondrocytes . For determination of efficiency of viral infection, cultured chondrocytes were detached by trypsinization and replated onto poly- l -lysine–coated dishes at a density of 2 × 10 6 cells/ml. After 15 min, adhered cells were washed with PBS twice, fixed with 3.7% neutralized formaldehyde solution for 5 min, and permeabilized by incubation with PBS containing 0.5% Triton X-100 for 15 min. Cells were incubated with hybridoma culture supernatant containing AMV-3C2, a mouse mAb against the gag protein of avian myeloblastosis virus and recognizing cells harboring avian Rous sarcoma or leukemia virus. Detection of bound antibody was obtained by the ABC method using Histofine kits (Nichirei). RT-PCR was carried out as described . 1 μg of whole cellular RNA was reverse transcribed by Superscript reverse transcriptase (GIBCO BRL) and random hexamers. After reverse transcription, samples were amplified for the indicated number of cycles using Elongase (GIBCO BRL) at the following conditions: 10 s at 95°C for denaturation and 1 min at 60°C for annealing and extension. The sequences of chick MGP primers were based on the report by Wiedemann et al. 1998 and were: 5′ GAGTGAGGCACAGCAAGAGACA 3′ and 5′ AGGAGGTTACCCTGAGGTCCAA 3′, generating a 402-bp fragment. The chick cytoplasmic β-actin primers were as follows: 5′ AAGGAGAAGCTGTGCTACGTCG 3′ and 5′ CTTCTGCATCCTGTCAGCAATG 3′, generating a 309-bp fragment . Chick embryo fibroblasts transfected with RCAS-MGP or control insertless RCAS plasmids were grown to confluency in monolayer. Small cell layer fragments, ∼200–300 μm 2 , were scraped off the culture dish surface and were microsurgically implanted in the mesenchyme at the distal region of the leg bud of Hamburger-Hamilton stage 22–23 chick embryos . Eggs were returned to the incubator and allowed to develop further. At the indicated time points, embryos were removed from eggs, sacrificed, and examined visually or under a dissecting scope to determine the gross effects of experimental manipulations on limb development. Control and experimental limbs were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were processed for general histology by staining with hematoxylin and eosin stains and for histochemical detection of mineral by staining with 0.5% alizarin red S solution, pH 4.0, for 5 min at room temperature. Companion continuous 5-μm sections were processed for in situ hybridization using 35 S-labeled riboprobes as described previously to determine the gene expression patterns of MGP, type II collagen, and type X collagen. The cDNA clones used were the following: a full-length chick MGP clone prepared by RT-PCR (see above), the 0.8-kb chick type II collagen clone pDLr2, and the 0.197-kb chick type X collagen clone pDLr10 . In the first set of experiments, we determined whether interference with endogenous MGP function by treatment with the vitamin K antagonist, warfarin, causes excessive matrix mineralization regardless of the stage of maturation of chondrocytes or whether the effects are developmentally regulated. To this end, confluent multiwell cultures of immature and hypertrophic chick embryo sternal chondrocytes were treated with 10 μM warfarin for 4 d in medium containing supplements required for mineral deposition in culture, namely the phosphate donor β-glycerophosphate and the collagen synthesis cofactor ascorbic acid (hereafter called medium A). As controls, companion cultures were treated with warfarin plus vitamin K1 or vitamin K1 alone, or were left untreated. To detect mineralization, cultures were stained with alizarin red or subjected to cell layer–associated calcium content analysis. We found that warfarin treatment had no appreciable effects on mineralization in immature chondrocyte cultures ; these cultures all contained ∼1–1.5 μg/μg DNA of cell layer–associated calcium. However, the warfarin treatment markedly stimulated mineralization of the hypertrophic cultures ; cell-associated calcium content increased from ∼3.3 ± 0.4 μg/μg DNA in control to 11.2 ± 2.1 μg/μg DNA in warfarin-treated cultures. The warfarin stimulation of mineralization was counteracted by cotreatment with vitamin K1 , attesting to the specificity of warfarin effects. Vitamin K1 alone had no obvious effects in either hypertrophic or immature cultures . Vitamin K2 and K3 gave identical results (not shown); thus, the three vitamins were used interchangeably hereafter. To insure that the differential responses above were specific, reflected the cells' potentials to mineralize, and were not influenced by the type of mineralization cofactors used, similar confluent immature and hypertrophic cultures were grown for 4 d in medium supplemented with β‴γλυψ∈ροπηοσπηατ∈ ανδ ρ∈τινοιψ αψιδ ≲η∈ρ∈αφτ∈ρ ψαλλ∈δ μ∈διθμ Β≳> Ιν ∈αρλι∈ρ στθδι∈σ< ϕ∈ σηοϕ∈δ τηατ ρ∈τινοιψ αψιδ ισ α στρονγ∈ρ στιμθλατορ οφ μιν∈ραλιζατιον‴ρ∈λατ∈δ αψτιωιτι∈σ ιν ψηονδροψυτ∈σ ≲Ιϕαμοτο ∈τ αλ>< +′′×α≳> Ψθλτθρ∈σ ιν μ∈διθμ Β ϕ∈ρ∈ τρ∈ατ∈δ ϕιτη +″ mM warfarin, 10 mM vitamin K, or both as above. Even in this medium, warfarin treatment failed to stimulate matrix mineralization in immature cultures , but did so in hypertrophic cultures . Calcium content increased from 14.4 ± 2.7 μg/μg DNA in control hypertrophic cultures to 32.6 ± 4.2 μg/μg DNA after warfarin treatment. This stimulation was counteracted by cotreatment with vitamin K . Interestingly, the substantial mineralization seen in control hypertrophic cultures was partially counteracted by treatment with vitamin K , which decreased the cell layer-associated calcium content to 9.1 6 1.8 μg/μg DNA. To investigate whether higher doses of warfarin or longer treatment periods could actually induce mineralization in immature cultures, cultures in medium A and B were treated with 10, 20, or 30 μM warfarin for 4 d; parallel cultures were treated for 8 d. We found that neither the higher warfarin concentrations used nor the longer treatment period (not shown) resulted in mineralization of immature chondrocyte cultures. To determine the relationship between mineralization responses and endogenous MGP gene expression, cultures of immature and hypertrophic chondrocytes in medium A or medium B were treated as above and were processed for Northern blot analyses. We found that both immature and hypertrophic cultures contained obvious amounts of the 1.3- and 3.0-kb MGP RNAs . Cultures in medium A contained two to threefold more MGP transcripts than cultures in medium B . However, these basal levels of expression were not significantly affected by treatment with warfarin, vitamin K, or warfarin plus vitamin K . Since APase is an enzyme whose activity is closely associated with the mineralization process, we determined the activity of the phosphatase in control and warfarin-treated hypertrophic cultures. We found no change in APase activity per cell as a result of warfarin and vitamin K treatments or cotreatments in either medium A or medium B . This analysis also showed that warfarin treatment had no effect on cell number . Thus, MGP function in mineralization as revealed by warfarin interference depends on the stage of chondrocyte maturation, does not appear to be related to MGP expression levels, and does not involve changes in APase activity. We determined next whether MGP regulates only the quantity of mineral deposited by cells in their matrix or may also affect mineral nature and overall quality. Thus, we compared the mineral deposited by control and warfarin-treated hypertrophic chondrocytes by SEM, x-ray microanalysis, and FT-IR spectroscopy. Cells used for these experiments were grown in medium B because they formed more mineral than cells in medium A . When viewed by SEM, many crystals were observed on, and to be associated with, the cell surface in both control and warfarin-treated cells . When examined at a higher magnification, each crystal displayed a typical multibead organization, with beads averaging 200–400 nm in diameter . After SEM, multiple microscopic fields were subjected to x-ray microanalysis. Prominent phosphorus and calcium peaks were produced by crystals in both cultures . Integration of the peaks showed that the calcium/phosphorus molar ratio was ∼1.3–1.4, thus, resembling that of apatite in hypertrophic cartilage . To examine the mineral composition in greater detail, mineral samples from control and warfarin-treated cultures were examined by FT-IR spectroscopy . The samples elicited similar second derivative spectra in the v 1 v 3 phosphate region 1,200–950 cm −1 , with clear peaks at 1,160, 1,102, 1,028, 962, and 906 cm −1 that are characteristic of stoichiometric hydroxyapatite. Likewise, similar second derivative spectra were observed in the v 4 phosphate domain 650–500 cm −1 with strong peaks at 606 and 565 cm −1 attributable to phosphate ions in apatite, and in the v 2 carbonate region 890–850 cm −1 . Bands at 880, 875, and 872 cm −1 in the carbonate region indicated that CO 3 had at least in part substituted for OH and PO 4 , and that carbonate apatite was present in the mineralized cultures. Thus, qualitatively similar mineral is deposited by hypertrophic chondrocytes in the absence or presence of warfarin. In both cases, the mineral has morphological, organizational, and biochemical characteristics of apatite crystals displaying relatively low crystallinity, typical of mineralizing cells in culture as well as young bone tissue in vivo . We asked next whether the amount of MGP expressed by hypertrophic chondrocytes is a factor influencing the mineralization potential of the cells. To address this question, we constructed an RCAS-based expression vector encoding full-length chick MGP and used the resulting viral particles (RCAS-MGP) to infect freshly isolated hypertrophic chondrocyte populations. As a control, companion cells were infected with insertless RCAS viral particles. Cultures were grown for ∼1 wk until confluent, and were processed for RT-PCR to estimate the levels of MGP gene expression and for immunocytochemistry to determine the percentage of virally infected cells in control and RCAS-MGP cultures, using an antibody to the viral structural protein p19. We found that MGP gene expression was significantly higher in RCAS-MGP cultures (expressing both endogenous and virally encoded MGP) than in control cultures (expressing endogenous MGP only) . Housekeeping actin gene expression was identical in both cell populations . Immunocytochemistry revealed that ∼60% of the cells in both control and RCAS-MGP cultures were virally infected . To determine the effects of MGP overexpression on mineralization, companion multiwell cultures were maintained in mineralization medium A or B for 7 d (rather than 4 d as in the experiments above) to induce optimal mineralization. At the end of this period, cultures (in duplicate) were stained with alizarin red to reveal mineral deposition. Control cultures were clearly stained by alizarin red , indicating that the cells had mineralized their matrix and that infection by insertless virus had no deleterious effect on mineralization. In comparison, cultures infected with the RCAS-MGP virus exhibited significantly reduced alizarin red staining . When control cultures were treated with 10 μM vitamin K1 or K2 for the last 7 d of culture, mineralization was also reduced ; indeed, when RCAS-MGP cultures were similarly treated with vitamin K, mineralization was completely prevented . We asked next whether MGP overexpression in the developing chick limb would have similar inhibitory effects on the mineralization process of long bones. A small pellet of fibroblasts producing RCAS-MGP or control RCAS viruses was implanted in the vicinity of the mesenchymal condensations of tibia and fibula present in stage 22–23 (day 3.5–4.0) chick embryo hindlimb buds. We aimed to infect only these skeletal elements and to use uninfected femur and tarsal elements as an internal control. Embryos were reincubated until day 10–12, and longitudinal sections through the entire legs were prepared and processed for histology, histochemistry, and in situ hybridization. Analysis of alizarin red–stained sections from control day 12 embryo showed that the femur (fe), tibia (ti), and tarsal (ta) elements displayed their typical elongated morphology, and that their alizarin red staining diaphyses were undergoing mineralization and ossification . In contrast, in RCAS-MGP–infected embryos, the tibia failed to stain with alizarin red ; unexpectedly, the tibia was also significantly shorter and broader than the control. These changes were limited to the tibia since femur and tarsal elements in the RCAS-MGP leg were normal and stained with alizarin red . Higher magnification analysis of alizarin red–stained or hematoxylin/eosin–stained sections showed that the diaphysis of control tibia contained hypertrophic mineralizing cartilage and endochondral bone (not shown), was surrounded by a mineralized intramembranous bone collar , and was being invaded by marrow cells . In contrast, the diaphysis of RCAS-MGP tibia was entirely cartilaginous . There was no evidence of erosion or invasion by marrow cells, and there was a lack of a bone collar . In addition, the diaphyseal chondrocytes were not fully hypertrophic and displayed average cell diameters smaller than those in the control . To confirm and extend these observations, we carried out in situ hybridization on similar longitudinal sections of day 12 control and RCAS-MGP tibia, using probes encoding cartilage characteristic type II and X collagens and MGP. It should be noted that, because of their different lengths, about one half of the control tibia is shown in Fig. 10A–C , whereas the entire RCAS-MGP tibia is shown in Fig. 10D–F . In control tibia , transcripts encoding type II collagen were abundant in the epiphyseal articular layer and underlying growth plate containing proliferating, prehypertrophic, and hypertrophic chondrocytes; the transcripts were reduced in late posthypertrophic chondrocytes , as reported previously . Transcripts encoding type X collagen, a product typical of hypertrophic chondrocytes, were abundant in hypertrophic and posthypertrophic chondrocytes . MGP transcripts displayed more complex patterns reminiscent of those seen in mouse embryos ; they were prominent in the articular layer , in proliferating/prehypertrophic chondrocytes along the lateral side of tibia , and in posthypertrophic chondrocytes . Notably, MGP transcripts were essentially undetectable in the entire hypertrophic zone, either along its lateral side or its central region. However, in the RCAS-MGP tibia, the type II collagen transcripts were equally abundant from epiphysis to diaphysis and the type X collagen transcripts were quite scarce . This indicated that the respective downregulation and upregulation of these two genes, normally associated with chondrocyte hypertrophy, had not occurred and that chondrocyte maturation had indeed been inhibited. As expected, MGP transcripts were very prominent and present throughout tibia and neighboring tissues, reflecting the presence of both endogenous and virally derived transcripts . The results of the study provide clear evidence that MGP roles in endochondral ossification are under tight cellular control. In hypertrophic chondrocyte cultures, interference with GLA residue synthesis by warfarin treatment causes rapid and extensive mineralization; in immature chondrocyte cultures, a similar treatment does not cause mineralization. This differential response is not simply because of differences in MGP levels, since there is little difference in the levels of MGP gene expression in the two cell populations. We find also that mineral deposited by hypertrophic cells in the absence or presence of warfarin treatment is qualitatively similar and represents biological apatite. We infer from this observation that MGP mainly controls the degree of mineralization, rather than the quality or type of mineral deposited. Last, we find that virally driven overexpression of MGP in cultured hypertrophic chondrocytes causes a marked decrease in mineralization. This finding lends further strength to the inhibitor studies, and indicates that in mineralization-competent cells, the amount of MGP plays a critical role in the mineralization process. As might be expected, MGP overexpression in the limb bud in vivo causes a marked reduction in mineralization. However, we did not anticipate to also observe that such overexpression delays chondrocyte maturation, causes shortening and broadening of the skeletal elements, and blocks both endochondral and intramembranous ossifications. This data provides new evidence that MGP roles in skeletogenesis may extend beyond the regulation of mineralization. In our inhibitor studies, we examined the effects of warfarin on the mineralization of hypertrophic chondrocytes under two conditions, medium A and medium B. In earlier investigations, we showed that both conditions induce expression of many cellular activities associated with the terminal mineralization phase of maturation, including increased APase activity, decreased type X collagen gene expression, and shifts in energy metabolism . We now show that in either condition, warfarin treatment does not elicit major changes in MGP expression, APase activity, or cell number. Thus, because the treatment enhances mineralization in both conditions, it is clear that the charged carboxyl groups by themselves serve as critical sites for the regulation of mineral deposition by MGP. A previous study showed that osteocalcin is expressed by hypertrophic sternal chondrocytes in culture , suggesting that this protein and its carboxyl groups may also have contributed to the regulation of mineralization. Ongoing analyses of the absolute amounts, distribution, and timing of expression of osteocalcin and MGP should clarify the respective contributions of these proteins to mineralization in the hypertrophic chondrocyte cultures used here. Effort was extended in evaluating the quality of the mineral formed in hypertrophic cell cultures. FT-IR analysis, SEM, x-ray microanalysis, and measurements of calcium/phosphate ratios all show that the mineral deposited in the presence of warfarin treatment is indistinguishable from that deposited in untreated cultures and represents normal carbonate apatite. The only difference observed is that much more mineral is deposited in warfarin-treated cultures, lending support to our conclusion that a critical role of MGP is to control the initiation of mineralization and the amount of mineral deposited. This conclusion correlates well with previous pharmacological, biochemical, and genetic studies , indicating that MGP and other calcium-binding proteins act as strong inhibitors of the initiation phase of mineralization. Given the self-sustaining and self-promoting nature of the mineralization process, the presence of these strong inhibitors may be needed to restrict mineralization to the appropriate sites and times in the embryo and growing organism. Once the first seed crystals form, however, MGP and other calcium-binding proteins may have additional roles, including controlling size, rates of growth, directionality, and maturation of mineralization. A recent study has provided evidence that osteocalcin may have a role in mineral maturation in the growing animal . These considerations and our findings reiterate the idea that mineralization is complex and that multiple mechanisms are needed to regulate it during embryogenesis and postnatal life. How could MGP exert its control on the initiation phase of mineralization? Given its ability to bind calcium, the protein could simply serve as a competitor and limit the amount of calcium ions available to initiate calcification . Mineralization is thought to initiate in matrix vesicles and/or at focal sites of proteoglycan accumulation . MGP has actually been found to be associated with matrix vesicles isolated from articular and growth plate cartilage . In this location, MGP would be in an ideal topographical position to inhibit or compete with calcium influx into the vesicle's lumen and inhibit the vesicle's capability to initiate mineralization. Likewise, MGP could limit the amount of calcium ions available for binding to focally accumulated proteoglycans. It is important to note that there are additional mechanisms limiting mineralization. For example, we have shown previously that there actually are two structurally and functionally distinct types of matrix vesicles produced by chondrocytes . Others have shown that the focal accumulations of proteoglycans are restricted to hypertrophic cartilage and are not common in cartilages not involved in mineralization . Thus, MGP is part of multiple mechanisms that serve to control mineralization and allow it to occur only at appropriate sites, times, and levels in the organism. This conclusion correlates well with the phenotype of MGP-null mice . Lack of MGP was found to lead to excessive or ectopic mineralization of specific tissues and structures such as the growth plate, aorta, and tracheal rings. However, articular cartilage, a tissue rich in MGP , was not reported to be abnormally mineralized. Clearly, this tissue must possess multiple means by which mineralization is prevented, even in the absence of MGP. Multiple inhibitory means must also characterize the immature chondrocytes used in our study, which do not mineralize their matrix once treated with warfarin. On the other hand, susceptible tissues such as blood vessels must possess a strong potential to mineralize, and will do so simply in the absence of MGP. This would account for the relatively high frequency with which arteries undergo pathological calcification in conditions such as atherosclerosis . Our in vivo data show that virally driven misexpression of MGP causes severe alterations of limb skeletogenesis. We find that the MGP-overexpressing tibia is shorter and wider than its normal counterpart, and the maturation process of growth plate chondrocytes is severely inhibited. In addition, the diaphysis fails to be invaded by bone and marrow progenitor cells, to undergo endochondral ossification, and to be surrounded by an intramembranous bone collar. Interestingly, it was found in MGP-null mice that the growth plates of limb skeletal elements are not only more mineralized than those present in control animals, but are also disorganized and lack characteristic chondrocyte columns . It is clear that absence or overexpression of MGP are both incompatible with the normal processes of chondrocyte maturation, cartilage mineralization, and ossifications. It was shown previously in mouse embryo limb skeletal elements that the normal gene expression pattern of MGP is very complex ; MGP expression is low in the proliferative zone of growth plate, strong in the prehypertrophic zone, low again in the hypertrophic zone, and strong again in the posthypertrophic mineralizing zone . We show here that the MGP gene expression pattern in chick growth plates is equally complex. The significance and biological consequences of these highly distinctive patterns of expression have remained largely unclear. In view of the results shown here and those in the MGP-null mouse study, it is conceivable that the MGP expression patterns may not only be related to mineralization, but may also have roles in chondrocyte and cartilage development. In particular, the downregulation of MGP gene expression in zones of the growth plate, and in the hypertrophic zone in particular, may actually be required for chondrocyte maturation and the emergence of hypertrophic chondrocytes. By maintaining constant MGP expression and preventing modulations of expression, the virally driven MGP misexpression may have blocked the cells at more immature stages and inhibited their further development into hypertrophic cells. The absence of fully hypertrophic chondrocytes would explain the lack of both endochondral ossification and invasion by bone and marrow progenitor cells, since only hypertrophic mineralizing cartilage can serve as a substrate for these processes. The absence of fully hypertrophic chondrocytes would also account for the reduction in skeletal element length since hypertrophy is the main determinant of longitudinal growth . The lack of a diaphyseal intramembranous bone collar is harder to explain. It may be a direct consequence of widespread virally driven MGP expression acting on perichondrial tissues and inhibiting their differentiation into osteoblasts. Alternatively, it may be an indirect consequence of the failure of cartilage to become fully hypertrophic; it was suggested long ago that cartilage and perichondrial tissues are characterized by mutual interdependence and interactions, and that alterations in one tissue may have developmental repercussions in the other tissue . Whatever the explanation, the absence of an intramembranous bone collar would account for the increased width of the MGP-overexpressing skeletal element, given that the collar has a primary role in regulating the diameter of the shaft in developing long bones. We do not know yet how MGP exerts influences on chondrocyte and cartilage maturation beyond its roles in mineralization. MGP may act via mechanisms independent of its ability to bind calcium and apatite, i.e., mechanisms unrelated to those involved in mineralization. Alternatively, MGP may again use its calcium binding ability to influence chondrocyte maturation. A previous study provides indirect support for the latter possibility. It was found that increases in extracellular calcium concentrations selectively induce expression of type X collagen in chondrocyte cultures . Because type X collagen is a marker of maturing and hypertrophic chondrocytes, the finding indicates that extracellular calcium can favor the hypertrophic program. A similar increase in available calcium could occur in zones of the growth plate with low MGP expression, such as the hypertrophic zone; in other words, low MGP gene expression may signify increased free calcium available for cellular activities and for promotion and completion of the chondrocyte maturation program. | Study | biomedical | en | 0.999996 |
10579729 | A 700-bp DNA fragment from the 5′ region of the mouse perlecan cDNA was used to screen a genomic library derived from a mouse D3/129 embryonic stem (ES) cell line (a gift from J.S. Mudgett, Merck Sharp & Dohme, NJ) to isolate perlecan genomic clones. The targeting construct consisted of an 8-kb fragment containing exon 5, an expression cassette flanked by loxP sites in which the phosphoglycerate kinase promoter controls the expression of the neomycin (neo) gene and the Herpes simplex virus thymidine kinase (HSV-tk) gene, respectively, an 0.8-kb fragment containing exon 6 followed by a single loxP site and a 1.5-kb fragment containing exon 7 (for more detailed information contact: reinhard. [email protected]). The targeting construct was electroporated into R1 ES cells. ES cells culture, electroporation, isolation, and analysis of G418-resistant ES cell clones was carried out as described . Genomic DNAs were digested with EcoRI, and probed with an external 3-kb BamHI-EcoRI genomic DNA fragment . ES cell clones that had undergone homologous recombination were transiently transfected with a Cre expression plasmid (a gift from Dr. Werner Müller, University of Cologne, Germany) and selected in the presence of FIAU. Surviving ES cell clones were isolated and analyzed by Southern blot by cleaving genomic DNA with EcoRI, BamHI, or HindIII, respectively, and probed with an internal genomic DNA fragment . The generation of germline chimeras and breeding were carried out as described . The following primary antibodies were used for immunohistochemistry: rat anti-collagen type II (diluted 1:400; obtained from Dr. Rikard Holmdahl, Lund University, Sweden); rabbit anti-collagen type X (1:500; obtained from Dr. Bjorn Olsen, Harvard Medical School, Boston, MA); rabbit anti–laminin-1 (1:200); rabbit anti–perlecan antibodies against domain I (1:1,000), domain III-3 (1:500) and domain V (1:1,000); rat anti–nidogen-1 (1:30); mouse anti–β-tubulin isotype III (1:100; Sigma Chemical Co.); rabbit anti-nestin (1:2,000; obtained from Dr. Zaal Kokaia, Lund University, Sweden); mouse anti-reelin (1:1,000; obtained from Dr. André Goffinet, University of Namur, Belgium); and Ki-67 (1:50; Dianova, Hamburg, Germany). The following secondary antibodies were used: biotinylated goat anti–rabbit Ig, biotinylated goat anti–rat Ig (obtained from Vector Laboratories Inc.); FITC-conjugated donkey anti–mouse; and Cy3-conjugated goat anti–rat (both obtained from Jackson ImmunoResearch Laboratories, Inc.). Total RNA from E12.5 embryos was isolated as described . RT-PCR was carried out with a commercial kit (Titan RT-PCR; Boehringer Mannheim). The primer sequences were as follows: 5′-CCCGAATACAGGAAGATCCC-3′ specific for exon 5, and 5′-TCACAGGCGAACTCGTTAGGCTCA-3′ specific for exon 8. The amplification products were separated on 2% agarose gel, blotted onto nylon filters, and hybridized with a γ-[ 32 P]ATP–labeled oligonucleotide (5′-CAGACATGTCCCTGCAGTCAGGCC-3′) specific for exon 7. Confluent ES cells were grown overnight in serum-free medium. The medium was collected and the cells were lysed with RIPA buffer supplemented with proteinase inhibitors (5 mM NEM, 2 mM PMSF, 5 mM EDTA). Radioimmunoassays were performed with the medium and cell extract using either tissue-derived (laminin, perlecan) or recombinant proteins (nidogen-1) as reference inhibitors. Two assays specific for perlecan domains I and III-3 were used and gave similar results . For Northern blotting, cartilage samples were dissected from the limbs of normal and perlecan-deficient E17.5 embryos, and total RNA was prepared, separated, blotted, and probed as described . The oligo-labeled probes represented ∼500 bp of the mouse Col2a1, matrilin-3, and cartilage matrix protein (COMP) cDNAs, respectively. Signal intensities were measured from nonsaturated films using a Gel-ProAnalyzer (Media Cybernetics). For histological analyses, staged embryos were fixed in 4% fresh paraformaldehyde in PBS overnight, pH 7.2, dehydrated in a graded alcohol series, and embedded in paraffin (Paraplast X-tra; Sigma Chemical Co.). Sections were cut at 6–8 μm and stained with hematoxylin/eosin or cresyl violet (Nissl staining). To detect proteoglycans and calcification, bone sections were stained with Safranin orange and van Kossa . To detect proliferating cells, 5-bromo-2′-deoxyuridine (BrdU; Sigma Chemical Co.) and anti-BrdU mAb (Boehringer Mannheim) were used as described . Apoptosis was analyzed by the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end labeling (TUNEL) method using a commercial in situ cell death detection kit (Boehringer Mannheim) according to the manufacturer's instructions. For immunohistochemistry, embryos were either fixed in 4% buffered paraformaldehyde or in 95% ethanol/5% acetic acid overnight at 4°C, embedded in paraffin, and sectioned at 6–8 μm. Immunostaining was performed either by using the Vectastain ABC Elite kit (Vector Laboratories) or by using an immunofluorescence method described earlier . Skeletons of E17.5 embryos were prepared and stained as described by Aszódi et al. 1998 and photographed on a dissecting microscope. For light microscopy of heart tissue, 4% paraformaldehyde immersion-fixed embryos were postfixed with 2% osmium tetroxide in PBS for 2 h at 4°C. After a thorough wash in PBS for 3× 10 min, the embryos were block-stained with 1% uranyl acetate in 70% ethanol for 8 h. Afterwards the specimens were dehydrated, infiltrated with and embedded in araldite, cut with a glass knife (1–2 μm) on a Reichert ultramicrotome (Leica), and stained with methylene blue. Ultrastructural analysis of basement membranes and cartilage tissue was performed as described earlier . For scanning electron microscopy, E10.5 embryos were prepared as previously described with the modification that samples were critical point dried in a Balzer critical point dryer using 100% ethanol as the intermediate solvent. Embryos were mounted on aluminum holders, sputtered with 60 nm palladium/gold, and examined in a Jeol JSM-T330 scanning electron microscope. Electron microscopy after negative staining with anti–laminin-1 antibody with protein A coupled to colloidal gold was done as described by Roth et al. 1980 . The perlecan gene was inactivated in ES cells by homologous recombination using the cre/loxP technique. A neomycin/thymidine kinase (neo/tk) cassette flanked by single loxP sites was inserted into intron 5 and a single loxP site was inserted into intron 6 . Eight independently targeted ES cell lines were obtained. Two of them (clones 243 and 310) were transiently transfected with a cre recombinase expression plasmid, selected in FIAU, and analyzed for cre -mediated deletion. In three clones derived from 243 and 1 clone derived from 310, respectively, the sequence between the outermost loxP sites (including the neo/tk cassette, part of intron 5, exon 6, and part of intron 6) was deleted . One clone carrying the constitutive null allele from 243 and from 310, respectively, was injected into C57BL/6 blastocysts and transferred to pseudopregnant females. Chimeric males from both clones gave germline transmission of the mutated perlecan gene. To verify that the deletion of exon 6 in the perlecan gene leads to a constitutive null mutation, ES cells carrying a homozygous mutation were generated. Heterozygous ES cells were retransfected with the original targeting construct and again selected for neo expression. Afterwards, one of the clones with a recombination event on the wild-type allele was transiently transfected with the cre expression plasmid to obtain ES clones with a homozygous deletion of exon 6 . To test whether the mutant allele was transcribed, RT-PCR was performed with total RNA extracted from normal, heterozygous, and homozygous ES cells. The amplified fragment encompasses sequences in exons 5–8. The size of the amplified DNA fragment was 640 bp for the wild-type allele and 479 bp for the mutant allele, indicating that splicing occurs between exon 5 and 7 . Splicing of exon 5–7 leads to a reading frame shift and to the formation of a truncated perlecan protein consisting of the 116–NH 2 -terminal amino acids that make approximately half of domain I. To determine whether the mutant mRNA was translated, normal and homozygous ES cells were grown in serum-free culture medium, and the supernatant as well as the cell extract were subjected to a highly sensitive RIA using polyclonal antibodies against laminin-1, nidogen-1, or the NH 2 -terminal domain I of perlecan. Whereas normal and mutant ES cells produce similar amounts of laminin and nidogen-1, only normal but not mutant ES cells produce perlecan . These data show that although the mutant perlecan allele is transcribed, no truncated protein could be detected with a domain I–specific polyclonal antibody. Most likely, the truncated form of domain I is not properly folded and is consequently degraded intracellularly as soon as it is translated. This was confirmed by episomal transfection of human EBNA-293 cells with domain I lacking 38 COOH-terminal residues that did not produce any recombinant fragment (data not shown). Therefore, the mutation in the perlecan gene is designated as a loss of function mutation. Mice heterozygous for the mutation appeared normal and did not display any overt anatomical or behavioral abnormalities. No mice homozygous for the mutation were detected among 728 weaned progeny from heterozygous intercrosses ( Table ). Analysis of newborn offspring detected three homozygotes and several cannibalized mice among 98 neonates ( Table ). The three homozygotes showed exencephaly, chondrodysplasia, hemorrhage in several organs (see below), severe cleft palates, and died around birth. To determine when the remaining homozygotes die, embryos were examined from E9.5 to birth ( Table ). At E9.5, wild-type, heterozygous, and homozygous mutant embryos were represented in a normal Mendelian ratio ( Table ). Perlecan-null embryos at E9.5 were indistinguishable from wild-type and heterozygous littermates. E9.5 perlecan-null embryos formed brain vesicles, optic vesicles, branchial arches, otic pits, limb buds, a beating heart, and 20–25 somites (not shown). Therefore, development to E9.5 proceeds normally in the absence of perlecan. By E10.5, defects began to appear in perlecan-null embryos. Although they were still present in the expected percentage ( Table ) and were of normal size, ∼70–80% were dead as demonstrated by the absence of the heart beating and the presence of severe hemopericardium . 20–30% of homozygotes were alive and of normal appearance (not shown). By E11.5 and E12.5 the percentage of dead embryos with hemopericardium increased further, whereas some of the living embryos developed abnormally in the head region and survived to the perinatal period. At all stages analyzed, the placental development was unaffected by the loss of perlecan. The placental size, architecture, and the blood content were similar between normal and perlecan-null embryos (not shown). In addition, PECAM whole mount stainings of E9.5 and E10.5 embryos revealed that homozygotes had no defects in angiogenesis, sprouting, and remodeling to generate vessels of different sizes (not shown). At later stages of development (E13–E17), we could observe the formation of microaneurysms associated with bleedings in several tissues including lung, skin, and brain (not shown). These results indicate that a null mutation in the perlecan gene leads to two patterns of development. Many of the perlecan-null embryos die during an early crisis (E10.5–E12.5) characterized by hemopericardium and heart arrest. The few remaining mutants survive to the perinatal period ( Table ), but develop severe brain and skeletal defects (see below). At E10–11, many perlecan-null embryos manifested several signs of cardiac insufficiency characterized by intrapericardial hemorrhages , weak heartbeats, or cardiac arrest. To investigate these defects, wild-type and alive perlecan-null embryos were analyzed by immunohistochemical and ultrastructural methods. The high resolution analysis of plastic-embedded hearts derived from E10.5 wild-type embryos showed a continuous wall of several layers of cardiomyocytes covered on both sides by a single cell layer of endothelial cells forming the endocardium and epicardium . In a large number of perlecan-null embryos, the compact layer of cardiomyocytes was interrupted by small intercellular clefts that were often covered by an intact layer of endo- and epicardium . In a few embryos, the clefts in the myocardium were filled with endocardial cells . The defects were associated with blood cell leakage into the pericardial cavity . Adjacent to the myocardial defects, the pericardial tissue was thickened because of an increase in cell number and matrix deposition . No defects were observed in the blood vessels of affected hearts (not shown). At E9.5, thin and normally appearing BMs were observed around perlecan-null cardiomyocytes (not shown). However, at E10.5 normal cardiomyocytes were covered by a continuous BM , whereas perlecan-null cardiomyocytes lacked BM deposition or showed small patches of electron dense material on their cell surface . The formation of sarcomeres and tight junctions were normal in perlecan-null cardiomyocytes. Ultrastructural analysis of BMs in other tissues including skin and gut (not shown) revealed no abnormalities, suggesting that at E10.5 the BM defects are restricted to the heart where they are exposed to mechanical stress. To test whether the BM defects in the perlecan-null myocardium are associated with abnormal expression of BM proteins, immunostaining for perlecan , laminin-1 , and collagen IV (not shown) was performed on normal and perlecan-null cardiac tissue. With the exception of perlecan, all BM components were similarly expressed in mutant tissue . These data demonstrate that perlecan-deficient cardiac muscle cells lack BM or are covered by abnormal BM, which disrupts the integrity of the myocardium and leads to the formation of small clefts in myocardial tissue and finally to blood leakage into the pericardial cavity. About 80% of the perlecan-null embryos surviving the first crisis developed an exencephalic malformation that was first visible between E10.5 and E11.5. To test whether the absence of perlecan results in an abnormal closure of the neural tube, scanning electron microscopy was performed with normal and perlecan-null E10.5 embryos. All normal ( n = 3) and perlecan-null embryos ( n = 11) analyzed, displayed properly closed neuropores . This was also confirmed by histological studies on E9.5 and E10.5 cephalic regions derived from mutant embryos (not shown). Some perlecan-null embryos had holes in the fore- and midbrain and showed collapsed brain vesicles . The cephalic region of normal embryos was covered by an intact layer of ectodermal cells . However, at higher magnification, the surface ectoderm of seven out of nine homozygotes showed small clefts that were 20–30 μm in width that contained round cells with small extensions . In nullizygotes with severe defects, round cells with small extensions traversed the cephalic mesenchyme to reach the surface ectoderm . To exclude that the clefts were caused artificially during embryo handling, heads were incubated with colloidal gold-conjugated antibodies binding to laminin-1 and analyzed by scanning electron microscopy. Preparation artefacts occurring after antibody incubation were devoid of staining , whereas clefts already present in homozygotes before handling showed colloidal gold staining of the exposed BM . Histological analysis of brain sections from normal and perlecan-null E9.5 embryos revealed normal BM between neural tissue and mesenchyme (not shown). At E11.5, 70% of homozygotes showed areas in which the BM surrounding the telencephalic vesicles was disrupted , and the brain tissue had invaded into the cephalic mesenchyme and fused with the overlaying ectoderm . Immunostaining revealed that the ectopias contained many nestin-positive cells , but lacked βIII isotype tubulin-positive cells . In the ectopic region, the neuroepithelium appeared thickened and the cells in the ventricular zone region were round instead of elongated as observed in the neocortex of normal embryos . At E11.5, clusters of neuroepithelial cells were exposed to the amniotic cavity and formed small disruptions of 5–10 μm . Immunostaining for proliferative cells with Ki-67 antibodies and for apoptotic cells with TUNEL labeling revealed no abnormalities in E10.5 and E11.5 perlecan-null brain tissues, neither in ectopias nor in normal appearing areas of the neocortex (not shown). At later stages, several homozygotes without obvious exencephaly showed a ruffled brain surface because the marginal zone of the neocortex was studded with large ectopias associated with a severe distortion of the laminar architecture of the cortex . All perlecan-null embryos analyzed so far, including those without exencephaly, exhibited neuronal ectopias in the ventral telencephalic region of the brain, when examined at E11.5 and later stages . The ectopias appeared as small, compact clusters of βIII isotype tubulin-positive cells that had invaded the mesenchyme at areas where the basement membrane is disrupted as shown by immunostaining for laminin-1 . Immunostaining for laminin-1 and perlecan at E13.5 revealed that both molecules were expressed around brain vessels and in the leptomeninges surrounding the brain tissue . No perlecan expression was found in the brain parenchyma of normal mice . All perlecan-null embryos that did not exhibit apparent heart defects continued their intrauterine development but died perinatally. Between E15 and the newborn stage, these animals developed a severe osteochondrodysplasia characterized by dwarfism, cleft palate, short limbs, and a short and abnormally bended vertebral column . Approximately 80% of homozygotes had exencephaly and lacked calvarial bones . Homozygotes without exencephaly had a domed skull . Whole mount skeletons of E17.5 embryos showed that all bones were present in perlecan-null embryos, except in exencephalic embryos, which lacked frontal and parietal bones . Detailed inspection of the mutant skeleton showed that long bones were approximately half of the size of wild-types. In addition, the cortical bone was thickened. In the skull, the mandible and nasal bone were shortened, and the structures of the middle and inner ear were poorly developed . The bones of chondrocranium (occipital, sphenoidal, and ethmoidal) were shortened and undermineralized (data not shown). Histological analysis at various stages of development revealed that the cartilage anlage of all long bones occurred normally in the mutant mice (not shown). Between E13 and 14, the first alterations in size and shape of the perlecan-null long bones became apparent . Mutant bones had disorganized growth plates characterized by the absence of the typical columnar arrangement of hypertrophic chondrocytes. In addition, hypertrophic chondrocytes showed an atypical morphology, and growth plates were always dissociated from their epiphyses . Such gaps in the tissue were never observed in normal bones and, therefore, suggest a mechanical weakness of the mutant cartilage leading to damage during tissue processing. Perlecan-null bones had small marrow cavities. Safranin-O staining was reduced in perlecan-null cartilage, suggesting a decreased proteoglycan content . van Kossa staining revealed that normal bones showed clear mineralization in the longitudinal septa of the late hypertrophic zone , perlecan-null tissue had minimal or no mineral deposits in the matrix around hypertrophic chondrocytes, and the calcified trabecula were transversely oriented in perlecan-null bones . Immunohistochemistry showed expression of perlecan in cartilage as well as the surrounding mesenchyme of normal but not mutant mice . Matrix proteins including collagen types II, IX, X, and XI, aggrecan, matrilin-1 and -3, and COMP were expressed in homozygotes . The ultrastructure of cartilage tissue derived from an E17.5 limb showed that wild-type hypertrophic chondrocytes were electron lucent with a paucity of organelles in the cytosol . The wild-type chondrocytes also showed contacts with the surrounding matrix that was homogeneously filled with fibrillar collagen . In contrast, hypertrophic chondrocytes of perlecan-null mice displayed an increased density of organelles and distended cisternae of ER . The cytosol was enriched with free ribosomes and polysomes . The collagen fibrils in wild-type growth plate cartilage showed a random distribution, had uniform length and diameter, and formed a typical network . The perlecan-null growth plate cartilage lacked such collagen network and the fibrils were shorter in length . To test whether an increased expression of cartilage ECM genes was responsible for the high metabolic activity in perlecan-null chondrocytes, Northern assays were performed with the total RNA derived from cartilage of normal and homozygous E18 limbs. Analysis of optical densities of mRNA signals such as shown in Fig. 8 G revealed that Col2a1 expression was increased threefold, and matrilin-3 and COMP expression was increased fivefold in mutant cartilage. We have generated perlecan-deficient mice, and we demonstrate that perlecan is essential for embryonic development. It plays a major role for maintaining the integrity of basement membranes and cartilage matrix, but has no apparently critical function in basement membrane assembly, organogenesis, and mesenchymal cell migration. Perlecan is expressed throughout development. In the preimplantation period, it is found between blastomeres and on the external trophectodermal cell surface of blastocysts just before they become attachment-competent, suggesting that embryo-derived perlecan initiates implantation by attaching embryos to the uterine epithelium . Our results do not confirm such a role for perlecan. We found neither a delayed nor a reduced implantation rate of perlecan-null embryos, and the Mendelian distribution of genotypes was normal at all stages analyzed between E5.5 and E9.5. Perlecan was also thought to be important for BM assembly and function . These complex and highly ordered BM structures are composed of many constituents and serve as barriers, substratum for epithelial sheets, and sinks for growth factor storage and release . Despite the ability of perlecan to interact with several BM components, adhesion molecules involved in BM assembly, and growth factors , all BMs form in the absence of perlecan and appear morphologically normal. A likely explanation for this finding is that other heparan sulfate proteoglycans substitute for the loss of perlecan and the glycosaminoglycan chains attached to it. A possible candidate is agrin, which is present in most if not all BMs and can also bind growth factors, α-dystroglycan and BM components . Mice lacking the neuronal splice variant of agrin have defects in neuromuscular synaptogenesis but are otherwise apparently normal . The lack of a general phenotype in these mutants could be due to the very low expression of nonneuronal agrin or the functional substitution by perlecan. At E9.5 all perlecan-null embryos analyzed were of normal size, had similar pulse rates to their wild-type and heterozygous littermates, and showed no histological abnormalities. Between E10.5 and E12.5, ∼70–80% of the perlecan-null embryos died. When alive homozygotes were dissected, their hearts were of normal size and shaped with a well developed myocardium that was lined by endocardium internally and epicardium externally. However, the ventricles were suffused with blood leakage into the pericardial cavity. Cardiac muscle cells that normally differentiate at around E7.5–E8, form intercalated discs and an immature BM on the free cell surface . Their contractions are initially arrhythmic, but at around E9 they contract regularly and with high frequency. The intraventricular pressure significantly rises in the developing chicken heart between embryonic day 5 and 6 , which corresponds to E10–12 in mouse. At around E9, a thin but distinct BM had formed on cardiac muscle cells in normal and perlecan-null mice. In all perlecan-null embryos with hemopericardium, however, this BM had deteriorated. At E10.5, we observed small clefts in the myocardium that were often still lined by endocardium and epicardium. Ultrastructural analysis revealed striking abnormalities in their BMs. Either the lamina densa was completely absent or the cell surface was sparsely covered by densities of irregular shape and size. These alterations could be observed throughout the mutant hearts and were not restricted to the defects in the myocardium. Furthermore, some cardiomyocytes were lined by partly normal and partly abnormal BMs. Interestingly, the homozygotes that survived the E10.5 crisis also showed the same BM defect in their hearts. We further confirmed this defect by crossing homozygotes with a transgenic strain expressing the green fluorescence protein under the control of the cardiac actin promoter . When rhodamine-labeled dextran was injected into the atrium of such embryos using the patch clamp technique to control volume and pressure, the cardiac muscle wall of ∼70% of the homozygous embryos showed two to five leaking transmural channels, and the remaining 30% began to leak when the injection pressure was slightly increased to a level tolerated in normal or heterozygous control hearts (Bloch, N., unpublished observation). At present, we do not know why the heart of these surviving homozygotes does not develop holes in the myocardium. A likely explanation is that cell–cell contacts such as intercalated discs, which were normal in affected and unaffected homozygotes, sufficiently compensate for the BM defects. Altogether, our findings suggest that loss of perlecan is not crucial for the assembly of BMs on early contracting cardiomyocytes but for the maintenance of their structural and functional integrity when subjected to mechanical stress. All perlecan-null embryos that survive the first crisis develop brain anomalies. This was unexpected since we and others found no perlecan expression in the central nervous system . We used different polyclonal antibodies raised against domains I, III, and V of perlecan that revealed expression in the choroid plexus, the subendothelial BMs, and in the BM surrounding the brain. The brain defects were first visible at E10.5 in the anterior region of the expanding brain vesicle. At this stage, two well developed BMs separate the ectoderm and the brain tissue from the mesenchyme. The disruption of both BMs caused aberrant fusion of brain tissue with the overlying ectoderm. Using EM scanning, the BM defects appeared as small clefts, usually not >20–30 μm in width, which contained small round cells. This type of defect exposed brain tissue to amniotic fluid, which led to the destruction of the tissue and the development of exencephaly. Exencephaly can result from defects in neural tube closure, abnormal neuronal migration, or altered proliferation and/or apoptosis of neurons or neuronal precursor cells. We did not observe any of these three defects in perlecan-null embryos. All embryos analyzed by electron or light microscopy showed normal closure of the neural tube, and we could not find abnormal rates of neuronal proliferation and apoptosis in the expanding brain vesicles and other regions of the developing brain before, or at the time of, the onset of defects. When the exencephaly was fully developed we saw increased proliferation of neuronal and glial cells in some of the mutant brains that, however, may be a secondary effect of the amniotic fluid on brain cells. Finally, we found no evidence of abnormal neuronal migration as the cause of the exencephaly. The earliest neuronal cells born in the forebrain are Cajal-Retzius neurons and the subplate neurons that start to migrate at around E9.5-E10 . Most other neurons start to migrate around E12 from the ventricular zone along the radial glia to form the typical laminae of the cortex . The cells in the affected brain areas of perlecan-null embryos expressed neither reelin nor other neuronal markers such as βIII tubulin but stained strongly for nestin, suggesting that they are neuroepithelial cells. It was shown in earlier studies that the intraventricular pressure in the developing brain is much higher than the pressure in the amniotic cavity . In chick embryos, it was demonstrated that the release of the intraventricular pressure leads to the collapse of the brain vesicles and to the formation of ruffles similar to that seen in the perlecan-null brains. We observed that the disruptions in BMs occurred always in the polar area of the brain vesicles where the brain tissue is thin and, therefore, most vulnerable to the vesicular pressure and before the expansion of the neuroepithelium. Therefore, these data suggest that perlecan maintains BM stability by withstanding the tensile force exerted by the expanding brain vesicles. Since the BM has an important barrier function, particularly during development, where tissues are constantly remodeled, small gaps in the BM can lead to an abnormal expansion of neuroepithelial cells and eventually to tissue fusion. A similar process has been observed in mice lacking the laminin α5 chain, which develop small gaps in the BM of the developing hind buds, allowing mesenchymal cells to invade the overlaying ectoderm and eventuating in syndactyly . In addition to the exencephaly, all perlecan-null embryos developed small neuronal ectopias ventral to the medial ganglionic eminence in the developing forebrain. These ectopias that were first visible at E11.5, maintained a constant size and contained postmitotic neuronal cells. Similarly, as on the surface of the brain vesicle, the cells had migrated through the disrupted BM and invaded the surrounding mesenchyme. At present, we are not sure why these neurons fail to stop their migration. It is probable that the migrating neurons exert mechanical stress on the perlecan-null BM, which leads to small gaps allowing cells to invade the mesenchyme. The BM had small gaps that were very similar to those found on the surface of the brain vesicles and the ectopias were always found at the same location, i.e., ventral to the ganglion eminence where active remodeling, extensive migration, and expansion of cells is occurring. The surrounding mesenchyme, being a mechanically resilient tissue, would physically prevent the further aberrant migration of these neurons. All perlecan-null embryos surviving the first critical period between E10 and E12.5 develop chondrodysplasia characterized by disproportionate dwarfism, disorganization of the growth plate, cleft palate, and perinatal lethality most probably because of respiratory failure. During mouse development, perlecan mRNA is expressed in the precartilaginous tissues, accumulates until E14–E15 in all cartilage primordia, and then sharply decreases to background level . However, perlecan expression is not detectable in membranous bones . Despite the abundant deposition of perlecan in cartilage, its function during skeletal development is speculative and mainly based on in vitro experiments. French et al. 1999 demonstrated recently that 10T1/2 cells efficiently aggregate into dense cartilaginous nodules when cultured on perlecan, suggesting that perlecan promotes chondrogenic differentiation. We find that the cartilaginous analogue forms normally in perlecan-null mice, indicating that perlecan is not essential for the condensation and differentiation of prechondrogenic mesenchyme into cartilaginous blastema. During endochondral ossification, the formation of the cartilage model is followed by a complex process that includes the further differentiation of chondrocytes, matrix calcification, vascularization, and the replacement of cartilage by bone . In long bones, endochondral ossification takes place mainly in the epiphyseal growth plate. The perlecan-null bones showed mild changes in epiphyseal cartilage but severe abnormalities in the growth plate, suggesting that perlecan is important for its proper organization and function. Since the perlecan-null chondrocytes can undergo an apparently normal differentiation cascade characterized by the synthesis of lineage-specific matrix components such as type X collagen and of other differentiation genes such as BMP2 , Sox9 , and indian hedgehog (Aszódi, A., unpublished observation), these abnormalities are apparently not associated with an altered chondrocyte differentiation or maturation. In addition to the growth plate abnormalities, perlecan-null long bones exhibit a bended shape. The shape deformation begins shortly after the differentiation of hypertrophic chondrocytes. This finding might be explained by the abnormal hypertrophic matrix, which has a decreased resistance against mechanical forces arising from muscle contraction. Interestingly, the skeletal abnormalities observed in the perlecan-null mice resemble the phenotype of Col2a1 -deficient mice and Dmm (disproportionate micromelia) mice that carry a three-nucleotide deletion in the C-propeptide coding region of the Col2a1 gene, resulting in a severe reduction of type II collagen fiber network in cartilage . Like in Col2a1 -null or Dmm mice, the cartilage of perlecan-null mice is very soft and structurally disorganized. The long bones are shortened and thickened and the bony trabecula are oriented transversely to the long axis. The reduced Safranin-O staining of cartilage and the almost complete lack of mineralization in the late hypertrophic zone is observed in both perlecan-null and Col2a1 -null mice. Surprisingly, the ultrastructural analysis of perlecan-null cartilage revealed a lack of the typical collagen network. The reduced amount and the shortening of collagen fibrils suggests that perlecan plays a pivotal role in maintaining the collagen network. In contrast to the reduced collagen fibrils, the EM analysis revealed a dilated ER and a tremendous increase in free ribosomes and polysomes in the cytosol of chondrocytes, which indicates an unusually high biosynthetic activity in perlecan-null chondrocytes. This was further supported by Northern analysis showing that cartilage tissue derived from perlecan-null embryos expressed three to five times more mRNA coding for Col2a1, matrilin-3, and COMP. Apparently, perlecan-null chondrocytes try to compensate for the loss of cartilage ECM by increasing its synthesis. Since the de novo synthesis does not compensate for the disintegrated matrix, the growth plates deteriorate and are unable to lay down a calcified matrix in the hypertrophic zone leading to insufficient endochondral ossification (template for endochondral bone). These experimental findings revealed a new function of perlecan during endochondral bone formation. A mechanistical explanation could be that perlecan protects the cartilage ECM possibly by binding and inactivating tissue proteases or by masking the ECM proteins and, hence, protecting them from protease attack. Interestingly, it has been shown that perlecan inhibits the proteolytic degradation of fibrillar Aβ , which is thought to play an important role for the accumulation and persistence of neurotoxic plaques in Alzheimer's disease. It is conceivable that similar mechanisms are responsible for the inhibition of proteolysis by perlecan in developing cartilage and Alzheimer's disease. | Study | biomedical | en | 0.999997 |
10587346 | The mouse B cell lymphoma CH27 (H-2 k , IgM + , FcγRIIB1 − ) was maintained in DME supplemented as previously described 41 and containing 15% FCS (15% complete media [CM]). The A20 B lymphoma line (H-2 d , IgG 2a + , FcγRIIB1 + ) stably transfected with either a human μ wild-type (A20μWT) or a mutation that generated a premature stop codon at amino acid K 595 , resulting in the deletion of the cytoplasmic tail (A20μCytoΔ; reference 12), was maintained in 15% CM containing 600 μg/ml G418. Goat Fab anti–mouse μ chain; rabbit anti–human IgG plus IgM (H and L chain); horseradish peroxidase (HRP)-conjugated goat anti–mouse IgG plus IgM (H and L chain); HRP-conjugated rabbit F(ab′) 2 anti–mouse γ chain and anti–human μ chain; and FITC-conjugated goat anti–mouse γ chain, anti–human γ chain, and anti–human μ chain were purchased from Jackson ImmunoResearch. Cholera toxin B subunit (CTB) conjugated to HRP and methyl-β-cyclodextrin were obtained from Sigma Chemical Co. Antibodies specific for mouse CD45R and Lyn were obtained from PharMingen. The goat polyclonal antibody specific for actin was obtained from Santa Cruz Biotechnology, and the rat mAb YL1/2 specific for tubulin 42 was a gift from Dr. Douglas T. Fearon (University of Cambridge, UK). RC20H phosphotyrosine-specific recombinant antibody conjugated to HRP was purchased from Transduction Labs. Polyclonal antibodies specific for H2-M were generated in rabbits using a peptide representing the cytoplasmic tail domain of H2-M (residues 224–245) containing a T cell epitope derived from tetanus toxoid (residues 582–599). The mouse IgG 2a hybridoma 17.3.3s producing an mAb specific for I-E k , the mouse IgG 2a hybridoma HB3 (MK-D6) producing an mAb specific for I-A d , and the rat IgG 2a hybridoma RI7 217.1.3 producing an mAb specific for the transferrin receptor (TfR) were obtained from the American Tissue Type Culture Collection. The rat hybridoma 79a3, producing an IgG 1 mAb specific for the cytosolic domain of mouse Igα, was generated and characterized in the Pierce laboratory at Northwestern University. All hybridomas were maintained in the Pierce laboratory, and mAbs were purified from culture supernatant by affinity chromatography. Goat Fab anti–mouse μ was iodinated using the iodine monochloride method to a specific activity of 0.5–1.0 × 10 7 cpm/μg as described 43 . More than 85% of the 125 I–Fab was precipitated by 10% TCA, indicating little free 125 I. Unlabeled Fab competed with 125 I–Fab for binding to the surface of CH27 cells, indicating that iodination did not affect the binding properties of the Fab. Lipid rafts were isolated using modified lysis conditions and flotation on discontinuous sucrose gradients 20 36 37 . In brief, cells (10 8 ) were washed with ice-cold PBS and lysed for 30 min on ice in 1% Triton X-100 in TNEV containing protease and phosphatase inhibitors (TNEV: 10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 5 mM EDTA; CLAP [2.5 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin A in DMSO] and 1 mM sodium orthovanadate). The lysis solution was further homogenized with ten strokes in a Wheaton loose-fitting dounce homogenizer. Nuclei and cellular debris were pelleted by centrifugation at 900 g for 10 min. For the discontinuous sucrose gradient, 1 ml of cleared supernatant was mixed with 1 ml of 85% sucrose in TNEV and transferred to the bottom of a Beckman 14 × 89 mm centrifuge tube. The diluted lysate was overlaid with 6 ml 35% sucrose in TNEV and finally 3.5 ml 5% sucrose in TNEV. The samples were centrifuged in an SW41 rotor at 200,000 g for 16–20 h at 4°C. 1-ml fractions were collected from the top of the gradient. A 50-μl sample of each fraction from the discontinuous sucrose gradient was incubated with 50 μl of substrate solution (5 mg/ml 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 0.1 M citrate-phosphate buffer, pH 4.0, with 0.015% H 2 O 2 ). The absorbence was measured at 405 nm on an ELISA plate reader. Cells (2 × 10 8 ) were washed with ice-cold HBSS + (13 mM CaCl 2 , 50 mM KCl, 5 mM MgCl 2 .6H 2 O, 4 mM MgSO 4 , 1.38 M NaCl, 56 mM glucose, and 200 mM Hepes, pH 7.4) and incubated in 0.2 mg/ml N -hydroxysuccinimide long chain–biotin (Pierce Chemical Co.) in HBSS + for 15 min at 4°C. Additionally, an equal volume of 0.2 mg/ml biotin was added, and incubation at 4°C was continued for another 15 min. The biotinylation reaction was quenched with ice-cold DME/BSA, washed extensively, and resuspended in 5 ml DME/BSA. For metabolic labeling, cells (2 × 10 8 ) were starved for 30 min in Met − /Cys − DME with 5% dialyzed FCS (5% labeling media) and labeled for 15 min with 200 μCi/ml 35 S–70% methionine/30% cysteine (NEN Express). Cells were washed twice with ice-cold HBSS + and resuspended in 1 ml of 0.5 mg/ml 3,3′-diaminobenzidine (DAB) in HBSS + with or without 0.1% H 2 O 2 . The cells were incubated for 45 min at 4°C, washed with ice-cold HBSS + , and lysed in 1 ml of lysis buffer for 30 min on ice. For studies on class II colocalization, 1% Triton X-100 lysis buffer was used (1% Triton X-100, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide, and CLAP). For studies on colocalization with biotinylated surface proteins, RIPA lysis buffer was used (1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS in PBS with 0.02% sodium azide and CLAP). Cellular debris and aggregated protein polymers were pelleted from the lysate with a 30-min 14,000 g microfuge spin before immunoprecipitation. Cells (10 7 ) were surface biotinylated as described above and washed twice in ice-cold PBS. Phosphatidylinositol-specific phospholipase C (PI-PLC; Sigma Chemical Co.) was added at a concentration of 1 U/ml PBS and incubated at 37°C for 1 h. Cells were pelleted and the supernatant collected. The cells were then washed twice with ice-cold PBS before lysis in 1 ml 1% Triton X-100 lysis buffer. The lysates were cleared of debris as described above. The cell lysates and the supernatants were immunoprecipitated for human Ig, and biotinylated material was visualized by immunoblotting as described below. Cell lysates were precleared of nonspecific proteins and endogenous Ig by incubation with a 30% slurry of protein A–Sepharose or protein G–Sepharose (Amersham Pharmacia Biotech) at 4°C for 1 h. Antibodies (10 μg) and beads (50 μl) were added to the cleared lysate and incubated overnight. The beads were washed three times with lysis buffer and once with PBS. Samples were eluted from the beads by either reducing and boiling for 5 min or incubating with a cocktail lacking reducing agent at room temperature for 30 min. The samples were subjected to 10% SDS-PAGE. Gels with metabolically labeled samples were dried and exposed to film, and gels with biotinylated samples were transferred onto Millipore Immobilon PVDF (polyvinylidene difluoride) membrane. The membranes were blocked for 1 h at 25°C in a buffer containing 0.5% Tween-20, 18% glucose, and 10% glycerol in PBS (TGG) with 3% milk and 1% BSA. The blots were washed in PBS/0.1% Tween-20. A 1:1000 dilution of streptavidin–HRP (Amersham Pharmacia Biotech) in TGG containing 0.3% BSA was added and incubated at 25°C for 1 h. After washing with PBS/Tween-20, the blots were visualized with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). All films were quantified by densitometry. The present evidence indicates that the plasma membrane contains sphingolipid- and cholesterol-enriched microdomains, or lipid rafts, proposed to play a role in membrane trafficking and signal transduction. These microdomains are resistant to Triton X-100 detergent solubilization, allowing for their isolation in discontinuous sucrose density gradients. The location of the BCR in unactivated B cells and after BCR cross-linking with regard to lipid rafts was determined. The CH27 B lymphoma cells that were surface biotinylated were either untreated or treated with anti-Ig at 4°C for 15 min, washed, and warmed to 37°C for 0 or 30 min. The cells were lysed in 1% Triton X-100 lysis buffer on ice, and the lysates were subjected to discontinuous sucrose density gradient centrifugation. Individual fractions from the gradient were subjected to SDS-PAGE and immunoblotting. The position of the lipid rafts in the sucrose gradient was determined by the presence of the ganglioside G M1 , detected using G M1 -specific ligand CTB . As shown in Fig. 1 , G M1 is enriched in the fractions at the top of the sucrose gradient, fractions 3–6. There was no detectable G M1 in fractions 7–9 and only a small amount of G M1 in the solubilized material located at the bottom of the gradient, fractions 10–12, indicating a clear separation of the lipid rafts from the Triton X-100–soluble membranes and components. To determine where in the gradients the BCR resided, immunoblots were probed with antibodies specific for mouse IgM and an mAb specific for Igα. In untreated cells, Ig and Igα were found in the soluble fractions of the sucrose gradient and not in the detergent-insoluble lipid raft region, indicating that in resting cells the BCR is excluded from the lipid rafts . After BCR cross-linking, a significant portion of both the Ig and Igα are translocated into the lipid raft regions of the sucrose gradient. Both Ig and Igα are present in the lipid rafts immediately after cross-linking and warming to 37°C. Somewhat less Ig and Igα are detected in the lipid raft region 30 min after cross-linking. To determine where in the gradient fractions the surface BCR resided, the position of biotinylated sIg and sIgα was examined. The biotinylated CH27 lymphoma cells were either untreated or treated with anti-Ig and analyzed as described above. Biotinylated proteins were immunoprecipitated from the Triton X-100 detergent lysates using streptavidin–agarose, and the immunoprecipitates were analyzed by SDS-PAGE and immunoblot probing with antibodies specific for Ig or Igα. The distribution of the surface BCR is similar to that observed for total cellular BCR. In the absence of anti-Ig cross-linking, the biotinylated sIg is excluded from the lipid raft and is found in the detergent-soluble region of the gradient. Immediately upon cross-linking, the biotinylated sIg is found in the raft region, where it persists for at least 30 min. The behavior of biotinylated sIgα is similar to that of sIg, being excluded from lipid rafts in the absence of cross-linking by anti-Ig and included in lipid rafts upon cross-linking. Thus, cross-linking the BCR with anti-Ig results in the rapid translocation of the surface BCR into plasma membrane lipid rafts. The results of experiments described above indicate that upon cross-linking, the BCR is rapidly translocated into lipid rafts. As stated above, BCR cross-linking results in the initiation of a signal transduction cascade involving the phosphorylation of Igα/Igβ ITAM tyrosines. The Src family kinase Lyn is an essential protein tyrosine kinase involved in the initial phosphorylation events. The BCR response is ultimately terminated by dephosphorylation of BCR signaling components involving plasma membrane phosphatases such as CD45R. Thus, the location of Lyn and CD45R in membrane fractions was determined in unactivated and activated B cells. B cells were untreated or incubated with anti-Ig at 4°C for 30 min, washed, and warmed to 37°C for 0 or 30 min. The cells were lysed in 1% Triton X-100 lysis buffer and subjected to discontinuous sucrose gradient centrifugation, and the gradient fractions were analyzed by SDS-PAGE and immunoblotting for the presence of Lyn and CD45R. The kinase Lyn is concentrated in the lipid raft region of the gradient in resting cells and remains in rafts after BCR cross-linking, at least for the 30-min time course of this experiment . In contrast, the phosphatase CD45R is excluded from the lipid rafts in resting cells and remains excluded after BCR cross-linking . The location of H2-M, a class II–like protein that at steady state has been shown to reside primarily in the IIPLC in CH27 cells 44 , was also determined. As anticipated, H2-M was found in the Triton X-100–soluble membrane fractions in resting cells, and its presence in soluble membranes did not change upon BCR cross-linking. A portion of the cytoskeleton protein actin was found to be constitutively associated with lipid rafts . In contrast, tubulin was found to be completely excluded from the lipid rafts in both anti-Ig–treated and untreated cells. The presence of Lyn in the lipid raft suggests that the Igα present in the lipid rafts after BCR cross-linking may be phosphorylated. To determine the plasma membrane location of phosphorylated proteins, B cells were untreated or treated with anti-Ig at 4°C and warmed to 37°C for 0, 10, or 30 min. At the end of each time point, the cells were lysed in 1% Triton X-100 lysis buffer and subjected to discontinuous sucrose density gradient centrifugation. The gradient fractions were analyzed by SDS-PAGE and immunoblot probing for phosphotyrosine-containing proteins using the phosphotyrosine-specific recombinant antibody RC20H. In the absence of cross-linking, phosphorylated proteins are present in the Triton X-100–soluble membrane fractions, and only faint bands of phosphorylated proteins can be detected in the lipid raft region of the gradient , reflecting the constitutive level of protein tyrosine phosphorylation in CH27 cells. Upon BCR cross-linking, the intensity and number of phosphorylated proteins increase immediately in the fractions that contain detergent-soluble and -insoluble membranes and then return to the levels present in unactivated cells by 30 min. To determine if the phosphorylated proteins in the lipid rafts include either Igα or Lyn, immunoblots were reprobed with antibodies specific for Igα and Lyn. As shown, the Igα band at 34 kD and the Lyn bands at 53 and 56 kD align with phosphotyrosine-containing proteins . By immunoblotting, Lyn appears to be present in the lipid rafts in approximately the same amount in untreated and anti-Ig–treated cells throughout the 30-min chase; however, the phosphorylation state of Lyn appears to change with BCR cross-linking. Igα enters the raft after BCR cross-linking, with maximal translocation occurring 10 min after BCR cross-linking; by 30 min, the amount of Igα in lipid rafts decreases. At each time point, the Igα present in the raft region appears to be phosphorylated. It is difficult to determine if Lyn and Igα phosphorylation is restricted to the lipid rafts because of the complexity of the pattern of phosphorylated proteins in the Triton X-100–soluble fractions, although there are no obvious phosphoproteins aligning with Igα and Lyn. Taken together, these results suggest that after cross-linking, the BCR enters the lipid rafts, where the Lyn kinase is concentrated to facilitate initiation of signal cascades, including Igα and Lyn phosphorylation. The exclusion of CD45R from the lipid rafts indicates that only BCRs outside the lipid rafts would be targets of CD45R phosphatase activity. The results described above provide evidence that upon cross-linking, the BCR rapidly translocates into lipid rafts. To verify that the BCR that moved into lipid rafts was the BCR bound to anti-Ig as a surrogate antigen, B cells were untreated or treated with HRP–anti-Ig at 4°C for 15 min, washed, and warmed at 37°C for 0, 15, or 30 min. The cells were lysed in 1% Triton X-100 lysis buffer and subjected to discontinuous sucrose gradient centrifugation, and the HRP activity in the gradient fractions was measured . In untreated cells, there was little endogenous peroxidase activity detected in the lipid raft region of the gradient, and a small amount of endogenous peroxidase activity was detected in the soluble membrane fractions at the bottom of the gradient. In HRP–anti-Ig–treated cells, HRP activity was found to be highly concentrated in the lipid raft regions immediately after warming to 37°C. There appears to be a slight increase in the HRP activity in the lipid raft regions 15 min after warming to 37°C and a small decrease in HRP activity after 30 min. However, the enzyme activity was not measured in a quantitative fashion, so the small differences may not be significant. To gain a more quantitative measure of the translocation of the antigen bound to the BCR into lipid rafts, B cells were incubated for 1 h with monovalent 125 I–Fab–anti-Ig and subsequently left untreated or treated with rabbit anti–mouse Ig and warmed to 37°C for 0–30 min . In the absence of cross-linking, ∼12% of the 125 I–Fab–anti-Ig is located in lipid rafts. After cross-linking, this percentage increases to ∼26% and then decreases to 18% by 30 min after cross-linking. The presence of the 12% of 125 I–Fab in the lipid rafts in unactivated cells, even though sIg and Igα were not detected in lipid rafts by immunoblotting , suggests that the 125 I–Fab–anti-Ig preparation likely contained some aggregated Fab–anti-Ig or F(ab′) 2 –anti-Ig capable of cross-linking the BCR. Nevertheless, the increase in the percentage of 125 I–Fab–anti-Ig in rafts after anti-Ig treatment correlated with the translocation of biotinylated sIg and Igα and HRP–anti-Ig into lipid rafts. Using a nondisruptive chemical cross-linking technique, we previously showed that upon cross-linking the BCR is rapidly targeted from the plasma membrane through TfR-containing early endosomes to the IIPLC 45 . To determine if the BCR remains associated with components of the lipid raft during intracellular targeting, the same chemical cross-linking technique was used to follow the intracellular movement of the lipid raft ganglioside G M1 in untreated B cells and in B cells treated with anti-Ig. CTB–HRP was used to label G M1 at the B cell surface at 4°C and to follow the movement of G M1 into the cell. Any protein present in the same compartment as CTB–HRP will be polymerized into insoluble aggregates in the presence of membrane-soluble DAB and H 2 O 2 but not in the absence of H 2 O 2 . The insoluble polymers can be removed by centrifugation, and thus the absence of a protein in the lysate indicates its presence in CTB–HRP-containing compartments. As detailed elsewhere, the HRP-mediated polymerization of proteins depends on the presence of the protein, HRP, DAB, and H 2 O 2 in the same subcellular compartment, and nonspecific, promiscuous polymerization of proteins outside HRP-containing compartments is not observed 9 45 . The movement of G M1 from the plasma membrane into early endosomes and to the IIPLC was followed. To detect the movement of CTB–HRP into early endosomes, the TfR was monitored. TfR has been shown by others to be excluded from lipid rafts 35 . B cells were surface biotinylated and incubated at 4°C with CTB–HRP in the presence or absence of anti-Ig. The cells were washed and warmed to 37°C for varying lengths of time up to 120 min. At the end of each time point, the cells were exposed to the chemical cross-linking reagent DAB in the presence or absence of H 2 O 2 and lysed, and the lysates were centrifuged to remove insoluble polymers. TfR was immunoprecipitated from the cleared lysate and subjected to SDS-PAGE and immunoblot probing for biotinylated proteins using streptavidin–HRP. In cells in which the BCR was not cross-linked, CTB–HRP briefly contacts ∼50% of TfRs after warming to 37°C and by 30 min is in contact with a steady state level of ∼20% of the biotinylated TfRs . Because it is difficult to rule out the presence of aggregated or multimerized CTB in the CTB–HRP conjugate preparation, it is not possible to be certain that the observed contact of the CTB–HRP with TfR reflects the constitutive behavior of G M1 or whether the contact with TfR is induced by aggregated CTB–HRP. In B cells in which the BCR is cross-linked, CTB–HRP appears to contact more of the TfR upon warming to 37°C (>90%) and to stay in contact with the TfR longer (>60 min) before returning to steady state levels of 20% by 120 min. The kinetics of contact of CTB–HRP with the TfR after BCR cross-linking are similar to those previously observed for BCR contact with the TfR after BCR cross-linking 45 . To determine if any G M1 is targeted to the IIPLC, B cells were pulsed for 15 min with [ 35 S]methionine in the presence of CTB–HRP and in the presence or absence of anti-Ig. The cells were washed, warmed to 37°C, and chased for 60–180 min. Times were selected that allowed for the detection of newly assembled class II molecules that first bind peptide and adopt SDS-stable conformation in the IIPLC, ∼60–90 min after synthesis 9 46 . At the end of each chase time, the cells were treated with DAB in the presence or absence of H 2 O 2 to cross-link proteins present in the same compartment as the CTB–HRP. The lysates were centrifuged to remove insoluble cross-linked polymers, and class II molecules were immunoprecipitated from the cleared lysate. In untreated B cells, SDS-stable class II heterodimers first appear faintly at 60 min of chase and continue to increase over 150 min and then decrease by 180 min as the class II molecules exit the IIPLC and traffic to the plasma membrane . The intense band above the class II α/β dimers present at 60 min and decreasing thereafter has been previously determined to contain Ii dimers. Significantly, the amount of SDS-stable class II molecules formed was equivalent at each time point in the presence or absence of H 2 O 2 , indicating that CTB–HRP was not present in the peptide loading compartment. In contrast, in B cells in which the BCR was cross-linked, the number of SDS-stable class II molecules was decreased in the presence of H 2 O 2 beginning after 90 min of chase time. The reduction of SDS-stable class II molecules was maximal (∼30%) at 120–150 min and less by 180 min as class II molecules exited the peptide loading compartment. Taken together, these results indicate that in unactivated B cells, the CTB–HRP bound to G M1 briefly enters early endocytic TfR-containing compartments but is not targeted to the late IIPLC. Cross-linking the BCR, which, as shown previously, results in BCR targeting to the IIPLC 9 45 , results in a concomitant targeting of G M1 -bound CTB–HRP to the IIPLC. G M1 appears to stay in contact with the BCR en route to the IIPLC, as shown by the ability of CTB–HRP to cause Igα polymerization throughout the chase period (data not shown). Thus, the BCR appears to remain associated with a portion of the lipid raft G M1 as the BCR is targeted to the IIPLC. The results presented above showed that the BCR is excluded from lipid rafts in unactivated cells and upon cross-linking is translocated into lipid rafts and targeted to the IIPLC. The targeting of the BCR to the IIPLC was accompanied by the movement of a portion of the lipid raft G M1 to the IIPLC. To determine the relationship between residency of a receptor in lipid rafts and targeting to the IIPLC, we analyzed a human Ig, μCytoΔ, in which the cytoplasmic domain was deleted. Previous characterization showed that μCytoΔ stably transfected into mouse A20 cells (A20μCytoΔ) is expressed as a GPI-linked protein that does not associate with the Igα/Igβ complex 13 14 . As a GPI-linked protein, μCytoΔ is predicted to reside in lipid rafts. The behavior of μCytoΔ was compared with that of the endogenous mouse Ig in A20μCytoΔ cells and to that of a WT human Ig (μWT) in A20μWT cells. By flow cytometry, both μCytoΔ (cytoplasmic tail deletion) and μWT are expressed at levels similar to that of the endogenous mouse Ig in both cell lines . The staining is specific, and the cells do not stain using FITC-labeled antibodies specific for the human γ chain. The cell surface expression of μCytoΔ as a GPI-linked protein was verified by PI-PLC treatment of biotinylated cells followed by immunoprecipitation of human IgM and immunoblot analysis, probing for biotinylated proteins. Upon treatment with PI-PLC, μCytoΔ is released into the supernatant, in contrast to μWT, which remains cell associated . The lipid raft localization of μCytoΔ, μWT, and endogenous mouse IgG was determined using HRP-conjugated antibodies as described above. In brief, A20μCytoΔ or A20μWT cells were untreated or incubated with HRP-conjugated antibodies specific for either mouse IgG or human IgM for 1 h at 4°C. The cells were washed and warmed to 37°C for 30 min, lysed in 1% Triton X-100 lysis buffer, and subjected to discontinuous sucrose density gradient centrifugation. The HRP activity in the individual gradient fractions was measured. The endogenous peroxidase activity in the untreated cells is low in the lipid raft region of the gradient and only slightly higher in the soluble membrane fractions at the bottom of the gradient . The HRP-coupled antibodies specific for the endogenous mouse IgG were present in both the lipid raft region of the gradient and the soluble membrane region at the bottom of the gradient. The behavior of μWT was similar to that of mouse IgG after HRP–anti-Ig treatment, distributing both in the raft region and in the detergent-soluble region of the sucrose gradient . These patterns were similar to that observed for mouse IgM in CH27 cells. In contrast, HRP-conjugated antibodies specific for human IgM in A20μCytoΔ cells were located exclusively in the lipid raft region of the gradient. To independently confirm the location of μCytoΔ with regard to lipid rafts, A20μCytoΔ cells were either untreated or incubated with antibodies specific for mouse IgG or human IgM at 4°C for 1 h, washed, and incubated at 37°C for 30 min. The cells were lysed in 1% Triton X-100 in TNEV buffer and fractionated on a discontinuous sucrose gradient, and the fractions were subjected to SDS-PAGE and immunoblot probing for either mouse IgG or human IgM. The mouse IgG is not present in the lipid raft region of the gradient in unactivated A20μCytoΔ cells , and upon cross-linking, mouse IgG translocates to the lipid rafts. This result is similar to that described above for mouse IgM in CH27 cells and indicates that the heavy chain isotype of the sIg does not influence its behavior with regard to lipid raft localization. In cells treated with antibodies specific for human IgM, mouse IgG remains excluded from the lipid raft region. Thus, cross-linking μCytoΔ had no effect on endogenous mouse IgG. In contrast to the behavior of endogenous mouse IgG, human μCytoΔ is found nearly exclusively in the lipid raft region in unactivated cells . Upon cross-linking using antibodies specific for human IgM, the position of μCytoΔ in lipid rafts does not change. Moreover, the cross-linking and translocation of the mouse IgG into lipid rafts does not alter the position of μCytoΔ. To determine if the inclusion of μCytoΔ in the buoyant fractions was dependent upon the integrity of the lipid rafts and to determine if aggregation of the receptor alone dictates its behavior on sucrose gradients, A20μCytoΔ cells were treated with anti-Ig and the cholesterol-sequestering drug methyl-β-cyclodextrin that disrupts cholesterol-dependent rafts. In cyclodextrin-treated cells, μCytoΔ is no longer present in the buoyant region of the sucrose gradient but rather in the Triton X-100–soluble dense region of the gradient, even in the presence of cross-linking antibody . The effect of cyclodextrin was partially reversible after 3 h with the addition of cholesterol . Taken together, these results indicate that the localization in buoyant regions of the sucrose gradient is dependent on the integrity of the lipid rafts, and aggregation of sIg with anti-Ig alone does not result in appearance of the sIg in the buoyant gradient functions. To determine if the μCytoΔ is targeted to the IIPLC after cross-linking, A20μCytoΔ cells were pulsed with [ 35 S]methionine for 15 min in the presence of HRP-conjugated antibodies specific for either mouse IgG or human IgM. The cells were washed and incubated for 60–180 min at 37°C. At the end of each time point, the cells were treated with DAB in the presence or absence of H 2 O 2 and lysed, and the I-A d class II molecules were immunoprecipitated. The immunoprecipitates were subjected to SDS-PAGE without reducing or boiling the samples, conditions under which peptide-bound class II α/β dimers are stable. In cells treated with HRP–anti–mouse IgG, the number of class II α/β dimers is decreased after treatment with DAB plus H 2 O 2 as compared with DAB alone . The reduction in class II molecules is observed at 60 min, reaches a maximum of 40% at 120 min, and decreases thereafter as the class II molecules exit the IIPLC. Similar results were obtained by analyzing μWT in A20μWT cells using HRP–anti–human IgM, in which case 25% of newly synthesized SDS-stable class II molecules were cross-linked after 120 min of chase time. These results are in agreement with those previously shown for the contact of WT IgM BCR with newly synthesized I-E k in CH27 cells 9 45 . In contrast, in A20μCytoΔ cells treated with HRP–anti–human IgM, there was no reduction in the number of class II molecules in cells treated with DAB plus H 2 O 2 as compared with DAB alone . Moreover, in A20μCytoΔ cells treated with antibodies specific for mouse IgG and HRP-conjugated antibodies specific for human IgM, μCytoΔ was not targeted to the IIPLC (data not shown). Thus, μCytoΔ resident in the lipid raft did not accompany the WT mouse BCR to the IIPLC. These findings indicate that the human IgM that constitutively resides in the lipid rafts is not targeted to the IIPLC after cross-linking, in agreement with previous results showing that the μCytoΔ did not target antigen for class II processing 15 . Moreover, these results indicate that residency in the lipid rafts region alone is not sufficient for targeting to the IIPLC. The BCR plays two key roles in the B cell response to antigen. The first is to transmit signals through several intracellular pathways that ultimately dictate the fate of the B cell's encounter with antigen 1 2 3 . BCR signaling itself has been recently appreciated to be a complex phenomenon that is directly influenced both positively and negatively by B cell coreceptors, including CD19/CD21, CD22, CD40, and FcγRIIB 1 . How the components of the BCR signaling cascade and the coreceptors are organized on the plasma membrane to affect the final outcome is not known. In addition to signaling, the BCR physically transports antigen from the cell surface to the IIPLC 6 . The current evidence indicates that the signaling function of the BCR is required to specify both the targeting of the BCR to the IIPLC and the rate at which the targeting is achieved. How the signaling and targeting functions of the BCR are coordinated is not known. In this report, we show that after cross-linking, the BCR is rapidly translocated into lipid rafts that contain the Src family kinase Lyn, a key kinase in the initiation of the BCR signal transduction cascade, and exclude the phosphatase CD45R. Moreover, we show that phosphorylated Igα and Lyn are present in the lipid rafts after BCR cross-linking. Taken together, these results suggest that the recruitment of the BCR to the lipid rafts may represent an important, previously unappreciated event in BCR signaling, allowing the concentration of the BCR and the enzymes and adaptors of the signaling pathway. Upon BCR cross-linking, it has been observed that sIg becomes associated with the actin cytoskeleton and that cytoskeletal attachment is not dependent on Igα/β signaling 47 . Furthermore, sIg with cytoplasmic tail sequence KVK replaced with the Igα tail was shown to have reduced ability to associate with cytoskeletal actin 48 . Although the significance of cytoskeletal attachment remains unknown, we provide evidence that the WT BCR translocated into the lipid raft, along with the raft component G M1 , is subsequently targeted to the IIPLC in an accelerated fashion. Thus, the lipid raft may provide components necessary for the correct and accelerated targeting of the BCR to the IIPLC. Our result showing that μCytoΔ is constitutively localized in lipid rafts but is not targeted to the IIPLC suggests that raft localization alone is not sufficient for efficient internalization and proper intracellular targeting. Targeting may require initiation of signaling, a function for which μCytoΔ is deficient. However, rafts may provide a means for efficient cytoskeletal association of sIg either directly or through associated factors. Taken together, the results presented here are consistent with the hypothesis that lipid rafts serve as platforms for both signaling and trafficking of membrane receptors 16 . The molecular mechanisms by which the BCR is translocated to lipid rafts remain to be elucidated. The translocation of the BCR to lipid rafts was nearly instantaneous after cross-linking. The rapidity of the translocation was similar to that reported by Montixi et al. 36 , who showed that the engagement of the TCR triggers an immediate accumulation of the TCR and protein tyrosine kinases to lipid rafts, readily detectable within 10 s. Cross-linking the BCR may result in a conformational change of some sort in the BCR that allows its rapid translocation into lipid rafts by destabilizing its interaction with Igα/Igβ or the replacement of Igα/Igβ by some other membrane component. Indeed, Vilen et al. 49 recently provided evidence that after antigen binding, the sIg–Igα/Igβ complex is destabilized such that Igα/Igβ no longer coimmunoprecipitates with sIg. However, the results presented here show that both sIg and Igα enter the rafts, indicating that the BCR remains intact after cross-linking. A conformational change could also be the result of a specific phosphorylation event on one of the tyrosines within the Igα/Igβ ITAMs. Presumably, such a phosphorylation event is insufficient to initiate significant downstream signaling before translocation to the lipid rafts. The current evidence concerning the mechanism of the translocation of the TCR into lipid rafts upon TCR engagement suggests that phosphorylation of the TCR outside the lipid rafts promotes translocation. Alternatively, a conformational change could be induced by the clustering or aggregation of the BCR by cross-linking. Analysis of additional BCR constructs in which both the association of sIg with Igα and Igβ and the signaling potential of the Igα/Igβ complex are altered should be informative with regard to the molecular basis of the trigger that leads to BCR translocation. The BCR is similar to two other immune receptors in its behavior on the plasma membrane after cross-linking, namely the TCR and the IgE receptor, both of which become concentrated in lipid rafts after engagement. Given that these receptors share many common features in terms of the processes by which signal transduction cascades are initiated and regulated, the observation that similar strategies are used to allow concentration of the components of the signaling cascade is perhaps not surprising. As the composition of the lipid rafts is further characterized and the molecular mechanism underlying the triggering of these receptors to translocate to lipid rafts is defined, there are likely to be more commonalities discovered as well as interesting differences revealed. | Study | biomedical | en | 0.999999 |
10587347 | IL-2Rβ 2/− mice described previously 9 were maintained in our animal facility. All IL-2Rβ 2/− mice used in this study had been back-crossed at least 10 times to C57BL/6 (B6) mice, providing a pure genetic background. These mice were further crossed with B6/ CD45.1/CD45.1 congenic strain to make IL-2Rβ 2/− mice with the CD45.1 allotype marker. B6 recombination activating gene (RAG)-2 −/− (B6.RAG-2 −/− ) mice were provided by the Central Institute for Experimental Animals (Kawasaki, Japan) with the permission of Dr. F.W. Alt (Harvard Medical School, Boston, MA). B6 lpr/lpr mice were purchased from Japan SLC, Inc. B6 gld/gld mice were provided by Dr. K. Okumura (Juntendo University, Tokyo, Japan). TCR-β 2/− mice were provided by Dr. Y. Yoshikai (Nagoya University). Lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic mice were provided by Dr. R. Zinkernagel (Institute of Experimental Immunology, University Hospital, Zürich, Switzerland) and mated with RAG-2 −/− mice to generate TCR transgenic mice with RAG-2 −/− background. FITC-conjugated anti–mouse CD69 mAb (clone H1.2F3), PE-conjugated anti-CD62L antibody (clone MEL14), FITC- or biotin-conjugated anti-CD45.1 antibody (clone A20), FITC- or biotin-conjugated anti-CD45.2 antibody (clone 104), FITC- or PE-conjugated anti–Thy-1.2 antibody (clone 30-H12), PE-conjugated anti-B220 antibody (clone RA3-6B2), and FITC-conjugated anti–Gr-1 antibody (clone RB6-8C5) were purchased from PharMingen. FITC- or PE-conjugated anti-CD4 antibody (clone H129.19) and FITC- or PE-conjugated anti-CD8α antibody (clone 53-6.7) were purchased from Sigma Chemical Co. Cells were stained with antibodies for 20 min on ice, and analyzed using a FACSCalibur™ (Becton Dickinson). Biotin-conjugated antibodies were visualized by secondary staining with streptavidin-conjugated RED670 (GIBCO BRL). Bone marrow cells were obtained by flushing out the femoral bone marrow of 3-wk-old mice. In a combination of bone marrow cell preparation, T cells were depleted by treatment with anti–Thy-1.2 antibody and rabbit complement (ICN Pharmaceuticals, Inc.). A total of 2 × 10 6 bone marrow cells were intravenously injected into an irradiated (9 Gy) B6.RAG-2 −/− mouse. Lymph node cells from IL-2Rβ 2/− mice or spleen cells and lymph node cells from other types of mice were collected and passed through nylon wool columns. Some of the nylon wool–passed cells were analyzed before transfer. The remaining cells were either mixed or unmixed, and a total of 1–4 × 10 7 cells were injected intravenously to sublethally irradiated (4 Gy) B6.RAG-2 −/− mice. Spleen and lymph node cells were stained with biotin-conjugated anti-CD4 or anti-CD8 antibody, and secondarily incubated with streptavidin microbeads (MACS; Miltenyi Biotec). The following column work was performed according to the manufacturer's protocol (Miltenyi Biotec). RNA was extracted from collected cells using RNAzol (Tel-Test), and cDNA was created using the RNA LA PCR kit (Takara). 30 cycles of PCR reaction were performed under the following conditions: 94°C, 30 s; 60°C, 30 s; 72°C, 90 s. PCR primers used in this study were as follows: 5′ β-actin, TGG AAT CCT GTG GCA TCC ATG AAA C; 3′ β-actin, TAA AAC GCA GCT CAG TAA CAG TCC G; 5′ IL-2, TGA TGG ACC TAC AGG AGC TCC TGA G; 3′ IL-2, GAG TCA AAT CCA GAA CAT GCC GCA G; 5′ IL-4, CGA AGA ACA CCA CAG AGA GTC AGC T; 3′ IL-4, GAC TCA TTC ATG GTG CAG CTT ATC G; 5′ IFN-γ, AGC GGC TGA CTG AAC TCA GAT TGT AG; 3′ IFN-γ, GTC ACA GTT TTC AGC TGT ATA GGG; 5′ TNF-α, GGC AGG TCT ACT TTG GAG TCA TTG C; 3′ TNF-α, ACA TTC GAG GCT CCA GTG AAT TCG G; 5′ TNF-β, TGG CTG GGA ACA GGG GAA GGT TGA C; 3′ TNF-β, CGT GCT TTC TTC TAG AAC CCC TTG G; 5′ Fas ligand (FasL), GGT CAG CAC TGG TAA GAT TG; 3′ FasL, GAG TTC ACC AAC CAA AGC CT; 5′ granzyme B, GCC CAC AAC ATC AAA GAA CAG; 3′ granzyme B, AAC CAG CCA CAT AGC ACA CAT; 5′ perforin, GTC ACG TCG AAG TAC TTG GTG; and 3′ perforin, AAC CAG CCA CAT AGC ACA CAT. The statistical analysis was performed using StatView J-4.5 software. To investigate the mechanism of abnormal development of multiple hematopoietic cells in IL-2Rβ 2/− mice, we first examined bone marrow chimeric mice reconstituted with IL-2Rβ 2/− bone marrow cells. When lymphocyte-deficient RAG-2 −/− mice were reconstituted with IL-2Rβ 2/− bone marrow cells, T cells arising from the transferred bone marrow cells later than 6 wk showed a markedly activated memory phenotype of CD69 + CD62L lo , CD44 hi , and CD45RB lo (data not shown), all of which were characteristic features of T cells in IL-2Rβ 2/− mice . These mice also showed phenotypes of decreased B220 + cells in lymphatic organs, increased Gr-1 + cells in bone marrow, and anemia (data not shown), all of which were observed in IL-2Rβ 2/− mice 9 , indicating that most phenotypes caused by IL-2Rβ deficiency were restored by bone marrow reconstitution. However, when RAG-2 −/− mice were reconstituted with a mixture of IL-2Rβ 1/+ and IL-2Rβ 2/− bone marrow cells, T cells arising from IL-2Rβ 2/− bone marrow cells, which were distinguished from IL-2Rβ 1/+ –derived cells by CD45 allotype-specific antibody and were shown to exist with a number similar to that in simple IL-2Rβ 2/− bone marrow chimera, showed no sign of activation . B cells, granulocytes, and erythrocytes were also normal in these mice (data not shown), and inflammatory bowel disease was never observed. This result indicates that IL-2Rβ 2/− T cells do not develop into an abnormally activated phenotype when they exist together with normal IL-2Rβ 1/+ cells, which may have the activity to regulate the abnormal activation of IL-2Rβ 2/− T cells. In mixed bone marrow chimeric mice, the percentages of cells derived from IL-2Rβ 1/+ or IL-2Rβ 2/− bone marrow were examined for some different lineage of hematopoietic cells, and the result was summarized in Table . Although the bone marrow cells of two different mice were mixed at a 1:1 ratio and were injected into host mice, the ratio of the origin in differentiated hematopoietic cells in the individual bone marrow chimeric mice varied. The first striking finding was the difference in the contribution of IL-2Rβ 1/+ (or IL-2Rβ 2/− ) cells between B cells or granulocytes and T cells. All the mixed bone marrow chimeric mice showed a higher contribution of IL-2Rβ 1/+ cells in T cells than in B cells or granulocytes (i.e., there were more IL-2Rβ 2/− cells in B cells and granulocytes than in T cells). This skewing of T cells to IL-2Rβ 1/+ cells was more striking in CD8 + T cells than in CD4 + T cells. This difference in CD4 + cells and CD8 + cells also reflected the difference in the CD4 + /CD8 + ratio between IL-2Rβ 1/+ –derived and IL-2Rβ 2/− –derived cells. The average ratio was 2.68 for IL-2Rβ 2/− –derived cells and 1.25 for IL-2Rβ 1/+ –derived cells. When bone marrow cells had been depleted with T cells ( Table , A ), or when bone marrow cells from a neonatally thymectomized IL-2Rβ 2/− mouse were used ( Table , B ), the skewing was consistently observed, excluding the possibility of skewing due to the difference in hematopoietic potential influenced by mature T cells infiltrating the bone marrow. These profiles of cell origin in mixed bone marrow chimeric mice ( Table ) are striking, but should be interpreted carefully. Although we mixed the same numbers of nucleated cells from IL-2Rβ 2/− and IL-2Rβ 1/+ bone marrow, the frequency of hematopoietic stem cells might have varied in individual donor mice, resulting in the diverse contribution rate of two different donors for individual chimeric mice. Skewing of B cells and granulocytes towards IL-2Rβ 2/− cells as a phenotype intrinsic to IL-2Rβ 2/− should be excluded for the following reason. In IL-2Rβ 2/− mice, the number of granulocytes (Gr-1 + cells) is greatly increased, whereas the number of B220 + cells is markedly decreased, both of which are secondary phenomena affected by abnormal T cells 9 . Therefore, the numbers of B cells and granulocytes should not have moved in the same direction when these cells were affected by an IL-2Rβ deficiency. The contribution of host-derived Gr-1 + CD45.1 − cells could be neglected, because no change was observed in the CD45.1 + /CD45.1 − ratio in Gr-1 + cells compared with that in B220 + cells. With these considerations in mind, we concluded that the IL-2Rβ 2/− /IL-2Rβ 1/+ ratio in B cells and granulocytes reflected the ratio in hematopoietec stem cells. Accordingly, the skewing of T cells towards IL-2Rβ 1/+ cells was a real event, and reflected either a decrease in IL-2Rβ 2/− –derived T cells or an increase in IL-2Rβ 1/+ –derived T cells. The CD4 + /CD8 + ratio was also significantly different between IL-2Rβ 1/+ and IL-2Rβ 2/− cells ( Table ). This difference was mainly caused by a decrease in the CD4 + /CD8 + ratio in IL-2Rβ 1/+ cells, because that ratio in mixed bone marrow chimeric mice was significantly lower than that in mice simply reconstituted with IL-2Rβ 1/+ bone marrow (1.25 ± 0.29, n = 10 vs. 1.84 ± 0.22, n = 5; P < 0.01, unpaired two group t test), whereas the CD4 + /CD8 + ratio of IL-2Rβ 2/− cells in mixed bone marrow chimeric mice was similar to that in simple IL-2Rβ 2/− bone marrow chimeric mice (2.68 ± 0.46, n = 10 vs. 2.74 ± 0.28, n = 5; P > 0.5). Therefore, an especially significant skewing of CD8 + T cells to IL-2Rβ 1/+ –derived cells was more likely to be due to an increase in IL-2Rβ 1/+ CD8 + cells, rather than to a decrease in IL-2Rβ 2/− CD8 + cells. We performed the examination of mixed bone marrow chimera using some different types of mutant mice as partners for IL-2Rβ 2/− . As shown in Fig. 2 , when bone marrow cells of B6 lpr/lpr mice which lacked the functional Fas molecule were mixed with IL-2Rβ 2/− , the resulting IL-2Rβ 2/− T cells were normal in their activation and memory phenotype. Functional FasL-deficient B6 gld/gld cells also had the same effect on the regulation of IL-2Rβ 2/− T cells. In contrast with these Fas/FasL mutant strains, when TCR-β 2/− bone marrow cells were mixed with IL-2Rβ 2/− and reconstituted the RAG-2 −/− host, the resulting CD4 + or CD8 + T cells were all IL-2Rβ 2/− –derived, and showed a striking activated phenotype similar to those in IL-2Rβ 2/− mice or simple IL-2Rβ 2/− bone marrow chimeras . T cells in LCMV-specific TCR transgenic mice with RAG-2 −/− background, which consequently express TCR molecules with a single specificity, showed no activity to regulate the activation of IL-2Rβ 2/− T cells ( Fig. 2 , TCR tg (RAG-2 −/− )). All of these chimeric mice were effectively reconstituted with each partner of bone marrow cells, because the percentages of mutant partner–derived T cells per total T cells were not significantly different from those of mice reconstituted with wild-type B6 and IL-2Rβ 2/− bone marrow ( P > 0.5 for every combination). Percentages of CD45.1 − cells (partners for IL-2Rβ 2/− ) per total T cells recovered from mixed bone marrow chimera of each combination were 60.5 ± 15.3 ( n = 5), 60.6 ± 10.5 ( n = 4), 58.2 ± 10.0 ( n = 4), 53.7 ± 5.1 ( n = 3), for B6, B6 lpr/lpr , B6 gld/gld , and TCR transgenic, respectively, and that of CD45.1 − cells per total B cells was 58.0 ± 11.5 ( n = 3) for TCR-β 2/− . These results indicated that functional T cells with an adequate TCR repertoire were required, but that Fas or FasL was unnecessary to regulate the abnormal activation of IL-2Rβ 2/− T cells. To investigate the mechanism of regulation of IL-2Rβ 2/− T cells in more detail, we performed a transfer of purified T cells to RAG-2–deficient host mice. Nylon wool column–passed lymph node cells (Thy-1 + cells > 90%) from IL-2Rβ 1/+ and IL-2Rβ 2/− mice were mixed with 1:1 ratio and transferred to RAG-2 −/− mice. Here, we used IL-2Rβ 2/− mice >6 wk, and almost all T cells from these mice were expressing activated memory phenotypes with CD69 + and CD62L lo . 7 d later, cells were recovered from the spleen and lymph nodes, and their origin was examined. As shown in the middle panels of Fig. 3 , recovered IL-2Rβ 2/− T cells were much fewer than IL-2Rβ 1/+ T cells. This phenomenon was consistently observed because the percentage of CD45.1 + (IL-2Rβ 2/− ) cells per total T cells was 2.6 ± 1.1% ( n = 10, from five independent experiments). When IL-2Rβ 1/− CD45.1 + T cells and IL-2Rβ 1/+ CD45.1 − T cells were mixed and transferred to the RAG-2 −/− host, no increase or decrease in either population was observed 7 d after transfer (data not shown), excluding the possibility that IL-2Rβ 1/+ T cells reacted to CD45.1 antigen, Neo r gene product, or some minor antigenic differences which had been carried from embryonic stem cells and had not been eliminated by back-crossing to B6 mice. In the recovered IL-2Rβ 1/+ T cells that had been transferred with IL-2Rβ 2/− T cells, the CD4/CD8 ratio was skewed towards a CD8 + dominant phenotype , whereas no such skewing was observed in IL-2Rβ 1/+ T cells simply transferred to RAG-2 −/− hosts without IL-2Rβ 2/− T cells. The CD4 + /CD8 + ratio of IL-2Rβ 1/+ cells recovered from mice transferred with a mixture of IL-2Rβ 1/+ and IL-2Rβ 2/− T cells was significantly lower (0.96 ± 0.27, n = 10, from five independent experiments; P < 0.001, unpaired two group t test) than that of donor IL-2Rβ 1/+ mice (1.85 ± 0.14, n = 5), whereas that from mice transferred with single IL-2Rβ 1/+ T cells was not (1.69 ± 0.36, n = 4, from four independent experiments; P > 0.3). Analyses of mice at several different time points after T cell transfer revealed that the change in the IL-2Rβ 2/− /IL-2Rβ 1/+ cell ratio was gradually taking place in vivo in the initial 6–7 d after T cell transfer to RAG-2 −/− host mice . At time points later than 7 d, the IL-2Rβ 2/− /IL-2Rβ 1/+ cell ratio was consistently low and not significantly changed from that of day 7 (data not shown). These results suggested that normal IL-2Rβ 1/+ T cells had the activity to eliminate abnormally activated IL-2Rβ 2/− T cells. T cells from some different types of mutant mice were used for the partner of IL-2Rβ 2/− T cells, and were transferred to RAG-2 −/− mice. 7 d later, recovered cells were analyzed for their origin, and percentages of recovered cell numbers per transferred cell numbers were calculated. As shown in Fig. 5T cells from B6 lpr/lpr mice and B6 gld/gld mice showed an almost identical action to B6 T cells in eliminating IL-2Rβ 2/− T cells. On the other hand, T cells from LCMV-specific TCR transgenic mice with RAG-2 −/− background showed no such elimination activity. When normal T cells were separated into CD4 + cells and CD8 + cells, and their activity was measured independently, CD8 + cells showed a significantly stronger effect than total T cells or CD4 + T cells alone. Interestingly, CD8 + T cells showed a markedly increased recovery rate when CD8 + T cells were purified and transferred with IL-2Rβ 2/− T cells. Our study of mixed T cell transfer showed an elimination of IL-2Rβ 2/− T cells by normal T cells, and an especially strong elimination activity in CD8 + T cells suggested a cytotoxic mechanism in this process. Accordingly, we next examined the expression of genes involved in T cell cytotoxic activity. T cells, which were prepared from normal B6 (IL-2Rβ 1/+ ) mice and IL-2Rβ 2/− mice, were mixed in equal numbers and transferred to B6.RAG-2 −/− host mice. 7 d later, B6-derived T cells were recovered and purified by sorting cells stained with anti-CD45.2 and anti-CD4/CD8 antibodies, and the expressions of IL-2, IL-4, IFN-γ, TNF-α, TNF-β, FasL, granzyme B, and perforin were analyzed by the reverse transcription PCR method. The expression in T cells recovered from mice transferred with IL-2Rβ 2/− T cells was compared with that in T cells simply transferred without IL-2Rβ 2/− T cells and recovered. As shown in Fig. 6 A, recovered T cells expressed all the genes tested, including TNF-α, TNF-β, FasL, granzyme B, and perforin. Among these, the expression levels of TNFs, especially TNF-β, were higher in cells transferred with IL-2Rβ 2/− T cells . These results indicated the possibility that normal T cells eliminated IL-2Rβ 2/− T cells by using these molecules associated with T cell cytotoxic activity. It was surprising to find that a disruption of one of the components of the IL-2/IL-2 receptor system caused a significant activation and increase of T cells 8 9 14 15 16 . This finding proved that the IL-2/IL-2R system is important for regulating and maintaining the homeostasis of immune systems, although the precise mechanism of such regulation was not clarified. Studies on IL-2–deficient and IL-2Rα–deficient mice have demonstrated some insights into the possible mechanism. T cells in IL-2–deficient mice were shown to be more resistant to apoptosis induced by reactivation or Fas triggering 11 . IL-2Rα–deficient T cells were also shown to be apoptosis resistant 12 17 . This failure of spontaneous death may be a possible mechanism explaining the expansion of abnormally activated T cells under the condition in which IL-2 or IL-2R never functions. However, studies on IL-2Rβ–deficient mice revealed that T cells in these mice were normally sensitive to Fas-mediated or superantigen-induced cell death 13 . This finding indicated the existence of another essential mechanism causing the accumulation of abnormally activated T cells in IL-2Rβ–deficient mice. In this study, we have demonstrated by two different experimental systems that normal T cells regulate the abnormal activation of IL-2Rβ 2/− T cells. Our experimental result of bone marrow transplantation appears to be a “prevention of abnormal activation,” while that of T cell transfer seems more like an “elimination of already activated cells,” possibly demonstrating that two different mechanisms are involved in regulating abnormal T cells. However, these two experiments showed some coincidental results: (a) lack of Fas or FasL molecules does not affect regulatory activity; (b) cells with transgenic TCR lack regulatory activity; and (c) CD8 + cells from IL-2Rβ 1/+ mice are increased when they regulate IL-2Rβ 2/− T cells. These coincidences may indicate that the two experimental systems reflected the same event, and it would only be logical to consider that a common mechanism worked to express two different-looking phenomena. Based on this assumption, the suppression of activation observed in bone marrow–transplanted mice could be maintained by continuous elimination of activated T cells. Depletion of T cells lacking IL-2Rβ, observed in the T cell transfer experiment, is not due to mere “general weakness” of those cells compared with normal IL-2Rβ 1 T cells, because IL-2Rβ 2/− T cells, although their number is slightly reduced, do coexist with IL-2Rβ 1 T cells in the mixed bone marrow chimeric mice, and do not decrease when transferred with TCR transgenic T cells. Therefore, lack of IL-2Rβ itself cannot trigger the elimination; however, some sign of the activated state could do so. Krämer et al. also performed a study of mixed bone marrow transplantation using IL-2–deficient mice, and found that IL-2 −/− –derived T cells did not develop into an abnormal state in mixed chimera containing 30% IL-2 + lymphocytes 18 . Although their finding is similar to our observation in mixed bone marrow chimera of IL-2Rβ 2/− and IL-2Rβ 1 cells, it is unclear whether the same mechanism works for the normalization of mutant T cells in the mixed bone marrow transplantation system of IL-2 −/− and that of IL-2Rβ 2/− , because no information, such as the detailed distribution of the lymphocyte population in IL-2 −/− mixed bone marrow chimera and the result of the mixed T cell transfer experiment for IL-2 −/− , is provided. Furthermore, in the case of the IL-2 −/− system, because IL-2 could be secreted from normal T cells and act on IL-2 −/− T cells, it is impossible to identify whether the normalizing mechanism is a paracrine action of IL-2, or the regulatory activity of wild-type T cells. In our case, because IL-2 could never act on receptor-deficient T cells, a regulatory mechanism other than the action of IL-2 on the activated T cells must be working between normal T cells and IL-2Rβ–deficient T cells. By adding the system of the T cell transfer experiment, we have postulated the mechanism to be the elimination of activated T cells. The experiment using TCR transgenic mice showed a coincidental result in bone marrow transplantation and T cell transfer. T cells with transgenic TCR showed no activity to regulate activated IL-2Rβ 2/− T cells. This result may indicate that α/β-type functional TCR is directly involved in the process of regulation and/or elimination as the recognition molecule on the regulatory T cells. However, it is also possible that T cells in TCR transgenic mice are functionally monotonous, and may not include the regulatory T cell population. A significant increase in Vβ12 TCR–using cells in CD8 + T cells that had eliminated IL-2Rβ 2/− T cells in mixed T cell transferred mice (our unpublished observations) may indicate that CD8 + T cells bearing Vβ12 TCR are the key population responding to IL-2Rβ 2/− T cells. However, the increase in Vβ12 + T cells is still small (control = 1.92 ± 0.10% vs. posttransfer = 3.20 ± 0.40%, n = 3; P < 0.01), suggesting that the responsible T cells are more likely to be multiclonal. What molecules are involved in the process for the regulatory cells to recognize abnormally activated T cells remains an important question. They could be T cell activation–linked molecules potentially presented to the regulatory cells by the activated cells. IL-2Rβ 2/− T cells express increased levels of many cytokines, including IL-2, IL-4, IL-10, IFN-γ, TNF-α, and TGF-β ( 13 ; our unpublished observations), and cell surface molecules such as CD69 and CD44. These molecules could be working for recognition by the regulatory T cells. The additional question arises as to what molecules are working in the effector phase. We performed an analysis of the expression of several genes which may possibly be involved in the effector phase of cytotoxic T cells 19 20 . A Fas–FasL deficiency causes expansion of T cells and autoimmune-like abnormalities, suggesting a possible involvement of this system in the regulation of activated T cells 21 . Although FasL expression was clearly identified in the T cells eliminating IL-2Rβ 2/− T cells , our study proved that Fas and FasL interaction was not indispensable in the regulation and/or elimination of IL-2Rβ–deficient T cells . Similarly, expression of TNFs, perforin, and granzyme B was observed, and the level of TNF-β expression was elevated in T cells eliminating IL-2Rβ 2/− T cells. Mice lacking the activity of TNFs, perforin, or granzyme B molecules show no sign of an increase in activated T cells 22 23 24 , suggesting that the defect of a single molecule may be indecisive in the regulatory activity. Taken together, these molecules involved in the cytotoxic T cell function may compensate for one another, and IL-2Rβ may be the key molecule controlling the central function or development of cells using these molecules. Our study indicated that CD8 + cells are more effective regulators against the activated IL-2Rβ 2/− T cells. The regulatory cells found in this study may be related to the suppressor T cell subsets that have been described in previous reports. Both CD8 + and CD4 + subsets were reported to include suppressor T cells 25 26 27 , and the suppressor activity is sometimes related to Fas–FasL dependency 28 . In our study, however, the regulatory activity is stronger in CD8 + cells than in CD4 + cells, and Fas–FasL independent. CD4 + cells alone are also sufficient to achieve the regulatory activity , indicating the heterogeneity of the responsible cells. This elimination by CD4 + cells is not due to any minor contamination of CD8 + cells, <5% of which remain after purification with anti-CD8 antibody and MACS, because a small number of CD8 + T cells (one tenth of IL-2Rβ 2/− cells) is not sufficient to eliminate IL-2Rβ 2/− T cells (data not shown). The “regulatory T cells” described in more recent studies seem to expand to both the CD4 + and CD8 + populations 29 30 31 32 33 34 35 , indicating that a variety of cells perform the immunoregulatory activity. CD4 + regulatory cells described in several studies constitutively express CD25 (IL-2Rα 36 37 38 39 40 ). These CD25 + CD4 + regulatory cells suppress the IL-2 production from CD4 + CD25 − cells, and only respond to a high dose of IL-2. CD38 + CD4 + regulatory cells proliferate when subject to TCR stimulation in the presence of IL-2 41 . Although the cell surface phenotypes are varied, these findings of IL-2R expression and responsiveness to IL-2 may agree with our data, further indicating the importance of the IL-2/IL-2R system in the regulation of T cell activity. The importance of the regulatory T cells found in our analysis is that they have the activity to eliminate abnormally activated T cells. Such elimination activity has not been reported in the majority of previously described regulatory T cells, except CD8 + regulatory T cells, which were shown to eliminate activated Vβ8 + CD4 + cells in a Qa-1–restricted manner 42 . The elimination of activated IL-2Rβ 2/− T cells may be related to cytotoxic activity, because such elimination is related to CD8 + population, and the eliminating cells express molecules related to the cytotoxic effector function. Further characterization and purification of the T cells regulating IL-2Rβ 2/− cells by the in vitro culture system may establish the mechanism of this elimination process. In this study, we investigated the relationship between IL-2Rβ 2/− T cells and normal T cells, and found a mechanism by which normal T cells could regulate activated IL-2Rβ 2/− T cells. We propose that IL-2Rβ is essential for the development of the regulatory TCR-α/β T cells that effectively eliminate activated T cells. Lack of such regulatory activity may result in an accumulation of activated T cells such as those observed in IL-2Rβ–deficient mice. | Study | biomedical | en | 0.999997 |
10587348 | A BALB/c-derived genomic fragment including the C/EBPγ gene was provided by Dr. S. Nagata (Osaka University, Osaka, Japan). As the long arm of the homology region, an 8.8-kb genomic fragment including the first exon, the first intron, and part of the second exon was used. The neomycin resistance gene derived from pMC1Neo-poly(A) 15 was inserted into the targeting vector to disrupt the basic DNA binding and leucine zipper regions. A short arm of the homology region was amplified by PCR. The MC1 herpes simplex virus thymidine kinase 16 was inserted 5′ upstream of the homologous region. E14-1 embryonic stem cells, which were derived from 129/SvJ (129) mice, were transfected with a linearized targeting vector by electroporation. Homologous recombinants were identified among double-resistant clones against G418 and gancyclovir by PCR and Southern blot analysis. Generation of chimeras and mutant mice was essentially as described 17 . Splenic B and T cells were purified from B6 spleen cells using Magnetic Cell Sorter (MACS ® ; Miltenyi Biotec) with B220 and CD3 microbeads, respectively. For purification of NK cells, DX5 + cells were first prepared from B6 spleen cells using MACS ® with biotinylated DX5 and streptavidin microbeads. Then the cells were stained with anti-CD3 and anti-DX5. CD3 − DX5 + cells were sorted with EPICS Elite (Coulter Immunology) and used as NK cells for reverse transcriptase (RT)-PCR. Total RNAs were extracted from tissues or cells using Sepazol-RNA I (Nacalai Tesque). Oligo-dT–primed reverse transcription was performed with Superscript-RT (GIBCO BRL). Then cDNAs were subjected to PCR analysis. Primers for C/EBPγ were as follows: sense primer, 5′-GGCCGCTCGGAGTGGAGGCCGTCTGGG-3′; antisense primer, 5′-ACGTTGTCTGCGAGGCTGTGCGCATGC-3′. The length of the amplified product was 547 bp. These primers cannot amplify the genomic DNA because sense and antisense primers are located in the first and second exons, respectively. Cycling conditions were 35 cycles of 94°C for 30 s, 65°C for 30 s, and 75°C for 1 min. Primers for β-actin were as follows: sense primer, 5′-CCCACACTGTGCCCATCTAC-3′; antisense primer, 5′-TACGGATGTCAACGTCACAC-3′. Cycling conditions were 24 cycles of 94°C for 15 s and 60°C for 30 s. The C/EBPγ genotype of neonates obtained by intercrossing heterozygous mutant mice was determined by PCR. The splenocytes were taken from the neonates within 12–18 h after birth and used as sources for hematopoietic stem cells. They were injected intravenously into recombination activating gene (RAG)2 −/− B6 mice 18 that had received 12 Gy from an X-ray irradiation system, MBR-1520R (Hitachi Medical Corp.), before transfer. The recipient mice were given 1 mg/ml neomycin sulfate and 1,000 U/ml polymixin B in their drinking water after irradiation and analyzed 6–10 wk after reconstitution. To evaluate the chimerism in NK cells, we used surface marker Ly9.1, the allele of which is carried by 129 but not B6 mice, as used by Ogasawara et al. 19 . The genetic background of the donors in our experiments was a mixture of 129 and B6. Therefore, chimeras, of which the donor was Ly9.1 + , were used for our experiments. In all reconstituted mice used, >99% of CD3 − DX5 + cells were positive for Ly9.1 expression (data not shown). Single-cell suspensions from thymus, spleen, or cultured cells were incubated with anti-CD16/32 (PharMingen) to minimize nonspecific staining. They were then stained with cocktails of mAbs conjugated to FITC, PE, biotin, or Cy-Chrome for 20 min at 4°C. The biotinylated Abs were developed with streptavidin conjugated to PE or Cy-Chrome (PharMingen). All mAbs, with the exception of PE-labeled anti-IgD (Southern Biotechnology Associates), were purchased from PharMingen. Flow cytometric analysis was performed using a FACSCalibur™ with CELLQuest™ software (Becton Dickinson). Fresh splenocytes (10 5 cells per well) were cultured in complete RPMI 1640 with 10 μg/ml anti-IgM (Zymed Labs.) plus 0.5 μg/ml anti-CD40 (PharMingen), 10 μg/ml LPS (055:B5; Sigma Chemical Co.), 20 ng/ml IL-2 (Genzyme Corp.), 0.1 μg/ml anti-CD3 (PharMingen) plus 0.1 μg/ml anti-CD28 (PharMingen), and 2.5 μg/ml Con A (Sigma Chemical. Co.) in 96-well plates. 48 h later, they were pulsed with 0.2 μCi of [ 3 H]thymidine (NEN Research Products) and cultured for a further 15 h. Splenocytes from bone marrow chimeras were incubated with 2 ng/ml IL-12, 20 ng/ml IL-18, and 2 ng/ml IL-12 plus 20 ng/ml IL-18 or 500 U/ml IL-2 for 24 h, and their cytotoxic activities against YAC-1 cells were measured as described previously 20 21 . Spontaneous cytotoxic activity was measured by incubating splenocytes with 51 Cr-labeled YAC-1 cells in the absence of cytokines for 4 h. Splenic cells at 10 5 cells per well in 96-well plates were cultured in complete RPMI 1640 in the presence or absence of 2 ng/ml IL-12 and/or 20 ng/ml IL-18 for 24 h. Amounts of IFN-γ in harvested supernatants were measured by ELISA using Duoset (Genzyme Corp.) according to the manufacturer's instructions. The lowest detection limit of ELISA is 10 pg/ml. Semiquantitative RT-PCR for IFN-γ was performed as described previously 22 . Newborn spleen cells were cultured in complete RPMI 1640 with 300 ng/ml IL-15 (Genzyme Corp.) at 10 6 cells per well in 24-well plates. 10 d later, cells were harvested, washed four times, and used for further analysis. For cytotoxic activities, harvested cells were incubated with labeled YAC-1 cells in the absence of cytokines for 4 h. Their cytotoxic activities against YAC-1 cells were then measured as described previously 20 . For IFN-γ measurement, harvested cells were cultured at 10 5 cells per well in 96-well plates with 2 ng/ml IL-12 and 20 ng/ml IL-18 for a further 24 h. Then, amounts of IFN-γ in the culture supernatants were measured with ELISA. Total RNAs were purified from 10-d–cultured newborn spleen cells with IL-15, reverse transcribed, and amplified. Primers and amplifying conditions for IL-12Rβ1, IL-12Rβ2, and IL-18R were described previously 21 . Newborn spleen cells cultured with IL-15 for 10 d were stimulated with 2 ng/ml IL-12 or 20 ng/ml IL-18 for 20 min and lysed in buffer containing 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, and 10 mM β-glycerophosphate. For signal transducer and activator of transcription (STAT)4 activation, the cell lysates were immunoprecipitated with anti-STAT4 Ab (Santa Cruz Biotechnology) and Protein A–Sepharose (Amersham Pharmacia Biotech). The immunoprecipitates were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antiphosphotyrosine 4G10 (Upstate Biotechnology Inc.) or anti-STAT4 Ab. For c-Jun NH2-terminal kinase (JNK) activation, the cell lysates were separated on SDS-PAGE, followed by blotting with antiphospho-JNK Ab (Promega Corp.) or anti-JNK1 Ab (Santa Cruz Biotechnology). Proteins bound to the Abs were visualized using the enhanced chemiluminescence system (Dupont). The C/EBPγ genomic locus was disrupted by inserting the neomycin resistance gene into the second exon . This insertion resulted in disruption of the basic and leucine zipper domains, which are essential for DNA binding and dimer formation. Homologous recombinants were obtained through a double selection with G418 and gancyclovir. Targeted clones were injected into B6 blastocysts to generate chimeric mice, which were bred to achieve germline transmission. By mating heterozygous mutants, homozygous mutants were obtained at a frequency of the expected Mendelian ratio (22.6% of littermates). C/EBPγ expression was detected in wild-type newborn spleens and livers, but not in those of homozygous mutants as expected . General appearance at birth was indistinguishable among C/EBPγ 1/+ , C/EBPγ 1/− , and C/EBPγ 2/− mice. However, only 11% of homozygous mutants could survive >60 h, although they were healthy until 12 h after they were born ( Table ). These results indicate that C/EBPγ is involved in early neonatal survival but not embryonic survival. By histological examination, C/EBPγ 2/− mice at 24 h showed emphysematous changes in their lungs (data not shown). However, it is not clear at the time of this writing whether the lung lesions alone can account for the high mortality of homozygous mutants at the early neonatal stage. C/EBPγ is expressed in T and B cells 9 . Furthermore, NK cells, which belong to a distinct lymphoid lineage from T and B cells 13 14 , also expressed C/EBPγ mRNA . To investigate in vivo roles of C/EBPγ in these lymphoid lineage cells, we generated bone marrow chimeric mice by transferring newborn spleen cells into lethally irradiated, RAG2 −/− B6 mice. Thymocytes from C/EBPγ 2/− chimeras showed normal T cell development . Splenocytes from C/EBPγ 2/− chimeras also showed normal population of CD4 + 8 − and CD4 − 8 + T cells . Furthermore, analysis of surface markers such as B220, IgM, and IgD revealed that B cell maturation was not disturbed in C/EBPγ 2/− chimeras . In addition to surface phenotype, proliferative responses to mitogens or stimulating Abs were not significantly different between C/EBPγ 1/+ and C/EBPγ 2/− chimeras . Taken together, these results suggest that C/EBPγ is not essential for functional T and B cell development. NK cells can be identified as CD3 − IL-2Rβ 1 , CD3 − NK1.1 + , or CD3 − DX5 + cells by flow cytometry. However, the NK1.1 analysis in the chimeras is limited, because only some mice in a mixed genetic background of 129 and B6 carried the NK1.1 allele, of which expression is detected on B6 but not 129 NK cells. Therefore, the NK cell population was analyzed for IL-2Rβ and DX5 expression. Splenic CD3 − IL-2Rβ 1 cells in C/EBPγ 2/− chimeras were detected at levels equivalent to those in C/EBPγ 1/+ chimeras . Furthermore, the frequency of CD3 − DX5 + cells was also comparable between C/EBPγ 1/+ and C/EBPγ 2/− chimeras . Percentages of CD3 − DX5 + cells were larger than those of CD3 − IL-2Rβ 1 cells in both control and C/EBPγ 2/− chimera splenocytes, as described previously 23 . Next, NK cytotoxicity was measured by YAC-1 cell–killing activity in the absence or presence of various cytokines . Spontaneous cytotoxicity of C/EBPγ 2/− chimera splenocytes (1.0% at 100:1) was impaired as compared with that of control chimera splenocytes . IL-12 and/or IL-18 act on NK cells and can enhance their cytotoxic activity 21 . When stimulated with these cytokines, C/EBPγ 2/− chimera splenocytes also showed impaired killing activity as compared with control splenocytes . IL-2 is another stimulatory cytokine for NK cell activity 24 . Decreased killing activity of C/EBPγ 2/− chimeras was also observed in the presence of IL-2 . Poly (I:C)-stimulated C/EBPγ 2/− chimera splenocytes also showed lower cytotoxic activity than control splenocytes (data not shown). NK cells constitutively express both functional IL-12R and IL-18R, whereas naive T cells do not 21 25 26 . Therefore, splenic IFN-γ production by stimulation with IL-12 and IL-18 is dependent on NK but not T cells. To evaluate the ability of NK cells from chimeric mice to produce IFN-γ, splenocytes were cultured with or without IL-12 and/or IL-18. 24 h later, cell-free supernatants were harvested and assayed for IFN-γ production with ELISA. Under this condition, C/EBPγ 2/− chimera splenocytes produced much lower amounts of IFN-γ than C/EBPγ 1/+ chimera splenocytes . IL-12 or IL-18 can induce IFN-γ production at the transcriptional level in NK cells 27 . Consistent with reduced IFN-γ production, induction of IFN-γ mRNA was markedly decreased in C/EBPγ 2/− chimera splenocytes . Taken together, these results suggest that C/EBPγ is required for induction of IFN-γ by IL-12 and/or IL-18 in NK cells. NK cell population was analyzed in newborn splenocytes. Both CD3 − IL-2Rβ 1 and CD3 − DX5 + cells were equivalently detected in C/EBPγ 1/+ and C/EBPγ −/− newborn splenocytes . IL-15 can stimulate NK cell activity and proliferation and is essential for NK cell development 28 29 . Adult bone marrow cells can generate NK cells when cultured with IL-15 28 30 . Wild-type newborn spleen cells could also give rise to CD3 − IL-2Rβ 1 DX5 + cells in the presence of IL-15 . In this culture condition, harvested cell numbers from control (C/EBPγ 1/+ , n = 5, and C/EBPγ 1/− , n = 3) and C/EBPγ 2/− ( n = 7) mouse spleen cells were 5.7 ± 4.1 × 10 5 and 4.9 ± 6.7 × 10 5 per well, respectively. Surface phenotype of cultured C/EBPγ 2/− cells was identical to that of wild-type cells . However, NK cells generated from C/EBPγ 2/− spleens showed impaired cytotoxic activity against YAC-1 cells as compared with those from C/EBPγ 1/+ spleens . Furthermore, C/EBPγ 2/− NK cells produced lower amounts of IFN-γ in response to IL-12 plus IL-18 than C/EBPγ 1/+ and C/EBPγ 1/− NK cells . Taken together, two major NK cell activities, cytotoxic activity and IFN-γ production, were impaired in both C/EBPγ 2/− chimera splenocytes and C/EBPγ 2/− NK cells generated in the presence of IL-15 in vitro. To determine if impaired induction of IFN-γ by IL-12 and IL-18 is caused by decreased expression of these receptors, we examined their mRNA expression in C/EBPγ 1/+ and C/EBPγ 2/− NK cells. IL-12Rβ1, IL-12Rβ2, and IL-18R expression was equivalent in C/EBPγ 1/+ and C/EBPγ 2/− NK cells . IL-12 can induce tyrosine phosphorylation and activation of STAT4 31 32 . This pathway was not impaired in C/EBPγ 2/− NK cells . IL-18 can cause phosphorylation and activation of JNK 33 . Equivalent JNK phosphorylation was observed in C/EBPγ 1/+ and C/EBPγ 2/− NK cells when stimulated by IL-18 . Taken together, these results suggest that signaling pathways proximate to these cytokine receptors are intact in C/EBPγ 2/− NK cells. In this study, we demonstrate that C/EBPγ 2/− NK cells have defects in IFN-γ production and cytotoxicity. It has been shown that several regulatory elements such as the activator protein (AP)-1 or NF-κB sites are essential for IFN-γ gene expression by IL-12 and/or IL-18 34 35 . Although NF-IL6 is a candidate for heterodimerizing with C/EBPγ 36 , it seems unlikely that the heterodimer plays an essential role. First, no C/EBP sites have been shown to be important for IFN-γ induction. Second, splenic IFN-γ production in response to IL-12 and/or IL-18 is not impaired in NF-IL6 −/− mice (our unpublished data). It is possible that C/EBPγ plays a critical role in IFN-γ gene regulation by dimerizing with AP-1 components. AP-1 components are activated by IL-12 or IL-18 and essential for both IL-12– and IL-18–induced IFN-γ promotor activation 34 . Fos or Jun is shown to require C/EBPγ in order to efficiently bind to the regulatory element in the IL-4 promotor 12 . Although further studies are necessary, our results suggest that C/EBPγ regulates IFN-γ gene expression. At present, the possibility that C/EBPγ is necessary for expression of other gene(s) critical for IFN-γ gene induction cannot formally be excluded. IFN-γ does not seem to be involved in cytotoxic activity of NK cells, because NK activity is not remarkably impaired in IFN-γ 2/− mice 21 . Therefore, impaired IFN-γ production cannot account for decreased cytotoxic activity of C/EBPγ 2/− NK cells. Although CD18 is important for NK cells to recognize target cells 37 , surface expression of CD18 was not decreased in C/EBPγ 2/− chimera splenocytes (data not shown). Furthermore, IL-12 and IL-18 could induce expression of a critical cytolytic mediator, perforin, in C/EBPγ 2/− chimera splenocytes (data not shown). These results indicate the presence of other target(s) of C/EBPγ that are involved in the cytotoxicity of NK cells. It is noteworthy that in spite of NK cell dysfunction, NK cell generation is intact in the absence of C/EBPγ. This means that C/EBPγ should play critical roles in a late maturation step rather than in an early developmental step. In contrast, mutant mice established so far manifested impaired NK cell generation. The IL-15 receptor consists of IL-15Rα, IL-2Rβ, and the common γ chain 38 39 40 41 42 . Deficiency of one of these components caused reduction of NK cell numbers in vivo and impaired NK cell expansion in vitro 43 44 45 . Janus kinase (JAK)-3 and STAT5 are essential for IL-15 signaling components. JAK-3 −/− or STAT5 −/− mice also showed impairment of NK cell generation 23 46 47 48 49 . Furthermore, IFN regulatory factor (IRF)-1 was found to be critical for expansion of NK cells 19 . This can be explained by impaired IL-15 production in the bone marrow microenvironment 19 . In these mutants, deficient NK cell generation is caused by disturbance of IL-15 signaling or decreased IL-15 production. Membrane lymphotoxin (LT)α also plays essential roles for NK cell development by acting independently of IL-15/IRF-1 or upstream of the IL-15/IRF-1 pathway 50 . NK cell generation was severely impaired in LTα 2/− mice. Furthermore, NK cells, which were generated in vitro with IL-15 from LTα 2/− bone marrow cells, showed intact cytotoxic activity. These characteristics in LTα 2/− mice are distinct from those in C/EBPγ 2/− mice. In addition, there are two more mutants with impaired NK cell development, the mechanism of which is not yet clear. One is deficiency of a winged helix-turn-helix transcriptional factor, Ets-1 51 . The other is deficiency of Id2, an inhibitor for transcription factors with helix-loop-helix domains 52 . At present, little is known about the molecular mechanisms that regulate NK cell functions or development. Our study clearly reveals that C/EBPγ is critically involved in functional NK cell maturation. Identification of the target genes regulated by C/EBPγ will clarify the molecular mechanism of NK cell functions. | Study | biomedical | en | 0.999997 |
10587349 | RPMI 1640, HBSS, and penicillin/streptomycin were from StemCell Technologies, Inc. Wortmannin, l -α-phosphatidylinositol, PMSF, leupeptin, pepstatin A, and aprotinin were purchased from Sigma Chemical Co. LY294002 and microcystin were from Calbiochem Corp. Protein A–agarose and electrophoresis reagents were purchased from Bio-Rad Laboratories. [γ- 32 P]ATP was from Nycomed Amersham Plc. The following mAbs were used: 3C10 (mouse IgG2b, anti-CD14 mAb; a gift from Dr. W.C. Van Voorhis, University of Washington, Seattle, WA); W6/32 (mouse IgG 2a , anti–HLA class I mAb; American Type Culture Collection); UB93-3 (mouse mAb to PI 3-kinase; Upstate Biotechnology Inc.) and 9A7 (rat IgG 2b , anti-VDR; Chemicon International). Other Abs used included anti-CD14 and anti-CD11b, both murine IgG 1 . Murine IgG 1 isotype control MG100 was from Caltag Laboratories. The promonocytic cell line THP-1 was from the American Type Culture Collection. The promonocytic cell line U937 transfected with cDNA, encoding the entire coding region of either wild-type bovine PI 3-kinase subunit p85α (Wp85α) or mutant bovine p85α, (Δp85α) has been described 37 . The mutant has a deletion of 35 amino acids from residues 479–513 of bovine p85α and the insertion of two other amino acids (Ser-Arg) in the deleted position. Mutant p85α competes with native p85 for binding to essential signaling proteins, thereby acting as a dominant negative mutant 38 . THP-1 and U937 cell lines were cultured in RPMI 1640 supplemented with 10% FCS (HyClone), 2 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cell density was maintained at a concentration of <5 × 10 5 /ml. Peripheral blood mononuclear cells were isolated as described previously 39 . Monocytes were allowed to adhere for 1 h at 37°C in a humidified atmosphere with 5% CO 2 . Nonadherent cells were removed by three washes with HBSS. Adherent cells were immediately treated and used for cell surface phenotype analysis. To measure the expression of surface molecules, cells were incubated with specific mouse mAb or irrelevant isotype-matched IgG (10 μg/ml) for 30 min, then washed twice and labeled with FITC-conjugated F(ab′) 2 sheep anti–mouse IgG (Sigma Chemical Co.) for 30 min. Cells were then washed twice and fixed in 2% paraformaldehyde in staining buffer. All staining and washing procedures were performed at 4°C in HBSS containing 0.1% NaN 3 and 1% FCS. Cell fluorescence was analyzed using a Coulter Elite flow cytometer. Relative fluorescence intensities of 5,000–10,000 cells were recorded as single-parameter histograms (log scale, 1,024 channels, 4 log decades), and the mean fluorescence intensity (MFI) was calculated for each histogram. Results are expressed as MFI indices which correspond to MFI of cells + specific Ab/MFI of cells + irrelevant isotype-matched IgG. Cell lysates for analysis of PI 3-kinase were prepared in 20 mM Tris, pH 8.0, 1% Triton X-100, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM Na 3 VO 4 , 5 mM NaF, 100 nM microcystin, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin. Aliquots of lysates adjusted for protein concentration (300–500 μg protein) were incubated for 2–4 h at 4°C with UB93-3 mAb (anti–PI 3-kinase), and immune complexes were adsorbed onto protein A–agarose for 30–60 min. The complexes were washed twice with lysis buffer and three times with 10 mM Tris-HCl, pH 7.4. PI 3-kinase activity was measured as described 22 40 . In brief, immunoprecipitates were incubated for 10 min at 4°C with 10 μg of sonicated (3 times for 20 s in an ultrasonic cell disrupter; Branson Sonic Power Co.) l -α-phosphatidylinositol in 10 μl of 30 mM Hepes, to which was added 40 μl of kinase assay buffer (30 mM Hepes, 30 mM MgCl 2 , 200 μM adenosine, 50 μM ATP, and 10 μCi of [γ- 32 P]ATP). Reactions were carried out for 15 min at room temperature, and stopped by the addition of 100 μl of 1 N HCl and 200 μl of chloroform/methanol (1:1, vol/vol). Lipids were separated on oxalate-treated silica TLC plates using a solvent system of chloroform/methanol/water/28% ammonia (45:35:7.5:2.5, vol/vol/vol/vol). Plates were exposed to X-ray film at −70°C. Incorporation of radioactivity into lipids was measured by excising the corresponding portions of the TLC plate, followed by liquid scintillation counting. Alternatively, cell lysates were incubated overnight at 4°C with 9A7 mAb (anti-VDR), and immune complexes were adsorbed onto protein A–agarose, washed in lysis buffer containing 200 mM NaCl, and assayed for PI 3-kinase activity as described above. RNA isolation, cDNA synthesis, and PCR conditions were as described previously 41 . Sequences (5′ to 3′) of oligonucleotide primers used in PCR amplifications were as follows: CD14 sense, CCC AAG CTT GGG CAG AGG TTC GGA AGA CTT ATC G; CD14 antisense, GGG GTA CCC CTT GAC CGT GTC AGC ATA CTG CC 29 ; Cdk inhibitor p21 sense, TTC TCC TTT TCC TCT CTC C; p21 antisense, TCT ACT CCC CCA TCA TAT ACC; β-actin sense, CAC CCC GTG CTG CTG ACC GAG GCC; β-actin antisense, CCA CAC GGA GTA CTT GCG CTC AGG 41 . Controls included in the reverse transcription (RT)-PCR reactions were no RNA and RNA without RT, and different cycle numbers of PCR reactions were performed to ensure linear cDNA amplification. Phosphorothioate-modified oligonucleotides (S-oligos) to VDR and p110 subunit of PI 3-kinase were synthesized by Life Technologies, Inc. The oligonucleotides were phosphorothioate-modified to limit intracellular degradation, and purified by high-performance liquid chromatography to remove incomplete synthesis products. 21-mer sequences, including the presumed translation initiation site of human cDNA sequences corresponding to the VDR 42 and the α isoform of the p110 subunit of PI 3-kinase 43 , were made in both sense and antisense orientations with the following sequences: VDR sense, 5′-ATG GAG GCA ATG GCG GCC AGC-3′; VDR antisense, 5′-GCT GGC CGC CAT TGC CTC CAT-3′; p110 sense, 5′-ATG CCT CCA AGA CCA TCA TCA-3′; and p110 antisense, 5′-TGA TGA TGG TCT TGG AGG CAT-3′. The sequences were selected by screening for uniqueness using Blast 227, and were also tested for lack of secondary structure and pairing by using Primer Software (v. 2.0). 3–7 × 10 6 THP-1 cells were suspended in 500 μl RPMI containing 2.5% lipofectAMINE (Life Technologies, Inc.) and 5 μM S-oligo, and incubated on a rotary shaker for 4 h at 37°C. In addition, fluorescent-modified antisense and sense and flow cytometry were used to monitor S-oligo incorporation into cells. After this incubation, the medium was brought up to 5–10 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO 2 . To examine a potential association between the VDR and PI 3-kinase, D 3 -treated cells were lysed in the buffer used to prepare cell lysates for PI 3-kinase assay, then immunoprecipitated with specific mAb to VDR. Immunoprecipitates were washed with lysis buffer containing 200 mM NaCl to minimize nonspecific protein–protein interactions. Samples were then analyzed by SDS-PAGE and immunoblotting with Abs to the p85 PI 3-kinase subunit as described previously 40 . To measure the effects of cell treatments with antisense S-oligos on protein levels of VDR, whole-cell lysates prepared by boiling in Laemmli buffer were subjected to SDS-PAGE and immunoblotting with the mAb 9A7. Membranes were developed by enhanced chemiluminescence (ECL) as described 44 . Initial experiments involving immunofluorescence and flow cytometric analysis defined the experimental conditions for D 3 -induced CD14 expression by THP-1 cells. THP-1 cells maintained in complete medium expressed nearly undetectable levels of CD14 on the surface, and treatment with hormone caused a dose- and time-dependent increase in CD14 expression . A response was obtained in the presence of as little as 0.1 nM D 3 , and at 100 nM, virtually all cells expressed CD14 at an ∼50-fold increase in MFI. The kinetics of induction of CD14 in response to 100 nM D 3 is shown in Fig. 1 B. CD14 expression increased progressively with time and was maximal (55-fold increase in MFI index, average of 2 separate experiments) after 48 h. Since a 24-h exposure to 100 nM D 3 was sufficient to induce a high level of surface CD14 (53-fold increase in MFI index, average of 2 separate experiments) in nearly 100% of cells, subsequent experiments were carried out under these conditions. Other experiments examined whether the CD14 response of cells to D 3 (100 nM, 24 h) was serum dependent. Results obtained in three independent experiments using RPMI 1640 alone showed that THP-1 cells were fully responsive for CD14 induction (MFI index = 104.4 ± 5.1, mean ± SEM). Addition of FCS had only a limited additive effect (MFI index = 125.0 ± 14.1). Conversely, addition of insulin growth factor (IGF)-I a serum component known to influence cell differentiation 30 , to D 3 minimally inhibited CD14 expression. These observations suggest that in THP-1 cells, D 3 is capable of inducing CD14 in the absence of any serum factors, and that D 3 delivery to cell surface is independent of serum vitamin D binding protein 45 46 . In light of the important roles played by PI 3-kinase in regulating the differentiation of a variety of cell types 47 48 49 , the possibility that D 3 -induced monocytic differentiation involves PI 3-kinase was investigated. As shown in Fig. 2 A, incubation of serum-starved THP-1 cells with 10 nM D 3 brought about a significant increase in PI 3-kinase activity (3.07 ± 1.17 fold increase, mean ± SEM, n = 3), with a maximum response observed at 1 μM (6.59 ± 1.47 fold increase). Although PI 3-kinase activity was also significantly increased in IGF-I (100 ng/ml)–treated cells , this was not associated with increased CD14 expression . Time course experiments using 100 nM D 3 showed that PI 3-kinase activation was detectable by as early as 5 min and reached a maximum level (4.94 ± 0.72 fold increase) by 20 min. To address whether PI 3-kinase activation is required for D 3 -induced monocyte differentiation, serum-starved THP-1 cells were incubated with either wortmannin or LY294002 for 20 min before addition of hormone. Inhibitors were used at concentrations known to be relatively selective for inhibition of PI 3-kinase 48 50 . Incubation with inhibitors alone had no effect on basal expression of CD14 (data not shown). Preincubation with 5 nM wortmannin led to 65.7 ± 5.6% inhibition of D 3 -induced CD14 expression . Increasing the concentration of wortmannin to 50 nM inhibited CD14 expression by 86.0 ± 8.5%. LY294002, an inhibitor of PI 3-kinase that acts via a distinct mechanism from that of wortmannin, when used at 1.5 μM reduced D 3 -induced CD14 expression by 66.8 ± 11.9%. CD14 expression decreased further in the presence of 15 μM LY294002 (88.1 ± 5.1%). In contrast to abrogation of D 3 -induced CD14, neither wortmannin nor LY294002 had significant effects on the expression of HLA class I molecules (data not shown), indicating that the effects of neither wortmannin nor LY294002 were due to nonspecific toxicity. The PI 3-kinase inhibitors LY294002 and wortmannin also attenuated D 3 -induced cell surface expression of CD11b by THP-1 cells. While incubation with inhibitors alone had no effect on basal levels of CD11b expression, preincubation with 5 nM wortmannin inhibited the response to D 3 by 41 ± 7% ( Table ). A higher concentration of 50 nM increased inhibition to 100 ± 12%. LY294002 inhibited D 3 -induced CD11b surface expression by 48 ± 10% at a concentration of 1.5 μM, and by 88 ± 14% at a concentration of 15 μM ( Table ). Surface expression of CD14 and CD11b was also examined in human peripheral blood monocytes. As shown in Table , LY294002 and wortmannin markedly inhibited D 3 -induced expression of both CD14 and CD11b in these cells. Inhibition of PI 3-kinase has been shown to affect the transport of some cell surface molecules 51 , and thus was a possible mechanism to explain the effects of PI 3-kinase inhibitors on D 3 -induced CD14 expression. However, when total RNA was isolated and RT-PCR was performed using primers for CD14 and β-actin , the results showed that mRNA levels for CD14 were markedly reduced in cells incubated with either wortmannin or LY294002 . These findings indicate that inhibition of PI 3-kinase results in attenuation of D 3 -induced CD14 gene expression at a pretranslational level. Treatment of immature myeloid cells with D 3 induces the expression of the Cdk inhibitor p21, and the latter has been shown to regulate gene expression and to promote myleoid cell differentiation 52 . Experiments were carried out to examine whether the induction of p21 in response to hormone also involved PI 3-kinase. As shown in Fig. 3 C, THP-1 cells expressed low levels of p21 mRNA in the basal state. As expected, p21 expression was significantly induced in response to incubation of cells with D 3 . However, in contrast to the findings with CD14 and CD11b, preincubation of cells with either wortmannin or LY294002 had no effect on hormone-induced expression of p21. These findings suggest that PI 3-kinase selectively regulates monocyte differentiation, independent of any effects on p21. The role of PI 3-kinase in regulating monocyte differentiation was investigated further by inhibiting the synthesis of the p110 catalytic subunit of PI 3-kinase. THP-1 cells were incubated in the presence of antisense S-oligo complementary to the p110 translation initiation region (including the ATG initiation codon), and then assayed for both PI 3-kinase activation and CD14 expression in response to D 3 . Flow cytometric analysis of cells exposed to fluorescein-modified antisense S-oligo, under the same conditions used for unmodified S-oligos, revealed that THP-1 cells readily incorporated foreign DNA (data not shown). As shown in Fig. 4 A, antisense S-oligo to p110 mRNA significantly attenuated D 3 -induced PI 3-kinase activity (% inhibition = 87.1 ± 5.6, mean ± SEM, n = 3), whereas at the same concentration, the control sense S-oligo had no apparent effect on the PI 3-kinase response. In parallel with this effect on PI 3-kinase activation, pretreatment with antisense S-oligo (and not with the control, sense oligo) almost completely inhibited hormone-induced surface expression of CD14 . In addition, RT-PCR results shown in Fig. 4 C demonstrated that D 3 -induced mRNA levels for CD14 were markedly diminished in cells treated with antisense S-oligo to p110 mRNA. The PI 3-kinase requirement for D 3 -induced CD14 expression was also examined in U937 cells transfected with a dominant negative mutant of p85 (Δp85). Stable transfection of these cells with Δp85 resulted in a significant reduction of stimulated PI 3-kinase activity 22 37 . Exposure of Δp85 U937 to D 3 (100 nM, 48 h) led to only a marginal increase in surface expression of CD14 above baseline. In contrast, cells transfected with wild-type p85 showed significant induction of CD14 expression . In addition, as observed with THP-1 cells, RT-PCR experiments showed that attenuation of PI 3-kinase in U937 cells resulted in markedly reduced response to D 3 for induction of CD14 mRNA . Taken together, these findings strongly suggest that PI 3-kinase plays a central role in D 3 -induced monocyte differentiation, and indicate that PI 3-kinase activation is required to induce CD14 expression. Many responses to D 3 , such as induction of expression of osteopontin, osteocalcin, calbindin, and 24-hydroxylase, are brought about by a mechanism involving binding of the VDR to a specific VDRE in the corresponding promoters 53 . Since no VDRE has been identified within the CD14 gene promoter 28 , this raised the question as to whether the VDR plays any role in regulating D 3 -induced CD14 expression. To address this question, THP-1 cells were incubated overnight with antisense S-oligo specific to VDR mRNA. This resulted in significant attenuation of the level of VDR protein in THP-1 cells as detected by Western blotting . In contrast, pretreatment of cells with the control, sense S-oligo had no apparent effect on the level of VDR protein. In parallel with the reduction of VDR protein, D 3 -induced surface expression of CD14 was markedly attenuated . In addition, cells were treated with S-oligo antisense specific for VDR mRNA before D 3 , followed by RNA extraction and RT-PCR. The results showed that the induction of CD14 mRNA was markedly attenuated in antisense-treated cells . Taken together, these findings strongly suggest that the VDR is essential for D 3 -induced CD14 gene expression. Given the evidence that both PI 3-kinase and the VDR were essential for induction of CD14 in response to D 3 , the question examined next was whether the VDR is required for activation of PI 3-kinase. THP-1 cells were treated with antisense S-oligos as described above to reduce VDR protein expression before incubation with D 3 (100 nM, 20 min). Cell lysates were then prepared and assayed for PI 3-kinase activity. The results in Fig. 7 A show that pretreatment with antisense to VDR almost completely abrogated (92.9% inhibition, average of two separate experiments) PI 3-kinase activation in response to D 3 . In contrast, treatment with control sense S-oligos to VDR had no inhibitory effect. The evidence that the D 3 receptor was required for both PI 3-kinase activation and CD14 expression in response to D 3 suggested the possibility that this might involve a signaling complex containing both the VDR and PI 3-kinase. To test this hypothesis, THP-1 cells were incubated with D 3 , and immunoprecipitates prepared with mAb to the VDR were assayed for PI 3-kinase activity. The results shown in Fig. 7 B indicate that immunoprecipitates of the VDR prepared from cells activated with D 3 contained PI 3-kinase activity. To examine further whether the VDR associates with PI 3-kinase upon D 3 stimulation, aliquots from anti-VDR and anti-p85 (PI 3-kinase) immunoprecipitates of D 3 -treated cells were subjected to SDS-PAGE and Western blotting. Blots were probed with mAb to p85 and developed by ECL. The results shown in Fig. 7 C indicate that Abs to p85 reacted in anti-VDR immunoprecipitates with a band that presumably corresponds to the p85 subunit of PI 3-kinase. This association was only observed in cells treated with hormone, and not in untreated cells. Taken together, the findings suggest that D 3 treatment induces the formation of a signaling complex containing the VDR and PI 3-kinase. This results in activation of the lipid kinase, and is required for monocyte differentiation and induction of CD14 expression. The steroid hormone vitamin D 3 is known to induce immature, myeloid precursor cells to differentiate into mature monocytic cells. This process is accompanied by high-level expression of mRNA and protein for CD14, and other markers such as CD11b 27 28 29 . How these D 3 -initiated events are regulated is not completely understood. Cellular responses to D 3 have primarily been attributed to activation of the VDR, which acts as a transcription factor modulating the expression of a variety of genes 53 . In contrast to genomic signaling, several reports have implicated alternative, nongenomic mechanisms of action for D 3 involving initiation of signaling at the cell membrane 54 55 56 . The objective of this study was to examine signaling events in response to D 3 in order to define further how this hormone regulates myeloid cell differentiation. The principal conclusions drawn from the experiments reported are that D 3 -induced expression of CD14 and CD11b requires both PI 3-kinase and the VDR. Moreover, this process appears to involve the formation of a PI 3-kinase–VDR signaling complex. The conclusion that D 3 -induced CD14 expression is PI 3-kinase dependent is based on several lines of evidence, including: (a) in vitro kinase assays with immunoprecipitated PI 3-kinase, showing that incubation of cells with D 3 leads to activation of the enzyme ; (b) D 3 -induced CD14 expression is abrogated in THP-1 cells incubated with the PI 3-kinase inhibitors, wortmannin and LY294002 ; (c) an antisense strategy to downregulate PI 3-kinase also attenuated D 3 -induced CD14 expression ; and (d) expression of a dominant negative mutant of PI 3-kinase (Δp85) in U937 cells also completely abrogated D 3 -induced expression of both CD14 mRNA and protein . Similarly, CD11b induction in response to D 3 was also attenuated by wortmannin and LY294002 in both THP-1 cells and in human peripheral blood monocytes. Taken together, these findings establish that D 3 -induced CD14 and CD11b expression are regulated by PI 3-kinase, and that they are consistent with a nongenomic mechanism of hormone action. Expression of the Cdk inhibitor p21 is induced by D 3 , and this is associated with the differentiation of immature myeloid cells, including the expression of CD14 52 . Therefore, it was possible that inhibition of PI 3-kinase may have abrogated hormone-induced expression of p21 proximally, leading to secondary, indirect effects on the expression of CD14 and CD11b. However, the findings that neither wortmannin nor LY294002 inhibited the induction of p21 while at the same time they inhibited the expression of CD14 and CD11b indicated that this was not the case. These findings suggest that PI 3-kinase selectively regulates these monocyte differentiation markers independent of any effects on p21. They also suggest that D 3 -induced signaling initiated through the VDR appears to involve at least two separate pathways, one leading to p21 induction that is independent of PI 3-kinase, and a second pathway that is PI 3-kinase dependent and which regulates at least the induction of CD14 and CD11b. Moreover, although p21 has been shown to be involved in regulating monocyte differentiation 52 , results from the present study suggest that it is not sufficient by itself for induction of monocyte differentiation in response to D 3 , and that other, PI 3-kinase– and VDR-regulated elements are required. Induction of CD14 expression in response to D 3 is regulated at the level of gene transcription 28 29 and the CD14 promoter, particularly from bp −128 to −70, has been shown to be critical for CD14 expression in response to D 3 28 . However, this region does not contain a canonical VDRE 28 . Rather, the CD14 promoter contains two GC boxes that bind the nonreceptor, transcription factor Sp1, and this interaction is believed to be essential for CD14 expression 28 57 . In light of this molecular data, activation of CD14 transcription would not be expected to be a direct response to D 3 signaling through the VDR. Nevertheless, experiments that used VDR antisense S-oligo to downregulate expression of the endogenous VDR before D 3 stimulation showed that the VDR is required for D 3 -induced expression of CD14 mRNA . The obligate requirement for the VDR in this response was also supported by the finding that activation of PI 3-kinase in response to D 3 was markedly attenuated in cells treated with VDR antisense . Moreover, a signaling complex containing both the VDR and PI 3-kinase was identified in D 3 -treated cells , suggesting that a functional cytosolic VDR is a prerequisite for PI 3-kinase activation in response to D 3 . These findings appear to identify a novel, nongenomic mechanism of action for the VDR. It is of interest to note that the hormone-induced formation of a VDR–PI 3-kinase signaling complex is reminiscent of the finding that activation of the Raf-MAP kinase pathway by D 3 in keratinocytes involves an association of the VDR with the adaptor protein p66 shc 58 . Together, these findings suggest that other models of nongenomic signaling involving the VDR may yet be identified. The observations that PI 3-kinase regulates CD14 expression in response to D 3 and that the VDR is directly involved in this process do not exclude the possibility of a component of genomic action by the VDR, despite the absence of a canonical VDRE within the CD14 promoter. One possibility to consider is that upon incubation of cells with D 3 , the VDR may translocate to the nucleus towards D 3 -responsive elements, and enhance the activity of the known CD14 transcription factor Sp1. Two observations lend support to this hypothesis: (a) the finding of transcriptional synergism between the VDR and Sp1 using a construct of the VDRE, the binding sites for Sp1, and a luciferase reporter gene 59 ; and (b) the presence of a VDRE-like sequence at the distal Sp1 site within the CD14 gene 60 . The findings that D 3 -induced monocyte differentiation for CD14 and CD11b expression requires PI 3-kinase are consistent with previous reports in which this lipid kinase has been implicated in regulating the differentiation of various cell types, including immature myeloid cells 49 61 62 63 . For example, indirect data, based solely on the use of inhibitors, suggested a potential role for PI 3-kinase in regulating the differentiation of the myeloid leukemia cells HL-60 63 . In addition, when FDC-P1 myeloid cells that express the M-CSF receptor c- fms were treated with M-CSF, there occurred the rapid formation of a complex between c- fms and several signal transduction proteins, including PI 3-kinase 49 . Other studies have suggested that the differentiation of FDC-P1 cells is regulated by the activities of both phospholipase C-γ and PI 3-kinase in response to M-CSF stimulation 61 . In addition, stimulation of type III receptor tyrosine kinases in the same cell line was observed to lead to PI 3-kinase–dependent monocytic differentiation 62 . Thus, the steroid hormone D 3 appears to share with M-CSF the property of PI 3-kinase activation during the induction of monocyte differentiation. It is of interest to compare D 3 with phorbol esters that also act to promote monocyte differentiation. For example, PMA induces the expression of the monocyte differentiation markers CR3 (CD11b/CD18) and p150,95 64 , and modulates several functional properties of myeloid precursor cells, such as intracellular adhesion molecule 1–dependent adhesion 22 , phagocytosis 44 65 , and bactericidal activity 66 . However, PMA does not activate PI 3-kinase (Hmama, Z., and N. Reiner, unpublished data), and PMA-induced cell differentiation is resistant to PI 3-kinase inhibition 22 . Consistent with the findings presented above, although low concentrations of D 3 induce an ∼50-fold increase in CD14 surface expression, optimal doses of PMA resulted in only marginal increases (Hmama, Z., and N. Reiner, unpublished data). Regarding signaling mechanisms regulating PMA-induced cell differentiation, several reports have implicated the protein kinase C–Raf-MAP kinase pathway 33 67 68 . MAP kinase activity has been also shown to be activated by D 3 in keratinocytes, enterocytes, and in the promyelocytic cell lines HL60 and NB4 34 58 69 . To examine whether the MAP kinase pathway may be involved in THP-1 cell differentiation, D 3 -treated cells were examined for tyrosine phosphorylation of p42 and p44 MAP kinase isoforms, and assayed for MAP kinase activity using myelin basic protein as substrate. The results obtained (data not shown) indicated that MAP kinase is not activated in response to D 3 in THP-1 cells. The mechanism by which PI 3-kinase becomes activated in a complex with the VDR is not presently known. At least one well-established mechanism for activation of PI 3-kinase is known to involve interactions of the Src homology 2 domain of the p85 regulatory subunit with tyrosine phosphorylated proteins, including both receptor- and nonreceptor-protein tyrosine kinases 35 . In the VDR, the only potential sites of phosphorylation correspond to Ser 51 70 and Ser 208 71 , and the relevance of these residues in VDR-mediated D 3 signal transduction is unknown. Thus, it is not presently clear how a direct interaction of the VDR with the p85 subunit may lead to activation of PI 3-kinase. On the other hand, a multimolecular complex involving the VDR and a tyrosine-phosphorylated adaptor protein could potentially be involved in D 3 -induced activation of PI 3-kinase. At present, it is not clear how VDR-activated PI 3-kinase would be targeted to the membrane compartment to mediate cellular responses, given the cytosolic and nuclear localization of the VDR. One possibility to consider is that there is a small, membrane-associated population of VDR molecules that induces translocation of PI 3-kinase to the membrane compartment. A second possibility is that once activated, the kinase may simply diffuse to the vicinity of the inner leaflet of the plasma membrane. Third, it is also possible that VDR-activated PI 3-kinase may act on nonmembrane-associated substrates to mediate its effects. During the course of these studies, it was observed that IGF-I failed to induce CD14 surface expression , despite it ability to activate PI 3-kinase . Therefore, PI 3-kinase activation in response to D 3 appears to be necessary, but not sufficient to bring about CD14 expression. These findings suggest the possibility that induction of CD14 in response to D 3 may involve VDR-mediated signals that bifurcate to involve nongenomic effects of PI 3-kinase, perhaps acting in concert with a genomic mechanism of action. In this respect, cross-talk between nuclear receptor–mediated signaling and nongenomic actions have been described in D 3 -treated osteoblasts 72 . In summary, the findings presented demonstrate a novel pathway in which D 3 signaling for myeloid cell differentiation involves PI 3-kinase activation and signal complex formation with the VDR, which is itself shown to be required for cell differentiation. This pathway is essential for D 3 -induced expression of CD14 and CD11b, and attributes important functional roles to PI 3-kinase and the VDR in monocyte differentiation. | Other | biomedical | en | 0.999995 |
10587350 | IL-1β and IFN-γ were purchased from Genzyme. S -nitroso- N -acetylpenicillamine (SNAP), N -methyl arginine (L-NMA and D-NMA), actinomycin D (ActD), trichostatin A (TSA), 5-aza-2′-deoxycytidine (AzadC), dithiothreitol (DTT), β-mercaptoethanol (β-ME), and protease inhibitors were obtained from Sigma Chemical Co. Sodium nitroprusside (SNP) was from Fluka. 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride (AMT), S -ethylisothiourea hydrobromide (EIT), and l - N 6 -(1-iminoethyl)-lysine hydrochloride (L-NIL) were from Tocris Cookson, Ltd. 3-morpholinosydnonimine hydrochloride (SIN) was from ICN Iberica. Rediprime ® DNA labeling system, [ 32 P]dCTP, and cold and 3 H-labeled S -adenosylmethionine were from Nycomed Amersham plc. Glutathione (GSH) and poly deoxyinosine-deoxycytosine (poly dI-dC) were from Boehringer Mannheim. Restriction enzymes were from Promega or New England Biolabs. All other reagents were of the best quality commercially available. Insulin-producing rat RINm5F (RIN) cell, Jurkat T cell, and mouse leukemic monocyte-macrophage cell (RAW 264 cell) lines were grown in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B under 5% CO 2 at 37°C. Human lymphocytes were obtained from peripheral blood of healthy donors as reported previously 18 . Total RNA was extracted from cell lines or fresh peripheral lymphocytes by the guanidine phenol method. RNA was reverse transcribed using random hexamers, and the cDNA was amplified using specific primers. PCR amplification of the CGG repeats at the FRAXA site and KH domains was carried as reported previously 18 19 . Amplification of hypoxanthine phosphoribosyltransferase gene ( HPRT ) in RIN cells was assessed using murine primers. Human specific primers were used for HPRT mRNA analysis in Jurkat T cells and fresh peripheral lymphocytes 7 . Reverse transcription (RT)-PCR of ATP -ase or glyceraldehyde 3-phosphate dehydrogenase gene ( GAPDH ) was used as control. PCR products were visualized on agarose gel stained with ethidium bromide. Northern blot of FMR1 gene was performed using 10 μg total RNA and 10 ng/ml of human FMR1 cDNA probe labeled with [α- 32 P]dCTP. Hybridization conditions were: 16 h at 42°C in 50% formamide, 6× saline-sodium phosphate-EDTA (SSPE), 5× Denhardt's solution, 0.5% SDS, 100 μg/ml herring sperm DNA. Wash conditions were: 2× SSPE, 0.1% SDS at room temperature and 0.1× SSPE, 0.1% SDS at 55°C. DNA MeTase expression was assayed with the same protocol using a specific 5-kb cDNA probe. Northern blot of iNOS and GAPDH was assayed by standard procedures. Western blot analysis of DNA MeTase was performed using 20–40 μg of nuclear protein extract resolved on 5% SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and subjected to immunodetection using a 1:2,000 dilution of primary antibody and an enhanced chemiluminescence detection 13 . DNA samples were prepared from cultured cell lines by standard procedures. 10 μg of genomic DNA was digested overnight with the restriction enzymes EcoRI-EagI or HindIII-SacII, EagI and SacII being sensible to methylation. Restriction fragments were separated by electrophoresis on 0.8% agarose gel, Southern blotted, and hybridized with radiolabeled StB12.3 probe as described previously 20 . DNA MeTase activity was determined in nuclear protein extracts by the assay developed by Adams et al. 21 with minor modifications. Cells were lysed in buffer containing 20 mM Tris-HCl, pH 8, 137 mM NaCl, 5 mM MgCl 2 , 5 mM EDTA, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 100 μg/ml RNase. After centrifugation, nuclear extracts were prepared by resuspension of the crude nuclei in high salt buffer. 15–25 μg of proteins was incubated for 2 h at 37°C with 4 μg of poly (dI-dC) as template and 5.25 μM 3 H-labeled S -adenosylmethionine (1 μCi; Amersham Pharmacia Biotech) as methyl donor. Reactions were stopped, proteins extracted, and DNA template was recovered by ethanol precipitation. RNA was removed by resuspension of the precipitates in NaOH; DNA was spotted on Whatman filters, dried, and then washed with trichloroacetic acid (5%) followed by ethanol, then ether. Filters were placed in the scintillation mixture, and DNA MeTase activity was determined by scintillation counting. Results were expressed as cpm per microgram of protein; all experiments were performed in duplicate. Background levels were determined in assays where poly (dI-dC) was omitted. Statistical analyses were performed using Student's t test. Lactate dehydrogenase (LDH) and pyruvate kinase (PK) were measured by standard procedures in the 12,000 g supernatant of Jurkat T cell homogenate as described previously 22 . Hexokinase (HK) was measured in the homogenate of Jurkat T cells as reported elsewhere 23 . Statistical analyses were performed using Student's t test. Cellular proliferation was determined by a colorimetric assay system using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) following the manufacturer's instructions (Cell Proliferation Kit I; Boehringer Mannheim). Fragile X syndrome, the most common form of hereditary mental retardation 24 , results from repression of the FMR1 gene due to the expansion of the CGG repeats in its first exon and methylation of the 5′ CpG island. The latter alteration appears to be the primary cause of the disease, since hypermethylation of the CpG island in the active X chromosome is only observed in affected individuals, whereas there are cases with full expansion of the CGG repeats but with an unmethylated island that do not manifest the syndrome 25 26 . Furthermore, in vitro reactivation of the FMR1 gene by demethylating agents has been reported recently 27 . We have observed a marked inhibitory effect of IL-1β on FMR1 gene expression in RIN cells assessed by RT-PCR of both KH domains and CGG repeats . Inhibition of FMR1 expression was appreciable after 12 h of incubation with IL-1β, and complete suppression of the gene resulted with longer exposures . Since IL-1β is known to be a powerful stimulus for induction of NOS in RIN and other cell types 28 29 , we investigated whether NO acted as a mediator of FMR1 repression. Fig. 1 b shows that SNP, an NO donor, mimics the action of IL-1β, and that the IL effect is fully prevented by the simultaneous addition of L-NMA, an inhibitor of NOS activity. This preventive effect was not observed when we used D-NMA (not shown). To further demonstrate that IL-1β exerts gene silencing via NO production, we used specific iNOS inhibitors such as AMT, EIT, and L-NIL and found that all of them also prevented the action of IL-1β . To determine if FMR1 mRNA stability was altered by NO, ActD was used to inhibit RNA synthesis. As shown in Fig. 1 d, the time course of FMR1 mRNA degradation was not modified by the presence of SNP. Thus, production of NO by IL-1β or addition of NO precursors can produce FMR1 gene silencing. In preliminary experiments, we have observed that IFN-γ, which induces NO synthesis, as well as NO donors can also inhibit FMR1 expression in a monocyte-macrophage cell line (RAW 264 cells). To elucidate the mechanisms underlying the effect of NO on gene regulation, we studied the expression of FMR1 in human cells (Jurkat T cells and fresh lymphocytes) where the complete map of the FMR1 promoter is known 30 . Although these cells are not stimulated by IL-1β, the application of NO precursors (500 μM SNP and 100 μM SIN) resulted in complete suppression of FMR1 expression , thus allowing a more detailed analysis of the repression of FMR1 gene by NO. Since it has been reported that NO can produce DNA damage by disruption of nucleotide bonds 31 , we tested if the effect of NO could be explained by direct interaction with CG sites, particularly abundant in (CGG) n repeats and CpGs of FMR1 . The fact that we were unable to induce CG cleavage in the FMR1 gene by NO donors in cellular and cell-disrupted preparations (not shown) led us to hypothesize that NO could be part of a signaling pathway regulating methylation of the CpG island in the FMR1 promoter region. This was tested by Southern blot using methyl-sensitive restriction enzymes and the StB12.3 probe currently applied to study the length and the methylation status of FMR1 in fragile X patients 20 24 . Fig. 2 b shows that treatment of Jurkat T cells with SNP or SIN produced full CpG island methylation (lanes 2, 4, and 6), which was totally prevented when the cells were treated with the demethylating agent AzadC in the presence of any of the NO donors (lanes 3 and 5). In the experiments where we applied an NO donor (or IL-1β) plus AzadC, we incubated the cells for 24 h as has been recommended when using the demethylating agent 7 . After incubation with and washout of SIN, addition of AzadC also reverted the methylating action of NO (lane 7), indicating that the effect of AzadC was not due to direct chemical interaction with SIN. CpG methylation induced by NO donors (SIN or SNAP) progressed with time after the initial 3 h of treatment, and full methylation was observed at 6 h . To further investigate the NO-dependent methylation process, we measured the activity of DNA MeTase, the major DNA methylating enzyme that produces 5′ methylcytosine 32 . Incubation of RIN cells with IL-1β increased DNA MeTase activity to about twice the level found in control cells ( P < 0.001, n = 3). A qualitatively similar effect was observed in Jurkat T cells exposed to an NO donor . These increases in activity are within the range of those recently published in transformed rodent cells overexpressing c- fos 33 . Since NO-induced methylation of the CpG island is abolished by the presence of the demethylating agent AzadC , it is expected that addition of this product would prevent FMR1 silencing induced by either IL-1β or NO. Fig. 3 a illustrates that FMR1 suppression produced by IL-1β was almost completely reverted by incubation with the demethylating agent. It is also shown that AzadC did not diminish the level of expression of iNOS induced by IL-1β. Similarly, demethylation also prevented FMR1 suppression resulting from exposure of the cells to NO donors . These data indicate that NO-dependent FMR1 gene silencing results from methylation of the CpG island, an effect mediated by activation of DNA MeTase. The major mammalian DNA MeTase is a large protein with an NH 2 -terminal putative regulatory domain comprising two thirds of the protein with eight cysteine residues, and a COOH-terminal catalytic domain with the adenosylmethionine binding region and a proline-cysteine catalytic center 34 . The signals and mechanisms involved in regulation of DNA MeTase activity are poorly understood. The NH 2 terminus is unnecessary for catalysis, but its cleavage from the COOH terminus causes a large stimulation of the initial velocity of methylation of unmethylated DNA 34 . The NH 2 terminus contains a major phosphorylation site (serine 514) although its relevance in catalysis is uncertain, since treatment of the enzyme with phosphatases and kinases results in no significant effect on the catalytic rate 35 . We assayed whether the effect of NO on DNA MeTase activity was due to activation of guanyl cyclase, by incubation of Jurkat T cells with 2 mM dibutyryl cGMP for 24 h. cGMP had no effect on either DNA MeTase activity or the expression of FMR1 gene (not shown). The Ras signaling pathway has been shown to increase DNA MeTase transcription 36 , and recently it has been suggested that fos may transform cells through alterations in DNA methylation 33 . Therefore, we tested if the expression of DNA MeTase could be altered by exposure of Jurkat T cells to an NO donor. Northern and Western blot analyses, shown in Fig. 4 , a and b, respectively, indicate that NO does not affect the expression of the major human DNA MeTase . We have not studied the expression of the recently described DNA MeTase3A and DNA MeTase3B 37 ; however, it is very unlikely that they mediate the effects of NO described here, since these two enzymes are present mainly in embryonic tissues 37 . Moreover, NO was able to activate DNA MeTase in a nuclear protein extract (see below), thus strongly suggesting that the regulation of the enzyme by NO is not dependent on transcription. The Western blot in Fig. 4 b also shows that NO did not modify the magnitude or size of any of the bands obtained with the polyclonal antibody against the DNA MeTase, thus indicating that cleavage of the NH 2 -terminal regulatory domain is probably not involved in the mechanism of action of NO. However, the presence of several cysteine residues in the protein suggested the possibility of a direct reaction of NO with thiols. Fig. 5 a shows that application of an NO donor (SNAP) to a nuclear protein extract induced a dose-dependent increase of DNA MeTase activity. In addition, sodium nitrite and peroxynitrite at different concentrations did not increase the enzymatic activity. On the contrary, high concentration of peroxynitrite drastically inhibited the reaction. Fig. 5 b illustrates the time course of DNA MeTase activation induced by 50 μM of different NO donors (SNAP, SIN, and SNP) in the nuclear protein extract. In all cases, after 3 h incubation with the NO donor a statistically significative increase in the DNA MeTase activity was observed ( P < 0.001, n = 3 for SNAP; P < 0.001, n = 2 for SIN and SNP); however, no change was obtained when expired SNAP or SIN was used (more than eight half-lives). Thiol oxidation independent of NO did not seem to play a role in the action of NO donors on DNA MeTase activity, since similar effects were seen when superoxide dismutase and catalase were added to the reaction mixture (not shown). Enhancement of DNA MeTase activity induced by SNAP was completely reversed by further incubation, after washout of the NO donor, with reducing agents such as DTT, GSH, or β-ME . These results strongly suggest that NO, either directly or through mediators present in the nuclear extract, regulates DNA MeTase activity possibly by nitrosation of some cysteines present in the protein. To evaluate the selectivity of the stimulatory effect of NO on DNA MeTase, we measured in cell extracts the activity of other enzymes, such as lactate dehydrogenase (LDH), hexokinase (HK), and pyruvate kinase (PK), encoded by genes that are constitutively expressed in all cells and are involved in housekeeping functions. Cell extracts were incubated for 3 h in the absence or presence of the stimulus. Basal activities (given by mean ± SD) were 0.02 ± 0.006 ( n = 6) pmol/h/μg protein for DNA MeTase, 110 ± 22 ( n = 5) pmol/min/μg protein for LDH, 12 ± 5 ( n = 3) pmol/min/μg protein for HK, and 27 ± 11 ( n = 3) pmol/min/μg protein for PK. Table shows that whereas 50 μM SNAP induced a marked increase in DNA MeTase activity, it had no effect on the activities of any of the other enzymes studied. Thus, in cell extracts, NO exerts a selective effect on DNA MeTase. Given that the inhibitory effect of NO on FMR1 expression can be explained by activation of DNA MeTase and methylation of the CpG island, we explored if a similar action is exerted on other genes, such as HPRT , known to contain a CpG island in the promoter region. Fig. 6 shows that exposure of RIN and Jurkat T cells to IL-1β (a) and NO donors (b), respectively, resulted in abolishment of HPRT expression. In both cases, demethylation with AzadC produced recovery of gene expression. NO-dependent methylation of CpG islands observed in transformed cells (such as RIN and Jurkat T cells) was also clearly apparent in fresh human lymphocytes. Fig. 6 c illustrates the silencing of FMR1 and HPRT genes by NO donors and the reversion of this effect by AzadC. The expression of genes such as GAPDH or Na/K ATP -ase, which do not contain CpG-rich promoters, is unaffected by NO . NO-induced DNA methylation was maintained for several hours after washout of the signal; however, it was a reversible phenomenon. In mitotically active cells (such as Jurkat T cells) previously exposed to NO donors, a unique and methylated band was observed by Southern blot 48 h after removal of the stimulus, whereas after 72 h two bands, methylated and unmethylated, were present . After 48 or 72 h incubation, we observed a clear increase in the number of cells . These results indicate that DNA methylation appears to be transient, due to either loss of methylation in the new generation of dividing cells or the existence of active DNA demethylation. This latter mechanism is suggested by the fact that reversibility of NO-induced methylation was also observed in nondividing cells such as quiescent fresh lymphocytes (not shown), and is in accordance with a recent report describing the existence of DNA demethylases in human cells 38 . DNA methylation causes repression of gene expression by promoting the condensation of chromatin. Methylated sites on DNA bind the 5-methylcytosine binding protein (MeCP2) that exists in a complex with Sin3A and histone deacetylase, resulting in a compact chromatin structure 6 . To investigate whether NO-mediated gene silencing requires histone deacetylation, we tested the effect of TSA, an inhibitor of deacetylases 33 . Fig. 7c and Fig. d , show that suppression of FMR1 expression (determined by both Northern blot and RT-PCR, respectively) was prevented by incubation of the cells with 2 μM TSA, hence supporting the view that NO-induced gene repression is due to increased recruitment of histone deacetylases by methylated DNA. NO is a broadly distributed signaling molecule involved in numerous physiological and pathophysiological processes 39 40 41 , but its action on the genome is poorly understood. Moreover, many actions of IL-1β are mediated by the induction of iNOS and the resulting NO production 42 43 . We show that by activating DNA MeTase, NO can induce methylation of 5′ CpG islands and, hence, repress gene expression. Methylation/demethylation of DNA is known to be associated with X-linked gene inactivation, imprinting, and fragile X syndrome 7 8 15 . Changes in DNA methylation are also observed during development, the acquisition of T cell cytokine production profiles, and tumorigenesis 9 10 11 12 13 14 17 , and several transcription factors actively promoting DNA demethylation have been reported 9 44 . Our findings provide the first case of FMR1 gene silencing in situations other than fragile X syndrome, although the methylation status in the two conditions shows some differences. Methylation induced by NO was lost with time, and TSA reversed the inhibitory effect on gene expression induced by NO (see above). In contrast, it has been recently reported that TSA has no effect on transcription in cells from fragile X patients due to additional modifications in histone–DNA association 45 . The marked repressive effect of IL-1β and NO on the expression of housekeeping genes, such as FMR1 and HPRT , with a CpG island in the promoter might be part of a general adaptive mechanism triggered in cells challenged by stressing situations. It has been reported that nuclear factor κB (NF-κB) in B cells can induce specific demethylation of the Igκ locus 46 , and in some cells, including RIN cells, IL-1β receptor stimulation induces a cascade that activates NF-κB 47 48 . In this work, we have shown that IL-1β clearly represses gene expression by a mechanism involving methylation. Our data indicate that the possible demethylating activity of NF-κB in RIN cells does not counterbalance the increase in activity of the DNA MeTase. Similarly, in Jurkat T cells the incubation with PMA plus a calcium ionophore , which induce NF-κB 49 , does not prevent the inhibitory action of NO on gene expression (results not shown). However, it could be speculated that IL-1β transitorily represses housekeeping genes having CpG islands via NO production and methylation while inducing tissue/stage-specific gene expression by activating demethylation via NF-κB. For instance, it has been reported that Ras induces an increase in demethylase activity in parallel to its induction of transcription of DNA MeTase 36 . Methylation of cytosine also gives an explanation for the high occurrence of genomic C–T transitions observed under exposure to NO 50 . The abundance of 5-methylcytosine in methylated DNA favors the transition to thymine by simple deamination, which can occur spontaneously and is potentiated by NO 51 . Finally, given the resemblance between certain viruses and housekeeping promoters 1 , the reported antiviral action of NO 52 could be explained by methylation-dependent silencing of the viral genome. Interestingly, DNA MeTase is a housekeeping gene without 5′ CpG island and, thus, its expression is insensitive to NO (see above). In contrast, it has a specific promoter containing activating protein (AP)-1, AP-2, and glucocorticoid response elements 53 , suggesting a potentially high level of regulation by cellular signal transduction pathways. In conclusion, we report here a novel action of IL-1β mediated by NO production. NO induces a posttranscriptional increase in the activity of DNA MeTase, resulting in CpG island methylation and suppression of gene expression. These results give new insights into the pathophysiological regulation of genes with CpG-rich promoters. | Other | biomedical | en | 0.999995 |
10587351 | The E2A- and E47-deficient mice have been described previously 4 15 . The β2-microglobulin (β2M) mice were purchased from The Jackson Laboratory. All mice were analyzed between 4 and 7 wk of age. Total thymocytes from E47- or E2A-deficient mice and wild-type littermates were dissected into RPMI medium containing 10% fetal bovine serum and 50 μM 2-ME and supplemented with glutamine, penicillin, and streptomycin. The cells were stained with antibodies to CD4 and CD8 or with propidium iodide (to determine viability) before culture (time = 0). The cells were then placed in culture at 37°C plus 5% CO 2 for 28 h before harvesting and staining with anti-CD4–PE and anti-CD8–FITC antibodies and propidium iodide to determine cell viability. The percentage of viable DP cells was determined by multiplying the number of viable cells at 28 h by the percentage of DP cells at 28 h and dividing this by the number of DP cells put into culture at time = 0. Staining of the cells has been described previously 4 . For two-color analysis, cells were stained with anti-CD4–PE (RM4-5; PharMingen), anti-CD8α–FITC (53-6.7; PharMingen), anti–TCR-α/β–FITC (H57-597; PharMingen), and anti-CD69–PE (H1.2F3; PharMingen). Hybridoma supernatant from the T3.70 line, provided by Dr. Ellen Robey (University of California, Berkeley, CA), was used for the Vα3 staining. For three-color analysis, cells were stained with anti-CD4–TriColor (CT-CD4; Caltag), anti-CD8α–PE (53-6.7; PharMingen), anti-CD69–FITC (H1.2F3), anti–heat stable antigen (HSA)–FITC (M1/69; PharMingen), and anti–TCR-α/β–FITC. Cells were analyzed on a FACScan™ (Becton Dickinson), and live cells were gated on the basis of forward and side scatter. For the kinetic analysis of thymic development, mice were continuously exposed to the thymidine analogue bromodeoxyuridine (BrdU; 0.8 mg/ml) in their drinking water for the indicated times. Thymocytes were labeled with antibodies extracellularly as described above using biotinylated anti-CD4 or anti–TCR-α/β (PharMingen) and PE-labeled anti-CD8 or anti-CD69 (PharMingen). Cells were then fixed and stained with FITC-labeled anti-BrdU (Becton Dickinson) as described previously 16 . The 16610D9 cell line was derived from a thymoma that developed spontaneously in a p53-deficient mouse and was adapted to culture. The cells were subcloned to generate the 16610D9 line, which was then cultured at 37°C plus 5% CO 2 in Optimem (GIBCO BRL) containing 10% fetal bovine serum and 50 μM 2-ME and supplemented with glutamine, penicillin, and streptomycin. The retroviral vector LZRSpBMN-linker-IRES-EGFP (S-003) was obtained from Hergen Spits (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The mouse Id3 cDNA that was cloned into the polylinker was generated by PCR of an Id3 cDNA with the following primers: Id3 forward, 5′-CCGGAATTCATGGACTACAAGGACGACGATGACAAGGGATCCATGAA-GGCGCTGAGCCCGGTG; and Id3 reverse, 5′-CCGGAATTCTCAGTGGCAAAAGCTCCTC, to generate FLAG-tagged Id3. The PCR product was cloned into pBSK, digested out of pBSK with EcoRI, and cloned into the EcoRI site in S-003. The φNX-eco packaging line 17 was transfected with the retroviral constructs by calcium phosphate precipitation. The transfected cells were switched to 16610D9 culture medium 24 h after transfection, and supernatants were harvested after an additional 24 h. Transduction of the 16610D9 cells was performed as described previously 18 . Cells were harvested 48 h after infection and analyzed by flow cytometry as described above using PE-conjugated antibodies (with the exception of CD4, which was TriColor conjugated), or lysed to make whole cell extracts (WCEs). To prepare WCE, cells were pelleted, washed once in cold 1× PBS, and the cell pellet was then frozen on dry ice for ∼15 min. The pellet was thawed, resuspended in 15 μl/10 6 cells cold buffer C (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF plus protease inhibitors) containing 1% NP-40, and vortexed vigorously for 2 min at 4°C. Debris was pelleted by spinning at high speed in a microfuge at 4°C, and the supernatant was removed as the WCE. Protein concentrations were determined using Bio-Rad's protein assay reagent as described by the manufacturer. Double-stranded DNA probes were end-labeled using T4 polynucleotide kinase and purified over a G25 Sepharose column. 15 μg of WCE was used in a gel shift assay as described previously 19 . The sequence of the μE5 oligo probe is as follows: 5′-TCGAAGAACACCTGCAGCAGCT-3′. Previous studies have established that mutant mice lacking both E12 and E47 exhibit abnormalities in thymocyte development 7 . To generate sufficient numbers of mice to further characterize the T cell phenotype, we analyzed E47-deficient mice, which have a significantly lower rate of postnatal lethality compared with the E2A −/− mice 15 . However, we note that the E47-deficient mice also express reduced levels of the E12 protein compared with control littermates 15 . We analyzed thymocytes derived from 4–6-wk-old E2A- or E47-deficient mice for the expression of CD4 and CD8 by flow cytometry. Wild-type and E47 or E2A heterozygous mice had virtually identical thymic profiles (data not shown). However, E2A- and E47-deficient thymi showed significant decreases in the percentage of DP thymocytes compared with their heterozygous littermates . In contrast, the proportions of both the CD4 and CD8 SP populations were increased an average of two- to threefold in the E2A- and E47-deficient thymi . Within the thymus, an increase in the relative percentage of mature SP thymocytes can arise through several mechanisms. For example, either aberrant proliferation of the SP populations, increased frequency of apoptosis within the DP population, or increased maturation of the DP cells could explain the phenotypical changes observed in the E2A- and E47-deficient mice. To begin to address these possibilities, we analyzed thymi from E2A-deficient mice for incorporation of BrdU and survival in vitro. E2A-deficient mice and control littermates were injected with BrdU, and the thymic subpopulations were analyzed for BrdU incorporation 24 h after injection. We found no increase in the percentage of SP thymocytes from E2A-deficient mice staining positive for BrdU compared with the control littermates (data not shown). Thus, the increased percentage of SP cells is not a result of abnormal proliferation within this population. To determine whether the absence of E2A promotes DP cell death, we isolated total thymocytes from E47- or E2A-deficient mice and wild-type littermates and analyzed the viability of the cells after 28 h in culture. Whereas an average of 52% of the wild-type DP cells was alive after the culture period, only ∼17% of the E47- and E2A-deficient DP cells survived ( Table , and data not shown). Similar percentages of wild-type or E2A-deficient CD4 + cells were alive after 28 h in culture, suggesting that the increase in cell death is specific to the DP population ( Table ). Thus, DP thymocytes lacking E2A activity display decreased survival in vitro. To address the possibility of an increase in DP thymocyte maturation, we further characterized the changes in the T cell populations by analyzing thymocytes from E2A- and E47-deficient mice for the expression of TCR-α/β and CD69. During normal thymocyte development, expression of both TCR-α/β and CD69 increases upon positive selection 20 21 22 23 24 . However, whereas SP thymocytes retain high levels of TCR-α/β expression, CD69 levels are subsequently downregulated upon completion of maturation 21 24 25 . Thus, TCR hi CD69 + cells represent those thymocytes in the process of positive selection, whereas TCR hi CD69 − thymocytes have completed the maturation process. We find that thymocytes derived from E2A- or E47-deficient mice have a three- to fourfold increase in the proportion of TCR hi CD69 − cells compared with their littermates . The absolute number of mature TCR hi CD69 − thymocytes is increased an average of 1.5–3-fold (data not shown). These data indicate that the absence of E47 leads to an increase in the proportion of SP cells that have completed thymocyte maturation, and suggest that E47 might have an additional role in regulating DP thymocyte differentiation. To further explore whether the absence of E47 leads to an increase in thymocyte maturation, E47-deficient mice were crossed to AND TCR mice which express a class II–restricted TCR transgene. Maturation of CD4 SP thymocytes is strongly enhanced in H-2 b mice expressing the AND TCR 26 . Thymocytes derived from E47-deficient mice and wild-type or heterozygous littermates carrying the AND transgene were analyzed by flow cytometry for the expression of CD4 and CD8. As expected, E47 +/+ ;AND mice contained a high fraction of mature CD4 + cells and a correspondingly lower fraction of immature DP thymocytes than nontransgenic thymi . Thymic profiles from E47 +/− ;AND mice were not significantly different from the E47 +/+ ;AND profiles . However, the percentage of mature CD4 SP cells was significantly higher (87 vs. 73%) in thymocytes derived from E47 −/− ;AND mice, and the proportion of DP cells was significantly lower . Importantly, we observed a similar trend in the peripheral T cell populations. The ratio of CD4 + to CD8 + splenic T cells increased from ∼22 in the wild-type or heterozygous mice expressing the AND TCR to 55 in the E47 −/− ;AND mice . In the lymph nodes, the ratio of CD4 + to CD8 + cells increased from 11 in the E47 +/+ ; AND mice to 69 in the E47 −/− ;AND mice . The increase in total percentage of T cells in the E47-deficient lymph nodes (98 vs. 55%) is due to the complete absence of B cells in the E2A- and E47-deficient mice 4 15 . Thus, there is a selective increase in only those SP cells expressing the relevant TCR, and this increase is reflected in the periphery. These data suggest that the absence of E47 can markedly enhance the positive selection of class II–restricted TCRs. To determine whether the absence of E47 also promotes increased selection of class I–restricted TCRs, we analyzed E47-deficient mice expressing the H-Y TCR transgene, which specifies reactivity with the male H-Y antigen presented by the H-2D b class I molecule 27 28 . Expression of the H-Y TCR transgene in females of the H-2 b background leads to positive selection of CD8 SP thymocytes 27 28 . In contrast, thymocytes in male mice expressing the H-Y TCR are deleted at the DP stage. Negative selection mediated by the H-Y TCR transgene was unaffected by the absence of E47 (data not shown). However, E47 −/− ; H-Y female mice displayed an increased percentage of mature CD8 SP thymocytes compared with the E47 heterozygous littermates . This phenotype was accompanied by a significant decrease in the proportion of immature DP thymocytes . Because of continued rearrangement of endogenous α chain genes in TCR transgenic mice, a fraction of the thymocytes will express a TCR composed of the transgenic β chain and an endogenous α chain 25 27 29 . To analyze the proportion of cells expressing the transgenic TCR-α/β, we stained total thymocytes with the T3.70 antibody, which reacts specifically with the transgenic α chain molecule 30 . Thymocytes isolated from E47 −/− ;H-Y mice contained almost twice the percentage of cells expressing high levels of both the transgenic α and β chains, consistent with a higher proportion of mature CD8 SP T cells . The increased percentage of mature CD8 + T cells observed in the E47 −/− ;H-Y mice is also reflected in the periphery. The ratio of CD8 + to CD4 + T cells is increased from ∼0.4 in the E47 +/− ;H-Y spleen to 1.2 in the E47 −/− ;H-Y spleen . A similar change in the CD8/CD4 ratio was observed in the lymph nodes . Therefore, like the E47 −/− ;AND mice, the E47 −/− ;H-Y mice display an increase in only the percentage of cells expressing the relevant TCR, and the relative proportions of CD4 and CD8 lineage cells are altered in the peripheral lymphoid organs as well. These changes in peripheral CD8/CD4 ratios from E47-deficient/TCR transgenic mice would not be expected if the increase in proportion of thymic SP cells was entirely the result of increased death within the DP thymocyte population. In addition, a selective increase in only the population of cells expressing the relevant TCR is not consistent with a defect in emigration of mature SP thymocytes from the thymus. Interestingly, in addition to the increase in CD8 SP cells, E47 −/− ;H-Y thymi contained elevated percentages of DP cells that expressed CD69 and high levels of TCR-α/β . Thus, a higher proportion of the DP population is undergoing positive selection in the absence of E47. Taken together, these data suggest that the absence of E47 results in increased selection to both class I– and class II–restricted T cell receptors. As described above, heterozygosity at the E47 locus does not perturb maturation of SP thymocytes expressing class II–restricted TCR transgenes. In contrast, we consistently observed increases in the proportion of CD8 + cells in female H-Y transgenic mice that were heterozygous for E47 compared with wild-type littermates . In most cases, the percentage of CD8 + cells was increased in the periphery of E47 +/− ;H-Y mice as well . Thus, maturation of CD8 SP cells in mice expressing the H-Y TCR transgene is influenced by the dosage of E47. In addition to the increased production of CD8 SP cells, female E47 −/− ;H-Y mice displayed an aberrant population of T cells in the peripheral lymphoid organs that expressed high levels of CD8 and intermediate to high levels of CD4 . To examine whether the peripheral DP (PDP) population expressed markers characteristic of mature T lineage cells, we analyzed this population for the expression of TCR-α/β and HSA. The PDP cells expressed HSA at a level similar to normal SP thymocytes, and significantly lower than the level of expression on thymic DP cells . However, the expression of HSA on the PDP population is significantly higher than the level observed on normal SP peripheral T cells . TCR-α/β expression on the PDP cells was identical to that of the mature SP T cells, with all of the cells expressing uniformly high levels of TCR . Taken together, the data suggest that the PDP cells in the E47 −/− ;H-Y mice are positively selected T lineage cells that fail to complete maturation before exiting to the periphery. As described above, the absence of E47 promotes the development of SP thymocytes. To determine whether the absence of E47 allows the development of SP cells in the absence of an appropriate signal, we bred the E47-deficient mice to β2M–deficient mice, which virtually lack MHC class I expression and produce very few mature CD8 SP thymocytes 31 32 . Although the E47 deficiency did not promote CD8 lineage development in the β2M-deficient background to wild-type levels, we consistently noted a small percentage of TCR hi CD8 SP cells in the E47 −/− ;β2M −/− thymi that were absent in the E47 +/− ;β2M −/− thymi . Furthermore, E47 −/− ;β2M −/− mice displayed a higher proportion of TCR hi CD8 SP cells in the peripheral lymphoid organs compared with the control littermates . The decreased CD4/CD8 ratio from 146 in the E47 +/− ;β2M −/− spleen to 37 in the E47 −/− ;β2M −/− spleen indicates that the absence of E47 allows for an increase in maturation of CD8 + cells even in the absence of the appropriate TCR–MHC interaction. Thus, the data suggest that the downregulation of E2A activity can promote thymocyte maturation. The steady state size of the thymic subpopulations is determined by the rate at which cells are generated, the rate at which they die, and the rate at which they emigrate from the thymus. To more directly assess the effect of an E47 deficiency on the production of mature SP T cells, we measured the kinetics of appearance of mature thymocytes using the thymidine analogue BrdU. We exposed E47-deficient mice and their heterozygous littermates to BrdU in their drinking water and then analyzed for the presence of BrdU and the expression of TCR-α/β and CD69 . Because cells expressing medium to high levels of TCR are not dividing, there is a lag in the appearance of BrdU in these populations. As described above, both the E47-deficient and control mice display this lag in labeling of mature thymocytes, demonstrating that the absence of E47 does not lead to the aberrant proliferation of the SP cells. Because the TCR med-hi populations are nondividing, the rate of appearance of BrdU in these populations reflects the rate at which these cells are produced. In mice deficient for E47, the rate of appearance of BrdU-labeled TCR hi CD69 − mature thymocytes is significantly enhanced, suggesting that the relative proportion of cells that develop into mature CD69 − T cells is increased in the absence of E47 . In contrast, E47-deficient mice display a decrease in the rate of appearance of BrdU in the TCR med CD69 + population . Taken together, these data suggest that the rate at which DP thymocytes mature into TCR hi CD69 − SP T cells is enhanced in the absence of E47. As shown in Table , E2A-deficient DP thymocytes show reduced survival in vitro. However, the data described above suggest that the absence of E47 also promotes thymocyte positive selection. To test directly whether lowering the activity of E47 promotes maturation, we used a retroviral transduction system to study the effects of inhibiting E2A activity in an immature DP T cell line. The mouse cDNA for Id3, a negative regulator of E2A binding activity, was cloned into the retroviral vector S-003 14 . This vector allows translation of both Id3 and enhanced green fluorescent protein (EGFP) from one retroviral transcript. Thus, retroviral transductants can be identified by the expression of EGFP. The S-003/Id3 and S-003 empty vector constructs were transfected into the φNX-eco retroviral packaging line, and supernatants from these cells were used to transduce virus into the T cells. The 16610D9 T cell line used was derived from a thymoma that developed spontaneously in a p53-deficient mouse and was adapted to culture. This T cell line expresses characteristics typical of DP thymocytes, including high levels of HSA, intermediate levels of TCR, and low levels of CD5 and CD44 . In addition, the 16610D9 cells express significant levels of E2A binding activity . Id3 was transduced into the 16610D9 line and analyzed 48 h after infection for the expression of HSA, CD69, CD44, CD5, TCR β chain, and CD4 and CD8. The early stages of positive selection are marked by increased expression of CD69, CD5, and TCR 20 22 24 25 . During the later stages of positive selection, CD44 is upregulated whereas HSA is downregulated 33 . In addition, T cells undergoing the initial stages of positive selection downmodulate both CD4 and CD8 expression 34 . At the time of analysis, ∼85% of both the control and Id3-infected cells were EGFP + . The ability of Id3 to inhibit E47 binding activity was assayed by electrophoretic mobility shift assay using an E-box probe. E47 and HEB binding activity was easily detectable in the control transduced cells, but dropped to 10–20% of that level in the Id3 transductants . Thus, overexpression of Id3 is sufficient to inhibit E47 binding activity within the 16610D9 cells. The infected cells were then analyzed by flow cytometry for the expression of the indicated markers. Strikingly, the Id3 transductants displayed altered expression of all the markers analyzed. In particular, expression of both TCR-β and CD5 increased dramatically in the cells lacking E47 binding activity, but was unchanged in the nontransduced cells . In addition, the Id3 transductants clearly upregulated CD69 and CD44 expression, as evidenced by an increase in both the mean linear value of the histogram and the median linear value of the histograms . On the other hand, HSA expression was marginally downregulated in the Id3-expressing cells . Furthermore, we detected a dramatic downmodulation of CD4 and CD8 coreceptor expression in Id3-transduced cells . Thus, these data clearly show that Id3-transduced T cells acquire phenotypic characteristics of thymocytes undergoing positive selection. Taken together, these data demonstrate that lowering the level of E47 activity promotes T cell differentiation in vitro and are consistent with a key role for E47 in promoting DP thymocyte maturation in vivo. Immature thymocytes have the ability to undergo two distinct fates. A DP thymocyte can be selected to mature into either a CD4 + or CD8 + T cell, or alternatively it may undergo cell death. Although a large number of signaling molecules have been implicated in thymic selection, their nuclear targets have remained largely unknown. Here, we show that the activity of one particular transcriptional regulator, E47, is important in regulating thymocyte positive selection. Our data indicate that the relative level of E47 can influence thymocyte selection to both the CD4 and CD8 lineages. In the absence of E47 activity, the rate of production of TCR hi CD69 − thymocytes is enhanced, resulting in increased percentages of SP thymocytes. In a TCR transgenic background, a deficiency in E47 results in an increase in positive selection of only those T cells expressing the relevant TCR. In addition, inhibition of E47 activity within a DP T cell line induces the cells to differentiate. Based on these observations, we propose that the downregulation of E2A activity promotes thymocyte maturation. Mice deficient for E2A display an increased percentage of mature SP thymocytes coupled with a decreased proportion of DP cells. Here we show that the E2A-deficient mice exhibit increases in DP cell death in vitro. Although it is likely that a decreased rate of DP survival could contribute to the observed phenotype, it is doubtful that the increase in proportion of SP cells is solely a result of increased death within the DP population. Our kinetic data demonstrate that the absence of E2A activity additionally promotes thymocyte maturation. Furthermore, E47-deficient TCR transgenic mice display a corresponding increase of SP T cells expressing only the relevant TCR in both the thymus and the peripheral lymphoid organs. The appearance of DP T cells expressing characteristics of positively selected cells in the periphery of E47 −/− ;H-Y mice also indicates that the absence of E2A affects DP thymocyte maturation. Moreover, our data show that CD8 + T cells have the ability to mature in the absence of class I, albeit with low efficiency, when E47 is lacking. Finally, inhibition of E47 activity within a DP T cell line alters the expression of markers that correlate with DP maturation. Taken together, these data demonstrate that a downregulation of E47 activity promotes thymocyte maturation and that a combination of increased DP cell death and enhanced maturation contributes significantly to the phenotype observed in the E47-deficient mice. This raises the question of how E47 is involved in positive selection. TCR–MHC interactions that induce positive selection must have two important effects. These signals must rescue the cells from apoptosis and promote maturation. Recent data indicate that the restoration of E47 activity in E2A-deficient DP thymoma cell lines leads to programmed cell death 18 . It is possible that the continued presence of E2A activity in DP thymocytes that have received a signal through the TCR contributes to the induction of apoptosis. Thus, only those cells that downregulate E2A activity survive. We note that the thymic phenotype of E47-deficient mice shows similarities to the bcl-2 transgenic mice, including a higher percentage of CD8 SP cells and an alteration in the CD4/CD8 ratio 7 35 36 37 38 . Like bcl-2 expression, decreased E47 activity might prolong the survival of the DP thymocytes, thus leading to enhanced production of CD8 + cells. However, as mentioned above, DP thymocytes lacking E2A show an increase in cell death in vitro, whereas bcl-2 expression prolongs DP thymocyte survival 36 38 . Additionally, expression of bcl-2 promotes CD8 + thymocyte maturation in MHC class I–deficient mice and in class II–restricted TCR transgenic mice, whereas the absence of E47 does not . Finally, in contrast to the E47-deficient mice, the excess CD8 + thymocytes that develop in the bcl-2 transgenic mice do not survive in the periphery 35 36 . Recently, activation of the Notch signaling pathway has been shown to upregulate several markers that correlate with DP maturation. In particular, a constitutively active fragment of Notch was capable of upregulating bcl-2 expression in DP thymomas and rendering these cells resistant to glucocorticoid-induced apoptosis 39 . Interestingly, like the E47-deficient mice, activated Notch1 transgenics have increased percentages of CD8 + thymocytes 40 . However, a significant population of CD8 + thymocytes can develop in Notch1 transgenic mice lacking expression of class I MHC, whereas only a small CD8 + population can develop in class I–deficient mice lacking E47 40 . Nonetheless, it is possible that Notch signaling regulates E2A activity. In fact, others have shown that activated Notch1 and Notch2 effectively inhibit E47 activity in transient transfection assays 41 . It is conceivable that the lack of E47 activity lowers the threshold of avidity required for positive selection. This model has been proposed to explain the phenotype of the activated Notch1 transgenic mice 39 . For example, the absence of E47 might allow for the development of T cells that would normally die by neglect because of an insufficient affinity for MHC. It is thought that CD8 lineage commitment requires weaker signals from the TCR compared with the strength of the signal required for CD4 lineage commitment 42 43 . If less stringent signals are required for commitment to the CD8 lineage, then a slight lowering of the threshold affinity of positive selection may result in an even greater increase in CD8 + cells compared with CD4 + cells. Such a model might explain the decrease in the CD4 + /CD8 + thymocyte ratio in E2A-deficient mice and the finding that more CD8 + T cells develop in MHC class I–deficient mice that also lack E47 expression. Thus, the absence of E47 might allow for the development of some T cells that would normally die by neglect. The data described above suggest that E47 activity is downregulated during the final stages of thymocyte maturation. A key step during the transition of a DP to SP thymocyte is the termination of TCR-α rearrangements. Previous studies have suggested a link between E2A activity and RAG expression, which is also downregulated in immature thymocytes upon interaction of the TCR with the appropriate MHC–peptide complex 44 . For example, expression of both RAG-1 and RAG-2 can be activated in a pre-T cell line by the ectopic expression of E47 9 . Additionally, E2A and HEB have been implicated in regulating RAG transcription in committed T lineage cells 11 . Consistent with this idea, E2A-deficient thymocytes show decreased levels of both RAG-1 and RAG-2 transcripts (Bain, G., unpublished observations). However, RAG levels are not completely abolished, and TCR V(D)J recombination proceeds in E2A-deficient mice, presumably due to the presence of an HEB homodimeric complex detectable in E2A-deficient thymocyte extracts 7 . We would like to suggest that an interaction of a TCR with the appropriate peptide–MHC complex results in the downregulation of total HLH activity, which leads to the abatement of RAG expression 44 . We also note that lowering the DNA binding activity of E47 in a DP cell line leads to the activation of CD5, CD69, and TCR expression and the downmodulation of CD4, CD8, and HSA levels. Thus, our observations indicate that E47 directly or indirectly regulates the expression of a wide variety of genes that are characteristic of thymocyte maturation. | Study | biomedical | en | 0.999998 |
10587352 | Adult C57BL/6 (B6) mice were purchased from SLC. B6Ly5.1 mice were maintained in our animal facility. Embryos at various stages of gestation were obtained from time-mated pregnant B6Ly5.1 mice. The following antibodies were used: PE–anti-Thy1.2 (5a-8; Caltag), biotinylated anti-NK1.1 and PE–anti-NK1.1 (PK136; PharMingen), PE–anti-CD45 (30F11.1; PharMingen), PE–anti-CD122 (TM-β1; PharMingen), FITC–anti-CD3∈ (145-2C 11 ; PharMingen), allophycocyanin (APC)–anti-CD3 (CT-CD3; Caltag), FITC–anti-CD25 (PC61; PharMingen), PE–anti-CD44 (IM7.8.1; Caltag), APC–anti-CD8 and FITC–anti-CD8 (YTS169.4; Caltag), APC–anti-CD4 and PE–anti-CD4 (GK1.5; Caltag), FITC anti–TCR-γ/δ (GL-3; Caltag), and PE anti–TCR-α/β (H57-597; Caltag). Anti-FcR (2.4G2) and anti-Ly5.2 (AL1-4A2, donated by Dr. I.L. Weissman, Stanford University, Stanford, CA) were labeled with FITC in our laboratory. Anti-TER119 (established by Dr. T. Kina in our laboratory), anti-CD44 (IM7.8.1), anti-B220 (RA3-6B2), anti–Mac-1 (M1/70), and anti–Gr-1 (RA3-8C5) were biotinylated in our laboratory. Anti–c-kit (ACK-2; donated by Dr. S.-I. Nishikawa, Kyoto University) and anti-Ly5.1 (A20-1.7; donated by Dr. Y. Saga, Banyu Seiyaku, Tokyo, Japan) were conjugated with Cy5 using a labeling kit (Biological Detection Systems). For biotinylated antibodies, Cy5-streptavidin (Caltag) was used as the secondary reagent. Recombinant murine (rm)IL-7 was donated by Dr. T. Sudo (Basic Research Lab, Toray, Kanagawa, Japan). Commercially available recombinant murine stem cell factor (rmSCF), rmIL-2, and rmIL-15 (all from Genzyme) were also used. FcR − and FcR + subpopulations of CD44 + CD25 − FT cells were obtained from fetuses at 12 d postcoitum (dpc) as described previously 19 . To isolate CD44 + CD25 − and CD44 + CD25 + cells in the lineage marker (Lin) − CD3 − CD4 − CD8 − population, 14-dpc FT cells were stained with biotinylated Lin (anti-TER119, anti–Mac-1, anti–Gr-1, anti-NK1.1, and anti-B220), washed, and then stained with a mixture of Cy5-streptavidin, APC–anti-CD4, APC–anti-CD8, APC–anti-CD3, FITC–anti-CD25, and PE–anti-CD44. Cells were subsequently sorted using a FACS Vantage™ (Becton Dickinson). In sorting CD122 + cells, 14-dpc FT cells were stained with the same mAbs as above except that PE–anti-CD122 was used instead of PE–anti-CD44. The method for high oxygen submersion (HOS) culture has been described in detail elsewhere 27 . Single dGuo-treated lobes were placed into wells of a 96-well V-bottomed plate (Nalge Nunc International), to which progenitors were added. The plates were centrifuged at 150 g for 5 min at room temperature, placed into a plastic bag (Ohmi Oder Air Service), and the air inside was replaced by a gas mixture (70% O 2 , 25% N 2 , 5% CO 2 ). The plastic bag was incubated at 37°C. The cultures were maintained in RPMI 1640 medium (GIBCO BRL) supplemented with 10% FCS (M.A. Bioproducts), l -glutamine (2 mM), sodium pyruvate (1 mM), sodium bicarbonate (2 mg/ml), nonessential amino acid solution (0.1 mM; GIBCO BRL), 2-ME (5 × 10 −5 M), streptomycin (100 μg/ml), and penicillin (100 U/ml). Culture medium was also supplemented with various cytokines to promote the growth of NK cells (see Results). Medium was replaced by half every 5 d. After 10 d of culture, cells inside and outside the FT lobe were harvested from each well. One fifth of each sample was stained with FITC–anti-Ly5.2, PE anti–Thy-1, and Cy5–anti-Ly5.1 to be screened for the presence of progenitor type (Ly5.1 + ) cells and expression of Thy-1 on these cells. The samples containing Ly5.1 + cells were selected for further analysis. The remaining four fifths of cells from the selected samples were divided into four groups. One group was stained with FITC–anti-CD3∈, PE–anti-NK1.1, and Cy5–anti-Ly5.1, the second with FITC–anti-CD8, PE–anti-CD4, and Cy5–anti-Ly5.1, and the third with FITC anti–TCR-γ/δ, PE anti–TCR-α/β, and Cy5–anti-Ly5.1. Surface phenotype was analyzed by a FACS Vantage™. The remaining group was used for the analysis of TCR gene rearrangement. The cytotoxic assay was carried out as reported previously 28 . Yac-1 cells were used as target cells. In brief, target cells were labeled with PKH67 green fluorescence dye (Sigma Chemical Co.) and mixed with various numbers of effector cells. The mixture was incubated at 37°C for 4 h in a V-bottomed 96-well microplate. Culture medium was the same as that used in HOS cultures. Dead cells were stained with propidium iodide red fluorescence dye (Sigma Chemical Co.), and the proportion of propidium iodide–stained cells (dead cells) among PKH67-stained cells was determined by analyzing with a FACS Vantage™. One fifth of the cells harvested from each sample (see preceding section) was subjected to PCR analysis. To prepare genomic DNA, 3,000 cells were resuspended in 20 μl of 1× PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, and 1.5 mM MgCl 2 ) including 0.45% NP-40, 0.45% Tween 20, and 1.2 μg of Proteinase K (Sigma Chemical Co.), and incubated at 55°C for 1 h, then 95°C for 10 min. Samples of these disrupted cells were used as templates for PCR amplification. Primers were: 5′ of Dβ2, 5′-GCACCTGTGGGGAAGAAACT-3′; 3′ of Jβ2.6, 5′-TGAGAGCTGTCTCCTACTATCGATT-3′; Vγ 4 , 5′-AGTGTTCAGAAGCCCGATGCA-3′; 3′ of Jγ 1 , 5′-AGAGGGAATTACTATGAGCT-3′. The reaction volume was 20 μl, containing 5 μl of the cell extract, 1.5 μl of 10× PCR buffer, 0.16 μl of 25 mM dNTPs, 4 pmol of each primer, and 0.6 U of Taq polymerase (Life Technologies). Thermocycling conditions were as follows: 5 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 60°C for D-Jβ rearrangement or at 55°C for V-Jγ rearrangement, 2 min at 72°C, and finally 10 min at 72°C. The PCR products were applied to a 1.2% agarose gel, electrophoresed, and stained with ethidium bromide. We first tried to establish a culture system capable of generating T and NK cells. This was attained by modifying the MLP assay system that was effective in investigating the developmental capability of progenitors toward T, B, and myeloid lineages 20 . Since FT contains a small number of NK cells as well as NK progenitors 29 , it is expected that dGuo-treated FT lobes support the differentiation of NK cells. Cytokines IL-2 and IL-15 were used for promoting the growth of NK cells in FT organ culture. FT cells from 12-dpc fetuses (100 cells) of B6Ly5.1 mice, which are exclusively CD44 + CD25 − , were seeded to each well of a V-bottomed 96-well plate, in which a dGuo-treated FT lobe (B6) had been placed. IL-2, IL-15, or a cocktail of these and other cytokines was added, and the plate was incubated under HOS conditions for 10 d. Cells were harvested, counted, and assayed for expression of T and NK cell markers on donor-derived (Ly5.1 + ) cells. Table indicates that the cell recovery increased slightly by the addition of cytokines, and in all groups >90% of the recovered cells were Thy-1 + . Without cytokines, ∼40% were CD3 + NK1.1 − (T cells), whereas only 3% were CD3 − NK1.1 + cells (NK cells). Addition of IL-2 or IL-15 dramatically increased the proportion of NK cells, while the proportion of T cells declined reciprocally. The effect of IL-2 or IL-15 in supporting the generation or growth of NK cells is compatible with a previous report 30 . Addition of IL-7 and SCF does not show any prominent influence on the cell recovery or proportions of T and NK cells. However, these factors are supportive for improving the seeding efficiency in the single progenitor assay (data not shown). In the following experiments, we used a cocktail of IL-2 (25 U/ml), IL-7 (50 U/ml), and SCF (10 ng/ml) as the agent modifying FT organ culture to evenly support the development of NK and T cells. The time course of T and NK cell generation in FT organ cultures with or without cytokine cocktail was investigated. FT cells from 12-dpc fetuses (100 cells) were cultured with a dGuo-treated lobe in the presence or absence of a cytokine cocktail (IL-2, IL-7, and SCF). At various days after the culture, cells were harvested, counted, and analyzed for their surface phenotypes. Numbers of CD3 + NK1.1 − and CD3 − NK1.1 + cells were plotted against culture days . The results indicate that the generation or growth of NK cells is highly dependent on exogenous cytokines, and that the NK cell generation in cytokine-supplemented cultures preceded T cell generation by ∼1 d. Fig. 1 C shows the representative flow cytometric profiles for expression of NK1.1 versus CD3, CD4 versus CD8, and TCR-α/β versus TCR-γ/δ on cells generated in cultures with or without the cytokine cocktail. These results indicate that the cytokine cocktail used here enhanced the generation of NK cells without modulating T cell generation. We next investigated whether the NK1.1 + cells generated in cytokine-supplemented FT organ cultures were functional as NK cells. Cells were harvested from cytokine-supplemented FT organ cultures seeded with 12-dpc FT cells (100 cells), and CD3 − NK1.1 + cells and CD3 − NK1.1 − cells were isolated with a flow cytometer. These cells were examined for killer activity by culturing with the NK-sensitive cell line Yac-1 at various E/T ratios. Fig. 2 shows that NK1.1 + cells but not NK1.1 − cells are cytotoxic, indicating that the NK1.1 + cells generated in the cytokine-supplemented FT organ culture are functional NK cells. Individual cells in the CD44 + CD25 − population from 14-dpc FT (B6Ly5.1) were seeded in the wells in which a dGuo-treated FT (B6) lobe had been placed . The culture medium was supplemented with IL-2, IL-7, and SCF. After 10 d of culture under HOS conditions, cells were harvested from each well for flow cytometric analysis. Expression of T and NK cell markers was examined by gating Ly5.1 + cells. Surface phenotypes of cells derived from three types of progenitors identified in this experiment are shown in Fig. 3 B. The progenitors generating NK1.1 + cells but not CD3 + cells were determined to be p-NK , whereas those generating only CD3 + cells were determined to be p-T (middle panels). In addition, the presence of p-T/NK was shown unambiguously (right panels). The T cells generated from a p-T as well as from a p-T/NK express CD4 and/or CD8 as well as TCR-α/β or TCR-γ/δ. The generation of NKT-like cells expressing both CD3 and NK1.1 was seen in the case of p-T/NK. Such NKT-like cells were also generated from p-T but not from p-NK when the culture period was extended (data not shown). In our previous study investigating the developmental potential of thymic progenitors toward T, B, and myeloid lineages, it was indicated that a large majority of the progenitors in FT cells was committed to the T cell lineage 21 . Because the culture conditions used in this study were unable to support the generation of NK cells, both p-T/NK and p-T should have been defined as “p-T.” Subpopulations of 12- and 14-dpc FT cells were used as the progenitor source. These subpopulations were sorted, and the single cells (total 50 cells in each group) were cultured for 10 d in cytokine-supplemented FT organ culture as in the preceding section. Cells were recovered from each well, and analyzed with a flow cytometer. Three types of progenitors were found to be present in these subpopulations, and these three types were the same as those seen in Fig. 3 B. The distribution of different types of progenitors in subpopulations of 12- and 14-dpc FT cells is shown in Fig. 4B and Fig. C , respectively. It was found that all T cell progenitors in the most immature CD44 + CD25 − FcR − (FcR − ) population of 12-dpc FT were p-T/NK, and the frequency of the p-T/NK in this population is equivalent to that determined to be “p-T” in our previous investigation performed with the MLP assay 21 . Quite a large number of p-NK also exists in this population. A small number of p-T appears at the next CD44 + CD25 − FcR + (FcR + ) stage, but p-T/NK and p-NK still predominate in this population. In the CD44 + CD25 − population of 14-dpc FT, the proportion of p-T becomes higher than in 12-dpc FT. It is also found that p-NK is the largest in number in this population. A prominent increase of p-T was seen at the CD44 + CD25 + stage. Of interest is that p-T/NK and p-NK still exist in this stage. At the CD44 − CD25 + stage, most of the cells no longer express progenitor activity, although a very small number of p-T can be seen (data not shown). The results shown in Fig. 4 suggest that the NK potential is retained in the T cell progenitor until just before the TCR-β gene rearrangement begins. In contrast, p-NK can split from p-T/NK at any stage of early T cell development in the FT. It is well known that NK cells do not express TCR on their surface, nor do they rearrange TCR genes 3 . However, because the NK cells generated in the present study are artificially induced from thymic progenitors by culturing them with a thymic lobe in the presence of exogenous cytokines, the possibility could not be ruled out that the progenitors that had already initiated their TCR gene rearrangement were induced to become NK cells. DNA was extracted from NK and T cells derived from single p-T/NK, from NK cells derived from single p-NK, and from T cells derived from single p-T. Rearrangement of TCR-β and TCR-γ genes was examined by PCR using the primer pairs shown in Fig. 5 . It is shown that multiple rearrangements had occurred at the Dβ-Jβ and Vγ-Jγ regions in all T cells derived from single progenitors. In marked contrast, TCR gene rearrangement was not observed in NK cells regardless of origin, indicating that the split to the NK progenitors ceases before rearrangement of the TCR-β gene. To ensure the existence of p-NK in the FT, we tried to isolate a population containing exclusively p-NK. For isolation of p-NK, we used an mAb to CD122 (IL-2/15Rβ), because it has been suggested that the NK progenitors express this molecule 6 31 . Lin − CD44 + CD25 − cells from 14-dpc FT can be separated into CD122 − and CD122 + subpopulations . In this experiment, anti-NK1.1 mAb is included in the lineage (Lin) markers. Cells enclosed in the rectangles were sorted, and served for the single cell assay to determine NK and T cell generating potential. The results, shown in Fig. 6 B, indicate that the CD122 − population contains all three types of progenitors, whereas only p-NK were detected in the CD122 + population. Moreover, p-NK were highly enriched in the CD122 + population: 17 out of 50 cells examined were p-NK. Failure to detect any p-T/NK or p-T indicates that the frequency of these progenitors in this population is <1/50. Such a low frequency of p-T in the CD122 + population was confirmed by culturing single cells with a dGuo-treated lobe in the absence of exogenous IL-2 (data not shown). On the other hand, previous studies in which 10 3 CD122 + FT cells were cultured with a dGuo-treated FT indicated that this population retains the potential to generate T cells 6 32 . Thus, the CD122 + population might contain a very small number of p-T, but the large majority of CD122 + progenitors are NK lineage committed. We have previously shown that only progenitors restricted to T, B, or myeloid lineage, but not multipotent progenitors, exist in the FT 21 . However, the assay system used in these experiments was not designed to support the generation of NK cells. In this study, we succeeded in establishing a new assay system capable of determining the developmental potential of a single progenitor toward T and/or NK lineages. By examining individual cells with this assay system, the existence of bipotent p-T/NK in FT was unambiguously shown. The unipotent p-T and p-NK detected in this study are also considered to reflect the commitment status in vivo for the following reasons: (a) p-T or p-NK are enriched or purified in subpopulations of FT cells; (b) the addition of IL-2 to the culture medium made p-NK come into view without reducing the frequency of progenitors with T cell potential (our unpublished data), indicating that the progenitors detected as p-NK are distinct from p-T/NK or p-T; (c) detection of p-T/NK but not p-T in the FcR − subpopulation of 12-dpc FT indicated that the NK potential retained by a p-T/NK–type T cell progenitor is very efficiently expressed in this assay; and (d) addition of higher doses of IL-2 (50–100 U/ml) did not increase the frequency of p-NK or p-T/NK (data not shown), strongly suggesting that the progenitors detected as p-T retain no NK potential. With this clonal assay, we clarified that all of the T cell progenitors previously determined in the CD44 + CD25 − FcR − population of 12-dpc FT 21 were bipotent p-T/NK . It has recently been shown that a population capable of generating both T and NK cells is present in fetal spleen and fetal blood of 13–16 dpc fetuses 33 34 , and we have found that a large number of p-T/NK exist in 12-dpc fetal liver (FL; our unpublished data). These findings strongly suggest that the p-T/NK that emerged in prethymic tissues have migrated into the FT. This study further showed that the earliest CD44 + CD25 − FcR − population contained quite a large number of p-NK in addition to p-T/NK. This is in marked contrast to the detection of only a very small number of p-T at early stages . It is likely that the early thymic p-T/NK generate p-NK through uneven cell division, retaining their T/NK bipotent activity. However, the possibility cannot be ruled out that some of these p-NK are of extrathymic origin, as the p-NK have also been detected in FL (our unpublished data). A rapid increase in the proportion of p-T, which are unable to generate NK cells, between the CD44 + CD25 − and CD44 + CD25 + stages may indicate that progenitors are committed to the T cell lineage immediately before TCR-β gene rearrangement. The predominance of p-T at the CD44 + CD25 + stage is compatible with previous findings that the CD44 + CD25 + population scarcely retains NK potential 9 17 . Consistent with this is the finding that the expression of T cell lineage–restricted molecules becomes prominent at this stage 19 35 . It remains to be clarified whether the increase of p-T in this population is due to multiple uneven splits of p-T from p-T/NK or to the proliferation of p-T themselves at this stage. It is well known that NK cell development is independent of the thymus, and NK activity in nu/nu mice is reported to be higher than in normal euthymic mice 4 . Thus, a question to be answered is why the progenitors migrating into the thymus retain NK potential. It is possible that the thymus supplies NK cells to peripheral lymphoid tissues during fetal through neonatal ages, since NK cells are generated first in the FT during ontogeny 29 36 . An alternative interpretation could be that the NK progenitors or a small number of NK cells generated in the FT play a part in inducing the architecture of the thymic microenvironment. Such an idea is based on recent findings that progenitors expressing surface lymphotoxin, which retain NK potential, participate in forming lymph nodes and Peyer's patches 37 38 . However, it seems unlikely that NK progenitors are indispensable for the construction of the thymic environment, since isolated p-T can produce T cells in the dGuo-treated FT lobe . Moreover, thymic T cell development is normal in lymphotoxin knockout mice 39 and Id2 knockout mice 40 that lack lymph nodes and Peyer's patches. There may be no necessity for splitting off NK potential before entering the thymus, since NK potential does not seem to disturb T cell generation in the thymus. On the other hand, it is unlikely that the role of p-NK is to supply NKT cells, as NKT-like cells are generated from p-T but not from p-NK . More detailed investigations are necessary to clarify the role of p-NK or NK cells in the thymus. Fig. 7 illustrates the stages of commitment of p-T/NK to p-T and p-NK revealed by this study. All T cell progenitors in FL are T/NK bipotent, although p-NK also exist in FL (our unpublished data). The developmental potential toward T and NK lineages is intimately kept together until immediately before TCR-β gene rearrangement begins. Nevertheless, p-NK are generated at a rather broad range of differentiation stages, including prethymic stages. Delivery of p-NK in the FT begins at the earliest CD44 + CD25 − FcR − stage, and continues up to the CD44 + CD25 + stage. In contrast, commitment to the T cell lineage, or the delivery of p-T, occurs mainly at the CD44 + CD25 + stage, which is immediately before TCR-β gene rearrangement begins. Such an intimate association of NK potential to the T cell lineage may provide circumstantial evidence for the proposal that T cells have evolved from a prototype of NK cells. To date, no signals, either extracellular or intracellular, mediating commitment of p-T/NK to p-T and p-NK have been determined. It has previously been shown that high doses of IL-2 and IL-15 may interfere with the cell fate choice of progenitors between T and NK lineages 6 . Knockout of IL-2/15Rβ 41 or IL-15Rα 42 gene results in the complete absence of NK cells. Targeting of the IFN regulatory factor 1 (IRF-1) gene, which is indispensable for IL-15 production, also brings about deletion of NK cells 43 . However, the role of cytokines or cytokine receptors seems to be mostly related to the promotion or inhibition of the growth of precursors and/or mature cells rather than to lineage commitment. Some transcription factors (GATA-3, TCF-1) have been reported to be required for T cell development 44 , but it has not yet been clarified whether these molecules are also involved in NK cell development. Molecules that seem to be more directly involved in the branching point between T and NK lineages are natural dominant negative helix-loop-helix factors Id2 and Id3. Transduction of the Id3 gene into CD34 + cells from human FL was found to result in enhancement of NK cell generation at the cost of T cell generation 45 . In the Id2-deficient mice, NK cell development was found to be severely depleted without affecting T cell development 40 . A transcription factor, Ets-1, was also found to play an important role in the generation of NK but not T cells 46 . A combination of these genetic investigations and the single progenitor assay established in this study will contribute significantly to the elucidation of molecular and cellular mechanisms underlying the commitment and differentiation of T and/or NK cells. We are currently setting up such an experimental system. | Study | biomedical | en | 0.999997 |
10587353 | The 3.L2 T cell clone and hybridoma and their maintenance and specificity were previously described 23 24 . The T cell hybridoma 3A9 was described in reference 25. Cells from the murine B cell line CH27, or Hi7, an I-E k –transfected fibroblast line, were used as APCs. Anti-human (h)CD8α antibodies were RPA-T8 (PharMingen) for FACS ® analysis and OKT-8 (American Type Culture Collection [ATCC]) for cross-linking. OKT-8 was purified from tissue culture supernatants using protein A. Rabbit anti–Zap-70 was the gift of A.C. Chan (Washington University, St. Louis, MO). Anti-ζ polyclonal antiserum 777 11 and biotinylated 4G10 (Upstate Biotechnology Inc.) were used for immunoblotting. Phoenix E cells were obtained from ATCC with permission from G. Nolan (Stanford University, Palo Alto, CA). T cell lines were established from splenocytes from 2.102 TCR–transgenic mice bred to recombination activating gene (RAG)-1 −/− mice 26 . For maintenance of 2.102 cell lines, 2 × 10 5 2.102 T cells were restimulated biweekly with 5 × 10 6 irradiated B6.AKR splenocytes, 1 μM peptide Hb(64–76), and 33 U/ml IL-2. For subcloning of 2.102 lines, 2.5 × 10 5 B6.AKR splenocytes, 1 μM Hb(64–76), and 33 U/ml IL-2 was incubated in round-bottomed wells. Antagonist assays of T hybridoma lines were performed as previously described 14 with Hi7 cells as APCs. Proliferation of 2.102 lines was performed as follows: 5 × 10 4 CH27 cells were prepulsed with Hb(64–76), treated with mitomycin C (Sigma Chemical Co.) for 2 h at 37°C, washed three times, and then incubated with 3 × 10 4 2.102 T cells. Thymidine was added after 48 h. 3.L2 and 3A9 cells were transfected by electroporation and selected with Zeocin (Invitrogen Corp.). Phoenix E cells were transfected using CaCl 2 for production of high titer retroviral supernatants 27 . 2.102 T cells were infected with retroviral supernatants 3 d after activation 27 . The chimera between the extracellular and transmembrane part of human CD8α and intracellular murine ζ was made similar to that described in reference 28 by PCR using the primers T7 and acim8z (5′-TCC TGC TGA ATT TTG CTC TGT TGC AGT AAA GGG TGA TA-3′) and T3 and cim8z (5′-TAT CAC CCT TTA CTG CAA CAG AGC AAA ATT CAG CAG GA-3′). pcDNAZeo (Invitrogen Corp.) or the retroviral green fluorescent protein (GFP)-RV 27 were used as plasmids. Tyrosines were replaced by phenylalanines using PCR as in references 11 and 29 . A stop codon was introduced after seven amino acids of intracellular ζ with the primers 5′-TTC AGC AGG AGT TAA GAG ACT GCT GCC-3′ and 5′-AAG TCG TCC TCA ATT CTC TGA CGA CGG-3′. Transfected 3.L2 hybridoma cells (2 × 10 7 ) were incubated with 5 μg OKT-8 on ice and activated at 37°C for 5 min. Cells were lysed in 500 μl lysis buffer 17 . hCD8-ζ was precipitated using OKT-8 and protein A–Sepharose and analyzed by phosphotyrosine blotting using biotin–4G10 (Upstate Biotechnology Inc.) and horseradish peroxidase–streptavidin (Southern Biotechnology Associates Inc.). The activation of 3.L2 T cell clones with peptide-pulsed Hi7 cells was previously described 17 . Phosphotyrosine bands were quantitated using ImageQuant (version 1.1; Molecular Dynamics). The TCR ζ chain is constitutively phosphorylated in resting peripheral T cells. To investigate constitutive as well as induced TCR-ζ phosphorylation, we first examined the 3.L2 T cell clone. 3.L2 is specific for peptide Hb(64–76)/I-E k 23 . Peptide Hb(64–76) is derived from an allelic form of murine hemoglobin protein, amino acids 64–76. In rested 3.L2 T cells, ζ was phosphorylated, as apparent in a phospho species of 21 kD termed p21 . Stimulation of 3.L2 with fully stimulating agonist peptide Hb(64–76) presented on APCs augmented ζ phosphorylation and led to the appearance of an additional phospho species of 23 kD called p23 . The 3.L2 TCR can also interact with a spectrum of peptide ligands related to Hb(64–76) that have a one–amino acid substitution at position 72 or 73 of the original protein sequence 23 24 . The response of the 3.L2 TCR to peptide Hb(64–76) can be antagonized by such specific analogue ligands . We used four analogue peptide ligands of Hb(64–76), termed D73, I72, A72, and G72. These peptides had the ability to inhibit proliferation of 3.L2 in a dose-dependent manner, as previously observed . This was a specific effect, as the control peptide E72 did not inhibit proliferation, although it can bind to I-E k as well as peptides D73, I72, A72, and G72. Exposure of 3.L2 to the four antagonist ligands D73, I72, A72, and G72 also induced an increase in ζ phosphorylation . However, more phosphorylation of p21 than p23 was induced, resulting in a change in the ratio of p23/p21. This type of ζ phosphorylation has previously been termed altered ζ phosphorylation and is induced by many antagonist ligands. Lastly, the control peptide E72 induced no augmentation in ζ phosphorylation of the 3.L2 T cell . Antagonism of the 3.L2 T cell only occurs at certain concentrations of agonist and antagonist ligands. For the experiment shown in Fig. 1 b, the agonist Hb(64–76) was used at the low but stimulating concentration of 10 −7 M. At this dose, only very little ζ phosphorylation was induced by Hb(64–76) . Peptides G72, A72, I72, and D73 strongly induced antagonism when they were used at higher concentrations of 10 −5 –10 −4 M. At such concentrations, the antagonist peptides induced very strong ζ phosphorylation . Thus, at concentrations required for antagonism, antagonist peptides induced more ζ phosphorylation than the agonist peptide. This correlated with inhibition of T cell activation rather than activation. In addition, stronger antagonist peptides induced more ζ phosphorylation than weaker antagonist peptides at the same concentration . Therefore, the strength of the altered ζ phosphorylation signal correlated with the strength of T cell inhibition. We recently reported that the six tyrosines of the TCR ζ chain are not phosphorylated to an equivalent degree 11 . Two main tyrosines, B1 and C2, are specifically phosphorylated in resting T cells . Upon activation with fully stimulating ligands, A1 and A2 next become phosphorylated. Tyrosines B2 and C1 are phosphorylated last, resulting in the full phosphorylation of ζ. In contrast, after stimulation with antagonist ligands, only phosphorylation of the basal sites B1 and C2 is markedly increased. A1 and A2 are also inducibly phosphorylated, albeit only slightly. The final phosphorylation of B2 and C1 is absent. Thus, A1 and A2 phosphorylation is necessary but not sufficient for the subsequent phosphorylation of C1 and B2, respectively. In conclusion, the majority of phosphorylated ζ molecules induced by antagonist ligands have only B1 and C2 phosphorylated. These findings raise the question of whether the distinct phosphorylated ζ species found in resting T cells or after stimulation with either agonist or antagonist ligands have an effect on T cell activation. To directly test whether the partially phosphorylated species found in T cells can inhibit T cell activation, we substituted multiple tyrosines in ζ with phenylalanine to prevent phosphorylation of these sites. This resulted in proteins that could only be phosphorylated in the pattern reflecting ζ phosphorylation as identified in T cells 11 . In particular, we designed mutants that allowed for the precise phosphorylation found in resting T cells or phosphorylation induced by antagonist or agonist peptides . We designed these mutants as chimeric proteins with hCD8α to provide a specific extracellular means for antibody cross-linking and for quantifying expression 28 . The construct with four substitutions from tyrosine to phenylalanine was termed 4F, and it retained only two intact tyrosines for phosphorylation, B1 and C2 . In resting T cells, these two main tyrosines were found to be phosphorylated with a high degree of specificity. In addition, stimulation with antagonist peptides led to a marked augmentation of phosphorylation at B1 and C2 when compared with resting T cells. Therefore, the 4F mutant not only reflected the phosphorylation pattern of resting T cells but also the main pattern induced by antagonist ligands. The construct 3F, with three tyrosine mutations, reflected an intermediate species. It allowed for the additional phosphorylation of tyrosine A2. A2 has been found to be phosphorylated to a minor degree in resting T cells. This was most likely because phosphorylation of A2 varied depending on the resting state of T cells 11 . Phospho-A2 was also induced by antagonist ligands. Thus, we could test a possible function of A2, B1, and C2 phosphorylation with the 3F mutant. The 2F mutant allowed the additional phosphorylation of A1. One ITAM, ITAM A, could now be fully phosphorylated, but B2 and C1 were mutated. A1 and A2 phosphorylation is required for further phosphorylation of B2 and C1. However, after stimulation with antagonist peptides, A1 and A2 are found to be slightly phosphorylated without the subsequent phosphorylation of B2 and C1. Thus, the 2F mutant represents a minor species present in antagonist-activated cells but not in resting cells, and it was important to test its function. 2F also reflects an intermediate species in cells activated with agonist ligands, where A1 and A2 are phosphorylated to an extensive degree before ζ then becomes fully phosphorylated. Several control proteins were designed: a protein without mutations in ζ, termed Wt, and two proteins with no tyrosines available for phosphorylation, termed 6F and Stop. 4OF was another control that reflected a pattern of phosphorylation not found in T cell activation. The mutants were transfected into a hybridoma derived from the 3.L2 T cell. Several clones for each construct were positive for surface expression of hCD8α. Transfected 3.L2 cells with similar hCD8-ζ expression levels were further analyzed . All tested transfectants retained their ability to respond to the Hb(64–76)/I-E k peptide in a dose-dependent manner by IL-2 production , indicating that their endogenous 3.L2 receptor was intact after the transfection procedure. Transfection with the different hCD8-ζ mutants did not significantly affect the sensitivity of TCR-mediated IL-2 production for any of the transfectant clones tested. We also determined an intermediate dose of Hb(64–76) that stimulated IL-2 production within the linear part of the dose–response curve for each transfectant . Next, we stimulated the transfected cell lines through their endogenous 3.L2 TCRs with an intermediate dose of Hb(64–76) peptide and APCs. This resulted in TCR-mediated IL-2 production within the linear portion of the dose–response curve . At the same time, we cross-linked the hCD8-ζ proteins by adding anti-hCD8α antibodies and measured IL-2 production . The experiments shown in Fig. 2 , right panels, and Fig. 3 were performed on the same day for each transfectant, so the degree of peptide stimulation indicated with the dotted lines in Fig. 3 can be directly compared with the dose–response curve in Fig. 2 . Such treatment led to an augmentation of IL-2 production in the Wt-transfected cell lines . IL-2 production was also induced when the cell line was stimulated through the Wt hCD8-ζ mutant alone (data not shown). This indicated that unmutated intracellular ζ induced T cell activation, consistent with previous observations 1 2 3 . No effect was observed when the 6F or Stop mutants were engaged , as tested in two independent clones for 6F and one clone for Stop. Thus, the presence of the hCD8-ζ molecule without tyrosines had no effect on T cell activation. Interestingly, the IL-2 response to the TCR ligand was inhibited when the 4F protein was cross-linked in two independent clones . Inhibition of the 3.L2 response was dose dependent, with 100% inhibition occurring at the highest concentration of OKT-8. The 3F mutant was also inhibitory in two independent transfectants . Cross-linking of 2F augmented the IL-2 response induced through the endogenous TCR . It also induced IL-2 production when cross-linked alone (data not shown). Thus, this mutant had an activating effect on T cell activation, consistent with previous studies of single intact ITAMs 1 . This indicated that the presence of one intact ITAM in 2F overcame the inhibitory effect of tyrosines B1 and C2. The effect of all hCD8-ζ mutants was maximal when TCR-mediated IL-2 production was intermediate and within the linear portion of the dose–response curve. When TCR-mediated stimulation was saturated, inhibition as well as augmentation of IL-2 production was not noticeable (data not shown). Thus, the concentration requirements for stimulation were similar when inhibition was mediated by TCR antagonism and by hCD8-ζ mutants. In conclusion, we identified two inhibitory ζ mutants, 4F and 3F. These proteins contained two or three partially impaired ITAMs and lacked intact ITAMs. Thus, we demonstrated that the ζ species that represented phosphorylation in resting T cells can inhibit T cell activation. Such species are also strongly induced by antagonist ligands. Next, we tested whether the inhibitory effect was specifically due to tyrosines B1 and C2 or whether the combination of other tyrosines could also inhibit T cell activation. B1 and C2 were mutated to phenylalanine, as was ITAM A. This left tyrosines B2 and C1 intact . Three independent transfectants were examined. The presence of hCD8-ζ 4OF did not alter the sensitivity of the 3.L2 TCR . Cross-linking of 4OF could also inhibit TCR-mediated T cell activation in all three clones. Inhibition was as efficient as with the 4F and 3F mutants. This indicated that the inhibitory effect was not dependent on tyrosines B1 and C2. Rather, other hemi-ITAMs could also inhibit T cell activation when intact ITAMs were absent. To test whether the inhibitory effect of ζ mutants was also observed in other cell lines, we transfected 4F and 6F into the 3A9 T cell hybridoma. 3A9 is specific for peptide HEL(48–62)/I-A k 25 , a peptide derived from hen egg lysozyme, amino acids 48–62. Two positive clones for each construct were identified by expression of hCD8α, and their responsiveness to HEL/I-A k was confirmed (not shown). Cross-linking of 4F but not 6F led to an inhibition of TCR-induced IL-2 production in a dose-dependent fashion (data not shown) in both independent clones tested for each construct. Thus, the inhibitory effect of partially phosphorylated ζ species was not limited to the 3.L2 T cell. We next examined the tyrosine phosphorylation state of the hCD8-ζ 6F, 4F, and Wt proteins in the transfected 3.L2 hybridoma lines. Untransfected 3.L2 or 3.L2 transfected with 6F, 4F, or Wt was incubated with anti-hCD8α antibodies and left on ice or antibody cross-linked . Cells were then lysed, the hCD8-ζ mutants were precipitated, and tyrosine phosphorylation of the chimeric hCD8-ζ protein was analyzed. In contrast to 6F, both 4F and Wt were tyrosine phosphorylated. Interestingly, 4F and Wt were both constitutively tyrosine phosphorylated to a similar extent before cross-linking of hCD8 . This was reminiscent of constitutive phosphorylation of ζ in resting T cells. The electrophoretic mobilities of 4F and Wt differed from each other. This might be because point mutations in ζ can affect its mobility significantly 11 . Alternatively, hCD8-ζ Wt could be phosphorylated to a higher degree than 4F, giving rise to a lesser mobility. In this case, the extent of phosphorylation of the hCD8-ζ Wt protein in the hybridoma cell line might be higher than that of endogenous TCR-ζ in resting nonimmortalized T cells. Phosphorylation of hCD8-ζ Wt significantly increased with activation, whereas phosphorylation of 4F was unchanged. This might be due to the binding of Zap-70 to fully phosphorylated ITAMs in hCD8-ζ Wt, which can protect tyrosines from dephosphorylation 30 . Binding of Zap-70 to phospho-4F might be impaired because no fully phosphorylated ITAMs are present, resulting in the rapid dephosphorylation of phospho-4F. Alternatively, the effect of cross-linking of the constitutively phosphorylated 4F mutant might be exerted through aggregation or association with the endogenous TCR and not through an increase in phosphorylation. All transfected cell lines expressed hCD8-ζ protein . hCD8-ζ appeared as two separate bands in immunoblotting, most likely due to glycosylation 28 . In conclusion, the remaining intact tyrosines in the inhibitory mutant 4F were phosphorylated. As 6F had no effect on T cell activation and was not phosphorylated, the inhibitory effect of 4F was most likely exerted through phosphorylated tyrosines. We next examined whether ζ mutants could also inhibit proliferation of nonimmortalized T cell lines. We used T cells from 2.102 TCR–transgenic mice on a RAG-1 −/− background 26 . 2.102 T cells are specific for Hb(64–76)/I-E k but express a different α/β TCR than 3.L2. We transduced such splenocytes by retroviral infection techniques using a bicistronic retrovirus coexpressing hCD8-ζ mutants and GFP 27 . This enabled us to identify transfected T cells by the expression of GFP without antibody cross-linking of the hCD8α mutants. We infected activated 2.102 splenocytes with retroviral supernatants containing empty vector or hCD8-ζ Wt, 6F, and 3F on two independent occasions. GFP-positive splenocytes were FACS ® sorted, expanded, and subcloned. Cells were then analyzed for the expression of hCD8α and GFP by FACS ® . Cells transduced with vector alone expressed GFP, indicating that the infection procedure was successful. Cells transduced with hCD8-ζ Wt, 6F, and 3F expressed both GFP and hCD8α. All cell cultures responded to Hb(64–76)/I-E k by proliferating with similar responsiveness, indicating that the endogenous 2.102 receptor was still intact (data not shown). We next examined the effect of the hCD8-ζ mutants. T cells were either exposed to a stimulating dose of Hb(64–76)/I-E k alone or simultaneously exposed to Hb(64–76)/I-E k and increasing doses of anti-hCD8α . Such treatment had no effect on the vector alone or 6F-transduced cells . Thus, neither the presence of anti-hCD8α antibodies nor of extracellular hCD8α had an effect on T cell proliferation. Cross-linking of hCD8-ζ Wt induced an increase in proliferation, indicating an activating effect . When the 3F mutant was cross-linked, proliferation to peptide-pulsed APCs was inhibited in a dose-dependent manner . This demonstrated that partially phosphorylated ζ species could also inhibit T cell activation in nonimmortalized T cells. Here we demonstrate that the type of TCR-ζ phosphorylation present in resting T cells can inhibit T cell activation. Thus, the constitutive phosphorylation of the TCR complex may not reflect a low level of ongoing T cell activation. Rather, it may serve to prevent any unintended T cell activation. TCR-ζ phosphorylation induced by antagonist ligands can also inhibit T cell activation. Our analysis therefore also supports the concept that TCR antagonism can occur through the generation of an inhibitory signal within the TCR complex. Our findings raise questions about the mechanism of T cell inhibition. We think that the inhibitory effect can be exerted through the action of proteins that specifically bind partially phosphorylated ζ. Such proteins could associate with ζ through single SH2 domains or through other protein–protein interactions. During full T cell activation, the stronger, cooperative binding of the tandem SH2 domains of Zap-70 to doubly phosphorylated ITAMs might displace these proteins. One possibility is that binding proteins could be inhibitory phosphatases that are brought into the vicinity of the endogenous TCR when inhibitory hCD8-ζ mutants are antibody cross-linked. Alternatively, binding proteins could be positively acting kinases present in limiting amounts. Through their association with ζ, kinases might become sequestered and then might no longer be available to mediate T cell activation. A similar mechanism has been identified for antagonist inhibition of the high-affinity receptor for the Fc portion of IgE, Fc∈RI 31 . We overexpressed chimeric proteins and antibody cross-linked their extracellular portion to study the role of partially phosphorylated ITAMs. Similar approaches have been widely used to establish the function and importance of intact ITAMs and their signal amplification ability 1 2 3 . In such studies, a multitude of ζ ITAM mutants were examined for their potential to stimulate T cells 1 2 5 6 7 . Here we further extended the use of this experimental system and expressed such chimeras in cells with functional endogenous TCRs of known specificity. This enabled us to study the previously unaddressed question of whether mutant ITAMs can also inhibit T cell activation. We used hCD8-ζ chimeras because similar proteins with human intracellular ζ have been previously employed 1 28 . hCD8-ζ does not associate with components of the endogenous TCR complex before antibody cross-linking 28 . It therefore does not interfere with regulation of TCR signaling in the absence of cross-linking. In addition, the chimeric protein could homodimerize, much like endogenous ζ. Ardouin et al. 9 have recently published a report that T cells from a P14 TCR–transgenic mouse lacking full ζ ITAMs functioned normally. The mutant ζ, a 1- ,b 1- ,c 1- , had the amino terminal tyrosine of each ITAM converted to a phenylalanine. These findings clearly showed that intact ζ ITAMs are not needed for T cell development and function and that the four CD3 ITAMs are sufficient. In our system, we did not examine the identical mutant, because we have previously not found a TCR-ζ species phosphorylated simultaneously at tyrosines A2, B2, and C2 in T cells 11 . Our 3F mutant was the most similar but with different tyrosines remaining. Based upon our findings, we would have predicted that the a 1- ,b 1- ,c 1- mutant would be inhibitory; however, this was not observed. One possible explanation was that only certain tyrosine residues were inhibitory. Arguing against this is our result with the 4OF mutant. An alternative explanation is that the remaining tyrosines in the ζ molecule must be phosphorylated to be inhibitory. Consistent with this, the a 1- ,b 1- ,c 1- mutant was not found to be phosphorylated, even upon pervanadate stimulation. We studied the function of TCR-ζ, although TCR-ζ ITAMs are not essential for T cell development and function, and CD3ζ −/− mice reconstituted with ζ lacking all three ITAMs appear largely normal. Clearly, the 10 TCR/CD3 ITAMs have overlapping functions, but we contend that their number and ordered phosphorylation collectively provide the TCR with a mechanism by which it can discriminate between closely related ligands. In the ζ ITAM-less mice mentioned, the selection of T cells with specific TCRs is altered, and autoreactive T cells can be found 10 . One difference between the components of the TCR complex is that CD3∈, CD3γ, or CD3δ is not constitutively phosphorylated in resting peripheral T cells, whereas CD3ζ is. T cells from mice expressing normal CD3∈, CD3γ, and CD3δ but mutated CD3ζ without ITAMs would therefore lack the inhibitory signal from partially phosphorylated ζ. Thus, the autoreactivity found in such mice might not only be due to altered thymic selection but also to increased peripheral reactivity. To elucidate the mechanism of TCR antagonism, two different known TCRs have recently been expressed on the same T cell 32 33 34 . “Cross-antagonism” of the two TCRs would indicate that an inhibitory signal leads to TCR antagonism. The outcome of the experiment varied, and cross-antagonism was observed in some but not all cases. This can be explained if TCR antagonism is induced by differing mechanisms 35 . Alternatively, depending on the expression level and spatial separation of the two expressed TCRs, a local inhibitory signal through one α/β TCR may or may not affect signaling through a second TCR. TCR antagonism can occur in the absence of functional ζ 9 36 . We think that CD3∈, CD3γ, or CD3δ can substitute for ζ ITAMs in the absence of ζ, as these proteins can also substitute for ζ in T cell development. It remains to be addressed whether CD3∈, CD3γ, or CD3δ can become partially phosphorylated after stimulation with antagonist ligands. CD3∈ was not phosphorylated after stimulation with altered peptide ligands 17 18 19 , but we recently found that stimulation of the 3.L2 TCR with antagonist ligands induces phosphorylation of CD3∈ (Kersh, E.N., and P.M. Allen, unpublished results). Different cell lines might therefore differ in their efficiency to induce CD3∈ phosphorylation, or certain antagonist ligands might be more effective in inducing CD3∈ phosphorylation. Our analysis of the 2F mutant provided insight into the hierarchy of stimulatory versus inhibitory ITAMs. In the 2F mutant, functional ITAM A mediated T cell activation despite the presence of inhibitory tyrosines B1 and C2. This result was unanticipated, because in the 4F mutant, B1 and C2 alone had the ability to inhibit TCR-mediated IL-2 production. TCR-mediated IL-2 production is most likely also the result of the phosphorylation of functional ITAMs but within the TCR complex. However, the effect of such phospho-ITAMs in the TCR complex could be inhibited by the presence of B1 and C2 in the 4F mutant. We think that the decision over activation or inhibition is made based on a concentration difference of fully or partially phosphorylated ITAMs: for inhibition, partially phosphorylated ITAMs must far outnumber fully phosphorylated ITAMs. By overexpression of 3F and 4F, fully phosphorylated ITAMs in the endogenous TCR complex were outnumbered. However, in the 2F mutant, B1 and C2 could not outnumber ITAM A, and therefore activation resulted. In conclusion, we find that basal TCR-ζ phosphorylation in resting peripheral T cells inhibits T cell activation, as do the subsequent first phosphorylation events and ζ phosphorylation induced by antagonist ligands. These findings add to our understanding of the complexity of signal initiation at the TCR and strengthen our earlier interpretation that sequential ζ phosphorylation steps increase the fidelity of T cell activation 11 . | Study | biomedical | en | 0.999997 |
10587354 | HSV-1 strain NS, a low passage clinical isolate, was the parental strain for the mutant viruses. NS-gCnull virus has the entire gC protein coding sequence replaced by a lacZ expression cassette under the control of the HSV-1 infected cell protein 6 early promoter, as previously described 23 . NS-gCΔC5/P virus has a deletion of the gC domain involved in blocking C5 and P binding to C3 9 21 . This deletion was constructed from the NS BamH1 “I” fragment by cutting with Nsi1 and EcoR1 and cloning this fragment (nucleotides 94,911–96,751) into pGEM7Z (Promega Corp.) to create plasmid pLW1. An Nhe1–EcoR1 fragment (nucleotides 96,276–96,751) was excised from pLW1 and cut with Apa1 to delete 91 gC amino acids (amino acids 33–123; nucleotides 96,407–96,679). The Nhe1–EcoR1 fragment deleted of gC amino acids 33–123 was inserted back into pLW1, and then the Nsi1–EcoR1 fragment from pLW1 was cloned back into the BamH1 I fragment to create a flanking sequence vector with a deletion of the gC C5/P domain. gCΔC3 has a deletion of 92 amino acids (gC amino acids 275–367), prepared by cutting plasmids B29 and H71 32 with BglII, religating the 5′ portion of B29 with the 3′ portion of H71, and then cloning this fragment into the EcoR1–EcoRV site of the BamH1 I fragment. The construction adds a 12-mer BglII linker after gC amino acid 275 and creates a flanking sequence vector that has a deletion of two gC C3 binding regions, resulting in a net loss of 88 amino acids. A flanking sequence vector to construct gCΔC5/P,C3 was prepared by replacing the BamH1 I EcoR1–EcoRV fragment in the gCΔC5/P flanking sequence vector with the EcoR1–EcoRV fragment of the gCΔC3 flanking sequence vector. Recombinant viruses were prepared in Vero cells by cotransfection using NS-gCnull virus DNA and the various flanking sequence vectors. Recombinant viruses were identified by anti-gC immunoperoxidase staining of infected cells 21 . Virus pools were prepared after three rounds of plaque purification. Vero cells were infected at an MOI of 5 for 20 h at 37°C, and then virus from supernatant fluids was purified on a 5–65% sucrose gradient 23 . The visible virus band was collected, dialyzed against PBS at 4°C, and stored at −70°C. Virus titers were determined by plaque assay on Vero cells. Vero cells were inoculated at an MOI of 5, and at 1, 4, 8, 12, 20, and 24 h after infection, cells plus supernatant fluids were harvested, cells were lysed by sonication, and viral titers were determined by plaque assay. DNA was digested with BamH1 and HincII, electrophoresed in 1.2% agarose gels, transferred to Immobilon-S membranes (Millipore Corp.), and UV cross-linked. A biotin-labeled gC-1 fragment spanning the entire protein coding sequence (nucleotides 96,276–97,945) was used as probe, and bands were detected by chemiluminescence (New England Biolabs) 6 . Purified virus was run on 10% SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp.), and visualized using polyclonal rabbit anti-gC antibody and horseradish peroxidase–conjugated goat anti–rabbit IgG (Amersham Pharmacia Biotech) 23 . Neutralization assays were performed by incubating 10 4 –10 5 PFU of purified virus with HSV-1 and -2–seronegative human serum as the source of complement for 1 h at 37°C. As a control, complement was inactivated by heat treatment at 56°C for 30 min. Virus titers were then determined by plaque assay on Vero cells. Complement neutralization was calculated as the difference in titers between virus incubated with active or heat-inactivated serum 23 . Vero cells were infected at an MOI of 2 for 20 h, and then cells were removed using cell dissociation buffer (GIBCO BRL), incubated with C3b-coated erythrocytes for 1 h at 37°C, and viewed by microscopy for rosettes. Cells with four or more bound erythrocytes were considered positive 23 . Human C3 was purified according to Hammer et al. 33 with modifications. 20 parts human plasma were treated with 1 part inhibitor containing 1 M KH 2 PO 4 , 0.2 M Na 4 EDTA, 0.2 M benzamidine, and 1 mM PMSF. C3 was precipitated from plasma first with 4.5% polyethylene glycol (PEG) and then with 12% PEG at 0°C. The pellet was dissolved in buffer A (20 mM Na 2 HPO 4 , pH 7.4) and loaded onto a DEAE 40HR column (5 × 6.3 cm; Waters) preequilibrated with buffer A. The bound proteins were eluted using a 0–0.5 M NaCl linear salt gradient. Fractions containing C3 were detected by SDS-PAGE and immunodiffusion, dialyzed against buffer A, loaded onto a Mono Q HR 10/10 column (Amersham Pharmacia Biotech), and eluted with a linear salt gradient to 0.5 M NaCl. Homogeneous C3 fractions were identified by SDS-PAGE and immunodiffusion, pooled, and dialyzed against PBS (10 mM Na 2 HPO 4 and 145 mM NaCl, pH 7.4). The purified C3 contained 80% native C3 and 20% C3(H 2 O), as determined by analyzing the protein sample on a Mono S column (Amersham Pharmacia Biotech) 34 . The flank model and the C3 knockout mice derived from C57BL/6 mice have been described previously 26 35 . In brief, purified virus was scratched onto the flank that was shaved and chemically denuded. Disease at the inoculation site first appeared on day 3 and was scored as follows: erythema or swelling with no vesicles was assigned 0.5 points; individual vesicles were scored as 1 point each with a total maximum daily score of 5. If lesions coalesced, up to 5 points were assigned based on the size of the lesions. Zosteriform disease first appeared on day 4 or 5 and was scored similarly, except that the maximum daily score was 10 because more lesions could be counted over the larger skin area involved. C3 reconstitution experiments were performed by intraperitoneal injection of C3 knockout mice using 3 mg of human C3 per mouse. C3 knockout mouse serum was assayed for total hemolytic complement activity before and after reconstitution with human C3 as described 36 . Features of wild-type gC and three gC mutants are shown in Fig. 1 . Wild-type gC has four C3 binding regions (light gray), each of which is required to bind C3 32 . The gC domain that interferes with C5 and P binding to C3 is located at the NH 2 -terminal region between residues 33 and 123 9 21 . gCΔC5/P mutant contains a deletion of gC amino acids 33–123. gCΔC3 mutant has a deletion of amino acids 275–367 that includes C3 binding regions II and III. This deletion removes a disulfide-bonded cysteine pair 37 to minimize the effect of the deletion on gC conformation. gCΔC5/P,C3 mutant has both domains deleted that interact with complement. The mutant gC proteins were recombined into virus, and the size of the gC DNA was evaluated by Southern blot analysis. HSV-1 was cut with BamH1 and HincII to cut once within the gC gene and probed with sequences spanning the entire gC protein coding sequence. Fig. 2 A (left blot) shows two gC bands for wild-type NS and smaller upper bands for gC mutant viruses NS-gCΔC5/P, C3 and NS-gCΔC3; the right blot shows the smaller upper band for gC mutant NS-gCΔC5/P. Bands were of the expected size for each of the gC mutant viruses. Western blots were performed on purified virus to demonstrate that mutant gC glycoproteins are incorporated into the virion and are of the expected size . The double mutant virus NS-gCΔC5/P,C3 makes the smallest of the three mutant proteins. NS-gCΔC5/P has a deletion of 91 amino acids and on SDS-PAGE runs smaller than NS-gCΔC3, which has a deletion of 88 amino acids. However, the difference in migration of the two glycoproteins was greater than expected based on amino acid size. A likely explanation is that five N-linked glycosylation sites and numerous predicted O-linked glycosylation sites lie within the deleted domain of NS-gCΔC5/P, whereas one N-linked and few O-linked glycosylation sites lie within the deleted fragment of NS-gCΔC3. The kinetics of virus replication and peak titers achieved for each gC mutant virus were similar to those of wild-type virus when evaluated by single-step growth curves (result not shown). gC protein expression at the infected cell surface was confirmed for each mutant virus by immunoperoxidase staining, and the ability of gC to bind C3b was determined by rosetting assays. Mutant viruses NS-gCΔC3, NS-gCΔC5/P,C3, and NS-gCnull lack the C3 binding domain and fail to form C3b rosettes, whereas NS and NS-gCΔC5/P mutant viruses have the C3 binding domain and form rosettes . The results suggest that the gC mutations have not drastically altered protein conformation, as mutant virus NS-gCΔC5/P formed rosettes, and each mutant virus incorporated gC into the envelope and expressed gC at the infected cell surface. Previously, we reported that HSV-1 lacking the entire gC protein is rapidly neutralized by human complement, showing 50% neutralization within 2 min and 100–5,000-fold neutralization by 1 h, the higher number occurring if fresh complement was added during the hour-long incubation. In contrast, wild-type virus is resistant to complement (less than or equal to twofold neutralization; reference 23). The panel of gC mutant viruses was incubated with HSV-1 and -2 antibody–negative human serum as source of complement for 1 h or complement-inactivated serum as a control, and viral titers were determined. A glycoprotein gE mutant virus derived from NS 6 served as a control for the effects of removing a glycoprotein from the virion envelope. gE lacks complement interacting domains. Complement has a small effect on NS and NS-gEnull viruses . gC mutant virus lacking the C5/P blocking domain was relatively resistant to complement (0.43 log 10 , P = 0.22 or 0.76, comparing NS-gCΔC5/P with NS or NS-gEnull, respectively). The gC mutant virus lacking the C3 binding domain was somewhat more susceptible to complement (0.66 log 10 , P = 0.01 or 0.1, comparing NS-gCΔC3 with NS or NS-gEnull, respectively); however, gC mutant virus lacking both domains was even more susceptible to complement (1.58 log 10 , P ≤ 0.001, comparing NS-gCΔC5/P,C3 with NS or gE null virus) and significantly more susceptible than either single gC mutant virus . We conclude that both gC domains are important in complement regulation, as the gC double mutant virus was more susceptible to complement than either single mutant virus alone. Studies were performed to determine if the complement-interacting domains are virulence factors in vivo using the murine flank model 26 35 . Fig. 5A and Fig. B show inoculation site and zosteriform disease scores, respectively, for complement-intact mice infected with 5 × 10 4 PFU of NS or gC mutant viruses. Disease scores at the inoculation and zosteriform sites are lower for mutant virus NS-gCΔC5/P than for NS ( P ≤ 0.02 for days 4–8 at the inoculation site, and P < 0.001 for day 5 at the zosteriform site), supporting the conclusion that the C5/P domain is a virulence factor. Even greater differences were detected comparing NS-gCΔC3 or double mutant virus NS-gCΔC5/P,C3 with NS ( P < 0.001 for days 5–8 at the inoculation and zosteriform sites). Notably, NS-gCΔC3 virus caused significantly less disease than NS-gCΔC5/P virus ( P < 0.001 for days 6–8 at the inoculation and zosteriform sites). We conclude that both gC domains have a significant effect on disease severity; however, the C3 domain is more important because the mutant virus lacking this domain is significantly less virulent than the C5/P mutant virus and is as impaired as the double mutant virus. Experiments were performed in C3 knockout mice to determine if interactions between gC and complement account for the lowered virulence of gC mutant viruses. Virulence of gC mutant viruses should be similar to wild-type virus in C3 knockout mice, as C5, P, or C3 cannot become activated. Fig. 5C and Fig. D show that virulence for each of the mutant viruses is comparable to wild-type virus in C3 knockout mice. As further evidence for the importance of the interaction between gC and complement, C3 knockout mice were reconstituted with C3. Total hemolytic complement activity was undetectable in serum from C3 knockout mice; however, after injection of human C3, total hemolytic complement activity was restored to levels detected in complement-intact animals (result not shown). Fig. 5E and Fig. F show that the gC double mutant virus NS-gCΔC5/P,C3 is markedly less virulent in C3 knockout mice reconstituted with C3, whereas the virulence of wild-type virus is unchanged (comparing NS and gC double mutant virus: inoculation site lesions, P < 0.05 on day 3, P < 0.01 on day 4, and P < 0.001 on days 5–8, whereas for zosteriform lesions, P < 0.001 on days 5–8). Fig. 6 shows photographs of flank lesions on day 6 after infection. The left panels show NS, and the right panels show NS-gCΔC5/P,C3, demonstrating that lesions caused by NS are comparable in the presence or absence of complement, whereas lesions produced by the gC mutant virus are greatly affected by complement. These experiments illustrate the important role of gC–complement interactions in pathogenesis. Experiments were performed using NS and gC double mutant virus NS-gCΔC5/P,C3 to compare disease scores over a range of inocula. In Fig. 7 , the disease scores are expressed as the average cumulative disease scores from days 3–8 after infection 26 . In complement-intact mice, infection with 5 × 10 5 PFU of the gC double mutant virus produced a disease score of 19.3 ± 1.9 at the inoculation site . Producing a comparable disease score required 50-fold less wild-type virus. Infection with 5 × 10 4 PFU of gC double mutant virus resulted in a disease score of 8.9 ± 0.9, which also correlated with the disease caused by 50-fold less wild-type virus. Similarly, ∼50-fold less NS than gC mutant virus was required to produce comparable zosteriform disease . In marked contrast, in C3 knockout mice, no significant differences were noted comparing wild-type and gC mutant viruses . We conclude that domains on gC that interact with complement account for a 50-fold effect on virulence in complement-intact animals, whereas in C3 knockout animals, the domains have no effect. Notably, NS disease scores are virtually identical in complement-intact and C3 knockout mice , which indicates that gC complement–interacting domains are remarkably effective in protecting wild-type virus from complement attack. We have previously defined a role for gC in binding C3 8 9 19 , accelerating the decay of the alternative pathway C3 convertase 20 and interfering with the interaction of C5 and P with C3b 9 21 . We showed that gCnull virus is highly susceptible to complement neutralization even in the absence of antibodies and that gCnull virus is less virulent in mice and guinea pigs 23 26 . We now address the contribution to pathogenesis of two gC domains that interact with complement by constructing gC mutant viruses lacking the C5/P blocking domain, the C3 binding domain, or both domains. The murine studies indicate that each of the single mutant viruses is significantly less virulent than wild-type virus; however, the C3 binding domain mutant virus is clearly the more important, as this mutant virus was as attenuated as the gC double mutant virus. Fig. 8 A presents a model of complement inhibition mediated by gC domains. In vivo, NS-gCΔC5/P mutant virus is only moderately less virulent than wild-type virus, suggesting that gC affects more than C5 and P binding to C3b, such as accelerating the decay of the alternative pathway C3 convertase, C3bBb 20 , or perhaps other C3-mediated functions yet to be determined . The deletion of the C3 binding domain eliminates gC binding to C3b , rendering the C5/P domain ineffective because it lacks proximity to C3b and cannot hinder C5 and P binding. Therefore, a C3 binding domain mutant virus should be as impaired as a double mutant virus . The model explains our in vivo results, but it fails to explain why the C3 binding mutant virus was not as impaired as the double mutant virus in neutralization assays performed in vitro. A possible explanation is that the main effect of gC in vivo may be on a function other than neutralization, such as preventing complement lysis of infected cells 24 . Our results definitively demonstrate that gC is a virulence factor because it regulates complement activation. This conclusion is based on observations that gC mutant viruses are significantly less virulent than wild-type virus in complement-intact mice, whereas virulence is comparable to that of wild-type virus in C3 knockout mice. Proof for the requirement for C3 was shown by C3 reconstitution experiments in which C3 reduced virulence of the gC double mutant virus but had no effect on wild-type virus. The magnitude of the gC effect was addressed by evaluating lesion scores using inoculation titers ranging from 5 × 10 5 to 5 × 10 2 PFU. In complement-intact mice, 50-fold more gC double mutant virus was required to cause disease comparable to that caused by wild-type virus. In vitro studies indicate that gC is also involved in the initial stage of virus attachment to cells by binding to heparan sulfate 38 39 . In the absence of gC, gB can mediate this effect 38 , but a role for gC in attachment has not been established in vivo. Our results indicate that the domains deleted from the gC mutant viruses do not play an important role in attachment, because in C3 knockout mice, the mutant viruses are as virulent as wild-type virus. Of particular interest is the C5/P deletion mutant virus. This mutant virus lacks gC amino acids 33–123, identified as an important domain for gC-mediated attachment to heparan sulfate in vitro 40 . Yet this virus shows no difference in virulence from wild-type virus in C3 knockout mice, suggesting that this domain is not important for virus attachment in vivo. Disease at the inoculation site develops by day 3 after infection, and zosteriform disease generally appears on day 5 6 26 . We scored inoculation site and zosteriform disease separately so that we could better evaluate early stages of infection. Interestingly, disease scores at the inoculation site of the C3 single and double mutant viruses did not differ from wild-type virus until day 5. This result suggests that the complement effect on gC mutant virus cannot be detected until rather late after infection. Several explanations are possible. If complement lyses infected cells rather than inhibiting cell free virus, it may take several days until enough cells are infected to create an observable effect. Another possible explanation is that complement concentrations may be too low at the inoculation site early in infection, whereas at later time points, virus-induced tissue injury leads to an increase in vascular permeability and higher levels of complement proteins. Additional considerations include that complement may be mediating its effects by stimulating chemotaxis or other proinflammatory events that may take several days to develop, or that antibodies may be required to augment the effects of complement. This last explanation seems unlikely in view of in vitro results that demonstrate that antibodies are not required for complement-mediated neutralization or lysis of infected cells 23 24 . We have previously described a humoral immune evasion domain on glycoprotein gE that inhibits IgG Fc-mediated functions 4 5 6 . We showed that gE blocks complement-enhanced antibody neutralization, reducing the effectiveness of antibody 100-fold in vitro and offering highly significant protection to the virus or virus-infected cell against antibodies in vivo 6 . The results of this study indicate that complement has little effect on wild-type virus, as disease scores of wild-type virus are virtually identical in C3 knockout and complement-intact mice. Immune evasion mediated by gC and gE may underlie the long held view that humoral immunity is relatively unimportant in HSV-1 pathogenesis 41 . These immune evasion molecules render the Fc domain of antibody and the complement cascade far less effective in host defense. These observations have important implications for vaccines and chemotherapy. They explain why antibodies and complement are poorly protective against HSV infection and provide potential targets for therapies to block HSV-1 immune evasion and render the virus more susceptible to innate and acquired immunity. | Study | biomedical | en | 0.999994 |
10587355 | RAG-2 −/− mice were bred and maintained in our animal facility 34 . Timed-pregnant RAG-2 −/− mice were used on day 14 of gestation. The SL-12β.12 cell line is a pre-T cell line derived from a spontaneous SCID mouse–derived thymoma that stably expresses functionally rearranged TCR β chain at the cell surface with endogenous pTα to form a pre-TCR 48 49 . The cell line was maintained in complete media (HG-DME media containing 10% FBS, 1% penicillin/streptomycin, 1% glutamine, 1% sodium pyruvate, 1% Hepes, 0.1% 2-ME, and 0.1% gentamicin [GIBCO BRL] supplemented with 0.5 mg/ml geneticin [GIBCO BRL]). Cells were kept in a humidified atmosphere of 5% CO 2 at 37°C. SL-12β.12 cells (5 × 10 6 ) were incubated at 4°C with 10 μg/ml biotinylated anti–TCR-β mAb (H57-597) for 30 min in DMEM supplemented with 0.1% BSA. Cells were then washed, resuspended in 100 μl DMEM/0.1% BSA containing 25 μg/ml avidin (Sigma Chemical Co.), and incubated at 37°C. Stimulation was stopped by the addition of 3 ml ice cold PBS followed by lysis in buffer containing 50 mM Tris, pH 7.4 (Boehringer Mannheim), 0.5% Triton X-100 (Boehringer Mannheim), 150 mM NaCl (ICN Biomedicals Inc.), 1 mM EDTA (BDH Chemicals Ltd.), 1 mM sodium orthovanadate (ICN Biomedicals Inc.), and Complete Inhibitor Cocktail (Boehringer Mannheim). Control cells (unstimulated) were treated similarly; however, avidin was not added before lysis. Lysates were resolved on 10% SDS-PAGE under reducing conditions and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Amersham Pharmacia Biotech). Phosphorylated ERK-1 (p44 MAPK) and ERK-2 (p42 MAPK) and total ERK-2 proteins were detected using phospho-p44/22 MAPK (Thr202/Tyr204) mAb (New England Biolabs Inc.) and total p42 MAPK antibody (New England Biolabs Inc.), followed by horseradish peroxidase–conjugated goat anti–rabbit IgG (Santa Cruz Biotechnology). Blots were revealed using Supersignal West Pico chemiluminescent substrate (Pierce Chemical Co.). All electroporations were carried out using a BTX Electro Cell Manipulator 600 apparatus. SL-12.β12 cells were electroporated at 450 μF, 186 Ω, 300 V, attaining a time constant of ∼30 ms. 3 × 10 6 cells were transfected with desired plasmid DNA (up to 40 μg), as indicated in the figure legends. Each sample was transfected with PathDetect reporter plasmids and 1 μg plasmid encoding β-galactosidase (pCMV-β-gal). The addition of a fixed amount of β-galactosidase plasmid allowed for the control of transfection efficiency during the experiment. The β-galactosidase activity was used to index the luciferase signal detected, as we were able to assay for luciferase and β-galactosidase activity within the same sample 50 . In brief, cells were washed in electroporation media and resuspended at 1.2 × 10 7 cells/ml (250 μl per transfection). DNA and cells were combined in 4-mm sterile cuvettes (Bio-Rad Labs.) and incubated on ice for 10 min. The cells were electroporated with the conditions noted above and incubated on ice for a further 10 min. Transfected cells were put into fresh complete media and incubated for 24 h at 37°C, with or without the addition of exogenous stimuli, as indicated in the figure legends. The cells were then lysed and analyzed for luciferase and β-galactosidase activity. Fetal thymic lobes from timed-pregnant RAG-2 −/− mice at day 14 of gestation were placed on nucleopore filters resting on Gelfoam rafts (Upjohn) soaked in fetal thymic organ culture (FTOC) media (complete media supplemented with a further 5% FCS; five to six lobes per filter) 51 . The thymic lobes were incubated at 37°C for 6 h in a humidified incubator with 5% CO 2 to allow them to adhere to the filters. After this time, the filters were briefly removed from the Gelfoam rafts, and thymi were immediately transfected by gene gun bombardment with a Helios gene gun set at 200 psi (Bio-Rad Labs.), with the desired DNA bound to gold particles (1 μg gold) . The amount of DNA loaded per microgram of microcarriers is referred to as the DNA loading ratio . The filters carrying the transfected thymic lobes were then placed back on the Gelfoam rafts and incubated at 37°C for the indicated time . For short-term biochemical readout assays, the thymic lobes were incubated for a further 16–20 h, and then a single-cell suspension was prepared and the cells were lysed and assayed for luciferase and β-galactosidase activities as described below. For long-term developmental progression studies, the transfected thymic lobes were incubated for 7 d, with the FTOC media being changed once during the incubation. After this time, a single-cell suspension of the lobes was prepared, and the thymocytes were analyzed by flow cytometry as described below. FITC-, PE–, and APC-conjugated anti–mouse CD4, CD25, and CD8 were used for flow cytometric analysis (purchased from PharMingen). Staining of cells was carried out as described previously 51 . In brief, thymic cell suspensions were washed in FACS buffer (HBSS containing 1% BSA and 0.1% sodium azide) and incubated with antibodies (1:300 dilution) for 30 min on ice. The cells were then washed in FACS buffer before analysis. Stained cells were analyzed with a FACSCalibur™ flow cytometer (Becton Dickinson). Analysis was performed using CELLQuest software (Becton Dickinson); the data were live gated by size and lack of propidium iodide uptake. Cells transfected with the PathDetect reporter plasmids (Stratagene Inc.) were assayed for luciferase and β-galactosidase activities using the Dual-Light reporter gene assay system (Tropix; Perkin Elmer-Applied Biosystems) as previously described 50 . In brief, the cells were lysed in lysis buffer (40 mM tricine, pH 7.8, 50 mM NaCl, 2 mM EDTA, 1 mM MgSO 4 , 5 mM dithiothreitol, and 1% Triton X-100). Supernatant (25 μl) was combined with an equal volume of luciferase reaction buffer (30 mM tricine, pH 7.8, 3 mM ATP, 15 mM MgSO 4 , 1 mM coenzyme A, and 10 mM dithiothreitol), and after addition of 1 mM luciferin, the sample was immediately assayed for luciferase activity with a Lumat LB 9507 Luminometer (Fisher Scientific Co.). To assay for β-galactosidase activity, Galacton-Plus (substrate for β-galactosidase; Tropix) was added to each tube after the luciferase assay was completed. The tubes were incubated for 30–60 min at room temperature, and then Accelerator-II (Tropix) was added to each tube and the samples were immediately assayed for β-galactosidase activity, measured as light emission with the Lumat LB 9507 Luminometer. Results represent the average luciferase activity indexed for β-galactosidase activity. To aid in deciphering the signaling pathways that lead to the differentiative and proliferative events of β-selection, the phosphorylation status of ERK-1/2 was investigated by performing Western blot analyses using an antiphospho-ERK-1/2 mAb. A SCID mouse–derived pre-T cell line, SL-12.β12, preincubated with biotinylated anti–TCR-β (H57-597) mAb, was stimulated for 1–30 min upon cross-linking with avidin. Engagement of the pre-TCR complex resulted in a rapid and transient phosphorylation of ERK-1/2 . Phosphorylation was observed as early as 1 min but declined almost to background levels 5 min after stimulation; subsequently, a secondary, weaker level of phosphorylation was evident between 10 and 30 min . To control for protein loading, the total amount of ERK-2 was assessed by Western blot with an anti–ERK-2 antibody . To study whether the decline in phosphorylation of ERK-1/2 at 5 min was specific to pre-TCR activation, we performed a similar experiment with variants of the SL12 cell line expressing comparable levels of TCR β chain with either a TCR α chain (mature TCR) or a pTα chain (pre-TCR), as determined by flow cytometry (data not shown). The TCR β chain on both cell lines was cross-linked for 1–30 min, and the phosphorylation status of ERK-1/2 was determined. In both cases, there was an attenuation of ERK-1/2 phosphorylation levels at 5 min, indicating that this phosphorylation pattern is characteristic of the SL-12 cell line (data not shown). These results indicate that engagement of the pre-TCR on SL-12β.12 cells leads to the phosphorylation of ERK-1/2, demonstrating the activation of the Ras/Raf/MEK/ERK signaling cascade. We sought to determine if the phosphorylation of ERK-1/2 observed upon cross-linking of the pre-TCR leads to their activation. To this end, we employed a reporter plasmid system (described in Materials and Methods) that allows detection of ERK-1/2 activation within the cell. This system utilizes two plasmids: a fusion activator plasmid (pFA-Elk), which encodes for the transactivation domain of the ERK substrate, Elk-1 (residues 307–427), fused with the DNA-binding domain of GAL4; and a luciferase reporter plasmid (pFR-Luc), which encodes for the luciferase gene under the control of five GAL4 binding elements. Hence, in transfected cells, phosphorylation of the Elk-1 fusion protein can be read in the form of luciferase activity. Accordingly, we transfected SL-12β.12 cells with pFR-Luc, either alone or together with pFA-Elk. The transfected cells were then stimulated with immobilized anti-TCR-β mAb or with PMA and ionomycin. Cells transfected with pFR-Luc alone displayed background luciferase activity (31 ± 18 relative light units [RLUs]). However, luciferase activity increased almost 850-fold above background levels in cells transfected with pFR-Luc and pFA-Elk . This elevation in luciferase activity may reflect low-level constitutive ERK activity within SL-12β.12 cells , as it was blocked by the addition of the MEK1 inhibitor, PD98059 (data not shown). Importantly, anti–TCR-β mAb stimulation of pFR-Luc/pFA-Elk–transfected SL-12β.12 cells resulted in a fivefold increase in luciferase activity compared with unstimulated cells . This elevation in luciferase activity was abrogated by the addition of PD98059 (129% inhibition). These data indicate that engagement of the pre-TCR complex results in the activation of ERK and subsequent phosphorylation of the ERK-1/2 substrate, Elk-1 . Activation of pFR-Luc/pFA-Elk–transfected cells by the addition of PMA/ionomycin induced a 20-fold stimulation in luciferase activity over unstimulated cells ; again, addition of PD98059 curtailed this activity (data not shown). As a control to demonstrate that ERK-1/2 activation can be efficiently detected with these reporter plasmids, pFR-Luc/pFA-Elk–transfected cells were also transfected with a plasmid encoding a constitutively active MEK1, pFC-MEK1. This serine/threonine kinase is responsible for the phosphorylation and activation of ERK-1/2. These cells showed maximal luciferase activity irrespective of PMA/ionomycin treatment , indicating that this reporter system provides a highly sensitive method for detecting ERK activation. Taken together, these results confirm that the ERK signaling cascade is activated upon engagement of the pre-TCR, as shown by the phosphorylation of ERK-1/2 and the phosphorylation/activation of the ERK-1/2 substrate, Elk-1 , in the SL-12β.12 cell line. An important application of the above reporter plasmid system would be to detect specific pre-TCR–mediated signals as they occur within an intact thymus. Therefore, an accelerated DNA/particle bombardment delivery system (gene gun) was adapted to transfect thymocytes in FTOC 52 . This approach allows transfected cells to respond to normal microenvironmental stimuli encountered during differentiation within the thymic milieu 52 53 . We have previously used this method to study transcriptional regulation events in the developing fetal thymus 52 . The preservation of structural and functional integrity of gene gun–transfected thymi was tested by allowing FTOCs to complete a program of β-selection. Thus, RAG-2 −/− mouse–derived FTOCs were gene gun transfected with control DNA and cultured for 7 d, either alone or in the presence of anti-CD3 mAb . Transfected FTOCs were then analyzed by flow cytometry for evidence of differentiation to the DP stage 17 18 19 . This analysis revealed that the thymocytes from anti-CD3–treated RAG-2 −/− gene gun–transfected FTOCs progressed to the DP stage, as indicated by upregulation of CD8/CD4 . This differentiative event involved the loss of CD25 cell surface expression and proceeded through a CD8 + immature SP stage . These results indicated that thymocytes and stromal elements within gene gun–transfected thymic lobes remain viable. To determine the long-term developmental capacity of transfected thymocytes, RAG-2 −/− thymic lobes were gene gun transfected with plasmids encoding either constitutively active Lck (Lck[F505]) or a productively rearranged TCR β chain. It has been previously shown in RAG −/− mice that introduction of transgenes encoding a kinase-active Lck or a functionally rearranged TCR β chain induced thymocyte proliferation and differentiation toward the DP stage of development 39 41 . Indeed, these events were also observed in gene gun–transfected RAG-2 −/− FTOCs . Flow cytometric analysis of Lck(F505)- and TCR-β–transfected FTOCs after 7 d revealed that thymocytes differentiated from the DN stage to the DP stage, as evidenced by upregulation of CD4 and CD8 surface expression and downregulation of CD25 surface expression . The apparent difference in the levels of CD8 surface expression in active Lck-transfected FTOCs was due to the fact that the experiment shown was analyzed separately and a different set of fluorochrome-labeled antibodies was used. These phenotypic changes are consistent with differentiation events that occur after completion of β-selection . Our finding that gene gun transfection of RAG-2 −/− FTOCs with a functional TCR β chain resulted in the normal developmental progression of DN thymocytes indicated that a functional pre-TCR complex was generated 39 . These data imply that after gene gun transfection, the formation of the pre-TCR results in signaling events that occur during normal β-selection in vivo. Because the cellular signaling processes that underlie β-selection are not well defined, we combined gene gun–mediated transfection of FTOCs with the reporter plasmid system. In this way, the intracellular signaling cascades that occur after pre-TCR formation can be monitored in a biological time frame and within a relevant in vivo setting. We hypothesized that gene gun–mediated transfection of RAG-2 −/− mouse–derived FTOCs with a plasmid encoding a functionally rearranged TCR β chain, together with reporter plasmids, would allow us to capture de novo β-selection signaling events that occur after the generation of the pre-TCR. Our results show that compared with FTOCs transfected with pFR-Luc alone , there was a slight elevation in luciferase activity in FTOCs transfected with pFR-Luc/pFA-Elk . This level of luciferase activity may represent endogenous ERK-1/2 activity present in transfected FTOCs. To directly test our hypothesis, RAG-2 −/− FTOCs were gene gun transfected with reporter plasmids (pFR-Luc/pFA-Elk) together with a plasmid encoding a functional TCR β chain. Our analysis revealed an almost sixfold increase in luciferase activity in FTOCs transfected with a TCR-β plasmid as compared with FTOCs transfected with the reporter plasmids alone . A substantial luciferase activity was also observed in FTOCs transfected with a constitutively active MEK1 plasmid together with the reporter plasmids . The level of stimulation observed in MEK1-transfected FTOCs demonstrates the sensitive nature of this assay. Several reports have suggested that the Ras/Raf/MEK/ERK signaling cascade is involved at the β-selection stage 13 42 43 54 . However, our data provides the first direct evidence for coupling of the pre-TCR complex to the ERK signaling cascade in vivo. Lck is thought to play an essential role as a mediator of β-selection events 15 17 24 41 55 . Therefore, we investigated whether Lck could activate the ERK signaling cascade in FTOCs. To this end, FTOCs were gene gun transfected with a kinase-active Lck together with the reporter plasmids. Fig. 5 shows that a sixfold increase in luciferase activity was observed in FTOCs transfected with constitutively active Lck . This level of luciferase activity is comparable to the stimulation observed in TCR-β–transfected FTOCs . To address whether the observed ERK activation after the pre-TCR complex formation was dependent on Lck activity, we took advantage of a plasmid encoding a dominant negative Lck (Lck[R273]). This mutant form of Lck has been shown to cause a dramatic block in T cell development at the DN stage 15 . Fig. 5 shows that transfection of RAG-2 −/− FTOCs with the reporter plasmids together with plasmids encoding a TCR β chain and a kinase-negative Lck led to an attenuation of luciferase activity, as compared with FTOCs transfected with TCR-β alone . These results indicate that expression of the pre-TCR complex by developing thymocytes activates the ERK signaling cascade and that this activation is directly dependent on Lck during thymocyte differentiation within the thymus. In this study, we demonstrate that formation and/or engagement of the pre-TCR complex results in the activation of the ERK signaling cascade. We have shown this in two distinct systems: (a) in a SCID mouse–derived pre-T cell line, SL-12β.12 48 , in which engagement of the pre-TCR leads to phosphorylation and activation of ERK-1/2; and (b) within the thymus, where formation of the pre-TCR in developing thymocytes induces ERK activation and phosphorylation of the downstream substrate, Elk-1. Furthermore, our findings confirm the central role of the nonreceptor tyrosine kinase Lck in β-selection by demonstrating that ERK activation is strictly dependent on Lck function in vivo. The ability to introduce reporter plasmids into developing thymocytes in vivo provides an important diagnostic tool for the detection of signaling events during T cell development in the thymus . The significance of this method is highlighted by the fact that, in the absence of the thymic microenvironment (i.e., cell suspension), thymocytes fail to respond to normal developmental cues and undergo rapid programmed cell death 56 57 . Indeed, transfection of thymocytes from RAG −/− mice with reporter plasmids revealed that cells in suspension fail to respond when cotransfected with a plasmid encoding a TCR β chain (data not shown). Thus, thymocytes in cell suspension appear to be unresponsive to pre-TCR–mediated signals, possibly due to the lack of stromal elements to assist in this signaling. These results are consistent with previous data showing that anti-CD3 mAb–mediated differentiation of RAG −/− thymocytes to the DP stage in FTOC does not occur when these thymocytes are treated in cell suspension 58 . The notion that the Ras/Raf/MEK/ERK signaling cascade plays a central role during β-selection is supported by data obtained from mice bearing targeted genetic deficiencies of key signaling molecules such as Lck/Fyn, ZAP-70/Syk, LAT, or SLP-76 24 25 26 28 29 30 31 . These mice displayed a block in T cell development at the DN stage, suggesting that these molecules are essential for differentiation to the DP stage of development 24 25 26 28 29 30 31 . However, several contradicting reports using transgenic mice carrying either constitutively active or dominant negative members of these signaling molecules have left the involvement of this pathway unresolved. Studies introducing constitutively active Lck, Ras, and Raf into RAG −/− mice also indicate that the Ras/Raf pathway is crucial at this stage of development 41 42 43 . In contrast, studies using transgenic mice carrying dominant negative forms of Ras (Ha-Ras N17 ) 45 , Raf 46 , and MEK-1 47 failed to support a role for these signaling molecules during β-selection. In this regard, it is important to note that mice carrying a dominant negative Lck transgene displayed varying degrees of DN to DP thymocyte development arrest directly correlating with the levels of transgene expression 15 . Thus, the conflicting results obtained using dominant negative molecules may reflect their ability to be sufficiently expressed at the appropriate stage of development. In addition, studies using retroviral infection of thymocytes with dominant negative MEK-1 showed a perturbation in the transition of thymocytes from the DN to DP stage, suggesting that the inhibitory effectiveness of dominant negative molecules appears to be dependent on the experimental system used 54 . Although several studies have indirectly supported the importance of the Ras/Raf/MEK/ERK-mediated signals downstream of the pre-TCR, in our system, the fact that the detection of ERK activation is predicated by the expression of the pre-TCR within individually transfected developing thymocytes directly links the pre-TCR to the activation of these kinases. Thus, this study provides the first evidence for the pre-TCR being responsible for the generation of specific downstream signals within the thymus and shows that Lck acts proximally to the nascent pre-TCR complex, leading to the subsequent activation of ERK . The transcription factor Elk-1 has been shown to be phosphorylated by ERK-1/2, although more recently it has been demonstrated that JNK (c-Jun NH 2 -terminal kinase) also has the ability to phosphorylate and activate Elk-1 59 60 61 . However, mouse models with gene-targeted disruptions of key signaling components of the JNK signaling pathway (JNK2 and stress-activated protein kinase kinase 1) did not reveal any abnormalities in β-selection events 62 63 64 . In this regard, our data with the SCID mouse–derived pre-T cell line SL-12β.12 show that engagement of the pre-TCR results in ERK phosphorylation and activation of the ERK substrate, Elk-1 , and that this can be blocked by addition of the MEK1 inhibitor, PD98059. Together, these data indicate that, in this setting, Elk-1 is specifically activated by ERK. Pre-TCR signals can be experimentally bypassed by introducing constitutively active mutants of certain signaling components, inducing β-selection–mediated events. Interestingly, the introduction of an activated form of Ras (Ha-Ras V12 ) or Raf-1 (Raf-CAAX), although allowing the differentiation of RAG −/− thymocytes to the DP stage, failed to establish allelic exclusion at the TCR-β locus when introduced into wild-type mice 43 65 . This indicates that although certain β-selection outcomes are mediated by the Ras/Raf pathway, distinct pre-TCR–derived signals upstream of Raf are responsible for the control of allelic exclusion. Therefore, it is interesting to note that RAG −/− FTOCs retrovirally infected with constitutively active MEK1 do not differentiate to the DP stage of development 54 . This finding suggests that Raf may activate additional signaling pathways enabling developing thymocytes to undergo full differentiation to the DP stage. Therefore, the intrathymic signal detection system developed here will allow for detailed studies of distal signaling events activated by the pre-TCR, facilitating the elucidation of potential signaling branchpoints. | Study | biomedical | en | 0.999997 |
10587356 | The Y153 Saccharomyces cerevisiae strain (auxotrophic for adenine, uracil, leucine, and tryptophan) as well as the bait and trap expression vectors, pAS1 and pACT, respectively, have been described 25 . The Rlk and Itk chimeric bait constructs were generated by cloning full-length cDNA, PCR-amplified using primers encoding NcoI and BamHI sites, into the NcoI and BamHI sites in the multiple cloning site of pAS1. The Itk cDNA in pcDNA1 (Invitrogen) was a gift of Dan Littman (New York University, New York, NY). A mouse T cell lymphoma MATCHMAKER cDNA library in pACT (Clontech) was screened using the Rlk bait as per the manufacturer's protocol. Full-length clones were identified by screening a custom-made Uni-ZAP mouse day 16 fetal thymic cDNA library (Stratagene) using a cDNA probe derived from the positive yeast clone, 2.3.2. Human embryonic kidney HEK293 cells were maintained in DMEM (GIBCO BRL) supplemented with 10% FCS, penicillin/streptomycin (100 μg/ml), and 2-ME. RIBP-HA cDNA was generated by cloning full-length (the short form) RIBP cDNA into pcDNA3 (Invitrogen) modified by ligation of a double-stranded oligonucleotide encoding a Kozak sequence and an HA tag between its HindIII and NotI sites (a gift from the laboratory of Harinder Singh, University of Chicago), between NotI and XbaI sites. Lck cDNA in the PEF expression vector was obtained from the laboratory of David Straus (University of Chicago). Transient transfections were carried out using a standard calcium phosphate precipitation method. Medium was exchanged 24 h after transfection, and cells were harvested 48 h after transfection. HEK293 cells were harvested and lysed in 1 ml of lysis buffer (0.5% Triton X-100, 50 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA [pH 8.0], 1 mM sodium vanadate, 10 μg/ml leupeptin, 10 μM aprotinin, 1 mM PMSF). Aliquots of the lysates (400 μg) were precleared at 4°C with 50 μl of protein G–agarose beads (GIBCO BRL) coated with 2.5 μg of rat IgG (Southern Biotechnology Associates). Immunoprecipitations were performed by incubating the precleared lysate with 50 μl of protein G–agarose beads precoated with 2.5 μg of rat high-affinity anti-HA Ab (Roche Diagnostics). Preclears and immunoprecipitates were washed four times in lysis buffer. All steps were performed at 4°C. The preclears and immunoprecipitates were boiled in 50 μl of 2× reducing SDS sample buffer and subjected to SDS-PAGE on a 10% gel. Proteins were subsequently transferred to a polyvinyldifluoride membrane preblocked in a 10% nonfat milk/1× TBST (Tris-buffered saline/Tween: 10 mM Tris, 150 mM NaCl, 0.05% Tween-20) solution. Blots were probed with a rabbit polyclonal anti-Itk antiserum (a gift from the laboratory of Dan Littman) solution (1:5,000 dilution in 5% nonfat milk/TBST) and developed with a 1:8,000 diluted anti–rabbit Ig (Amersham Pharmacia Biotech). For anti-RIBP immunoblotting, a rabbit polyclonal anti-RIBP antiserum solution (1:1,000 dilution in 2% nonfat milk/TBST) was used to probe blots. The RIBP gene was mapped by Southern blot analyses of two sets of multilocus crosses: (NFS/N or C58/J × Mus musculus musculus ) × M . m . musculus and (NFS/N × Mus spretus ) × M . spretus or C58/J. Offspring of these crosses have been previously typed for >1,200 loci distributed over the 19 autosomes and the X chromosome 26 27 . Recombinational distances were calculated according to Green 28 , and gene order was established by minimizing the number of recombinants. For Northern blot analysis, total RNA was isolated from the indicated tissues and cell types using TRIzol (GIBCO BRL) according to the manufacturer's protocol or as described previously 23 . Northern blots were probed for the presence of RIBP mRNA using 32 P-labeled cDNA probes derived from either full-length RIBP or RIBP 1–208 (encoding RIBP from the NH 2 terminus to the end of the SH2 domain) by random priming, using a Prime-It II kit (Stratagene). The RIBP transcript was detected as a band ∼1.7 kb in size, migrating faster than the 18S rRNA band. For Southern blot analysis, genomic DNA was prepared from embryonic stem (ES) cell clones by standard methods, and digested with the indicated restriction endonucleases. The DNA was denatured and, after gel electrophoresis and membrane transfer, hybridized to either a 5′ or 3′ 32 P-labeled RIBP cDNA probe . Bands representing either the endogenous or targeted RIBP allele were detected based on size, as described in the text and in the legend to Fig. 4 B. The targeting construct was electroporated into the R1 ES cell line, derived from the 129/Sv mouse strain, using standard techniques 29 . Homologous recombination at the RIBP locus was detected by Southern blot analysis using 5′ and 3′ probes binding outside the RIBP regions used in the targeting construct . Targeted ES cell clones were used for blastocyst injection, and the resulting chimeric animals were backcrossed to C57BL/6 mice. Agouti offspring carrying the targeted RIBP allele were interbred to generate progeny homozygous for the targeted RIBP allele. Progeny were typed by Southern blot analysis of tail genomic DNA , and subsequently, by PCR analysis, using primer pairs which either amplified endogenous sequence between exons 2 and 3 or sequences in the neo cassette. Single cell suspensions from thymi, lymph nodes, and spleens were prepared according to standard methods. Cells were then blocked with an anti-F c R mAb, 2.4G2, and stained with the Abs indicated in the text. Abs used in these experiments were conjugated to biotin or one of the following fluorochromes: FITC, PE, or Cy-Chrome. Ab-stained cells were analyzed on a FACScan™ (Becton Dickinson) flow cytometer. Whole lymph node cells, splenocytes, or a 1:1 mixture of lymph node or splenic T cells cultured with T cell–depleted, irradiated syngeneic splenic APCs, from wild-type (either progeny from heterozygous intercrosses or [C57BL/6 × 129] F2 mice; The Jackson Laboratory), heterozygous, or homozygous knockout (KO) mice were used in these experiments. Soluble anti-CD3∈ mAb (145-2C 11 ) with or without soluble anti-CD28 mAb (PV-1) was added to wells of round-bottomed 96-well plates (Costar) at the concentrations indicated in the text. When used, final concentrations of PMA and ionomycin were 10 ng/ml and 0.5 μM, respectively. Cells (2 × 10 5 ) were added to each well at a final concentration of 1 × 10 6 /ml and cultured at 37°C in DMEM supplemented with 10% FCS, penicillin/streptomycin (100 μg/ml), and 2-ME. After 40 h, individual wells were pulsed with [ 3 H]thymidine (1 μCi/well) for 8 h, for a total duration of 48 h of stimulation. Cell contents were then harvested onto fiberglass filters, scintillation fluid was added (25 μl/well), and counts were determined using a TopCount microplate scintillation counter (Packard). IL-2 ELISAs were performed on culture supernatants of activated cell cultures (described above). Nunc-Immuno plates (Nalge Nunc) were used for these assays. Plates were coated with an anti–IL-2 coating Ab overnight at 4°C, washed, and blocked in 2% BSA/PBS for 1 h at room temperature. Supernatants, diluted in 2% BSA/PBS, were incubated on the coated plates overnight at 4°C. The next day, plates were washed, then incubated with an anti–IL-2 detecting Ab for 1 h at room temperature. Plates were washed and incubated with a 1:5,000 dilution of streptavidin-horseradish peroxidase (Zymed), after which they were developed using TMB One-Step Substrate (Dako). Abs and IL-2 standards were all purchased from Endogen. Splenocytes from wild-type (either [C57BL/6 × 129] F2 described above, or when indicated, C57BL/6 mice), heterozygous, and homozygous KO mice were activated with the indicated concentrations of soluble anti-CD3 mAb with or without anti-CD28. At the indicated times, cells were harvested, and RNA was prepared as described above. Cytokine RNase protection assays were performed using the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen) according to the manufacturer's protocol. In brief, a 32 P-labeled RNA probe was synthesized via in vitro transcription from the mCK-1 plasmid template. Aliquots of 2.5 μg of total RNA per sample were hybridized overnight at 56°C to the probe. Samples were then digested with RNase A, after which they were treated with Proteinase K and phenol/chloroform extracted. Samples were then precipitated, resuspended in 1× loading buffer, heated briefly at 90°C, and subjected to electrophoresis on a 5% denaturing polyacrylamide gel. Gels were dried, and autoradiography was performed. Densitometry on the cytokine and housekeeping gene bands was performed using a Molecular Dynamics ImageQuant ® densitometer. The yeast two-hybrid system of detecting interprotein interactions was used to identify potential Rlk-binding proteins that might mediate or regulate Tec family kinase function. A cDNA encoding full-length Rlk was cloned into a yeast vector encoding the GAL4 DNA-binding domain, thus yielding a “bait” fusion protein consisting of Rlk physically linked to this DNA-binding protein. A mouse T cell lymphoma cDNA library was screened using the Rlk bait. This library consisted of “trap” cDNAs cloned into a yeast vector encoding the GAL4 transactivation domain. The T cell cDNA library was chosen as Rlk is highly expressed both in the thymus and in peripheral T cells. Two specificity criteria were used as measures of GAL4-mediated transcriptional activation: (a) histidine synthesis by the yeast, and (b) β-galactosidase synthesis. A total of 750,000 yeast colonies were screened. 86 of these colonies were identified as positive for bait–trap interaction by the first pass criterion of growth on histidine-deficient (His − ) medium supplemented with 3-aminotriazole, an inhibitor of weak endogenous histidine synthesis. Among the 86 colonies, 56 were positive for interaction with the Rlk bait fusion protein based on the second criterion of β-galactosidase synthesis (blue/white selection). The cDNA library plasmids isolated from the β-galactosidase–positive yeast colonies were subsequently transformed into bacteria to distinguish multiple trap cDNAs present within the same yeast colony during the original screening. A total of 65 unique cDNA clones were identified and subsequently retransformed into yeast. One clone, 2.3.2, was found to interact with Rlk with high specificity, and was chosen for further study. Initial sequence analysis demonstrated that clone 2.3.2 did not represent a full-length cDNA; consequently, a mouse fetal thymic cDNA library was screened using a probe derived from the positive yeast cDNA clone. This screen yielded a full-length cDNA that was used to examine interactions with Rlk and Itk. Yeast transformed with the full-length cDNA, termed RIBP, interacted specifically with both Rlk and Itk . We further examined whether the interaction of RIBP with Rlk depended on the kinase function of Rlk. An altered Rlk that contained a point mutation in the kinase domain (K309→R, denoted K309R) previously shown to abolish kinase activity (our unpublished observations) was incapable of binding RIBP . This suggests that the bait–trap interaction is critically dependent on the functionality and activity of the tyrosine kinase domain of Rlk. Human epithelial kidney (HEK293) cells were transfected transiently with constructs encoding HA-tagged RIBP and/or Itk to directly examine protein–protein interactions in mammalian cells. Cell lysates were immunoprecipitated with an anti-HA Ab, resolved by SDS-PAGE, and subjected to immunoblotting using an anti-Itk antiserum. As shown in Fig. 1 B, a specific interaction between RIBP and Itk was detected (top panel, lane 5). Coexpression of the Src family kinase, Lck, increased the amount of Itk coprecipitated with RIBP . This Lck-dependent augmentation of RIBP–Itk complex formation was not due to increased levels of Itk in the cell lysates or RIBP-HA in the immunoprecipitates . The results suggested that the interaction of RIBP with Itk is regulated, at least in part, by Src family kinase–specific phosphorylation of one or both proteins. Sequence and genomic analyses were performed on the RIBP gene product. RIBP contains an SH2 domain (highly homologous to the SH2 domain of Shc, 47% [data not shown]), a proline-rich region (PRR) capable of binding SH3 domains, and an NPIY substrate sequence for binding by phosphotyrosine-binding (PTB) domains 30 present within the PRR . Further analysis of the cDNA library identified a second RIBP transcript that represented an alternatively spliced form with an 8 amino acid sequence insert (VWASQQKA). Other potentially significant amino acid sequences within RIBP include two YXXP motifs, implicated in binding the SH2 domains of rasGAP, Abl, and Crk 31 , and one YXXV motif, potentially capable of binding the SH2 domain of the tyrosine phosphatase, SHP-2 32 . Additionally, RIBP contains three potential sites for N -myristoylation . These structural features and the absence of a catalytic domain suggest that RIBP functions as an adaptor protein. One adaptor protein, TSAd 33 , showed extensive sequence homology to RIBP. The human TSAd protein exhibits a 68% sequence identity and a 76% sequence similarity with RIBP. The sequence homology between RIBP and TSAd was greatest within the SH2 domain, where the two proteins share an 87% sequence identity . Furthermore, TSAd exists in two alternatively spliced forms, similar to RIBP, with a 10 amino acid insert present in the longer form. Although these results suggested that RIBP and TSAd may be species homologues, the sequence insertions in RIBP and TSAd are in different locations within the molecules. The 10 amino acid insertion in TSAd is located within the SH2 domain, whereas the 8 amino acid insertion in RIBP is located within one of the exons in the region between the SH2 domain and the PRR . Genomic linkage studies of the RIBP gene were performed by Southern blot analyses of two sets of multilocus crosses (described in Materials and Methods). Digestion of DNA samples from parental mice with HindIII produced RIBP fragments of 8.6 and 2.9 kb in M . spretus and 11.0 and 10.8 kb in NFS/N. BamHI produced fragments of 12.4 and 4.0 kb in NFS/N and 16.5 kb in M . m . musculus . Inheritance of the variant fragments was compared with that of other markers previously typed in these crosses. The RIBP locus was mapped to mouse chromosome 3 . Closest linkage was detected with Gba , for which no recombinants were detected in 171 mice, indicating that the genes are within 1.7 cM of one another at the upper limit of the 95% confidence interval. Similarly, no recombination was detected with Lmna in 103 mice, placing RIBP within 2.9 cM of Lmna . The RIBP gene maps to a region of mouse chromosome 3 with conserved synteny to human chromosome 1q21 34 35 , where both human GBA 36 and LMNA 37 , as well as TSAd 33 and Shc 38 , are located. Thus, the RIBP gene has a genomic localization congruent with that of the TSAd and Shc genes, supporting the possibility of gene duplication events in this region. Tec family members have been shown to have restricted tissue expression. In the cases of Rlk and Itk, expression is largely restricted to T cells, with Itk also expressed in NK cells and Rlk also expressed in mast cells. Northern blot analyses were performed on RNA samples isolated from various tissues in order to examine the tissue expression of RIBP. A 1.7-kb transcript representing RIBP was expressed in the thymus, lymph node, and to a lesser extent, in the spleen and bone marrow . In addition, analysis of multiple hematopoietic cell lines revealed that RIBP was expressed in T cell lines (e.g., BW.P and C57), but not expressed in a B cell lymphoma, A20, or a mastocytoma, P815 (data not shown). Northern blot analyses of thymocyte subpopulations demonstrated that RIBP mRNA was expressed in CD4 + CD8 + thymocytes , and in both CD4 + and CD8 + peripheral T cells . Additionally, although RIBP was expressed in TCR − thymocytes , little expression was detected in recombinase-activating gene (RAG)-2–deficient thymus . These data suggest that RIBP expression may be dependent on signal transduction through the pre-TCR, and consequently, that its expression levels vary during the course of T cell development. RIBP mRNA was found to be rapidly induced after T cell activation . RIBP expression was upregulated in the Th1 T cell clone, A.E7, in as little as 2 h after exposure to anti-CD3 . Similarly, RIBP mRNA was induced in another Th1 T cell clone, pGL10, when activated with anti-CD3 or ovalbumin in the presence of appropriate APCs (data not shown). The addition of exogenous IL-2 had no effect on RIBP expression, although it significantly increased [ 3 H]thymidine incorporation . In addition to T lymphocytes, NK cells stimulated with IL-2 were observed to express high levels of RIBP mRNA . Furthermore, RIBP was also expressed in the Th2 clones PL104 and PL3, as also shown in Fig. 3 B (bottom panel). RIBP KO mice were generated by disrupting the RIBP gene via homologous recombination. A targeting construct was generated that replaced exons 3 and 4 (encoding the SH2 domain) with a DNA segment encoding a neo gene. The targeting construct, depicted in Fig. 4 A, was introduced into the R1 ES cell line derived from the 129/Sv mouse strain. Southern blot analyses of drug-resistant ES cells demonstrated successful targeted disruption at the RIBP locus . Genomic DNA samples from control and targeted ES cells were digested with EcoRI and XhoI, resolved by gel electrophoresis, and hybridized to a 5′ RIBP cDNA probe. As seen in Fig. 4 B (top left panel), probing of control digested ES cell DNA revealed bands of the predicted size (3.8 kb). In contrast, DNA from targeted ES cell clones revealed bands migrating at 5.5 kb, consistent with the replacement of the third and fourth exons with the neo cassette. Similar results were observed using a 3′ RIBP cDNA probe of control versus targeted ES cell genomic DNA samples digested with Asp718 (an isoschizomer of KpnI) and XhoI . The neomycin-resistant ES cells (clone 2) were injected into C57BL/6 blastocysts using standard techniques 29 . Chimeric mice were bred to C57BL/6 mice to establish germline transmission, and heterozygous mice were interbred to obtain homozygous KO progeny. Southern blot analyses performed on tail genomic DNA samples from the progeny demonstrated successful targeted disruption at the RIBP locus . Initial studies revealed that homozygous KO mice lacked RIBP mRNA expression based on Northern blot analyses (data not shown). The lack of RIBP expression in the RIBP KO offspring was confirmed by immunoblot analyses using an RIBP-specific polyclonal antiserum. As seen in Fig. 4 C, lysates from wild-type C57BL/6 and heterozygous splenocytes activated with anti-CD3 contained significant amounts of RIBP protein migrating at ∼46 kD. In contrast, no RIBP protein was detected in activated T cells from homozygous RIBP-deficient animals. RIBP KO mice did not display any gross developmental abnormalities, and a normal Mendelian distribution of RIBP KO progeny was observed. The percentages of CD4 − CD8 − , CD4 + CD8 + , CD4 + CD8 − , and CD4 − CD8 + thymocytes did not differ between RIBP KO and wild-type or heterozygous (data not shown) control mice, and no significant qualitative or quantitative differences in lymph node and splenic T cell populations were evident . In addition, the CD4 + /CD8 + ratio in both lymph node and spleen (data not shown) was similar among the various groups. Moreover, no obvious differences in the sizes or cell yields of thymus, spleen, and lymph nodes isolated from RIBP KO mice were apparent compared with wild-type control mice (data not shown). These data suggest that RIBP does not have an essential role in T cell development or homeostasis of peripheral T cell populations. Given the findings that (a) RIBP expression is augmented by T cell activation, and (b) its binding partners Itk and Rlk have been shown to be requisite for T cell responses to activation stimuli 11 39 , studies to evaluate the functional status of RIBP KO T cells were undertaken. In response to TCR/CD3-mediated signals delivered by an anti-CD3 mAb, proliferation of RIBP KO T cells was considerably reduced relative to control T cell proliferation . This proliferative impairment was observed across the range of anti-CD3 concentrations tested, and ranged from an 80% reduction in proliferation at the lowest (anti-CD3 concentration) to a 50% reduction in proliferation at the highest (anti-CD3 concentrations). Although RIBP KO T cells displayed an apparent defect in TCR-mediated T cell activation, they were capable of receiving CD28-mediated costimulation . These data indicate that RIBP KO T cells are impaired in their ability to respond to TCR-mediated signals, whereas responsiveness to CD28 signals is less affected. To determine whether the RIBP KO T cells were defective in either proximal or distal components of the TCR signaling pathway, RIBP KO T cells were treated with either anti-CD3 or PMA and ionomycin, and proliferation was compared with that of wild-type T cells activated with the same stimuli. As shown in Fig. 6 B, RIBP KO T cell proliferation in response to anti-CD3 was 50% reduced compared with wild-type T cells. However, wild-type and RIBP KO T cells stimulated with PMA and ionomycin proliferated equivalently, suggesting that the inadequacy in TCR-mediated signaling in RIBP KO T cells lies upstream of pathways triggered by protein kinase C activation or intracellular Ca 2+ flux. These findings are consistent with those reported for T cells deficient in either Itk 11 or both Itk and Rlk 39 . RIBP KO splenocytes were stimulated with anti-CD3 and examined for cytokine production. Consistent with the reduced proliferative response , a significant reduction in the amount of IL-2 in the culture supernatant was observed . Furthermore, although CD28-mediated costimulation increased IL-2 production by both wild-type and KO T cells, the relative differences in IL-2 levels were maintained. These results were confirmed using an RNase protection assay to determine relative levels of IL-2 transcripts in activated RIBP KO versus wild-type T cells. As shown in Fig. 6 D, IL-2 mRNA was detected at 6 h in (C57BL/6 × 129) F2 wild-type T cells stimulated with anti-CD3. In contrast, little IL-2 mRNA was apparent in activated RIBP KO T cells at this time point . Densitometry analyses revealed that IL-2 mRNA levels in RIBP KO T cells were reduced by ∼80% relative to wild-type cells (data not shown). Also, consistent with the reduction in supernatant levels of IL-2 , the relative amounts of IL-2 mRNA from RIBP KO T cells activated with anti-CD3 and anti-CD28 were still significantly reduced compared with control T cells . In addition to a reduction in IL-2 levels, a pronounced decrease in IFN-γ production was observed in RIBP KO T cells . As was the case for IL-2, this reduction in IFN-γ production correlated with decreased IFN-γ mRNA levels , suggesting that RIBP affects lymphokine production at the level of transcription and/or mRNA stabilization. Little IFN-γ mRNA was observed in anti-CD3–activated RIBP KO T cells relative to wild-type T cells; densitometry revealed that this was a >90% reduction . However, anti-CD28 costimulation did restore the IFN-γ decrease observed in RIBP KO T cells, suggesting that this defect may be an indirect consequence of the reduced activation and IL-2 production. Finally, despite the reductions in IL-2 and IFN-γ production by RIBP KO T cells, no significant reduction in IL-4 production was observed . In fact, in some experiments, activated RIBP KO splenocytes produced more IL-4 than did control splenocytes. Thus, the lack of RIBP appears to more profoundly affect the ability of T cells to produce Th1-type cytokines than Th2-type cytokines. In this study, a yeast two-hybrid T cell cDNA library screen was used to search for proteins that could associate with the T cell–specific members of the Tec family of tyrosine kinases, Rlk and Itk. A novel adaptor molecule, termed RIBP, was identified that interacts with a site in Rlk and Itk regulated by tyrosine phosphorylation. RIBP is expressed in thymocytes, and at low levels in resting peripheral T cells. Expression of RIBP is upregulated after T cell activation, coinciding with the expression levels of one of its binding partners, Itk, and high expression levels are observed in activated NK cells. The restriction of RIBP expression to T cells and NK cells mirrors the expression patterns of several signaling molecules, including Itk 9 , linker for activation of T cells (LAT 40 ), which is also expressed in mast cells, and ZAP-70 41 . These results are consistent with the proposed common lineage of these cells 42 . Finally, genetic disruption of RIBP was found to significantly impair TCR/CD3-mediated proliferation, and IL-2 and IFN-γ production, although IL-4 production was unaffected. Furthermore, the TCR-mediated signal transduction defects observed in RIBP KO T cells were bypassed using the downstream mitogens, PMA and ionomycin. These results suggest that RIBP positively regulates TCR signaling by acting as a molecular link between Itk/Rlk and other components of the TCR signaling pathway. The consequences of this interaction include the regulation of TCR-mediated signal transduction, and consequently, lymphokine production. RIBP is a complex adaptor protein. The gene encodes a PRR that may bind SH3 domain–containing proteins such as other Src family kinases and other adaptor proteins. In addition, a tyrosine-phosphorylated NPIY sequence present in RIBP may bind PTB domain–containing proteins such as Shc. Proteins such as Shc have been implicated in TCR signal transduction via interactions with ZAP-70 43 and Cbl, an adaptor which is thought to negatively regulate ZAP-70 activation 44 45 and binds to the SH3 domain of Itk. The tyrosine-based protein binding motifs present in RIBP (YXXP and YXXV) may bind the SH2 domains of rasGAP, Abl, Crk, and SHP-2, molecules previously shown to regulate T cell signal transduction. Finally, RIBP contains an SH2 domain that may be involved in binding to other phosphotyrosine-containing proteins. In this regard, preliminary results indicate that the SH2 domain of RIBP is required for interaction with Rlk, whereas the PRR and tyrosine-based protein binding motifs are unnecessary (our unpublished observations). The molecular mechanisms that link RIBP, Rlk, and Itk, to the proximal event of TCR ligation are unclear. The finding that Lck coexpression augments the association of RIBP with Itk is consistent with a role for RIBP in the regulation of TCR signal transduction. Tyrosine phosphorylation of Itk, catalyzed by Lck or Fyn, is required for optimal Itk kinase activity 46 47 , and consequently, T cell activation. Phosphorylation of Itk occurs at Y511, a residue in the activation loop of the kinase domain of Itk 48 . Thus, RIBP could be functionally coupled to TCR engagement, as its binding to Itk is enhanced by the activity of this CD4/CD8 coreceptor–associated Src family kinase in vitro. RIBP may bind to both Itk and Rlk, raising the question as to its physiological partner and the relationship of its binding specificity to the functional deficit evident in RIBP KO T cells. The expression pattern of RIBP suggests that its physiological binding partner may be Itk in the periphery. Previous studies have shown that Rlk is highly expressed in resting T cells, but downregulated after T cell activation. In contrast, Itk gene expression is upregulated after T cell activation, similar to RIBP. Furthermore, the phenotype observed in RIBP KO T cells more closely resembles the phenotype of Itk-deficient T cells, which display diminished proliferation and IL-2 production in response to TCR ligation. In contrast, Rlk KO T cells have only minor deficits in TCR-mediated T cell activation 39 . However, there are circumstances when the binding of RIBP to Rlk may be physiologically relevant. For instance, both RIBP and Rlk are expressed in thymocytes. Although we have not identified any profound changes in T cell development in the RIBP KO or Rlk KO mice (our unpublished observations, and reference 39, respectively), there may be subtle, as yet unidentified effects of this interaction in the thymus. Moreover, both Rlk and RIBP are expressed in NK cells, and may play a role in this lymphocyte subset. In fact, recent evidence indicates that Itk and Rlk have overlapping functions in T cell activation, as mice deficient in both kinases have more severely diminished peripheral T cell function than mice deficient in either kinase alone 39 . Thus, further biochemical and breeding studies to generate RIBP/Itk and RIBP/Rlk double-KO mice will be needed in order to determine the differential effects of RIBP on the functions of Itk versus Rlk. It is important to emphasize that the functional impairment present in RIBP KO T cells was not as severe as that observed in Rlk/Itk double-KO T cells. There are two possible reasons for these findings. First, there may be functional homologues of RIBP that can partially compensate for the absence of RIBP in KO T cells. Second, not all of the intracellular effects on TCR signaling that Rlk and Itk mediate may require RIBP or any putative homologues. The fact that CD28-mediated costimulation partially compensates for the severely diminished proliferative response to TCR/CD3-mediated T cell activation in RIBP KO mice suggests that the RIBP KO T cells are capable of responding to the CD28-mediated costimulatory signal. Interestingly, Itk has been reported to be both a positive and negative regulator of CD28-mediated T cell activation. On the one hand, Itk is physically associated with CD28 after T cell activation 49 , and Jurkat T cells expressing CD28 cytoplasmic tail mutants incapable of recruiting Itk are suppressed in their ability to produce IL-2. However, Littman and colleagues demonstrated that T cells from Itk-deficient mice were hyperresponsive to CD28 costimulation 15 . Therefore, it remains to be determined in what context, if any, RIBP is involved in CD28 signal transduction. Finally, the results of this study support a role for RIBP in the regulation of Th cell subset differentiation. IL-2 and IFN-γ production were both significantly reduced in activated RIBP KO T cells , whereas IL-4 production was unaffected . Such a selective impairment in IFN-γ and IL-2 production suggests that RIBP may act in signaling pathways that promote Th1 differentiation. The lack of RIBP may reduce TCR-mediated signal strength via impairment of Itk and/or Rlk function, resulting in a selective loss of Th1 differentiation, as suggested by Bottomly and colleagues 50 . This may occur by generating decreased TCR signals that drive Th2 skewing. In fact, both Itk and Rlk have been implicated in T cell differentiation (Schaeffer, E., personal communication). Also, Rlk is selectively expressed in Th1-type T cells. These results may explain, at least in part, the finding that RIBP selectively affects Th1-type cytokine production. In conclusion, we have identified and functionally characterized a novel T cell adaptor molecule involved in the regulation of responses to T cell activation stimuli, specifically proliferation and lymphokine production. This adaptor, RIBP, appears to function in TCR/CD3-mediated signal transduction, consistent with previously reported data regarding its binding partners, the Tec tyrosine kinases Rlk and Itk. The binding of RIBP to Itk and Rlk may provide important biochemical links of these two important kinases with other components in the T cell activation machinery. Further molecular studies will be needed to determine the specific sites of action of RIBP within various T cell signal transduction pathways. | Study | biomedical | en | 0.999999 |
10587357 | Patients were eligible if they suffered from stage IV (i.e., distant metastases) cutaneous malignant melanoma that was not curable by resection and was progressive despite chemo(immuno)therapy. Further inclusion criteria were an expected survival ≥4 mo, Karnofsky index ≥60%, age ≥18 yr, measurable disease, HLA-A1 positivity, expression of Mage-3 gene shown by reverse transcriptase (RT)-PCR in at least one excised metastasis, and no systemic chemo-, radio-, or immunotherapy within 4 wk (6 wk in the case of nitrosurea drugs) preceding the first DC vaccination. A positive skin test to recall antigens was not required. Important exclusion criteria were active central nervous system (CNS) metastasis, any significant psychiatric abnormality, severely impaired organ function (hematological, renal, liver), active autoimmune disease (except vitiligo), previous splenectomy or radiation therapy to the spleen, organ allografts, evidence for another active malignant neoplasm, pregnancy, lactation, or participation (or intent to participate) in any other clinical trial. Concomitant treatment (chemo- or immunotherapy, corticosteroids, investigational drugs, paramedical substances) was prohibited. Palliative radiation or surgical therapy of selected metastases and certain medications (acetaminophen/paracetamol, nonsteroidal antiinflammatory drugs, opiates) to control symptoms were allowed. The study was performed at the Departments of Dermatology in Erlangen, Würzburg, and Mainz, Germany according to standards of Good Clinical Practice for Trials on Medicinal Products in the European Community. The protocol was approved by the Protocol Review Committee of the Ludwig Institute for Cancer Research (New York, NY) and performed under supervision of its Office of Clinical Trials Management as study LUD #97-001. The protocol was also approved by the ethics committees of the involved study centers. The study design is shown in Table . All patients gave written informed consent before undergoing a screening evaluation to determine their eligibility. Extensive clinical and laboratory assessments were conducted at visits 1, 5, and 8 ( Table ) and consisted of a complete physical examination, staging procedures, and standard laboratory values as well as special ones (pregnancy test, free testosterone in males, autoantibody profile, and antibodies to HIV-1/2, human T cell lymphotropic virus type I, hepatitis B virus, and hepatitis C virus). Patients were hospitalized and examined the day before each vaccination and were monitored for 48 h after the DC injections. Adverse events and changes in laboratory values were graded on a scale derived from the Common Toxicity Criteria of the National Cancer Institute, National Institutes of Health, Bethesda, MD. During prestudy screening, we tested a small amount of fresh blood to verify that appropriate numbers of mature DCs could be generated from the patient's monocytes 12 . Sufficient DC numbers could be successfully generated in all patients, but in some patients the test generation revealed that TNF-α had to be added to assure full maturation. To avoid repetitive blood drawings, we performed a single leukapheresis during visit 2 to generate DCs as described 13 . In short, PBMCs from the leukapheresis (≥10 10 nucleated cells) were isolated on Lymphoprep™ (Nycomed Pharma) and divided into three fractions. The first fraction of 10 9 PBMCs was cultured on bacteriological petri dishes coated with human Ig (100 μg/ml; Sandoglobin™; Sandoz GmbH) in complete RPMI 1640 medium (BioWhittaker) supplemented with 20 μg/ml gentamicin (Refobacin 10; Merck), 2 mM glutamine (BioWhittaker), and 1% heat-inactivated human plasma for 24 h to generate monocyte-conditioned medium (MCM) for later use as the DC maturation stimulus. The second fraction of 3 × 10 8 PBMCs was used for the generation of DCs for vaccination 1 and delayed-type hypersensitivity (DTH) test I. Adherent monocytes were cultured in 1,000 U/ml GM-CSF (10 × 10 7 U/mg; Leukomax™; Novartis) and 800 U/ml IL-4 (purity >98%; 4.1 × 10 7 U/mg in a bioassay using proliferation of human IL-4R + CTLL; CellGenix; expressed in Escherichia coli and produced under good laboratory practice conditions but verified for good manufacturing practice [GMP] safety and purity criteria by us) for 6 d, and then MCM was added to mature the DCs. MCM was supplemented in patients 04, 06, 09, 11, and 12 with 10 ng/ml GMP-rhu TNF-α (purity >99%; 5 × 10 7 U/mg in a bioassay using murine L-M cells; a gift of Dr. G.R. Adolf, Boehringer Ingelheim Austria, Vienna, Austria) to assure full maturation of DCs. Mature DCs were harvested on day 7. The third fraction of PBMCs was frozen in aliquots and stored in the gas phase of liquid nitrogen to generate DCs for later vaccinations and DTH tests. DCs for vaccinations were pulsed with the Mage-3A1 peptide 15 (EVDPIGHLY, synthesized at GMP quality by Clinalfa) as tumor antigen, and as a recall antigen and positive control, tetanus toxoid (TT) or tuberculin (if at visit 1 the DTH to TT in the Multitest Merieux was >10 mm; both purchased from the Bacterial Vaccines Department of the Statens Serum Institute, Copenhagen, Denmark). The recall antigen was added at 10 μg/ml for the last 24 h, and the Mage-3A1 peptide was added at 10 μM directly to the cultures for the last 8 h (if immunity to recall antigens was strongly boosted, the dose of recall antigen was reduced to 1.0 or 0.1 μg/ml or was omitted for the intravenous DC injections to avoid a cytokine release syndrome). On day 7, mature DCs were harvested, resuspended in complete medium, washed, and pulsed once more with Mage-3A1 peptide (now at 30 μM) for 60 min at 37°C. DCs were finally washed and resuspended in PBS (GMP quality PBS; BioWhittaker) for injection. DCs to be used for Mage-3A1 DTH tests were pulsed with Mage-3A1 (but no recall antigen); DCs that served as negative control in the DTH tests were not pulsed at all. An aliquot of the DCs to be used for vaccinations was analyzed as described 13 to assure that functionally active and mature DCs were generated. The features of the DCs are described in Results. Release criteria were typical morphology (>95% nonadherent veiled cells) and phenotype (>95% HLA-DR +++ CD86 +++ CD40 + CD25 + CD14 − and >65% homogenously CD83 ++ ). A total of five vaccinations (three into the skin followed by two intravenously) with antigen-pulsed DCs were given at 14-d intervals ( Table ). This design was chosen to explore the toxicity and efficacy of various routes in this trial. For vaccinations 1–3, 3 × 10 6 DCs were given subcutaneously at two sites (1.5 × 10 6 DCs in 500 μl PBS per site) and 3 × 10 6 intradermally at 10 sites (3 × 10 5 DCs in 100 μl PBS per site). The injection sites were the ventromedial regions of the upper arms and the thighs close to the regional lymph nodes and were rotated clockwise. Limbs where draining lymph nodes had been removed and/or irradiated were excluded. For intravenous vaccinations 4 and 5, a total of 6 and 12 × 10 6 antigen-pulsed DCs (resuspended in 25 or 50 ml PBS plus 1% autologous plasma) was administered over 5 and 10 min, respectively. Premedication with an antipyretic (500 mg acetaminophen/paracetamol p.o.) and an antihistamine (2.68 mg clemastinhydrogenfumarat i.v.) was given 30 min before intravenous DC vaccination. PBMCs were cultured in triplicate at two dose levels (3 × 10 4 and 1 × 10 5 PBMCs/well) plus or minus TT or tuberculin (at 0.1, 1, and 10 μg/ml) and pulsed on day 5 with [ 3 H]thymidine for 12 h. In all cases, the highest cpms were obtained with the highest doses of PBMCs and antigen and are shown in Fig. 2 . IL-4 and IFN-γ levels were measured in culture media by ELISA (Endogen, Inc.). In a separate plate, staphylococcal enterotoxin (SEA; Serva) was added at 0.5, 1, and 5 ng/ml, and proliferation was assessed after 3 d to provide a positive control for helper T cell viability and responsiveness. To quantitate antigen-specific, IFN-γ–releasing, Mage-3A1–specific effector T cells, an enzyme-linked immunospot (ELISPOT) assay was used as described 16 . PBMCs (10 5 and 5 × 10 5 /well) or in some cases CD8 + or CD4 + T cells (isolated by MACS™ according to the manufacturer, Miltenyi Biotec) were added in triplicate to nitrocellulose-bottomed 96-well plates precoated with the primary anti–IFN-γ mAb (1-D1K; Mabtech) in 50 μl ELISPOT medium per well. For the detection of Mage-3A1–reactive T cells, the APCs were irradiated T2.A1 cells (provided by P. van der Bruggen, Ludwig Institute of Cancer Research, Brussels, Belgium) pulsed with MHC class I–restricted peptides (Mage-3A1 peptide and the HIV-1 p17-derived negative control peptide GSEELRSLY) added at 7.5 × 10 4 /well (final volume 100 μl/well). After incubation for 20 h, wells were washed six times, incubated with biotinylated second mAb to IFN-γ (7-B6-1; Mabtech) for 2 h, washed, and stained with Vectastain Elite kit (Vector Labs.). For detection of TT-reactive T cells, TT was added at 10 μg/ml directly to the PBMCs (1 or 5 × 10 5 PBMCs/flat-bottomed 96-well plate). Assays were performed on fresh PBMCs. Spots were evaluated and counted using a special computer-assisted video imaging analysis system (Carl Zeiss Vision) as described 16 . The multiple microculture method developed by Romero et al. 17 was used to obtain a semiquantitative assessment of CTLp (precursors) specific for Mage-3A1 peptide. Aliquots of frozen PMBCs were thawed and assayed together. CD8 + T cells were isolated with magnetic microbeads (MACS™ separation columns; Miltenyi Biotec) and seeded at 10 4 /well in 96-well round-bottomed plates in RPMI 1640 with 10% heat-inactivated human serum. The CD8 − PBMCs were pulsed with peptide Mage-3A1 or the influenza PB1 control peptide VSDGGPNLY (10 μg/ml; 30 min at room temperature), irradiated (30 Gy from a cesium source), and added as an APC population at 10 5 /well together with IL-2 (10 IU/ml final) and IL-7 (10 ng/ml final) in a total volume of 200 μl/well. On day 7, 100 μl fresh medium was substituted, and peptide Mage-3A1 or PB1 (1 μg/ml final) and IL-2 (10 U/ml) was added. On day 12, each microwell was divided into three equal samples to test cytolytic activity in a standard 4-h 51 Cr-release assay on peptide-pulsed (10 μg/ml for 1 h at 37°C) T2A1 cells, nonpulsed T2A1 cells, and K562 target cells, respectively. All of the assays were performed with an 80-fold excess of nonlabeled K562 to block NK activity. Microwells were scored positive if lysis of T2A1 targets with peptide minus lysis without peptide was ≥12% and this specific lysis was greater than or equal to twice the lysis of T2A1 targets without peptide plus six as described 18 . We aimed at testing 30 microwells of 10 4 CD8 + T cells. Therefore, 1/30 positive wells equals at least one CTLp in 3 × 10 5 (i.e., 30 wells at 10 4 CTLp per well) CD8 + T cells (corresponding to ∼3 × 10 6 PBMCs). DTH to Mage-3A1 peptide was assessed by intradermal injection at two sites of each 3 × 10 5 Mage-3A1 peptide–loaded DC in 0.1 ml PBS. Negative controls were nonpulsed autologous DCs in 0.1 ml PBS and 0.1 ml PBS. DTH to seven common recall antigens (Multitest Merieux) including TT and tuberculin was performed on visits 1, 5, and 8 ( Table ). For recruitment into the study, Mage-3 gene expression in at least one metastatic deposit had to be demonstrated by RT-PCR as described 14 . Accessible superficial skin metastases were removed whenever possible after the vaccinations and subjected to Mage-3 RT-PCR as well as routine histology and immunohistology (to characterize cellular infiltrates). For analysis of the immune response, pre- and postimmunization values were compared by paired t test after logarithmic transformation of the data. Significance was set at P < 0.05. All 13 patients were HLA-A1 + , had proven Mage-3 mRNA expression in at least one excised metastasis, and suffered from advanced stage IV melanoma, i.e., distant metastases that were progressive despite chemotherapy and, in some cases, chemoimmunotherapy ( Table ). We offered DCs to all patients who fulfilled the inclusion and exclusion criteria, i.e., we did not select for subsets of patients. Two patients (numbers 01 and 03) succumbed to melanoma after two and three vaccinations, respectively. 11 patients received all five planned DC vaccinations in 14-d intervals ( Table ) and were thus fully evaluable. All vaccine preparations were highly enriched in mature DCs. More than 95% of the cells were large and veiled in appearance, expressed a characteristic phenotype by flow cytometry (HLA-DR +++ CD86 +++ CD40 + CD25 + CD14 − ), and acted as strong stimulators of an MLR at DC/T cell ratios of ≤1:300 13 . Most (mean 80%) expressed the CD83 mature DC marker 19 . These features were stable upon removal of cytokines and culture for one to two more days 13 . The DCs were pulsed with Mage-3A1 peptide as a tumor antigen and TT or tuberculin as a recall antigen. The latter were internal controls for immunization and possibly provided help for CTL responses 20 . No major (above grade II) toxicity or severe side effects were observed in any patient, including the two patients who were not fully evaluable. We noticed, however, local reactions (erythema, induration, pruritus) at the intracutaneous vaccination sites that increased with the number of vaccinations. In 9/11 patients, strong DTH reactions (induration >10 mm in diameter) were noted to DCs carrying a recall antigen . Elevation of body temperature (grade I and II fever) was observed in most (9/11) patients and was also related to pulsing DCs with recall antigen. The most striking example was patient 02, who progressively developed fever (up to 40°C) upon successive vaccinations but did not show a rise in body temperature when TT was omitted for the final (fifth) vaccination. We observed slight lymph node enlargement, clinically in 63% and by sonography in 83% of patients, after the intracutaneous DC injections. Interestingly, these were delayed, being inapparent during the 2 d of monitoring after vaccinations but detected when patients were investigated again the day before the next vaccination ( Table ). PBMCs that had been frozen before vaccination and 14 d after vaccination 5 were thawed and assayed together, as specified in the protocol ( Table ). In most patients, a significant boost of antigen-specific immunity developed to TT (and tuberculin in patient 10) . Supernatants from the proliferative assays contained large amounts of IFN-γ (mean 1,679 pg/ml, range 846–4,325) but little IL-4 (IFN-γ/IL-4, 317:1). In five patients, we studied the kinetics of the immune response to TT by IFN-γ ELISPOT analysis. We found an increase after the intracutaneous vaccinations ( P < 0.02) but a peculiar decrease after the intravenous vaccinations . Thus, comparing recall immunity before and after all five vaccinations as prespecified in the protocol ( Table ) obviously underestimated the extent of boosting. Aliquots of PBMCs, frozen before the first and after the third and fifth vaccinations, were thawed at the same time ( Table ) and subjected to a semiquantitative recall assay for CTLp . Before vaccination, CTLp frequencies were low or undetectable. In 8/11 patients, we found a significant expansion of Mage-3A1–specific CTLp as indicated by the increase (mean, eightfold; P < 0.008) of positive microcultures in the multiple microculture procedure employed for the semiquantitative assessment of CTLp 17 . Interestingly, in six patients, the CTLp frequencies were maximal after the three intracutaneous vaccinations but then decreased after the two additional intravenous vaccinations in all but one of these patients ( P < 0.026). Only in 1/11 patients did we observe an increase of CTLp to an irrelevant PB1 influenza peptide that served as a specificity control (not shown). We also tried to detect Mage-3A1–specific CTL effectors in uncultured fresh, nonfrozen PBMCs by performing ELISPOT analyses at 14-d intervals on all patients. A significant increase of Mage-3A1–reactive IFN-γ spot–forming cells was apparent only in patients 07 and 09 after the first and second vaccinations, respectively, but the frequency of Mage-3A1–specific effectors was very high (∼5,000 and 10,500/10 7 CD8 + T cells; not shown). Tests of DTH to Mage-3A1 peptide–loaded DCs yielded erythema and/or induration (>5 mm diameter) in 7/11 patients (not shown). The results were, however, equivocal due to the frequently observed background to nonpulsed DCs (up to 10 mm in diameter) and the variability from test site to test site. At the end of the trial, i.e., ∼2 wk after the fifth vaccination ( Table ), we observed temporary growth cessation of some individual metastases, but more intriguingly, in 6/11 patients, complete regression of individual metastases in skin, lymph nodes, lung, and liver . Resolution of skin metastases was found in three patients ( Table , patients 06, 07, and 08) and in two of them (06 and 07), it was preceded by local pain, itching, and slight erythema. The six regressing skin lesions of patients 06 and 07 ( Table ) were also excised and examined by immunohistology. Clusters of CD8 + T cells were seen around and in the tumor, the latter often necrotic, suggesting an immune attack . In patients 06 and 08, the metastases excised at study entry (four and two, respectively) proved to be Mage-3 mRNA + . However, all of the samples removed at the end (two and six, respectively) were Mage-3 mRNA − , suggesting immunoselection for antigen-negative tumor cells. Remarkably, significant infiltration of CD8 + T cells was not found in any of these lesions. In deciding on the source of DCs for this phase I trial, we selected mature , monocyte-derived DCs for the following reasons. Monocyte-derived DCs currently represent the most homogenous and potent DC populations, with several defining criteria and quality controls 12 13 21 . The method for generating production of these DCs is very reproducible and allows the cryopreservation of large numbers of cells at an identical stage of development 12 13 . Furthermore, these DCs can be produced in the absence of potentially hazardous FCS 12 13 21 . FCS exposure also leads to large syngeneic T cell responses in culture, so their clinical use 11 might produce nonspecific immunostimulatory effects. Unlike other investigators 9 10 11 , we chose to use mature rather than immature DCs for our first melanoma trial. The DCs that have been used with efficacy in animal experiments were primarily mature 3 8 . Mature DCs are much more potent in inducing CTL and Th1 responses in vitro (reference 22 and Jonuleit, H., A. Gieseke, A. Kandemir, L. Paragnik, J. Knop, and A.H. Enk, manuscript in preparation), and the DCs are also resistant to the immunosuppressive effects of IL-10 23 that can be produced by tumors 24 25 26 . Mature DCs also display an extended half-life of antigen-presenting MHC class I 26a and class II molecules 27 . Finally, mature DCs have a high migratory activity 21 and express CCR7 28 , a receptor for chemokines produced constitutively in lymphoid tissues 28 . Mature DCs, as used in this cancer therapy trial, have recently also been shown to rapidly generate broad T cell immunity in healthy subjects 28 . Mature DCs were loaded with only one melanoma peptide, Mage-3A1, to avoid uncertainties regarding loading of DCs with multiple peptides 11 of varying affinity and off rate. Successful loading was verified with a Mage-3A1–specific CTL clone and ELISPOT analysis (not shown). The Mage-3A1 peptide 15 was selected for several reasons. It is essentially tumor specific 2 and expressed in tumors other than melanoma 2 , and the Mage-3A1 epitope is likely a rejection antigen 14 . Moreover, the Mage-3A1 CTLp frequency is exceedingly low in noncancer patients (reference 18; 0.4–3 per 10 7 CD8 + T cells) as well as in cancer patients, even after peptide vaccination 14 . Thus, any induction or boost of Mage-3A1 CD8 + T cell responses would indicate a significant superiority in the adjuvant capacities of DCs. DTH assays with peptide-pulsed DCs were carried out as described by Nestle et al. 11 to detect Mage-3A1 immunity (not shown). However, we did not detect unequivocal DTH. This was due to the frequently observed background to nonpulsed DCs (possibly due to cytokine production by DCs) and the noteworthy variability from test site to test site. As Mage-3A1–specific T cells are CD8 + T cells and DTH assays typically detect primed CD4 + T cells, we suspect that DTH to MHC class I peptide–pulsed DCs may also for this reason prove not to be a sensitive or reliable way to monitor specific CD8 + T cell–mediated immunity. In contrast, we found sizable expansions of Mage-3A1–specific CTL precursors in PBMCs from a majority (8/11) of patients . This is an important proof of the principle of DC-based immunization, and it is also significant from the point of view that tumors can induce tolerance or anergy. It is very promising that CTLp expansions can be induced in far advanced and heavily pretreated stage IV melanoma patients. However, active Mage-3A1–specific effectors were generally not observed in ELISPOT assays, except for in two patients with high frequencies (>5,000/10 7 CD8 + T cells). Perhaps active CD8 + effectors were rapidly sequestered in the numerous metastases, as suggested by the biopsy studies illustrated in Fig. 6 . An alternative explanation is that looking for effectors in peripheral blood 14 d after a preceding vaccination might simply be too late. Interestingly, in six patients, CTLp had increased to their highest levels after the three intracutaneous vaccinations and then decreased ( P < 0.026) with subsequent intravenous immunizations . The decrease in CTLp might be due to emigration of activated Mage-3–reactive CTLs into tissues, tolerance induction, or clonal exhaustion via the intravenous route. We also observed decreased responses to recall antigens in the five patients that we studied before and after intravenous vaccination . The effect of the intravenous route requires additional study, as it may be counterproductive. In contrast, our results clearly demonstrate that the intracutaneous route is effective, so that the less practical intranodal injection propagated by other investigators 11 does not seem essential. It will, however, be necessary to compare subcutaneous and intradermal routes to find out if one is superior. We found regression of individual metastases in 6/11 patients when patients were staged 14 d after the fifth vaccination ( Table ). This percentage of responses was unexpected in far advanced stage IV melanoma patients who were all progressive despite standard chemotherapy and even chemoimmunotherapy. In the study by Nestle et al. 11 , chemotherapy was only given to 4/16 melanoma patients, and objective tumor responses were observed in 5/16. Therefore, we attribute the regressions to DC-mediated induction of Mage-3A1–specific CTLs. This interpretation is supported by the heavy infiltration with CD8 + T cells of regressing but not nonregressing (skin) metastases. The observation that all of the metastases in patients 06 and 08 that were excised at the end of the study were Mage-3 mRNA − (whereas those removed at the onset were uniformly positive) suggests immune escape of and selection for Mage-3 antigen–negative tumors. Immune escape might also have been responsible for the lack of tumor response in those nonresponders that had mounted a Mage-3A1–specific CTL response. After the end of the trial, surviving patients received further vaccinations with DCs and several tumor peptides (Mage-1, tyrosinase, and Mage-3) that were no longer part of the protocol. It is encouraging that 5/11 patients are still alive ( Table ) 9–17 mo after study entry, as the expected median survival in patients progressive after chemo(immuno)therapy is only 4 mo 29 30 . One of the initial responders (patient 06) has recently experienced a complete response and has now been disease free for 2 mo. It is interesting that Marchand et al. 14 have also observed that regressions, once they have started, proceed slowly and may take months to complete. In conclusion, the use of a defined DC vaccine combined with detailed immunomonitoring provides proof that vaccination with mature DCs expands tumor-specific T cells in advanced melanoma patients. In addition, we have found some evidence for the direct interaction between CD8 + CTLs and tumor cells as well as for escape of antigen-negative metastases. We are convinced that DC-mediated immunization can be intensified further to reveal the presence of expanded populations of effector cells. Efficacy might be increased at the level of the DC, e.g., by optimizing variables such as DC maturational state, route, dose, and schedule or by improving the short life span of DCs in vivo 31 32 ; at the level of the T cell, e.g., by providing melanoma-specific CD4 + T cell help 33 34 or IL-2 35 ; and by treating patients earlier in their disease course. | Other | biomedical | en | 0.999997 |
10587358 | Mononuclear cells were isolated by Histopaque (Sigma Chemical Co.) gradient centrifugation. Peripheral blood monocytes were then isolated from the mononuclear cells by either Percoll (Sigma Chemical Co.) gradient centrifugation 3 7 or countercurrent centrifugal elutriation (Beckman-Coulter) 7 33 . All experiments were performed on monocytes that were isolated both ways, except where noted. There were no differences in the results due to the method of isolation. Monocyte purity was >90% as determined by morphology, CD14 staining, and nonspecific esterase staining. Monocytes were differentiated in RPMI containing 20% heat-inactivated fetal bovine serum (FBS) plus 1 μg/ml polymyxin B sulfate (Sigma Chemical Co.) 4 5 in 24-well plates (Costar) except when noted. For transient transfections, 3 × 10 6 U937 cells were cultured in 100-mm plates, cotransfected for 4 h with 8, 6, or 4 μg of test plasmids and with 2 μg of CMV–enhanced green fluorescent protein (EGFP) expression plasmid (Clontech), using the FuGENE™ procedure (1:5 ratio of DNA/FuGENE™; Roche Biochemicals). Empty vector was added to transfections to yield a total of 10 μg of DNA per transfection. After transfection, cultures were washed, incubated in 20% FBS/RPMI for 12 h, and treated with hamster anti-Fas antibody (500 ng/ml, clone CH11; MBL) for an additional 12 h. U937 cells were collected, and EGFP-expressing cells were quantified by flow cytometry. Nonviable cells were excluded by propidium iodide (PI) incorporation. Histopaque/Percoll-isolated monocytes were cultured on 60-mm plates containing glass coverslips treated with acetic acid/ethanol (ETOH) 34 35 . At the indicated time points, cultures were fixed in 4% neutral buffered formalin for 5 min and subjected to two washes in PBS. Individual coverslips were treated with 0.5% NP-40 for 5 min, followed by two PBS washes. TdT enzyme and a cocktail containing dUTP conjugated to a fluorescein (FITC) were added to the coverslips according to the manufacturer's specifications (In Situ Death Detection kit; Roche Biochemicals). Nuclei were counterstained with Hoechst 33258 (Sigma Chemical Co.), and mounted for examination using mounting media for fluorescence (Kirkegaard & Perry). Specimens were examined and photographed on a Zeiss microscope equipped with a phase–contrast and epifluorescence optics. Pictures were recorded on Zeiss software. At the indicated time points, cultures were harvested in 0.02% EDTA and fixed in 70% ETOH overnight 36 37 38 . Cells were then stained with PI (Roche Biochemicals) as described previously 35 . The subdiploid peak, immediately adjacent to the G0/G1 peak (2N), was determined by flow cytometry using an EpicsXL flow cytometer (Beckman-Coulter) and system 2 software . Objects with minimal light scatter were excluded since they may represent debris and would have inappropriately enhanced our estimate of the subdiploid population 38 . For TUNEL analysis by flow cytometry, immediately isolated and 1-d monocytes were analyzed with the Apo-Direct™ apoptosis assay, according to the manufacturer's specifications (PharMingen). In brief, cultures were fixed in 1% paraformaldehyde, permeabilized in 70% ETOH for 1 h, and incubated with FITC-dUTP, TdT, and reaction buffer for 1 h. Cells were then analyzed for FITC-dUTP incorporation by flow cytometry. Fas expression was determined in immediately isolated monocytes and 7-d macrophages that were harvested in 0.02% EDTA, blocked in 50% human serum for 1 h, and then incubated with FITC-labeled anti-Fas antibody (clone UB2; Beckman-Coulter) or with FITC-labeled isotype control (Becton Dickinson). As additional negative controls, monocytes and macrophages were analyzed with FITC-conjugated anti-CD3 or anti–TCR-γ/δ antibodies (Becton Dickinson) and the appropriate isotype control. For surface FasL staining, monocytes or 7-d macrophages were blocked in 50% human serum for 1 h. Cells were then incubated in 33% human serum/33% goat serum with either rabbit anti-FasL (clone C-20; Santa Cruz Biotechnology), hamster anti-FasL (clone 4H9; Beckman-Coulter), or normal control IgG (Sigma Chemical Co.). Cells were then incubated with FITC-labeled goat anti–rabbit antibody (Kirkegaard & Perry) or rabbit anti–hamster antibody (Pel-Freez Biologicals). Mitochondrial permeability transition in macrophages after anti-Fas or TNF-α addition was analyzed by Rh123 (0.1 μg/ml; Molecular Probes). Rh123 was added to cultures for 30 min before analysis by flow cytometry, and live cells were determined by PI exclusion. Flow cytometry was conducted at the Robert H. Lurie Comprehensive Cancer Center, Flow Cytometry Core Facility of the Northwestern University Medical School. Monocytes or macrophages were incubated with either anti-FasL antibody (clone C-20), anti-FasL antibody (clone 4H9), anti-Fas antibody (clone CH11), control IgG (Dako), zVAD.fmk (Enzyme System Products), or TNF-α (R&D Systems) for 24 h. Cultures were harvested in 0.02% EDTA and examined for apoptosis. To inhibit Flip expression, phosphorothioate oligodeoxynucleotides were created to include the Flip initiation codon ( 24 ; Flip antisense oligonucleotide 5′-GACTTCAGCAGACATCCTAC-3′). Control nonsense phosphorothioate oligodeoxynucleotides have been described previously ( 39 ; 5′-TGGATCCGACATGTCAGA-3′). FITC-conjugated oligonucleotides (10 or 20 μM) were added to macrophages for 24 h. Cells were removed in 0.02% EDTA, fixed in 70% ETOH, stained with PI, and analyzed by flow cytometry. Parallel cultures were harvested for immunoblot analysis. Additionally, oligonucleotides in combination with either rabbit anti-FasL (clone C-20), hamster anti-FasL (clone 4H9), or control IgG (Dako) were also added to 7-d macrophages for 24 h. Cells were analyzed for DNA fragmentation by Cell Death ELISA (see below). Peripheral blood monocytes, isolated by Histopaque/Percoll gradient centrifugation, were differentiated in 20% FBS/RPMI/1 μg/ml polymyxin B and harvested for RNA preparation as described by Chomczynski et al. 40 . 1 μg of total RNA was incubated in reaction buffer containing oligo(dT) primer, Moloney murine leukemia virus reverse transcriptase (RT), reaction buffer, and RNase inhibitor for 1 h at 42°C according to the manufacturer's specifications (Clontech). The reaction was stopped by incubation at 94°C for 5 min. Primers specific for Flip were as follows: forward, 5′-GATGTCTGCTGAAGTCATCCATCA-3′; reverse for Flip L , 5′-CACTACGCCCAGCCTTTTGG-3′, and reverse for Flip S , 5′-AGTAGAGGCAGTTCCATG-3′. The reverse Flip S primer is within the 3′ untranslated region of Flip S , thus allowing for delineation between Flip L and Flip S . The PCR reaction was carried out with 5 U Taq polymerase (Perkin Elmer) in a total volume of 100 μl. Amplification was performed for 28 cycles (30 s denaturing at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C) in a thermal cycler. Control human β-actin primers were used under parallel conditions (Clontech). The 1,470-bp Flip L , 620-bp Flip S , and 838-bp β-actin amplified products were analyzed by 1.0% agarose gel electrophoresis and visualized under UV illumination after being stained with ethidium bromide. Whole-cell extracts were prepared from peripheral blood monocytes and differentiated in 20% FBS/RPMI/1 μg/ml polymyxin B. 25 or 50 μg of extract, as indicated, was analyzed by SDS-PAGE on 12.5% polyacrylamide gels, and transferred to Immobilon P (Millipore) by semidry blotting. Filters were blocked for 1 h at room temperature in PBS/0.2% Tween 20/5% nonfat dry milk. The filters were then incubated with rabbit anti-Flip 41 , which recognizes both Flip isoforms, with rabbit anti-caspase 8 antibody (Chemicon) or mouse anti-caspase 3 antibody (Transduction Laboratories) at 4°C in PBS/0.2% Tween 20/2% nonfat dry milk. Filters were washed in PBS/0.2% Tween 20/2% nonfat dry milk and incubated with either donkey anti–rabbit or anti–mouse secondary antibody (1:2,000 dilution) conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Visualization of the immunocomplex was performed using Enhanced Chemiluminescence Plus kit (Amersham Pharmacia Biotech). DNA fragmentation was detected using Cell Death ELISA Plus kit (Roche Biochemical) as recommended by the manufacturer. Mono- and oligonucleosomal DNA were detected in the cytoplasmic fraction of cell lysates. In brief, cell lysates were incubated in antihistone-coated microtiter plates. DNA attached to the bound histones was detected with peroxidase-conjugated anti-DNA antibody. After wash steps, substrate was added to the microtiter wells and color change was read at 405 nm by a microplate reader (Bio-Tek Instruments). Previous investigators demonstrated decreasing cell numbers over the first 3 d when monocytes were cultured in serum 32 42 43 44 . Here, we examined the occurrence of apoptosis during monocyte to macrophage differentiation in serum. TUNEL analysis, which measures DNA fragmentation, demonstrated peak apoptosis at 1 and 2 d after isolation, though few or no TUNEL-positive macrophages were observed at 0, 3, 7, and 14 d . Quantitative analyses of subdiploid DNA content in serum-treated monocytes revealed enhanced spontaneous apoptosis in the 0.5-, 1-, and 2-d cultures (12.1 ± 1.7, 26 ± 4.7, and 23 ± 9.0%, respectively). However, macrophages at 3, 7, and 14 d displayed minimal hypodiploid DNA content (<6%). To corroborate the quantification of apoptosis as determined by subdiploid DNA content, flow cytometry measuring FITC-dUTP incorporation (TUNEL) was performed. Since maximal apoptosis was exhibited in 1-d monocyte cultures, immediately isolated (day 0) and 1-d monocytes were examined. In the 1-d monocyte cultures, 25.8 ± 2% TUNEL-positive cells were detected , similar to the analysis of subdiploid DNA . Little or no TUNEL positivity (<1%) was observed in monocytes immediately after isolation. Furthermore, cell counting by trypan blue exclusion revealed a 50–60% reduction of monocyte cell numbers at 3 d after isolation (not shown), indicating that the apoptosis observed at 1 and 2 d resulted in the cumulative loss of cells. Consistent with previous reports 5 30 31 32 , >70% of serum-deprived monocytes underwent spontaneous apoptosis after 24 h (not shown). Collectively, these data demonstrate that in vitro–differentiated macrophages, unlike monocytes, undergo little or no spontaneous apoptosis. Monocytes cultured in serum undergo spontaneous apoptosis that may be mediated by the Fas–FasL pathway. Flow cytometric analyses of surface Fas and FasL revealed that Fas was present on essentially all the cells, although it was more strongly expressed on monocytes compared with macrophages . FasL was also detected on the surface of both monocytes and macrophages, though more FasL was expressed on macrophages . To confirm that the detection of Fas and FasL was specific, flow cytometric analysis using FITC-conjugated anti-CD3 and anti–TCR-γ/δ antibodies demonstrated comparable staining to the isotype control in both monocytes and macrophages . To define the mechanism(s) responsible for monocyte apoptosis, inhibitory FasL antibodies and the general caspase inhibitor zVAD.fmk were compared with TNF-α, which was previously shown to inhibit monocyte apoptosis 2 5 30 31 . Addition of either C-20 (82% inhibition) or 4H9 (41% inhibition, not shown) neutralizing anti-FasL antibodies to isolated monocytes in 20% serum significantly ( P < 0.02) inhibited spontaneous apoptosis compared with IgG-treated or medium control–treated (mock) monocytes . Similarly, TNF-α inhibited apoptosis by 62% ( P < 0.02) while the general caspase inhibitor zVAD.fmk partially blocked spontaneous apoptosis (41%, P < 0.01) in 1-d and 2-d (not shown) monocyte cultures. The contribution of caspase activation to Fas-mediated apoptosis in monocytes was further examined by immunoblot analyses. Reduced detection of the procaspases 8 and 3 was observed in extracts from 0 and 1-d monocytes compared with 7-d macrophages. The intermediate active form of caspase 3 (p24 45 ) was detected in extracts of monocytes on days 0 and 1, but not those from day 7 macrophages. As a control, Fas-agonistic antibody–treated Jurkat T cells also displayed the intermediate active caspase 3 (not shown). These data indicate that the spontaneous monocyte apoptosis that occurs in serum was attributed to Fas–FasL interaction and caspase activation. The ability of agonistic Fas antibody and TNF-α to induce apoptosis in macrophages was examined. Macrophages were resistant to spontaneous and agonistic Fas antibody–induced apoptosis as indicated by normal DNA content and mitochondrial membrane integrity, suggesting that a potent inhibitor of the death receptor–mediated pathway, not present in monocytes, may be upregulated in macrophages. As a control, agonistic Fas antibody induced apoptosis in Jurkat T cells and U937 cells (not shown). In certain cell types, Flip overexpression has been shown to directly inhibit Fas-mediated apoptosis 29 , indicating that Flip may function to promote survival during monocyte to macrophage differentiation. Previous investigations demonstrated that by day 3 in culture, monocytes begin to differentiate into macrophages based on morphology, cell surface markers including vitronectin (CD51 ), transferrin receptor (CD71; not shown), and cytolytic activity 46 . Immunoblot analyses performed on isolated peripheral blood monocytes harvested at 0, 1, 2, 3, 7, and 14 d revealed that Flip upregulation was associated with macrophage differentiation and reduced apoptosis, beginning on day 3 . Flip L was detected at 7 and 14 d of macrophage differentiation, whereas Flip S was highly expressed 3 d after isolation . The ratio of Flip L to Flip S on days 3, 7, and 14 varied between individuals. Although low levels of Flip were detected before day 3 in some individuals, Flip upregulation at 3, 7, and 14 d was observed in every individual examined ( n = 10). These data demonstrate that the decreased spontaneous apoptosis seen during and after macrophage differentiation is associated with Flip upregulation. To determine if the Flip expression observed at 3 d is transcriptionally regulated, RT-PCR analyses performed with specific primers that differentiate between the two Flip isoforms revealed minimal Flip S or Flip L mRNA transcripts in immediately isolated monocytes (Day 0). However, Flip S and Flip L mRNA were upregulated on day 3 . Equally expressed β-actin mRNA transcripts indicated that similar levels of mRNA were amplified in all samples. These data demonstrate that Flip is upregulated at the transcriptional level during monocyte differentiation into macrophages. Monocytes do not express Flip and are susceptible to Fas-mediated apoptosis, suggesting that forced expression of Flip may protect monocytes from apoptosis in response to Fas ligation. Since peripheral blood monocytes are difficult to transfect, the monocytic cell line U937, which expresses little or no Flip (not shown) 47 and undergoes apoptosis in response to agonistic Fas antibody 6 , was used. An established cell death assay 48 49 50 was performed using expression plasmids encoding Flip L , Flip S 24 , Bcl-2 35 , or empty vector transfected in combination with an EGFP expression plasmid. Flow cytometric analyses were used to determine changes in cell viability as indicated by the number of EGFP-positive cells. Equal levels of Flip S and Flip L expression were demonstrated by transient transfection of the expression plasmids in 293 cells (not shown). Expression vectors encoding Flip L or Flip S protected U937-transfected cells from Fas-mediated apoptosis in a dose-dependent manner . In addition, Flip S -transfected U937 cells displayed increased viability compared with Flip L -transfected cells (15-fold vs. 8-fold). On the contrary, Bcl-2 provided little protection (twofold) compared with empty vector–transfected U937 cells . To determine if Flip is functionally significant for macrophage survival, FITC-conjugated antisense oligonucleotides were created complementary to a sequence that included the start site of the Flip open reading frame 24 . Flow cytometry revealed similar uptake of the antisense and control nonsense oligonucleotides 39 in day 7 macrophages. Immunoblot analyses of oligonucleotide-treated macrophages demonstrated decreased expression of both Flip isoforms, using both 10 and 20 μM of the antisense oligonucleotides, compared with medium control (mock) or control oligonucleotide-treated macrophages . Flow cytometric analyses revealed that 40 ± 3% of the antisense oligonucleotide–treated macrophages underwent apoptosis compared with 1 ± 0.1 and 3 ± 0.5% of mock- and control oligonucleotide–treated macrophages, respectively. Furthermore, reduction of the procaspase 3 was observed in the antisense oligonucleotide–treated macrophages compared with mock or control oligonucleotide–treated cells, supporting the role of caspase activation in macrophage cell death. Inhibition of Fas–FasL interactions by two different antagonistic FasL antibodies (40% with C-20 and 67% with 4H9) suppressed macrophage apoptosis induced by Flip antisense treatment, as indicated by trypan blue exclusion (not shown) and by DNA fragmentation . Addition of FasL antibodies or control IgG had no effect on the viability of control oligonucleotide–treated cultures (not shown). These data demonstrate that Flip contributes to macrophage survival and document for the first time the functional significance of endogenous Flip expression, which is necessary for survival during in vitro monocyte to macrophage differentiation. Isolated monocytes are highly sensitive to apoptosis induced by Fas–FasL ligation, whereas differentiated macrophages are resistant. Our data document that Fas–FasL interactions, which mediate caspase activation, contribute to the continued reduction of monocytes during the first 48–72 h. As early as 2 h after isolation (day 0), before the appearance of hypodiploid DNA or TUNEL positivity, caspases were already activated. This could be the result of activation of caspases, where the inactive procaspase has been cleaved to form the active protease 51 . It is also possible that the increased procaspases detected in day 7 macrophages may be attributed to increased synthesis. While our data do not exclude this possibility, a previous study demonstrated caspase 8 activity in freshly isolated monocytes 52 , suggesting that the reductions seen in our study (days 0 and 1) were at least in part due to caspase activation. We also observed the intermediate cleaved caspase 3 (p24) isoform on days 0 and 1, but not day 7, indicating that activation had occurred at the early time points. Protection of monocytes from apoptosis by the caspase inhibitor zVAD.fmk further supports the interpretation that caspases were activated immediately after isolation . The fact that caspase activation appears to be initiated even during isolation may explain the lack of complete inhibition by neutralizing anti-FasL or zVAD.fmk. A decline in monocyte cell number by day 3 correlated with Flip expression. Both RT-PCR and immunoblot analyses demonstrated an upregulation in Flip expression by day 3. Although the ratios of the two isoforms, as determined by immunoblot analyses, varied between individuals at 3, 7, and 14 d, our data suggest that Flip expression was responsible for the protection against apoptosis observed on day 3 and thereafter. The incubation of macrophages with Flip antisense oligonucleotides reduced Flip expression by Western blot analyses and increased apoptosis as determined by hypodiploid DNA content. Furthermore, the concurrent reduction of procaspase 3 in the Flip antisense oligonucleotide–treated macrophages suggests that this process activated caspases. Additionally, the marked protection of U937 cells from Fas-mediated apoptosis by Flip, but not Bcl-2, supports the importance of Flip for macrophage survival. Recently, stably expressed Bcl-2 was shown to provide modest protection against Fas-mediated apoptosis in U937 cells 53 , which was comparable to that observed in our study. Decreased numbers of GFP-positive U937 cells were not seen even when higher concentrations of the Flip expression plasmids were used. However, in other cell types, overexpression of Flip ( 22 24 25 27 ; and data not shown) resulted in cell death, suggesting that the effects of Flip overexpression may be cell type specific. These data document an important role for Flip in the resistance of macrophages to Fas–FasL-mediated cell death. Surface Fas was expressed on essentially all day 7 macrophages, though less intensely compared with monocytes, suggesting that reduction in the amount of surface Fas on macrophages may have contributed to reduced apoptosis. However, in a recent investigation primary endothelial cells, which express low levels of Fas 54 55 comparable to those observed on macrophages, were sensitized to Fas-induced apoptosis by oxidized lipids 55 . Interestingly, the susceptibility of endothelial cells to Fas-mediated apoptosis induced by oxidized lipids correlated with Flip downregulation 41 even though the level of surface Fas remained unchanged. Fas–FasL interactions also mediated apoptosis in macrophages after Flip inhibition, since neutralization of FasL suppressed macrophage apoptosis in the presence of the Flip antisense oligonucleotides. These data demonstrate that Flip, and not reduced surface Fas, was responsible for the absence of Fas-mediated macrophage apoptosis. Activation of macrophages is an integral component of several host defense mechanisms, including activation of T cells, release of inflammatory cytokines, removal of virus-infected cells, and the antibody-dependent and -independent killing of tumorigenic cells 56 . Although macrophages are resistant to Fas-mediated apoptosis, they may be rendered Fas sensitive. TNF-α or a combination of TNF-α plus INF-γ sensitized mouse peritoneal macrophages to Fas-mediated apoptosis in vitro. Additionally, Th1 CD4 + T cells induced apoptosis of antigen-pulsed, IFN-γ–treated peritoneal macrophages, which was mediated by Fas–FasL interactions 57 . Studies have yet to determine if decreased Flip expression contributed to macrophage apoptosis under these conditions. FasL on macrophages may not induce apoptosis of T cells for which they serve as the APCs, because unactivated T cells strongly express Flip 24 58 59 . However, after stimulation, T cells downregulate Flip expression and undergo suicide, resulting in activation-induced cell death that is mediated by the Fas–FasL pathway 24 58 59 60 61 . Thus, depending on the environmental milieu, T cell and macrophage sensitivity to Fas-mediated apoptosis appears to be regulated by Flip expression. Circulating blood monocytes extravasate into tissues and differentiate into macrophages. Persistent expression of FasL by adenoviral delivery to endothelial cells prevented monocytes from emigrating into the inflamed tissue 54 , suggesting that Fas is functional on monocyte surfaces in vivo. In addition, mice carrying functional mutations of Fas–FasL displayed elevated macrophage cell numbers 11 , indicating the importance of Fas–FasL in regulating monocyte/macrophage homeostasis. Recently, we identified Flip expression in macrophages isolated from synovial fluid and in the synovial tissues (not shown) of patients with rheumatoid arthritis, demonstrating a potential significance for Flip expression in vivo. Additionally, animals with experimental arthritis also displayed increased numbers of synovial macrophages that were Flip positive (not shown). Thus, modulation of Flip expression may provide a novel therapeutic approach to diseases mediated by macrophages, such as rheumatoid arthritis or atherosclerosis. | Study | biomedical | en | 0.999996 |
10587359 | BALB/c mice were purchased from Bomholtgard. Mice deficient in FcR γ chains (FcRγ), which lack functional expression of both FcγR type I (FcγRI) and type III (FcγRIII), and wild-type littermates were developed as described previously 15 . The VDJH4C8-Cγ2a plasmid containing the complete 4C8 IgG heavy chain gene of the IgG2a isotype was constructed using the following DNA fragments: the rearranged VDJ region isolated from cDNA encoding the variable region of the heavy chain of the 4C8 mAb 16 , the promoter region isolated from pSV-Vμ1 17 , the heavy chain enhancer region isolated from pSVE2-neo 18 , and the Cγ2a region derived from the genomic clone, pIgH10 19 . Hybridomas secreting the 4C8 IgM and 34-3C IgG2a anti–mouse RBC mAbs were derived from unmanipulated NZB mice 2 4 . The 4C8 IgG2a class-switch variant was obtained by transfecting 4C8 heavy chain loss mutant cells by electroporation with the VDJH4C8-Cγ2a plasmid together with a pSVE2-neo plasmid containing the neomycin-resistant gene. After selection for resistance to neomycin and secretion of IgG antibodies, stable transfected cells secreting the 4C8 IgG2a variant were cloned by limiting dilutions. IgG2a anti-TNP (Hy1.2) and IgM anti-IgG2a 6 7 8 9 10 11 12 13 14 15 16 17 18 19 mAbs were used as control. Rat anti–mouse κ chain mAb (H139.52.1.5) was provided by Dr. M. Pierres (Centre d'Immunologie de Marseille-Luminy, Marseille, France 20 ). IgG mAbs were purified from culture supernatants by protein A column chromatography. IgM mAbs were purified by euglobulin precipitation from culture supernatants concentrated by 50% saturated ammonium sulfate precipitation, according to the method of Garcia-Gonzales et al. 21 . The purity of IgG and IgM was >90% as documented by SDS-PAGE. RNA was prepared from 4C8 IgG2a–transfected cells by RNeasy Mini Kit (Qiagen). The first strand of cDNA was synthesized with an oligo(dT) primer and 5 μg of total RNA. For amplification with Pfu DNA polymerase (Stratagene), the following primers were used: 5′-untranslated VH primer (5′-CAGTTCTCTCTACAGTTA-3′) and Cγ2a-CH1 primer (5′-GCCAGTGGATAGAC-3′) for the 4C8 heavy chain; 5′-untranslated Vκ primer (5′-CAGGGGAAGCAAGATGG-3′) and Cκ primer (5′-TGGATGGTGGGAAGATG-3′) for the 4C8 light chain. The nucleotide sequence corresponding to the variable region of the 4C8 heavy or light chain was determined by the dideoxynucleotide chain terminating method 22 . A flow cytometric assay was used to detect anti–mouse RBC activities of purified mAbs. 50 μl of various concentrations of purified mAbs was incubated with 20 μl of 1% RBC suspension, prepared from BALB/c mice, in 1% BSA-PBS for 30 min at 4°C. After washing three times with 1% BSA-PBS, the RBCs were incubated with biotinylated rat anti–mouse κ chain mAb (H139.52.1.5), followed by PE-conjugated streptavidin R (Caltag), and analyzed with a FACScan™ (Becton Dickinson). The presence of opsonized RBCs in mice injected with anti–mouse RBC mAb was detected by a similar flow cytometric assay, using biotinylated rat anti–mouse κ chain mAb. For in vitro and in vivo bindings of anti–mouse RBC mAb on biotinylated mouse RBCs, bound antibodies were revealed with PE-labeled F(ab′) 2 goat anti–mouse Ig conjugates (Jackson ImmunoResearch Labs). An indirect radioimmunoassay was also used to detect anti–mouse RBC activities of mAbs, as described previously 4 . In brief, 100 μl of various concentrations of purified mAbs was incubated with 10 μl of 25% RBC suspension in 1% BSA-PBS for 30 min at 4°C. After washing three times with 1% BSA-PBS, the RBCs were incubated with 125 I-labeled goat anti–mouse Ig in 1% BSA-PBS. Results are expressed as the radioactivity (in cpm) bound to RBCs. Complement-mediated lysis and hemagglutination assays were as described previously 4 . Autoimmune hemolytic anemia was induced by a single intraperitoneal injection of purified anti-RBC mAb into 2–3-mo-old mice. Blood samples were collected into heparinized microhematocrit tubes and centrifuged in a hematocentrifuge, and hematocrits (Ht) were directly determined after centrifugation, as described previously 4 . Mouse RBCs were biotinylated in vivo by an intravenous infusion of N -hydroxysuccinimide biotin (NHS-biotin; Boehringer Mannheim), according to the method described by Dale and Daniels 23 . In brief, 2 mg NHS-biotin was dissolved in 0.12 ml N,N -dimethylacetamide (Sigma Chemical Co.), incubated for 2 min in the dark at 37°C, and added to 3.2 ml sterile saline. 500 μg of NHS-biotin was then injected intravenously into mice. This in vivo biotinylation procedure resulted in essentially all the RBCs being biotinylated, as judged by staining with streptavidin Red 670 (GIBCO BRL) 24 h after the infusion of NHS-biotin. Livers and spleens were obtained at autopsy, processed for histological examination, and stained with hematoxylin and eosin (HE). The extent of in vivo RBC destruction by Kupffer cell–mediated phagocytosis was revealed by coloration of liver sections with Perls iron staining. The strategy for generating the recombinant IgG2a isotype-switch variant was to clone the heavy chain variable region (VH) gene from the 4C8 IgM anti–mouse RBC hybridoma and to join this gene to the already cloned mouse IgG2a heavy chain constant region gene. The recombinant IgG2a isotype-switch variant was then expressed in a heavy chain loss mutant of the 4C8 IgM hybridoma. Notably, the VH4C8 and Vκ4C8 sequences of the 4C8 heavy and light chain cDNA derived from a reverse transcription PCR amplification of mRNA isolated from the cells secreting 4C8 IgG2a switch variant were identical to the originally published sequence 16 . The in vitro binding activity of the 4C8 IgG2a switch variant, compared with its IgM isotype and the high-affinity 34-3C IgG2a anti–mouse RBC mAb, was investigated by flow cytometric analysis using a biotinylated rat anti–mouse κ chain mAb, followed by PE-conjugated streptavidin. Incubation of mouse RBCs with 0.1 ng of the 4C8 IgM or 34-3C IgG2a mAb resulted in substantial binding, whereas >250 ng of the IgG2a switch variant was required to show significant binding, with a maximal binding at 1 μg . Thus, mouse RBC-binding activity of the 4C8 IgG2a variant was at least 1,000 times less than that of its IgM isotype and the 34-3C IgG2a mAb. It should also be mentioned that the staining intensity obtained by the 4C8 IgG2a variant was far lower than that observed with the 4C8 IgM or the 34-3C IgG2a mAb. Marginal RBC-binding activities by the 4C8 IgG2a variant were confirmed with a radioimmunoassay . These results indicated that the 4C8 mAb has a low binding affinity, while its binding activity was markedly promoted by the pentameric high-avidity IgM isotype. Moreover, hemagglutination activity was only detectable with the 4C8 IgM mAb, and neither IgM nor IgG2a 4C8 mAb exhibited complement-mediated hemolysis in vitro in the presence of guinea pig, rabbit, or mouse serum (data not shown). Because of its markedly limited in vitro binding to mouse RBCs, we determined whether the 4C8 IgG2a variant was capable of binding to circulating RBCs in vivo. When analyzed by a flow cytometric assay 24, 48, and 72 h after a single intraperitoneal injection of 1 mg of the 4C8 IgG2a variant into BALB/c mice, opsonized RBCs were undetectable in the circulating blood . This was in marked contrast to the presence of bound antibodies on essentially all the circulating mouse RBCs between 24 and 72 h after the administration of 50 μg of the 4C8 IgM or 34-3C IgG2a mAb. Notably, the staining pattern obtained by in vitro incubation with the 4C8 IgG2a variant was indistinguishable between RBCs from mice injected with the 4C8 IgG2a and those from control mice, excluding a rapid and selective elimination of a subpopulation of circulating RBCs by the low-affinity 4C8 IgG2a (data not shown). Thus, the lack of detection of opsonized RBCs in the circulating blood further confirmed a low-affinity feature of the 4C8 IgG2a switch variant. Strikingly, mice injected with 1 mg of the 4C8 IgG2a variant developed a severe anemia with a decrease in Ht values down to 21% 4 d after the injection (see below). To verify the elimination of circulating RBCs by the 4C8 IgG2a variant, RBCs were in vivo biotinylated with an intravenous injection of NHS-biotin, and their clearance was then determined after the injection of 1 mg of 4C8 IgG2a or Hy1.2 IgG2a anti-TNP mAb. As shown in Fig. 3 B, biotinylated RBCs were gradually replaced by newly generated RBCs in control mice injected with Hy1.2 anti-TNP mAb, while the injection of the 4C8 IgG2a variant markedly accelerated this process. Although Ht values began to recover ∼5 d after the 4C8 IgG2a mAb injection and returned to almost normal levels by 8 d , the levels of biotinylated RBCs were further decreased. 8 d after 4C8 IgG2a mAb injection, biotinylated RBCs were hardly detectable (<1%), while ∼70% of circulating RBC were still biotinylated in control mice. This indicated that the 4C8 IgG2a variant indeed bound to circulating RBCs in vivo and efficiently eliminated them. Notably, the immunoreactivity of biotinylated RBCs, obtained from 24 to 72 h after in vivo biotinylation, to the 4C8 mAb was comparable to that of control RBCs, as judged by in vitro and in vivo binding analyses, and the kinetics and extent of anemia induced by the 4C8 mAb in biotin-treated mice were essentially identical to those of untreated mice . To compare the pathogenic activity of the 4C8 IgG2a variant with its IgM isotype and the high-affinity 34-3C IgG2a mAb, the development of anemia was analyzed by a single intraperitoneal injection of various amounts of purified mAb into BALB/c mice. Despite low-affinity binding to mouse RBCs, the 4C8 IgG2a mAb was highly pathogenic, and 50 μg was sufficient to cause significant anemia . This dose was comparable to that required for the induction of anemia by its IgM isotype and the high-affinity 34-3C IgG2a mAb. Histological examinations showed that erythrophagocytosis by Kupffer cells was the most remarkable pathological change associated with anemia induced by the 4C8 IgG2a isotype , as observed in mice developing anemia after the injection of the 34-3C IgG2a mAb 4 . Destruction of RBCs by phagocytosis was further documented by extensive iron deposits in Kupffer cells from mice injected with the 4C8 IgG2a variant . In contrast, the injection of 4C8 IgM mAb at a dose of 250 μg resulted in an enormous accumulation of agglutinated RBCs in the spleen, and in hepatic sinusoids accompanied by occasional necrosis of hepatic parenchymal cells , as shown previously 4 . To determine the contribution of FcγR-mediated erythrophagocytosis to the pathogenesis of the 4C8 IgG2a–induced anemia, the development of anemia was assessed in FcRγ-deficient mice lacking functional expression of both FcγRI and FcγRIII involved in phagocytosis of IgG2a-opsonized RBCs 13 14 . As shown in Fig. 6 , FcRγ-deficient mice were completely resistant to the pathogenic effect of 1 mg of the 4C8 IgG2a mAb, and failed to exhibit erythrophagocytosis, as documented by the lack of iron deposits in their Kupffer cells . In this study, we have demonstrated that the 4C8 anti–mouse RBC autoantibody, derived from lupus-prone NZB mice, has a low binding affinity to its corresponding self-antigen, yet its IgM and IgG2a isotypes exhibit a remarkably high pathogenic potency. Our results indicate that the pentameric form of a low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity, and that the capacity of the IgG2a isotype to interact with FcγR involved in erythrophagocytosis is responsible, despite its barely detectable RBC-binding activity, for its hemolytic activity. Demonstration of the high pathogenic potency of low-affinity autoantibodies highlights a remarkable role of Ig heavy chain effector functions, as opposed to a relatively minor role of autoantigen-binding affinities, in the pathogenesis of autoimmune hemolytic anemia. A striking feature of our analysis is that despite the lack of detection of significantly opsonized RBCs in the circulating blood, the 4C8 IgG2a variant with low binding affinity is able to induce a severe anemia as a result of peripheral destruction of circulating RBCs. This was documented by an efficient clearance of in vivo–biotinylated circulating RBCs and by Kupffer cell–mediated erythrophagocytosis. To our surprise, the amount of the IgG2a isotype-switch variant required to induce anemia was comparable to that of the high-avidity IgM isotype exhibiting >1,000 times stronger RBC-binding activity. This indicates that the pathogenic activity of low-affinity IgG anti-RBC autoantibodies is dramatically enhanced when provided with an appropriate Ig heavy chain effector function, namely the capacity to interact with FcγR involved in erythrophagocytosis. Thus, a rapid FcγR-mediated uptake by Kupffer cells of even very poorly opsonized RBCs present in hepatic microcirculation appears to be an extremely efficient mechanism for the development of anemia induced by IgG anti-RBC autoantibodies. An additional, unexpected observation was that the pathogenic potential of the low-affinity 4C8 IgG2a autoantibody was also comparable to the high-affinity 34-3C IgG2a mAb 4 . However, this comparison is only tentative in its interpretation because of possible differences in the specificity of the 4C8 and 34-3C mAbs 24 . Only experiments comparing IgG2a mAbs of the same specificity, but with different affinities, would provide definitive conclusions on this issue. Nevertheless, our results strongly suggest that RBC-binding affinities of Coombs' autoantibodies apparently play a relatively minor role for in vivo hemolytic activities of the IgG2a isotype capable of interacting efficiently with two different classes of FcγR (FcγRI and FcγRIII) involved in erythrophagocytosis 13 14 25 26 . It may be worth noting that although the pathogenicity of the 4C8 and 34-3C IgG2a mAbs was almost comparable at lower doses, it appears that the 34-3C mAb at higher doses is more pathogenic than the 4C8 IgG2a . Although we do not have a straightforward explanation for this phenomenon at present, it can be speculated that the IgG2a isotype of anti-RBC autoantibodies could mediate erythrophagocytosis through their binding to the high-affinity FcγRI, followed by their subsequent interaction with circulating RBCs. A significant binding of free IgG2a anti-RBC to FcγRI on phagocytic cells is likely to occur only when higher doses of IgG2a mAb are injected, because of the competition of circulating monomeric IgG2a without anti-RBC activity. Owing to its high-affinity RBC-binding capacity, even limited amounts of the cell-bound 34-3C IgG2a may efficiently capture circulating RBCs, thereby mediating erythrophagocytosis. In contrast, the ability to capture circulating RBCs by the low-affinity 4C8 IgG2a bound on Kupffer cells may be too poor to cause erythrophagocytosis by such a mechanism. Consistent with this hypothesis, our recent analysis on different FcγR-deficient mice has shown that the erythrophagocytosis caused by lower doses of the 34-3C and 4C8 IgG2a mAbs is primarily mediated by the low-affinity FcγRIII, while FcγRI is only involved in the development of anemia induced by higher doses of these mAbs (our unpublished results). In addition to the remarkable contribution of IgG Fc region mediating FcγR interaction to pathogenic activities of the low-affinity 4C8 IgG2a variant, these studies also suggest that the strong pathogenic activity of the IgM 4C8 isotype is dependent on its pentameric form, which promotes the binding and agglutination of RBCs. Consequently, the IgM isotype of the 4C8 mAb induces a different form of severe anemia, resulting from massive agglutination of RBCs in spleen and liver, which does not involve FcγR-mediated phagocytosis and complement activation 4 13 . Although the present study does not formally prove that the pentameric form of the 4C8 IgM antibody is crucial for the in vivo agglutination of RBCs, we have recently observed that the IgG3 variant of the 4C8 mAb exhibits neither FcγR-dependent erythrophagocytosis nor agglutination of RBCs in vivo (our unpublished results), supporting the importance of the IgM pentameric form for the in vivo agglutination of RBCs by this mAb. However, it should be stressed that we have previously observed that two of four IgM anti-RBC mAbs failed to induce anemia 4 . The lack of pathogenic activities by these two IgM autoantibodies is likely to be related to their specificities, since they are unable to induce agglutination of mouse RBCs in vitro. Thus, the surface density of corresponding self-antigen epitopes may be too low to promote high-avidity binding and agglutination of mouse RBCs in vivo by these IgM anti-RBC autoantibodies. Our demonstration here of the high pathogenic potency of low-affinity autoantibodies suggests that the affinity maturation of autoantibodies may not be a critical process for the generation of autoantibodies with immunopathologic consequences. This is consistent with the demonstration that low-affinity anti-IgG2a rheumatoid factors of the IgG3 isotype are able to induce remarkable pathology—immune complex–mediated skin vasculitis and lupus-like glomerulonephritis—owing to their cryoglobulin activity, a unique property of the IgG3 isotype 11 12 . These results provide new insight into the cellular basis for the generation of pathogenic autoantibodies of IgM and IgG isotypes. It has already been shown that a fraction of B cells expressing the low-affinity 4C8 autoantibody can escape clonal deletion in the bone marrow and can be activated to produce pathogenic autoantibodies in the periphery, as a result of nonspecific activation of B cells 27 28 . These autoantibodies may even be switched to IgG classes under certain conditions, possibly through the action of the cytokines, independently of the presence of autoantigen-specific T helper cells, as is the case of T cell–independent type II immune responses 29 . Genetic abnormalities present in certain autoimmune-prone mice may favor the switching of IgM to IgG isotypes, as shown by a spontaneous class switch from IgM to IgG2a autoantibody by B cells derived from lupus-prone (NZB × NZW)F1 mice in the absence of functional CD4 + T helper cells 30 31 . In addition, we have recently observed that the constitutive expression of the bcl-2 transgene in germinal center B cells is able to induce the spontaneous production of IgG autoantibodies (our unpublished results). Therefore, it is possible that autoreactive B cells can be generated as a result of somatic hypermutations in the germinal centers during immune responses against environmental antigens, and such B cells may persist, if they are defective in the process of apoptosis, which is likely to be one of the genetic defects present in autoimmune-prone mice 32 33 . Thus, although the presence of somatic mutations in Ig variable regions, including CDR, of autoantibodies is likely to be CD4 + T cell dependent, several autoantibody-producing B cells may not have been driven by autoreactive T helper cells and self-antigens. This idea is consistent with a limited affinity maturation of clonally related rheumatoid factors, despite extensive somatic mutations, derived from a Fas-deficient MRL- lpr/lpr mouse 10 . Clearly, in view of the high pathogenic potential of low-affinity autoantibodies and of the critical role of CD4 + T cells in the development of autoimmune diseases 34 , better understanding of the nature of help provided by CD4 + T cells would help elucidate the cellular basis central to the development of autoantibody-mediated autoimmune diseases. | Study | biomedical | en | 0.999997 |
10587360 | A PCR fragment encoding full-length murine BAFF was generated by reverse transcription–PCR using previously described sequence information 23 . First strand cDNA was synthesized from mouse lung polyA+ (Clontech) using oligo dT according to the manufacturer's protocol (GIBCO BRL). The PCR reaction contained 1× pfu buffer (Stratagene Inc.), 0.2 mM dNTPs, 10% DMSO, 12.5 pM primers, 5 units pfu enzyme (Stratagene Inc.), and the following primers with Not1 restriction sites 5′-TAAGAATGCGGCCGCGGAATGGATGAGTCTGCAAA-3′ and 5′-TAAGAATGCGGCCGCGGGATCACGCACTCCAGCAA-3′. The template was amplified for 30 cycles at 94°C for 1 min, 54°C for 2 min, and 72°C for 3 min, followed by a 10-min extension at 72°C. This sequence corresponds to nucleotides 214–1171 of the GenBank file AF119383. The PCR fragment was digested with Not1 and cloned into a modified pCEP4 vector (Invitrogen Corp.). The resulting vector was then digested with Xba1 to remove BAFF plus the SV40 polyA addition site sequence. This fragment was cloned into a pUC-based vector in which the promoter, a 1-kb blunt Bgl2–Not1 fragment containing the human ApoE enhancer and AAT (alpha antitrypsin) previously purified from the plasmid clone 540B (a gift from Dr. Katherine Parker Ponder, Washington University, St. Louis, MO) was further inserted at the EcoRV site. An EcoRV/Bgl2 fragment was purified from the final vector and used for the generation of transgenic mice. The injected offspring of C57BL/6J female × DBA/2J male F1 (BDF1) mice were backcrossed onto C57BL/6 mice. Techniques of microinjection and generation of transgenic mice have been previously described 27 . Animal experiments were approved by the Institutional Animal Care and Use Committee. Serum samples were subjected to reducing SDS-PAGE analysis using a linear 12.5% gel. MOPC-21 mouse IgG1 standard antibody was obtained from PharMingen. Total RNA from mouse liver was prepared and processed for Northern blot analysis using an isolation kit from Promega Corp. according to the manufacturer's guidelines. BAFF transgene-specific mRNA was detected using a probe spanning the SV40 polyA tail of the transgene construct and obtained by digestion of the modified pCEP4 vector with Xba1 and BamH1. The probe recognizes a 1.8–2-kd band corresponding to mRNA from the BAFF transgene. PCR analysis of tail DNA from BAFF transgenic (Tg) mice used 12.5 pM of the primers 5′-GCAGTTTCACAGCGATGTCCT-3′ and 5′-GTCTCCGTTGCGTGAAATCTG-3′ in a reaction containing 1× Taq polymerase buffer (Stratagene Inc.), 0.2 nM dNTPs, 10% DMSO, and 5 U Taq polymerase (Stratagene Inc.). 719 bp of the transgene was amplified for 35 cycles at 94°C for 30 s, 54°C for 1 min, and 72°C for 1.5 min, followed by a 10-min extension at 72°C. The presence of proteins in mouse urine was measured using Multistix 10 SG reagent strips for urinalysis (Bayer Corp., Diagnostics Division). Differential white blood cell counts of fresh EDTA-anticoagulated whole blood were performed with an Abbott Cell Dyne 3500 apparatus. For FACS ® analysis (Becton Dickinson & Co.), fluorescein–(FITC), CyChrome™–, and phycoerythrin (PE)-labeled rat anti–mouse antibodies: anti–B220, anti–CD4, anti–CD8, anti–CD43, anti–IgM, anti–CD5, anti–CD25, anti–CD24, anti–CD38, anti–CD21, anti–CD44, anti–MHC class II, anti– l -selectin, and hamster anti–Bcl-2/control hamster Ig kit were purchased from PharMingen. Production of recombinant Escherichia coli, as well as mammalian cell-derived mouse Flag-tagged BAFF, was performed as previously described for human BAFF 23 . All antibodies were used according to the manufacturer's specifications. PBL were isolated by density gradient centrifugation of EDTA-treated mouse blood over lymphocyte M (Cedarlane). FACS ® analysis of spleen, bone marrow, and mesenteric lymph nodes was performed effectively as described previously 28 . ELISA plates (Corning Glass Works) were coated overnight at 4°C with a solution of 10 μg/ml goat anti–total mouse Ig (Southern Biotechnology Associates, Inc.) in 50 mM sodium bicarbonate buffer, pH 9.6. Plates were washed three times with PBS/0.1% Tween and blocked overnight with 1% gelatin in PBS. 100 μl/well of serum serial dilutions or standard dilutions was added to the plates for 30 min at 37°C. Mouse Ig were detected using 100 μl/well of a 1-μg/ml solution of an alkaline phosphatase (AP)–labeled goat anti–total mouse Ig (Southern Biotechnology Associates, Inc.) for 30 min at 37°C. After a last wash with PBS/0.1% Tween, the enzymatic reaction was developed using a solution of 10 μg/ml of p -nitrophenyl phosphate (Boehringer Mannheim Biochemicals) in 10% diethanolamine. The reaction was stopped by adding 100 μl of 3 N NaOH/well. The optical density was measured at 405 nm using a spectrophotometer from Molecular Devices. Standard curves were obtained using purified mouse Ig purchased from Southern Biotechnology Associates, Inc. In the case of detection of rheumatoid factors, the plates were coated with normal goat Ig (Jackson ImmunoResearch Laboratories, Inc.) instead of goat anti–mouse Ig, and detection of mouse Ig was performed as described above. Detection of mouse isotypes in the RF assay was done using AP-labeled goat anti–mouse IgA, IgM, IgG2a, IgG2b, IgG1, and IgG3, as well as purified mouse IgA, IgM, IgG2a, IgG2b, IgG1, and IgG3 for standard curves (Southern Biotechnology Associates, Inc.). All statistical comparisons were performed by analysis of variance. The assay was performed as previously described 29 30 with the following modifications: ELISA plates (Corning Glass Works) were coated overnight at 4°C with 5 μg/ml of human C1q (Quidel) in 50 mM sodium bicarbonate buffer, pH 9.6. The plates were washed three times with PBS/0.1% Tween. 50 μl/well of 0.3 M EDTA was added to the plates plus 50 μl/well of serum serial dilutions or solutions of known concentrations of a standard immune complex (peroxidase-mouse antiperoxidase) from DAKO Corp. The plates were incubated 30 min at 37°C. The plates were washed three times with PBS/0.1% Tween. Mouse Ig in the immune complexes were detected using 100 μl/well of a 1 μg/ml solution of an AP-labeled goat anti–mouse Ig (Southern Biotechnology Associates, Inc.) as described above for the ELISA assays. Cryoglobulins were detected by incubating overnight at 4°C mouse serum diluted 1/15 in water and precipitates were scored visually. Detection of anti–single-stranded (ss) DNA antibodies was performed using NUNC-immuno Plate MaxiSorp plates (NUNC A/S). Plates were coated overnight at 4°C first with 100 μg/ml methylated BSA (Calbiochem Corp.), then with 50 μg/ml grade I calf thymus DNA (Sigma Chemical Co.) that was previously sheared by sonication, and finally digested with S1 nuclease. This DNA was used to coat plates for the anti–double-stranded (ds) DNA assays, but was additionally boiled 10 min and chilled on ice before coating plates for the anti–ssDNA assays. After blocking, serial dilutions of the serum samples were added and incubated at room temperature for 2 h. Autoantibodies were detected with goat anti–mouse IgG-AP (Sigma Chemical Co.) and developed as described above for the ELISA assays. Standard curves were obtained using known quantities of anti–DNA mAb 205, which is specific for both ss- and dsDNA 31 . Frozen sections of spleen and lymph nodes were subjected to immunohistochemical analysis as previously described 32 . Biotin-labeled antibodies rat anti–B220, anti–CD11c, and anti–syndecan-1, as well as unlabeled rat anti–CD4, anti–CD8α, and anti–CD8β were purchased from PharMingen. Biotin-labeled peanut agglutinin was obtained from Vector Laboratories, Inc. Horseradish peroxidase (HRP)–labeled mouse anti–rat Ig and HRP-streptavidin were purchased from Jackson ImmunoResearch Laboratories, Inc., and AP-labeled streptavidin from Southern Biotechnology Associates, Inc. In the case of immunohistochemistry on kidney tissue to detect Ig deposition, paraffin sections were used, dewaxed, and blocked using diluted horse serum from Vector Laboratories, Inc., followed by staining with HRP goat anti–mouse Ig from Jackson ImmunoResearch Laboratories, Inc. Detection was performed as previously described for frozen sections 32 . Full-length murine BAFF was expressed in transgenic mice using the liver-specific alpha-1 antitrypsin promoter with the APO E enhancer. The full-length version was chosen with the expectation that BAFF would be cleaved and act systemically or, if retained in a membrane-bound form, that local liver-specific abnormalities would be observed, possibly providing functional clues. We obtained 13 founder mice positive for the BAFF transgene. BAFF overexpression in the liver of transgenic mice was confirmed by Northern blot analysis (data not shown). An ELISA assay for murine BAFF is not available; however, we found that 2% serum from BAFF-Tg mice, but not from control mice, blocked the binding of mammalian cell-derived mouse soluble Flag-tagged BAFF to BJAB cells. Moreover, 5% serum from BAFF-Tg mice but not from control mice increased the proliferation of human B cells from PBL in the presence of anti–μ (data not shown). These data suggest that soluble BAFF is present in the blood of BAFF-Tg mice. In all BAFF-Tg mice examined histologically, the livers showed no abnormalities, indicating that local overexpression of BAFF did not induce any immunological or pathological events (data not shown). The transgenic mouse population was found to have more lymphocytes in the blood when compared with control negative littermates, reaching values as high as 13,000 lymphocytes/μl of blood . In contrast, the number of granulocytes per microliter of blood in both BAFF-Tg and control mice remained within normal limits . The elevated lymphocyte levels resulted from an expanded B cell subset, since FACS ® analysis, using anti–CD4 and –B220 antibodies, of peripheral blood cells (PBL) from 18 BAFF-Tg mice issued from six different founders showed increased B/T ratios . Likewise, combining the number of lymphocytes per microliter of blood with the percentage of circulating T cells, calculation of absolute numbers of CD4 circulating T cells revealed a 50% reduction of this T cell subset in BAFF-Tg mice when compared with control mice, and the same observation was made for the CD8 T cell subset (data not shown). All peripheral blood B cells from BAFF-Tg mice had increased MHC class II and Bcl-2 expression when compared with B cells from control mice , indicating some level of B cell activation in PBL of BAFF-Tg mice. T cells in the blood of BAFF-Tg mice did not express the early activation markers CD69 or CD25; however, 40–56% of CD4 or CD8 T cells were activated effector T cells with a CD44 hi , l -selectin lo phenotype versus only 8–12% in control littermates . Thus, BAFF-Tg mice clearly show signs of an expanded peripheral blood B cell compartment and global B cell activation along with T cell alterations. To see whether overexpression of BAFF in the transgenic mice was affecting the B cell compartment centrally in the bone marrow and peripherally in secondary lymphoid organs, we examined the spleen, bone marrow (BM), and mesenteric lymph nodes (MLN) from a total of seven BAFF-Tg mice and seven control littermates derived from four different founder mice by FACS ® analysis. The mature B cell compartment was analyzed by dual staining with anti–B220 and –IgM antibodies. Two representative BAFF-Tg mice and one control littermate are shown in Fig. 2 . The mature B cell compartment (IgM + /B220 + ) was increased in both the spleen and the MLN . Analysis of B220 + /IgM + B cells or the proB cell (CD43 + /B220 + ) and the preB cell (CD43 − /B220 + ) compartments in the BM showed that BAFF-Tg mice and control littermates were similar. The slight difference in the mature B cell percentage seen in Fig. 2 A (middle) was not consistent in all seven mice analyzed. These data indicate that overexpression of BAFF is affecting the mature B cell compartment in the periphery, but not progenitor B cells in the BM. We calculated the total number of B and T cells in the spleen, BM, and MLN ( Table ). The total number of B cells was at least sevenfold higher in the spleen and MLN of BAFF-Tg mice. The total number of T cells was also increased twofold in the spleen and MLN of these mice ( Table ). Total numbers of B and T cells in the BM in BAFF-Tg mice were similar to that of control mice ( Table ). The population of CD5 + B1 cells in the spleen, BM, and peritoneal lavages of BAFF-Tg mice was similar to that of control mice and only marginally increased in MLN (data not shown). Analysis by FACS ® of B cell subpopulations in the spleen revealed an increased proportion of marginal zone (MZ) B cells in BAFF-Tg mice when compared with control mice ( Table ). The population of follicular B cells remained equivalent in both BAFF Tg and control mice, whereas the fraction of newly formed B cells was slightly decreased in BAFF-Tg mice ( Table ). This result was also confirmed on B220 + splenic B cells using anti–CD38 vs. anti–CD24 antibodies or anti–IgM vs. anti–IgD antibodies and analyzing the CD38 hi /CD24 + and IgM hi /IgD lo MZ B cell population, respectively, as previously described 33 (data not shown). Immunohistochemical analysis using an anti–mouse IgM antibody revealed the expansion of the IgM-bright MZ B cell area in the spleen of BAFF-Tg mice when compared with control mice (data not shown). All BAFF-Tg B220 + splenic B cells also expressed higher levels of Bcl-2 (data not shown) and also MHC class II ( Table ) compared with splenic B cells from control mice, indicating that splenic B cells as well as peripheral blood B cells are in an activated state. At equal cell concentration, splenocytes isolated from BAFF-Tg mice survived longer in culture medium when compared with control splenocytes and the thymidine incorporation after 6 d of culture relative to the incorporation on day 0 was only decreased 20% in cultures with BAFF-Tg–derived splenocytes vs. 60% reduction with control splenocytes (data not shown). In vivo 5-bromo-2′-deoxyuridine (BrdU) incorporation for 4 d did not reveal any higher BrdU intake in BAFF Tg–splenic B cells when compared with control mice (data not shown). BAFF-Tg mice had large spleens , Peyer's patches (B), and lymph nodes (C). Immunohistochemistry showed the presence of enlarged B cell follicles and reduced periarteriolar lymphocyte sheath (or T cell area) in BAFF-Tg mice . Interestingly, few germinal centers were observed in nonimmunized control littermates (and is typical of this colony in general) and those present were small , whereas BAFF-Tg mice possessed numerous and large germinal centers in the absence of immunization . Staining with anti–CD11c for dendritic cells in the T cell zone and the marginal zone of control mice was considerably reduced in BAFF-Tg mice . Syndecan-1–positive plasma cells were almost undetectable in the spleen from control littermates , yet the red pulp of BAFF-Tg mice was strongly positive for syndecan-1 . Very similar observations were made with MLN . In the MLN of BAFF-Tg mice, the B cell areas were dramatically expanded , in contrast to the normal appearance, where B cell follicles were easily recognizable at the periphery of the node under the capsule with a typical paracortical T cell zone . The medulla of MLN from BAFF-Tg mice were filled with syndecan-1–positive cells that presumably are plasma cells . Therefore, analysis of secondary lymphoid organs in BAFF-Tg mice was consistent with the expanded B cell compartment seen by FACS ® analysis and indicates multiple cellular abnormalities and intense immune activity. The increased B cell compartment in BAFF-Tg mice suggested that the level of total Ig in the blood of these animals might also be increased. SDS-PAGE analysis of the serum showed that IgG levels were elevated in all BAFF-Tg mice, while the nontransgenic littermates displayed a normal pattern of serum proteins . By comparison with an IgG1 standard antibody, the levels of IgG in a nontransgenic mouse were ∼5–8 mg/ml, and these levels increased to at least 50 mg/ml in some BAFF-Tg mice (quantification was done with underloaded gels). In normal mice, the light chain band is smeared due to the polyclonal nature of the Ig and on this basis the elevated Ig levels in BAFF-Tg mice were also polyclonal in nature. IgM levels were visibly increased in these mice, albeit not as much as IgG, and this band is seen as a smear on top of a transferrin band in this gel. High serum Ig levels in BAFF-Tg mice were confirmed by ELISA , and the high levels of Ig seen in these mice led us to suspect the presence of rheumatoid factors, or autoantibodies directed against antigenic determinants on the Fc domain of IgG 34 . These antibodies could bind to the goat anti–mouse Ig used to coat the ELISA plates and give erroneously high values. ELISA plates were, therefore, coated with normal goat Ig and the binding of BAFF Tg Ig to normal goat Ig was measured. Fig. 6 C shows that sera from most BAFF-Tg mice contained Ig reacting with normal goat Ig, whereas only 2 of 19 control mice exhibited reactivity in the same assay. These RF were mainly of the IgM, IgA, and IgG2a isotypes (data not shown). Presence of RF can be associated with the presence of high levels of circulating immune complexes (CIC) and cryoglobulin in the blood 34 . To verify whether or not BAFF-Tg mice have abnormal serum levels of CIC, a C1q-based binding assay was used to detect CIC in the 21 BAFF-Tg mice analyzed above. Only five BAFF-Tg mice showed significantly high levels of CIC when compared with control mice; nonetheless, these mice corresponded to the animals having the highest total serum Ig and rheumatoid factor levels . We also observed precipitate formation when sera from BAFF-Tg mice, but not control sera, were diluted 1/15 in water, indicating the presence of cryoglobulin in these mice (data not shown). Thus, in addition to B cell hyperplasia, BAFF-Tg mice display severe hyperglobulinemia associated with the presence of RF and CIC. Initially, we observed kidney abnormalities reminiscent of a lupus-like disease in two of our founder mice. The presence of anti–DNA autoantibodies has also been described in SLE patients or the SLE-like (SWR × NZB)F1 (SNF1) mouse 31 . Anti–ssDNA autoantibody levels were detected in BAFF-Tg mice previously shown to have the highest level of total serum Ig . We analyzed the serum of two BAFF-Tg mice negative for antibodies against ssDNA (697-5 and 816-1-1) and three transgenic mice secreting anti–ssDNA antibodies (820-14, 816-8-3, and 820-7) for the presence of anti–dsDNA antibodies in parallel with five control littermates. BAFF-Tg mice also secreted anti–dsDNA; however, the levels of secretion did not always correlate with that of anti–ssDNA antibodies, as serum from BAFF-Tg mouse 697-5, which did not contain detectable levels of anti–ssDNA antibodies, was clearly positive for the presence of anti–dsDNA . Therefore, BAFF-Tg mice showing the most severe hyperglobulinemia secrete high levels of anti–DNA autoantibodies. Additionally, and also reminiscent of lupus-like nephritis, we detected immunoglobulin deposition in the kidney of six BAFF-Tg mice analyzed , three of these mice did not secrete detectable levels of anti–DNA antibodies (data not shown). All BAFF-Tg mice have proteinuria ( Table ). Sera from all BAFF-Tg mice, but not control mice, diluted 1/100, stained the nuclei of lymph node cells on tissue sections, indicating the presence of antinuclear antibodies in the serum of BAFF-Tg mice (data not shown). BAFF is a powerful cytokine affecting B cells, and has consequences for T cell and dendritic cell status. The nature of the expanded B cell subset in BAFF-Tg mice is still unclear, but seems to be restricted to mature B cells that have been activated. Overexpression of BAFF led to the emergence of autoimmune manifestations such as production of autoantibodies, proteinuria, Ig deposition in kidneys, and intense germinal center formation. Thus, BAFF ligand and its receptor on B cells form a novel immunoregulatory system. Whether a ligand is secreted or membrane-bound has profound biological ramifications. These mice were designed to express high levels of BAFF in the liver and, while it cannot be excluded that low level of expression somewhere in the immune system accounts for this unusual biology, we view it more likely that BAFF is secreted from the liver and acts at a distant site. We have indirect evidence for the presence of BAFF in the serum of transgenic mice and, moreover, injection of recombinant BAFF in normal mice led to some of the effects described here 26 . Well-defined secretion of a TNF family ligand with functional consequences in vivo has been observed only infrequently; e.g., TNF and lymphotoxin-α. BAFF, TWEAK, and APRIL are three relatively new ligands that possess canonical furin cleavage motifs in the stalk region and are readily secreted from transfected cell systems 23 35 36 . Whether such secretion in vitro actually predicts for a soluble ligand system is not clear, yet this BAFF-Tg mouse would indicate that secretion can occur at least from the liver and, thus, soluble BAFF ligand is capable of mediating biological events in vivo. Alternatively, facile cleavage may represent a mechanism that ensures a very transient localized signaling event. The in vitro analysis using recombinant soluble BAFF protein showed that BAFF costimulated B cell growth in conjunction with B cell receptor activation, yet by itself it did not stimulate proliferation of resting B cells 23 . If BAFF is truly a soluble mediator, then this observation is similar to that made originally for IL-2 and T cell growth, and prompts the question of whether BAFF is a B cell growth factor. The present data do not allow one to distinguish between a costimulatory action (e.g., analogous to the activity of CD28) and a true B cell growth factor-like activity. Regardless of the mechanism, these data suggest that expansion of the B cell compartment in these mice is the result of BAFF-induced proliferative stimuli, yet negative results with 4 d in vivo BrdU incorporation and increased ex vivo survival of splenocytes raised the possibility that this observation stems from an increased output from the bone marrow and/or a decreased death rate. The CD40 pathway clearly plays a major role in B cell regulation, inviting a comparison with the BAFF system. An increase in the size of the B cell population, enlarged spleens, lymph nodes, and Ig deposition in the kidney were also observed in CD40L-Tg mice 37 . Several aspects clearly distinguish these two mice; for example, CD40L-Tg mice develop inflammatory bowel disease (IBD), which was not observed in BAFF-Tg mice, and the alterations in the organization of the secondary lymphoid organs are very different 37 . In CD40L-Tg mice, but not BAFF-Tg mice, the organization of the thymus is altered, which presumably impairs proper T cell selection leading to IBD, as seen in other mouse models with thymic disfunction 38 . The difference between these two transgenic mice may be due in large part to the distinct distribution of these two ligands and their corresponding receptors. Transgene-expressed CD40L is a membrane-bound ligand primarily expressed on thymocytes and activated T cells, whereas transgenic liver-expressed BAFF has the characteristic of a soluble ligand and, therefore, can diffuse into multiple compartments. CD40 is expressed on a wide variety of cell types 20 , whereas expression of BAFF receptor is restricted to B cells and possibly monocytic cells 23 24 25 26 . Given the disparate phenotypes of the BAFF- and CD40L-Tg mice, it is fair to predict that BAFF and CD40L probably play distinct roles in normal animals as well. Our results on splenic architecture and elevated Ig levels in the serum of BAFF-Tg mice are consistent with those described in a recent study using short-term injection of soluble BAFF in normal mice 26 and tend to minimize possible developmental disturbances related to the expression of the BAFF transgene in our mice. However, chronic exposure of the transgenic mice to BAFF led to changes not paralleled in mice injected for 4 d with recombinant BAFF 26 . In both cases, serum IgM levels were elevated, yet the BAFF-Tg mice exhibited vastly increased IgG and IgA levels. The effects of short-term injections were interpreted as possibly stemming from activation of T-independent B cell events, whereas here the elevated IgG and IgA levels, the presence of non-IgM RF isotypes, and the extensive germinal center formation clearly indicate ongoing T cell–dependent B cell events. It is unclear at this point whether BAFF induces the expansion of both naive and activated B cells, as all B cells in these mice exhibit elevated expression of both MHC class II and Bcl-2 and hence show signs of activation. In preliminary experiments, anti–SRBC antibody titers after primary immunization were similar in BAFF-Tg mice and control littermates, suggesting that the original pool of naive SRBC-specific B cells was not expanded compared with that of control mice. In contrast, SRBC-specific IgG levels after a secondary response to SRBC were significantly higher in BAFF-Tg mice when compared with control mice (data not shown). This result supports a model where BAFF induces the proliferation and/or survival of B cells that had received an activating B cell receptor signal and this effect might only be detectable after secondary immunization or long-term monitoring of the immune response. The enlarged proportion of MZ B cells in the spleen is interesting as these cells are described as B cells in an activated state 39 and as such may be preferential targets for BAFF-induced proliferative/survival signals. Since the MZ contains memory B cells 40 , it is conceivable that memory B cells may be specific responders to BAFF-induced signals; this interpretation would be consistent with the stronger secondary response seen with SRBC in BAFF-Tg mice. Additional experiments will be required to define this aspect accurately. These results also raise the fundamental question of the physiological role of BAFF in normal individuals, and whether examining its function may answer remaining questions such as the nature of the mechanisms governing differentiation of B cells into plasma cells vs. germinal center B cells or plasma cells vs. memory B cells. Among the increased B cell populations in BAFF-Tg mice are emerging autoreactive B cells, secreting RF and anti–DNA autoantibodies. It is well known that tolerance to self antigens is never complete and autoreactive B cells, as well as low levels of rheumatoid factors, can be detected in normal individuals 3 . These autoreactive B cells are referred to as natural autoreactive B cells. Therefore, one possibility in BAFF-Tg mice is that the emergence of a large number of autoreactive B cells may reflect the expansion of occasional natural autoreactive B cells in response to BAFF-proliferative stimuli. If this was the case, one would predict that only higher levels of IgM RF would be detected 34 , yet high levels of IgA and IgG2a RF were observed indicating isotype switching in the RF-specific B cells. RF other than IgM are found in patients with autoimmune diseases such as rheumatoid arthritis 34 . Therefore, the population of RF-specific autoreactive B cells in BAFF-Tg mice is not only expanded but also activated, indicating a dynamic antigen-specific process leading to autoimmune manifestations rather than passive expansion. It cannot be excluded that BAFF-Tg mice have larger numbers of nonnatural self-reactive B cells that have not been identified or possibly larger numbers of foreign antigen-specific activated B cells. A number of studies have shown that the escape of autoreactive B cells from clonal deletion or functional inactivation (clonal anergy) alone is not enough to develop autoimmune disease 1 41 . Additional factors such as infection, cytokines, and costimulatory help from T cells are required. The presence of large germinal centers in secondary lymphoid organs of BAFF-Tg mice, higher total T cell numbers in the spleen and MLN, as well as the increased proportion of both CD4 and CD8 effector T cells in the periphery, and the quality of the RF isotypes strongly suggest the active participation of T cells in the immune reactions triggered in BAFF-Tg mice. Whether the enlarged population of activated effector T cells contains autoreactive T cells remains to be determined. One may question why BAFF-Tg mice do not show more severe pathological manifestations. Potential explanations include the expression of nonlethal BAFF levels in surviving BAFF-Tg founders, the absence of either pathogenic B cells or tissue-destructing antibodies as seen in some murine models of lupus and rheumatoid arthritis, respectively 6 42 and, finally, the H-2 b background of the BAFF-Tg mice, which may not favor the emergence of severe autoimmune manifestations. If overexpression of BAFF indeed initiates an active autoimmune reaction in our transgenic mice, we need to question why tolerance to self has failed. Downregulation of Bcl-2 expression in autoreactive B cells has been shown to be one way to sensitize these cell to cell death signals 43 . B cells in BAFF-Tg mice express higher levels of Bcl-2, indicating a possible protection against apoptotic signals, and also suggesting that BAFF, like CD40L, provides survival signals to B cells. One can speculate that this event coupled with a BAFF proliferative signal may explain the accumulation of autoreactive B cells in these mice. However, these changes alone are probably not sufficient to generate an autoreactive response and one potential answer may reside in the role of T cells. Increased numbers of effector T cells were directly observed in the periphery and there was an apparent reduction in the numbers of dendritic cells in the spleen of BAFF-Tg mice. Dendritic cells are believed to be essential for T cell tolerance to self 44 , and their deficit in BAFF-Tg mice may promote the emergence of autoreactive T cells. Thus, we hypothesize that the role of BAFF overexpression in impairing self-tolerance may rely on two mechanisms: promoting enhanced survival and proliferation of activated autoreactive B cells and suppression of the protective effects of dendritic cells against the emergence of autoreactive T cells. These experiments demonstrate that ectopic overexpression of BAFF was sufficient to initiate the expansion of the mature B cell compartment, resulting in lupus-like autoimmune manifestations. This transgenic mouse model potentially brings new insight into the etiology of autoimmune disorders, provides a novel framework for the investigation of autoreactivity, and potentially opens the door to new therapeutic strategies both for the treatment of some autoimmune disorders and the stimulation of humoral responses. | Study | biomedical | en | 0.999996 |
10587361 | The wild-type dihydrofolate reductase thymidylate synthase gene of P . berghei with a 2.2-kb upstream and 0.75-kb downstream region was cloned from the genomic library and subcloned into a plasmid vector, pBluescript II. Resistance to pyrimethamine was conferred to this gene by a single amino acid mutation (Ser 110 →Asp 110 ) using PCR 8 . The validity of this gene as a selectable marker was confirmed by transformation of parasites with this plasmid and subsequent selection by pyrimethamine as previously described 9 . The DNA fragment containing the 5′ portion of PbCTRP (2.05 kb) was subcloned into pBluescript II. The selectable marker gene was inserted into the MunI site of this fragment after ligation of EcoRI linkers to both ends. For the gene targeting experiment, the plasmid was completely digested with restriction enzymes XhoI and NotI to separate the linear targeting construct from the plasmid backbone. The gene targeting experiment was performed following the essentially same procedure as described by Menard et al. 10 . In brief, merozoites of P . berghei were transfected by electroporation with 40 μg of linearized targeting vector, injected intravenously into a rat, and selected by pyrimethamine. The selected parasites were further separated into the wild-type parasite population and disruptants by limiting dilution. The infected parasite population of each rat was determined by PCR and Southern blot analysis. Southern blot analysis was performed as previously described 1 . In brief, genomic DNA of the parasites was digested with restriction enzyme MunI, separated on a 0.7% agarose gel, and transferred to a nylon membrane. The blot was hybridized with a [ 32 P]dCTP-labeled HindIII/MunI-digested DNA fragment (0.8 kb) of PbCTRP . In rescue experiments, Southern blot analyses were performed with the same procedure. Radioactivity of the 2.5- and 6.5-kb bands was measured by the BAS 2000 system (Fuji Photo Film Co.), and the ratio of CTRP disruptants to wild-type parasites was estimated. After checking the number of exflagellated parasites in the infected blood (>50 per 10 5 erythrocytes), rats were subjected to bites of Anopheles stephensi mosquitoes for 30 min. The engorged mosquitoes were selected and maintained at 20°C. These mosquitoes were dissected 12 d after feeding, and oocysts in their midguts were carefully counted under a microscope with magnifications of 100 and 200. Fig. 1 a shows the targeting construct used in this experiment. It is composed of a selectable marker that confers pyrimethamine (antimalarial drug) resistance to parasites and CTRP sequences ligated at both ends. Merozoites of P . berghei were transfected with this construct by electroporation and intravenously injected into a naive rat. Integration of this construct into the CTRP locus by homologous recombinations resulted in disruption of this single-copy gene. The CTRP gene–disrupted parasites were selected in the rat by pyrimethamine. PCR and Southern blot analysis showed that the parasites selected with pyrimethamine were a mixture of wild-type parasites and CTRP disruptants . They were separated by limiting dilution and subsequent inoculation into a group of 20 rats. Out of 12 infected rats, 7 were infected only by CTRP disruptants and 4 were infected only by wild-type parasites. In these parasites, 4 disruptants and 3 wild-type parasite populations were used in the experiments described below . All seven parasite populations developed into mature ookinetes in vitro within 20 h. These ookinetes did not show any morphological differences from wild-type parasites with Giemsa staining under microscopic observation . Disruption of CTRP loci was further confirmed by immunocytochemistry . The infected rats were separately subjected to bites of Anopheles stephensi mosquitoes to assess the ability of these parasite populations to infect the insect vector. Before these mosquito challenges, all seven parasite populations showed normal exflagellation numbers in vitro (>50 exflagellations per 10 5 erythrocytes). Mosquitoes were dissected 12 d after feeding, and the number of oocysts in their midguts was counted ( Table ). In the wild-type populations, a total of 41 out of 48 mosquitoes was infected, and a total of 666 oocysts was found in the mosquito midguts. All infected mosquitoes had at least one oocyst containing clearly differentiated sporozoites. In contrast, no oocysts were found in 120 mosquitoes fed on the rats infected with CTRP -disruptant populations. We also examined the mortality rate of blood-fed mosquitoes (until day 12) in every parasite population. The mortality rates in the wild-type parasite populations (CTRP(+) 1–3) were 32.0, 33.3, and 50,0%, respectively. On the other hand, those in disruptants (CTRP(−) 1–4) were 18.8, 3.5, 20.0, and 9.5%, respectively. In total, the mortality rate of the mosquitoes in the CTRP disruptants was 13.9% (33 out of 237 mosquitoes), and the mortality rate in wild-type parasite populations was 36.7% (68 out of 185 mosquitoes). It has been reported that mosquito mortality rate increases after P . berghei infection by the damage of the midgut barrier by parasite penetration and after bacterial infection 11 . Therefore, the difference in mosquito mortality rate between wild type and disruptant might indicate that CTRP -disrupted ookinetes could not penetrate the midgut epithelium. The malarial ookinete develops from the zygote after fertilization in the mosquito midgut lumen. This is the only stage in the life cycle of Plasmodium that is diploid with heterologous chromosomes. Assuming that the PbCTRP gene from wild-type parasites would compensate for a disrupted CTRP gene when they mated heterologously in the mosquito midgut lumen, we performed the following rescue experiment. We prepared a rat infected with both wild-type parasites and CTRP disruptants. The proportion of CTRP disruptants to wild-type parasites in this rat was estimated as 5.5:1 by Southern blot analysis . Mosquitoes were infected by feeding on this rat. Parasites were fertilized, allowed to complete the sporogonic stage in these mosquitoes, and further transmitted to two other naive rats by bites from these mosquitoes (using 15 mosquitoes each). PCR and Southern blot analysis showed that these rats were infected with disruptants as well as wild-type parasites. The proportion of CTRP disruptants to wild-type parasites in these infected rats was estimated to be 0.68:1 and 0.58:1 by Southern blot analysis. These values are in good accordance with the figure calculated from the value in the original parasites, assuming that the heterozygous ookinete shows a normal phenotype (5.5:5.5 + 1 = 0.85:1). This indicates that these disruptants were rescued by mating with the wild-type parasites. This result also demonstrates that CTRP is not essential for other invasive stages, because fertilized parasites become haploid again after sporogony in the mosquito midgut. We further separated these parasites into wild-type parasite populations and disruptants by limiting dilution ( Fig. 3 , CTRP(+) 4–6 and CTRP(−) 5–8) and then performed mosquito infection experiments using the same procedure as in Table to confirm that the descended disruptants had really been rescued by mating with wild-type parasites. In the wild-type populations, a total of 1,951 oocysts was found in wild-type parasite populations (41 mosquitoes). However, no oocysts were found in the four CTRP disruptant populations (117 mosquitoes), indicating that rescued disruptants lost their infectivity again by separation from the wild-type parasites. The mortality rate was lower in those mosquitoes that fed on rats infected with CTRP disruptants (14.3%) compared with the wild-type parasite populations (30.2%). In this study, we performed targeted disruption of the PbCTRP gene. Disruption of the CTRP gene resulted in complete loss of ookinete infectivity. However, it did not apparently influence ookinete maturation and morphology. In addition, rescue experiments demonstrate that PbCTRP is essential only in the diploid stage. These results are comparable to those previously reported in the targeted disruption experiment of the TRAP gene. Considering the structural similarity between CTRP and TRAP, these molecules may play a related role in the machinery of the respective stages of active invasion. Although the mechanism of malaria parasite invasion of the vector mosquito is poorly understood, further studies aimed at identification of other molecular components interacting with CTRP as part of this invasion machinery will enhance understanding of the parasite–vector interactions. | Study | biomedical | en | 0.999998 |
10587362 | C57BL/6 mice (6–8-wk-old females) were acquired through the National Cancer Institute breeding program. Homozygotic mice genetically deficient for β2-microglobulin (β2m −/− ), MHC II (Abb −/− ), and perforin (B6. pfp −/− ), all in a C57BL/6 background, were obtained from Taconic Farms, Inc. Immunoglobulin μ chain (μMT −/− ) and gld / gld (B6. gld ) mice were acquired from The Jackson Laboratory. These mice were bred and kept in a pathogen-free Memorial Sloan-Kettering Cancer Center vivarium according to National Institutes of Health Animal Care guidelines. All mice entered the study between 7 and 10 weeks of age. B16F10/LM3 15 is a pigmented mouse melanoma cell line of C57BL/6 origin, derived from the B16F10 line, provided by Dr. Isaiah Fidler (M.D. Anderson Cancer Center, Houston, TX). The EL-4 cell line was derived from a C57BL/6 mouse lymphoma, and SK-MEL-188 is a human melanoma cell line. Tumor cell lines were cultured as described 15 . The human TRP-2 (hTRP-2) and mouse TRP-2 (mTRP-2) expression vectors (supplied by Drs. S.A. Rosenberg and J.C. Yang, National Cancer Institute, Bethesda, MD) were previously described 9 . These genes were cloned into the PCR3 vector, which was used as a control vector without inserts. The mouse GM-CSF gene, provided by PowderJect Vaccines Inc., was cloned into the WRGBEN vector 13 . The method of DNA immunization has been reported 13 . In brief, plasmid DNA encoding hTRP-2, mTRP-2, or GM-CSF was coated onto 1.0 μm gold bullets. Animals were immunized by delivering gold–DNA complexes using a helium-driven gun (Accell ® ; PowderJect Vaccines Inc.) into each abdominal quadrant (1 μg plasmid DNA/quadrant) for a total of four injections. NK cell depletion was performed as described 15 . Animals were injected intraperitoneally with mAb PK136 (anti–NK-1.1) 3 d before immunization, and every 4 d thereafter. Depigmentation experiments were performed as described 13 15 . In brief, after the final immunization, mice were shaved and depilated over the posterior flank and observed for 8 wk. Scoring of depigmentation was performed by dividing the abdomen into four equal quadrants. Quadrants were recorded as positive when they had estimated >50% depigmented hairs. Depigmentation was scored 0–4+ according to the number of quadrants that were depigmented in each mouse (e.g., 3+ if three of four quadrants are depigmented >50%). Antibody responses to syngeneic mTRP-2 were measured by immunoprecipitation, followed by Western blot assay as described 13 . B16F10/LM3 melanoma cells were lysed, followed by immunoprecipitation with anti–PEP-8, a rabbit polyclonal antisera raised against a carboxyl-terminal peptide of TRP-2 (from Vincent Hearing, National Cancer Institute). Lymphocytes (2 × 10 7 ) from draining inguinal lymph nodes were cocultured for 5 d with naive irradiated (3,000 rads) splenocytes (2 × 10 7 ), mitomycin C (100 μg/ml, 120 min at 37°C)-treated human SK-MEL-188 cells (4 × 10 5 ) as a source of antigen, and pristane-induced macrophages (2 × 10 6 ) from C57BL/6 mice. H-2K b –restricted lymphocyte lysis of EL-4 tumor cells pulsed for 1 h at 37°C with 4 μg mTRP-2 181–188 peptide (sequence VYDFFVWL) was determined by a 4-h standard 51 Cr release assay 16 . All mice were injected intravenously via the tail vein with 2 × 10 5 B16F10/LM3 melanoma cells. Tumor challenge was performed 5 d after the final immunization, unless otherwise indicated. Mice were killed at 14–22 d after tumor challenge, all lobes of both lungs were dissected, and surface lung metastases were scored and counted for all lobes under a dissecting microscope. Statistical analysis of tumor growth was performed using the two-sided Student's t test, assuming unequal variances and Wilcoxon Scores (Rank Sums). hTRP-2 has 90% homology and 83% identity to the amino acid sequence of C57BL/6 mTRP-2. DNA immunization with xenogeneic hTRP-2 decreased B16F10/LM3 lung metastases by ≥90% in tumor protection experiments . There was no significant evidence of tumor immunity after immunization with syngeneic mTRP-2 DNA compared with untreated mice or mice injected with control null vector . To assess the potency of DNA immunization using xenogeneic hTRP-2 DNA, mice were immunized 4 d after tumor challenge or 10 d after tumor challenge, when lung metastases were numerous and macroscopic. Immunization at 4 d decreased metastases by >80% . Therapeutic effects were observed 10 d after tumor challenge using immunization with hTRP-2 DNA plus recombinant mouse GM-CSF DNA as an immune adjuvant. Vaccination significantly decreased lung metastases by approximately half . No significant decrease in lung metastases was observed after treatment with hTRP-2 or mTRP-2 DNA, or GM-CSF DNA alone , although there was a trend towards decreased metastases with GM-CSF alone that did not reach significance ( P > 0.05). These results showed a requirement for xenogeneic antigen and the adjuvant effect of GM-CSF in the treatment of established tumors. We next determined whether immunization with mTRP-2 or xenogeneic hTRP-2 generated antibody and CD8 + T cell responses against syngeneic mTRP-2. 6 of 12 mice immunized with hTRP-2 had detectable IgG antibodies (IgG1 and IgG2b isotype) against mTRP-2 (data not shown). No autoantibodies against syngeneic mouse TRP-2 were generated after immunization with mTRP-2 (0/12). Generation of autoantibodies after immunization with hTRP-2 required both CD4 + and CD8 + T cells, because no autoantibodies were detected in mice deficient in MHC class I (0/11) or II molecules (0/10). CTL responses against TRP-2 were detected after immunization with xenogeneic hTRP-2, but not syngeneic mTRP-2 DNA. Specifically, CD8 + CTL from draining lymph nodes (supraclavicular nodes), stimulated in vitro for 5 d, recognized an MHC class I H-2K b –restricted peptide of mTRP-2 after immunization with hTRP-2 DNA . Interestingly, the H-2K b –restricted peptide of mTRP-2, mTRP-2 181–188 , is identical between mouse and human TRP-2, including the immediate flanking amino acid residues. Thus, this self-peptide in the context of self–TRP-2 DNA does not induce CTL responses, but presentation of the same peptide in the context of xenogeneic hTRP-2 is immunogenic. These results suggested that either antibody or CTL responses, or both, mediated tumor rejection. Roles for critical cell types were investigated by immunizing β2m −/− mice deficient in MHC class I and CD8 + T cells, MHC II −/− mice deficient in MHC class II and CD4 + T cells, Igμ 2/− mice deficient in mature B cells, and mice depleted of NK1.1 + cells, including NK cells . Both MHC class I and II molecules were required for tumor rejection, supporting a central role for both CD8 + and CD4 + T cells. Neither NK cells nor B cells were necessary for tumor immunity. Noticeably, mice deficient in B cells developed fewer baseline metastases compared with wild-type C57BL/6 mice, and were completely free of any detectable tumor after treatment with hTRP-2 (12 of 12 mice). This phenomenon of enhanced T cell–dependent tumor rejection associated with B cell deficiency has been reported previously 10 . These results showed that T cell immunity, including both CD8 + and CD4 + T cells, was required for tumor rejection, but antibodies were not. Signs of autoimmunity, manifested as depigmentation, were observed in mice immunized with hTRP-2, but not generally in mice immunized with syngeneic mTRP-2 . Depigmentation appeared 4–5 wk after starting immunization over depilated and shaved areas of the mouse coat, spreading to unshaved areas in most mice. Autoimmunity also required T cells, but not antibodies or NK cells, showing that tumor immunity and autoimmunity were coupled by a requirement for class I and II MHC expression leading to a requirement for T cells. CTLs have been proposed to be critical effector cells that mediate tumor rejection. Cytotoxicity of T cells can be mediated by exocytic granules involving perforin, or by cell membrane molecules that induce death of target cells. Tumor immunity proceeded in the complete absence of perforin in pfp −/− mice , whereas autoimmunity was mostly inhibited . Fas ligand was not necessary for either autoimmunity or tumor immunity . These results are consistent with perforin-mediated killing of normal melanocytes in hair follicles playing a central role in autoimmunity. However, tumor immunity could proceed in a perforin-independent manner. TRP-2 is recognized by CTLs of patients with melanoma 7 11 12 , and has also been defined as a potential tumor-rejection antigen in C57BL/6 mice 9 . Thus, TRP-2 provides a model for a differentiation antigen with relevance to a human cancer. Xenogeneic DNA vaccination is one strategy to immunize against potentially weak self-antigens. The approach using a xenogeneic source of antigen is well known to produce autoimmunity, but has also been used to induce tumor immunity against gp75 TRP-1 and another melanocyte/melanoma differentiation antigen gp100 13 14 16 17 18 . Other strategies have shown that syngeneic mouse gp75 TRP-1 expressed in insect cells 16 or by vaccinia virus 14 can also induce tumor immunity and trigger depigmentation. Expression of the syngeneic protein in the context of xenogeneic cells that may package the antigen in insoluble inclusion bodies (e.g., insect cells) or with strongly immunogenic viral proteins are alternative strategies, at least for inducing antibody-dependent immunity against tyrosinase family antigens. As noted above, the CTL response directed against the H-2K b -restricted peptide of mTRP-2, mTRP-2 181–188 , is remarkable because this peptide is identical between mouse and human TRP-2, including the immediate flanking amino acid residues. It is possible the distant amino acid residues alter processing of this peptide in hTRP-2, allowing more efficient presentation. Alternatively, amino acid differences in other peptides of hTRP-2 may provide T cell help, which in turn is sufficient to trigger CTL responses to the mTRP-2 181–188 self-peptide. The observation that only hTRP-2 DNA (but not mTRP-2 DNA) induced CD8 + T cell responses suggests that T cell tolerance was broken by xenogeneic DNA immunization, although we recognize this could reflect differences in efficiency of processing. Immunity against gp75 TRP-1 led to tumor protection and to depigmentation that was indistinguishable from autoimmunity induced by immunization against TRP-2 13 . Similar results were observed by Naftzger et al. 16 and Overwijk et al. 14 after immunization with syngeneic gp75 TRP-1 expressed by baculovirus in insect cells or in vaccinia virus, respectively. In gp75 TRP-1 systems, tumor immunity and autoimmunity were mediated by autoantibodies, and tumor immunity depended on NK cells and functional Fc receptors; neither tumor immunity nor autoimmunity required CD8 + T cells 13 14 15 16 17 . In contrast, tumor immunity and autoimmunity against TRP-2 required MHC class I molecules, and by implication CD8 + T cells, without a requirement for antibodies or NK cells. Thus, either autoantibodies or T cells can provide specificity for tumor immunity and autoimmunity. The final phenotype of tumor immunity and autoimmunity can be dependent either on antibodies or CD8 + T cells, but in both cases CD4 + cells are required. Vaccination against TRP-2 as late as 10 d after tumor challenge induced substantial decreases in tumor burden, whereas active immunization against gp75 TRP-1 at these later time points was ineffective 13 . We propose that this reflects in part the kinetics of T cell versus antibody responses, where effective T cell immunity can be generated over days, whereas effective antibodies may require weeks. Passive transfer of antibodies against gp75 TRP-1 had significant effects 4 and 7 d after tumor challenge 15 , consistent with this view. The potential coupling of tumor immunity with autoimmunity has been suggested by the clinical observation that patients with metastatic melanoma who develop vitiligo have a better prognosis and are more likely to respond to therapy 19 20 21 22 . The differences in mechanisms underlying tumor immunity and autoimmunity could be a consequence of fundamental differences in effector mechanisms used to kill tumor cells versus normal melanocytes. Alternatively, the differences could reflect the different tissue sites of melanocytic cells. In this scenario, the effector mechanisms in the skin require perforin, whereas other mechanisms are used in the lung. Mechanisms other than perforin or fas ligand may be involved in tumor rejection, but it is also possible that perforin and fas ligand provide redundant mechanisms for effector functions against tumors. Finally, the uncoupling of tumor immunity from autoimmunity in this model shows that one can inhibit autoimmunity and still be permissive for tumor immunity. This opens strategies for inducing immunity to treat cancer where autoimmunity can be inhibited while cancer immunity proceeds. | Study | biomedical | en | 0.999997 |
10601327 | To find out whether axons stimulate OPC proliferation, several groups have taken advantage of the fact that astrocytes, oligodendrocytes, and OPCs each have a characteristic antigenic phenotype . This allows the effects of transection on each cell type to be specifically investigated. Neonatal transection, for example, produces a large, more than a 10-fold decrease in the percentage of astrocytes that incorporate bromodeoxyuridine , suggesting that axons stimulate astrocyte proliferation, perhaps partly by releasing sonic hedgehog . In contrast, transection does not significantly alter the percentage of OPCs that incorporate BrdU . On the face of it, these findings suggest that axons regulate astrocyte, but not OPC, proliferation. The interpretation of these findings is not straightforward, however, because of an interesting difference in the behavior of astrocytes and OPCs when they withdraw from the cell cycle. Astrocytes do not alter their antigenic phenotype when they stop dividing, whereas OPCs do. When OPCs stop dividing, they quickly differentiate into oligodendrocytes, thus losing their OPC-specific markers. Thus, whereas the percentage of astrocytes that incorporate BrdU provides a meaningful index of their proliferation rate, the same may not be the case for OPCs. This point is vividly illustrated by measuring BrdU incorporation by purified OPCs in culture in response to different concentrations of their main mitogen PDGF (our unpublished observations). Regardless of PDGF concentration, the percentage of OPCs that incorporate BrdU does not vary. What varies, however, is the number of OPCs and oligodendrocytes. When PDGF concentration is high, most of the cells are OPCs and few are oligodendrocytes. When PDGF concentration is low, most of the cells become oligodendrocytes. Therefore, the failure of axotomy to influence the percentage of OPCs that incorporate BrdU does not necessarily mean that axons do not regulate their division. To reexamine the effects of axons on OPC proliferation, Barres and Raff 1993 measured the total number of OPCs and oligodendrocytes rather than the percentage that incorporated BrdU. When developing optic nerves are transected, the number of mitotic OPCs falls by 90% in 4 d. This same percentage reduction was obtained regardless of whether we measured the total number of mitotic OPCs per longitudinal section, the total number of BrdU1 OPCs per entire optic nerve, or the number of all OPCs per optic nerve. If the same experiment is performed in mutant mice whose axons do not degenerate after a transection , proliferation of oligodendrocyte precursor cells also decreases by 90%, raising the possibility that the proliferation depends on electrical activity in axons . Consistent with this possibility, intraocular injection of tetrodotoxin (TTX), which silences the electrical activity of retinal ganglion cells and their axons, decreases the number of OPCs by 80%. The effect of TTX is prevented by experimentally increasing the concentration of PDGF in the developing optic nerve, suggesting that axonal electrical activity normally controls the production and/or release of the growth factors that are responsible for the proliferation and/or survival of OPCs . Electrical activity could act indirectly by increasing glial production of mitogens such as PDGF or directly by increasing neuronal production or release of mitogens. Whatever the mechanism, these data provided strong evidence that axons control the rate of proliferation and/or survival of developing OPCs. In contrast, there is no evidence as yet that axons are required for OPC migration or differentiation into oligodendrocytes. It has recently been reported that axons are not necessary for OPC migration . Purified OPCs in culture differentiate constitutively into oligodendrocytes in serum-free medium lacking specific inducing signals . In the absence of axons, OPCs can differentiate into oligodendrocytes in vivo . Axons in embryonic and neonatal rats may instead inhibit the ability of OPCs to differentiate into oligodendrocytes. OPCs express Notch1 receptors and activation of the Notch pathway in purified OPCs in culture prevents their differentiation into oligodendrocytes . As neonatal axons express the Notch ligand Jagged1, which is downregulated concurrently with the onset of rapid oligodendrocyte generation, it is possible that axons help to regulate the timing of myelination by preventing oligodendrocyte differentiation during the first neonatal week. Similarly, developing Schwann cells express little of the myelin protein P0 before myelination in vivo, yet in culture purified Schwann cells in the absence of axons express a higher level of P0 . Thus, whereas axons are not required to stimulate differentiation of OPCs into oligodendrocytes, they may inhibit OPC differentiation before myelination. Oligodendrocytes require survival signals in vitro and during normal development in vivo to avoid programmed cell death . At least 50% of the oligodendrocytes produced in the optic nerve normally die within 2 to 3 d after they are generated . Most of the oligodendrocytes that survive for 3 or more days appear to survive for the lifetime of the organism. Experimentally increasing the levels of PDGF, IGF-1, CNTF, or NT-3 in the developing optic nerve greatly decreases the death and increases the number of oligodendrocytes that develop . These findings indicate that all of these signaling molecules are normally present in subsaturating amounts in the developing nerve and suggest that normally occurring oligodendrocyte death may reflect a competition for survival signals that are limited in amount or availability. As PDGF, IGF-1, NT-3, and CNTF are all produced by optic nerve astrocytes and all promote the survival of oligodendrocyte lineage cells in vitro and in vivo, it seems likely that astrocytes play a part in supporting the survival of this lineage in the nerve, at least during development. In addition, many studies have suggested that the survival of oligodendrocytes depends strongly on axons. There is general agreement that when the optic nerves are examined several weeks after a neonatal transection, very few oligodendrocytes and OPCs are found . This could be explained largely by the effects of axons in stimulating OPC division and/or survival or there could be additional effects of axons in promoting oligodendrocyte survival. The conclusion that axons promote oligodendrocyte survival has been drawn by nearly every investigator who has examined optic nerves after transection. For instance, using electron microscopy, Fulcrand and Privat 1977 observed cells with the characteristic ultrastructure of oligodendrocytes undergoing degeneration after postnatal transection. EM is not sufficient to identify degenerating oligodendrocytes unambiguously, however; but using an antigenic identification, David et al. 1984 also concluded that axons likely promote oligodendrocyte survival because of the severe loss of oligodendrocytes despite some continued OPC proliferation after neonatal transection. Oligodendrocytes in the rat optic nerve are normally generated predominantly between P5 and P45. Therefore, in order to determine whether axons help to promote oligodendrocyte survival, it is important to examine the effects of postnatal transection performed after P5. When P8 or P12 optic nerves are transected behind the eye so that the axons degenerate, the oligodendrocytes die . Within 3 d after transection, the number of cell undergoing apoptosis increases by more than fourfold, and many of the dying cells can be identified as oligodendrocytes based on expression of characteristic antigenic markers. P8 optic nerves have about 35,000 oligodendrocytes per nerve; when examined 10 d after a P8 transection on P18, the nerves contain only about 3,000 oligodendrocytes, compared with the 125,000 found in control P18 nerves . Thus not only are few new oligodendrocytes generated after a P8 transection, but >90% of the oligodendrocytes present at P8 die. The death of oligodendrocytes after P8 transection is prevented if the levels of IGF-1, CNTF , or neuregulins (NRG; P.-A. Fernandez, D. Tang, L. Cheng, A. Mudge, and M. Raff, manuscript submitted for publication) are experimentally elevated. How do axons regulate oligodendrocyte survival? Oligodendrocytes do not die if the optic nerve is transected in WLD mutant mice in which the axons do not degenerate and the ability of axons to promote oligodendrocyte survival does not depend on electrical activity in the axons . Purified neurons, but not neuron-conditioned culture medium, promote the survival of purified oligodendrocytes in vitro . These findings suggest that the axon-derived signal is contact-mediated and not dependent on electrical activity. Neuregulins have recently been proposed to be such a signal (see below). How does one reconcile the findings that both astrocyte-derived and axon-derived signals seem to promote oligodendrocyte survival? It is possible that signals from both sources collaborate to promote the survival of oligodendrocytes; alternatively, newly formed oligodendrocytes may depend on the astrocyte-derived signals while more mature oligodendrocytes may lose their dependence on astrocytes and come to depend solely on axons for their survival. Immature Schwann cells are also strongly dependent on axons for a survival signal . Interestingly, one week after PNS myelination, Schwann cells no longer depend on axons for their survival and may instead depend on autocrine signals . The same is true for oligodendrocytes, many but not all of which survive transection in adult optic nerves . These changes in survival requirements are reminiscent of the changes that occur in a number of types of neurons, including sensory DRG neurons and sympathetic neurons, that initially depend on target-derived signals for survival but lose this dependence in the adult . A tentative model for how a competition for axon-dependent survival signals may help to match oligodendrocyte and axon numbers during development has been proposed . Once an oligodendrocyte precursor cell stops dividing and begins to differentiate into an oligodendrocyte, its specific requirements for survival signals change: it rapidly loses its PDGF receptors, for example, so that PDGF can no longer promote its survival . It now has only 2–3 d to contact a nonmyelinated region of axon that provides new signals that are required for its continued survival. A cell that fails to find an axon will kill itself . Forcing oligodendrocytes to compete for axon-dependent survival signals that are limited in amount or availability would help to ensure that the final number of oligodendrocytes is precisely matched to the number (and length) of axons requiring myelination. Importantly, according to this model, newly formed premyelinating oligodendrocytes depend on astrocyte-derived signals for their survival for about the first 2 d, whereas after 3 d the oligodendrocytes are more mature and depend primarily upon an axon-derived signal. Much evidence supports such a model. The model explains why most developing oligodendrocytes that die do so 2 to 3 d after their generation and why most developing oligodendrocytes die after axotomy. It also explains why there appears to be a perfect matching between oligodendrocytes in the optic nerve and the number and lengths of axons ; in normal CNS white matter, all mature oligodendrocytes that survive seem to myelinate axons. Since the proposal of this model, several of its predictions have been tested. One prediction is that if the number of axons is experimentally increased, then the number of oligodendrocytes that survive will increase proportionally. This has been found to be the case . Another prediction is that oligodendrocytes that succeed in contacting axons will preferentially survive, while those that don't will die. This has been tested and found to be true . A last prediction is that if the number of oligodendrocytes generated is experimentally increased, increased death should reduce their numbers to normal. This has also been found to be the case, as overexpression of PDGF in transgenic mice initially leads to an enormous increase in oligodendrocyte numbers in the embryonic mouse spinal cord. All of the extra oligodendrocytes die, however, so that by a week or so after birth the number of oligodendrocytes is normal, as predicted by the model because the number of axons has not changed . In a recent paper, Bruce Trapp and his colleagues reported that rat optic nerve oligodendrocytes develop in the absence of viable retinal ganglion cell axons . To find out whether axons regulated oligodendrocyte development, they performed neonatal axotomy of the optic nerve. 7 d later, at P7, they found no change in the density of OPCs and a 50% decrease in the density of oligodendrocytes in optic nerve sections. However, because transection produces marked atrophy of the cut optic nerve, their data demonstrate a large decrease in the total number of OPCs and oligodendrocytes per axotomized nerve. Because they observed a fourfold decrease in the cross sectional area of the transected nerves, their OPC and oligodendrocyte density measurements indicate a fourfold reduction in the total number of OPCs and an eightfold reduction in the number of oligodendrocytes, by only one week after axotomy. The new findings of Ueda et al. 1999 therefore reconfirm the powerful role of axons in promoting the development of the oligodendrocyte lineage, although this is not the conclusion they draw. In fact, the presence of some oligodendrocyte lineage cells after neonatal transection is expected, as the early stages of the oligodendrocyte lineage (oligodendrocyte precursor cells and newly formed oligodendrocytes) are supported by astrocyte-derived signals such as PDGF. Ueda et al. 1999 also addressed whether axons help to promote oligodendrocyte survival. In these experiments they transected P4 nerves rather than P0 nerves in order to allow time for at least some oligodendrocytes to be generated, and then examined the nerves at P7. They found no change in the density of surviving oligodendrocytes or the percentage of oligodendrocytes undergoing apoptosis, suggesting to them that axons do not control oligodendrocyte survival. Interpretation of these findings, however, is limited by the fact that very few oligodendrocytes are normally found in the optic nerve at P4 . Thus nearly all of the oligodendrocytes examined between P4 and P7 would be newly formed oligodendrocytes, which do not yet depend on axons to survive . Clearly, for the effects of axons on oligodendrocyte survival to be examined meaningfully, axotomy must be performed at a later postnatal age after a significant number of oligodendrocytes have been generated, as was done previously . Nonetheless, Trapp and colleagues concluded that their data “argue against axonal regulation of optic nerve oligodendrogenesis.” In fact, whereas the new data of Trapp and colleagues provides evidence that axons do not strongly promote the survival of just born oligodendrocytes, their new studies do not address whether axons promote the survival of oligodendrocytes that are at least 2 to 3 d beyond their birthday or older, as suggested by the studies of Barres et al. 1992 , Barres et al. 1994 (see below). Ueda et al. 1999 suggest an alternative model for how oligodendrocyte numbers are controlled. They propose a density-dependent feedback mechanism where oligodendrocytes inhibit OPC expansion. Whereas such a mechanism might normally help to control the density of OPCs and newly formed oligodendrocytes in developing white matter and might explain the relatively unaltered densities of OPCs and newly formed oligodendrocytes observed after axotomy by Ueda et al. 1999 , it is not sufficient to explain how the final number of mature oligodendrocytes is determined. Even when the density of OPCs and newly formed oligodendrocytes is greatly increased by overexpression of PDGF, the final number of mature oligodendrocytes is unaltered . Moreover OPC proliferation continues in adult rodent optic nerves, but the number of mature oligodendrocytes does not change. During the past several years, neuregulins (NRGs) have emerged as a likely candidate for an axonal signal that promotes both Schwann cell and oligodendrocyte development. NRGs are a large family of proteins related to epidermal growth factor. They occur in multiple isoforms, some membrane bound and some soluble, that are encoded by at least four alternatively spliced genes. They were first identified as potent mitogens for Schwann cells and astrocytes in culture and called glial growth factor . In the developing CNS, NRGs are predominantly or entirely expressed by neurons, which target them to their axons throughout the CNS and PNS. NRGs promote the survival and proliferation of cells in the Schwann cell lineage by activating erbB2/erbB3 heterodimeric receptors . The entire Schwann cell lineage fails to develop in erbB3 deficient transgenic mice and in NRG-deficient transgenic mice . Although, in principle, this finding might be explained by the ability of neuronally derived NRG to instruct multipotential neural crest cells to become Schwann cells , it is unlikely to be the entire explanation since in erbB2-deficient mice Schwann cell precursor cells develop within the DRG but fail to migrate into the peripheral nerves . Thus the lack of Schwann cell development in these transgenic mice may be accounted for by the loss of functional axonal NRG signaling. Axons have been shown to promote the survival and proliferation of Schwann cell lineage cells in vitro and in vivo; NRGs mediate these axonal effects in culture experiments , as well as after axotomy of developing peripheral nerves . Similarly, the development of the oligodendrocyte lineage also depends on NRG signaling. In culture, NRG promotes the survival of oligodendrocytes and the proliferation of OPCs . When spinal cords from wild-type mice are cultured as explants, the oligodendrocyte lineage fails to develop if NRGs are neutralized . Moreover, the oligodendrocyte lineage does not develop in spinal cord explants obtained from NRG-deficient transgenic mice, but can be rescued by addition of recombinant NRG to the culture medium . The source of the spinal cord derived NRG that promotes OPC development is probably either motor neurons or ventral ventricular zone cells, both of which contain NRG immunoreactivity and are close to the site of OPC generation . These results clearly demonstrate the importance of NRG for either the differentiation or proliferation of OPCs. Recent experiments also provide strong support for the role of neuronally derived NRG in promoting oligodendrocyte survival as well. Retinal ganglion cells make NRG, which is targeted to their axons . The survival-promoting effect of DRG axons in vitro is strongly inhibited if NRG is neutralized. In the developing optic nerve, neutralization of NRG increases normal oligodendrocyte death, whereas delivery of exogenous NRG decreases it; moreover, the oligodendrocyte death induced by nerve transection is almost completely abolished by delivery of exogenous NRG (Fernandez, P.-A., D. Tang, L. Cheng, A. Mudge, and M.C. Raff, manuscript submitted for publication). These results suggest that NRG is one of the major signals used by RGC axons to promote oligodendrocyte survival in the developing optic nerve. In addition, other axonal signals are likely to participate. A recent study has suggested that integrin signaling helps to promote axon-mediated oligodendrocyte survival in DRG-oligodendrocyte co-cultures . This is interesting as in other tissues integrin signaling has been found to promote survival by enhancing responsiveness to trophic peptides . Thus it will be of great interest in future studies to explore whether NRG and integrin signaling are synergistic in promoting oligodendrocyte survival. In summary, axons powerfully control the development of myelinating glial cells. Recent studies have revealed that axons promote oligodendrocyte development by helping to drive the proliferation of OPCs and by promoting the survival of mature, myelinating oligodendrocytes. Axonally derived NRG is a likely candidate signal that mediates these effects. Together, these recent studies provide strong support for a model in which the number of myelinating cells is matched during development to the axonal surface area requiring myelination. | Study | biomedical | en | 0.999997 |
10601328 | PTP-SL, STEP, and ERK2 cDNA constructs have been previously described . pCαEV was provided by G.S. McKnight (University of Washington, Seattle, WA). pECE-HA-p38MAPK was provided by J. Pouysségur (Centre de Biochimie-CNRS, Nice, France). pRK5-GST-PTP-SL mammalian expression vectors were made by PCR with a primer containing a Kozak sequence followed by a start codon and the S . Japanicum glutathione-S-transferase (GST) sequence. Antibodies and reagents were used as described . Rabbit polyclonal anti-p38α (C-20) was purchased from Santa Cruz Biotechnology Inc. Dibutyryl-cAMP and okadaic acid (Boehringer Mannheim) were used at final concentrations of 2 mM and 1 μM, respectively. Forskolin (ICN Pharmaceuticals Inc.) was used at a final concentration of 40 μM, in the continuous presence of 1 mM IBMX (Sigma Chemical Co.). The PKA inhibitor H89 (Biomol) was used at a final concentration of 25 μM. When used, IBMX and H89 were added to the cells 30 min before stimulation. The bovine PKA catalytic subunit (cPKA) was purchased from Promega Corp. For PKA in vitro kinase assays, GST fusion proteins (1 μg) were incubated at room temperature during 1 h with 0.5 U/μl of cPKA in the presence of 2 μCi of γ-[ 32 P]ATP, 10 μM ATP, and 8 mM MgCl 2 (20 μl final volume). The reactions were stopped by adding SDS sample buffer and boiling, followed by SDS-PAGE and autoradiography. For in vitro association assays , GST fusion proteins were phosphorylated with cPKA as above, in the presence of 200 μM cold ATP, and then mixed with cell lysates and precipitated with glutathione-Sepharose, followed by immunoblotting. In vitro phosphatase assays were performed in 25 mM Hepes, pH 7.3, 5 mM EDTA, and 10 mM DTT (40 μl final volume), at 37°C, during the indicated times, as described in Zúñiga et al. 1999 . Rat fibroblast Rat-1, human embryonic kidney 293, and Simian COS-7 cell lines, were grown in DME containing high glucose supplemented with 5% (for COS-7 cells) or 10% heat-inactivated FCS. Cells were transfected using the DEAE-dextran method (COS-7 cells) or the calcium phosphate precipitation method (293 cells), and were harvested after 48–72 h of culture. In cells transfected with pCαEV, the expression of cPKAα was induced by incubation during the last 24 h of culture in the presence of 100 μM ZnSO 4 . For 32 P-labeling, transfected COS-7 cells were cultured for 4 h with phosphate-free DME 2% FCS in the presence of [ 32 P]inorganic phosphate (100 μCi/ml), and then cells were treated with dibutyryl-cAMP or forskolin plus IBMX during 1 h, or with okadaic acid during 30 min. HA-ERK2 or HA-p38α from transfected 293 cells were activated by cell treatment with EGF (5 min, 50 ng/ml) or sorbitol (30 min, 0.5 M), respectively. Cell lysis, precipitation with GST-fusion proteins, immunoprecipitation, and immunoblotting were done as described . COS-7 cells were processed for immunofluorescence as described . In brief, after transfection, cells were rinsed with IPBS buffer (1.5 mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl 2 , and 0.5 mM MgCl 2 , pH 7.4), and then fixed with methanol. Samples were incubated in blocking solution (IPBS 3% BSA), followed by incubation at 37°C for 90 min with the mixture of the anti-HA and anti–PTP-SL primary antibodies. After washing with IPBS, cells were incubated for 1 h at room temperature with a mixture of the anti-rabbit fluorescein isothiocyanate– and the anti-mouse tetramethylrhodamine B isothiocyanate–conjugated secondary antibodies, followed by washing with IPBS and mounting. PTP-SL and STEP tyrosine phosphatases contain a conserved KIM in their cytosolic noncatalytic regions that mediates association with MAP kinases . Since a consensus phosphorylation sequence for PKA occurs within the KIM , the phosphorylation of these two phosphatases by PKA was tested. GST-PTP-SL or -STEP fusion proteins, were incubated in vitro with cPKA in the presence of γ-[ 32 P]ATP, followed by SDS-PAGE and autoradiography. As shown, a strong phosphorylation of the PTP-SL and STEP fusion proteins was detected , whereas no phosphorylation took place with GST alone or a GST fusion protein containing a nonrelated PTP , indicating that PTP-SL and STEP are substrates of PKA. Substitution by alanine of the Ser 231 residue (S231A mutant), abolished the phosphorylation of PTP-SL by cPKA , demonstrating that this residue is the target of the kinase. Next, the in vivo phosphorylation of PTP-SL upon PKA activation conditions was investigated. Phosphorus 32 labeling was carried out on COS-7 cells transfected with plasmids encoding transmembrane (PTP-SL 1-549) or nontransmembrane (PTP-SL 147-549) PTP-SL isoforms, followed by treatment with PKA activators and immunoprecipitation with anti–PTP-SL antibody. Incubation in the presence of the cAMP analogous dibutyryl-cAMP or the adenylate cyclase activator forskolin increased the phosphorylation of wild-type PTP-SL isoforms , but not of the S231A mutants . Interestingly, the basal levels of phosphorylation were greatly diminished in the S231A mutants compared with the wild-type PTP-SL , indicating that PKA phosphorylates this residue under the normal cell growth conditions of COS-7 cells. Furthermore, cell treatment with the PP2A serine/threonine phosphatase inhibitor, okadaic acid (1 μM), induced the hyperphosphorylation of wild-type PTP-SL, but not of the S231A mutant . These results demonstrate that the Ser 231 residue of PTP-SL is a substrate of PKA, and suggest a role for PP2A in the in vivo dephosphorylation of such a residue. Next, the effect of PTP-SL phosphorylation by PKA on its association with MAP kinases was analyzed. GST-PTP-SL fusion proteins were phosphorylated in vitro by cPKA as above, in the presence of cold ATP, and the fusion proteins were incubated with Rat-1 cell lysates and precipitated with glutathione-Sepharose. Samples were resolved by SDS-PAGE and the presence of the MAP kinases ERK1/2 or p38α was detected by immunoblot using specific antibodies. Remarkably, the phosphorylation of GST-PTP-SL wild type by cPKA abrogated its association with both ERK1/2 and p38α ; however, no changes were observed with the GST-PTP-SL S231A mutant upon incubation with cPKA . To test the effect of PTP-SL phosphorylation by PKA on the association with the MAP kinases in vivo, GST-PTP-SL fusion proteins were overexpressed in 293 cells and precipitated in one-step with glutathione-Sepharose, followed by immunoblot analysis, as above. Treatment of cells with dibutyryl-cAMP or forskolin resulted in the lack of coprecipitation of ERK1/2 or HA-p38α with PTP-SL ; however, in dibutyryl-cAMP–treated cells that were preincubated with the PKA inhibitor H89, normal levels of association with the kinases were detected . Finally, the coprecipitation of ERK1/2 and p38α with the PTP-SL S231E mutant, which mimics a phosphorylated Ser 231 residue, was also tested. As shown, these MAP kinases did not associate in 293 cells with overexpressed GST-PTP-SL S231E , whereas association was efficiently detected with the GST-PTP-SL wild type, the S231A mutant or the C480S catalytically inactive mutant . The functional consequences of the phosphorylation of the Ser 231 residue of PTP-SL, on the dephosphorylation of ERK1/2 and p38α by the phosphatase, were analyzed using the S231E PTP-SL mutant. GST-PTP-SL wild type or S231E fusion proteins were mixed with pellets containing activated HA-ERK2 or HA-p38α, and phosphatase assays were carried out, followed by SDS-PAGE and immunoblot with the anti–phosphotyrosine 4G10 mAb. As shown, the tyrosine dephosphorylation of HA-ERK2 and HA-p38α by GST-PTP-SL S231E mutant was impaired compared with that shown by GST-PTP-SL wild type, whereas equal activities of both fusion proteins were measured towards the nonspecific p-NPP substrate . These findings demonstrate that phosphorylation of the Ser 231 residue of PTP-SL by PKA inhibits its association with ERK1/2 and p38α, and the subsequent tyrosine dephosphorylation of these MAP kinases. PTP-SL retains ERK2 in the cytoplasm in a KIM-dependent manner . To study the effect of phosphorylation of PTP-SL by PKA on its ability to retain MAP kinases outside of the nucleus, immunofluorescence analysis was performed on COS-7 cells cotransfected with HA-ERK2 or HA-p38α, and PTP-SL. Overexpression of HA-ERK2 or HA-p38α alone resulted in their accumulation in the nucleus ; however, in the presence of PTP-SL, the nuclear accumulation of these kinases was abolished, colocalizing with the phosphatase outside of the nucleus . Interestingly, neither the PTP activity nor the PTP domain of PTP-SL itself was required to retain HA-ERK2 outside of the nucleus, as observed by coexpression with PTP-SL catalytically inactive mutants (C480S or R486M) or with truncated PTP-SL forms lacking the PTP domain (PTP-SL 1-288) . On the other hand, upon coexpression with the PTP-SL S231E mutant, the cytoplasmic retention of HA-ERK2 or HA-p38α was significantly reduced, as compared with wild-type PTP-SL . Also, when cells coexpressing wild-type PTP-SL and HA-ERK2 or HA-p38α were treated with dibutyryl-cAMP, the nuclear localization of both MAP kinases was partially restored, and such an effect was prevented by cell preincubation with H89 . However, no effect was observed upon cell treatment with agents that activate other kinase pathways, such as EGF or PMA (data not shown). Furthermore, cotransfection with an inducible expression vector coding the Cα catalytic subunit of PKA (cPKAα), also favored the nuclear localization of these MAP kinases in the presence of PTP-SL . Remarkably, the effect of PKA activation on the colocalization of HA-ERK2 and HA-p38α with wild-type PTP-SL was not observed with the PTP-SL S231A mutant , demonstrating that phosphorylation of the Ser 231 residue of PTP-SL by PKA inhibits the in vivo association of PTP-SL with HA-ERK2 and HA-p38α, and favors the nuclear translocation of these kinases. PKA modulates the activity of MAP kinases in a cell type– and stimulus-specific manner by interfering with upstream events from signaling cascades activated through distinct Ras-like GTPases, including Ras, Rap1, and RalGDS . In addition, PKA activity favors the nuclear translocation of ERK1/2 in PC12 and hippocampal neurons, as well as in presynaptic sensory neurons from Aplysia . Our results, showing a crosstalk between the PKA and ERK1/2 and p38α kinases through the tyrosine phosphatase PTP-SL, support the existence of a novel mechanism by which PKA can regulate the activity of the MAP kinases and their translocation to the nucleus . Such a mechanism would involve the existence, in certain cell types, of a pool of inactive MAP kinases outside of the nucleus, which would be complexed with PTP-SL or other KIM-containing PTPs, including STEP and HePTP (see below). The dissociation equilibrium of the complex would depend upon the cell type– and the stimulus-specific conditions of PKA activity, and the lack of association would be favored by the PKA-mediated phosphorylation of the KIM regulatory residue on the PTP. Thus, upon conditions of PKA activation, both the tyrosine phosphorylation and the entry into the nucleus of the MAP kinases would be prevalent. It should be noted that the expression of PTP-SL and related isoforms is restricted to specialized areas of the brain, including the Purkinje cells in the postnatal cerebellum , suggesting the possibility of a differential regulation of MAP kinase functions by PTP-SL and PKA during brain development. The mutational analysis of the KIM of PTP-SL has revealed that the residues involved in the PKA phosphorylation consensus sequence are also crucial for the docking of this phosphatase with ERK1/2 . Such residues are conserved between the related tyrosine phosphatases PTP-SL, STEP, and HePTP, which have been found to associate with MAP kinases and regulate their activation . In this regard, while writing this manuscript, Saxena et al. 1999b have reported the negative role of PKA phosphorylation of the KIM of HePTP in the physical and functional association of HePTP with MAP kinases. Also, a tyrosine phosphatase from Drosophila , PTP-ER, has been found that inactivates MAP kinase, and that contains three KIMs with consensus phosphorylation sites for PKA . Finally, we have found that the retention of ERK2 outside of the nucleus is efficiently achieved by PTP-SL catalytically inactive mutants, as well as by truncated PTP-SL molecules lacking the PTP domain, demonstrating that this domain is dispensable in a such process. Thus, PKA-mediated KIM phosphorylations could have diverse regulatory effects on MAP kinase functions, depending on the functional properties of the affected KIM-containing molecule. The involvement of distinct kinases in the in vivo phosphorylation of PTP-SL is likely to exist, which ultimately could control the biological functions of this PTP. Thus, the Thr 253 residue of PTP-SL is phosphorylated in vivo by ERK1/2 upon EGF cell treatment in a manner dependent of docking through the KIM . Furthermore, the Thr 253 residue is also a putative PKC phosphorylation site, and PTP-SL is phosphorylated in vitro by this kinase (our unpublished observations). In this context, the binding of MAP kinases to the KIM of PTP-SL could mask the PKA phosphorylation motif by steric hindrance, hampering the phosphorylation of PTP-SL by PKA; conversely, phosphorylation of the KIM by PKA difficult the association of MAP kinases and the subsequent phosphorylation of the Thr 253 residue. The results presented here indicate a major regulatory role on the PTP-SL functions for the PKA-mediated phosphorylation of the Ser 231 residue; accordingly, the basal phosphorylation of PTP-SL in COS-7 cells is found predominantly in such residue . On the other hand, the functional significance of the phosphorylation of the Thr 253 residue by ERK1/2 remains elusive. The possibility exists that phosphorylation of Thr 253 regulates the dissociation of PTP-SL from MAP kinases, as it has been suggested for HePTP . However, the cytoplasmic retention of ERK2 by PTP-SL was efficiently achieved upon conditions of EGF-induced phosphorylation of Thr 253 (our unpublished observations). In addition, phosphorylation of this residue could account for the regulated binding of PTP-SL to other unidentified molecules. The participation of specific serine/threonine phosphatases in the in vivo dephosphorylation of the Ser 231 and Thr 253 residues of PTP-SL is also expected. In this regard, we have found that cell treatment with okadaic acid induces hyperphosphorylation of the Ser 231 residue, suggesting an active role for PP2A in the in vivo dephosphorylation of this key residue . Also, PP2A and PP2C have been shown to interfere with the activation of the MAP kinase pathways by affecting the phosphorylation of MAP kinases or upstream phosphorylation events . Thus, a complex network of kinases and phosphatases could be envisioned within the MAP kinase pathways, which integrate the different signals to generate specific cell responses. The importance of the assembly of the molecular components that regulate the activation of the MAP kinases has been recently outlined . The results reported here point to PKA as a major regulator of the physical and functional association between the ERK1/2 and p38α kinases and their inactivating tyrosine phosphatases. | Study | biomedical | en | 0.999998 |
10601329 | Two X chromosomes were used in this study: a normal sequence X chromosome marked with yellow ( y ) and white (w) ; and the FM7 balancer chromosome. The FM7 chromosome consists of three superimposed inversions: a nearly full-length paracentric inversion that moves most of the basal heterochromatin to the tip of the X and inverts all but the most distal bands of euchromatin; the dl-49 inversion; and a third inversion, broken in the distally located heterochromatin (region 20) and in the euchromatin at position 15 D. FM7 is as an excellent suppressor of exchange when heterozygous with a normal X chromosome. We monitor X and 4 th chromosome disjunction by crossing X/X; pol/pol females to attached XY/0; C(4)RM, ci ey R males. The frequencies of nondisjunction were calculated as described in Hawley et al. 1993 . Oocytes were prepared and examined as previously described with minor modifications . Egg chambers from 3–7-d-old females were extracted by quick pulses of a blender using a modified Robb's medium. The mixture was passed sequentially through a loose and fine mesh to separate late stage oocytes. The oocytes were fixed for 5 min on a rotator at room temperature in a hypertonic solution, therefore preventing hypotonic activation of the mature oocytes. After removal of the follicle cells, chorion, and vitelline membranes, the oocytes were permeabilized with 1% Triton X-100 in PBS. Oocytes were labeled with YL1/2 (1:200) and YOL1/34 (1:200) rat antitubulin mAbs (Accurate) and both Oligreen (Molecular Probes, Inc.; 1:10,000) and 1 MAB52 (1:500) anticore histone mouse monoclonal. In some experiments, oocytes were also labeled with MEI-S332 (1:500) guinea pig polyclonal antibody (generous gift of Drs. Tracy Tang and Terry Orr-Weaver, Massachusetts Institute of Technology, Cambridge, MA). Primary antibodies were then labeled with secondary antibodies (1:250) purchased from Jackson ImmunoResearch Laboratories, conjugated in the following manner: Cy2 to anti-mouse; Cy3 to anti-rat; and Cy5 to anti-guinea pig. Oocytes were examined using an MRC-1024 BioRad confocal microscope (Kalman collection), and spindle and chromatin lengths and widths were determined using BioRad 3D software. Spindle and chromatin lengths were determined from maximum intensity projected spindles. Spindle lengths were measured from pole to pole and chromatin length from the sites of the chromatin found closest to either pole. Image stacks were converted to maximum intensity projections and subsequently converted to Photoshop Images (Adobe Systems Inc.). Final images were produced on a dye sublimation printer (Tektronics Phaser 440). Although the spindles of FM7/X; α tub67C P40 / + oocytes are, on average, somewhat shorter than wild-type spindles (average spindle lengths are 12.2 versus 15.1 μm, respectively), confocal studies did not reveal obvious defects in spindle structure, as shown in Fig. 1 A. However, an examination of chromatin in FM7/X; α tub67C P40 / + prometaphase oocytes revealed a failure of the chromatin mass to elongate along the axis of the spindle . Chromatin masses in wild-type spindles appear almost spherical as spindle assembly initiates, but elongate as spindle assembly progresses. However, in α tub67C P40 / + oocytes, the chromatin remains almost spherical, even on fully elongated spindles. A plot of the chromatin mass length from oocytes with two wild-type copies of the α tub67C gene reveals a wide distribution of chromatin lengths . In contrast, a plot of the chromatin mass length from oocytes heterozygous for α tub67C P40 exhibits a very narrow distribution that is centered at a much shorter length . The chromatin mass ranged in length from 3.1–18 μm in control oocytes, whereas in FM7/X; α tub67C P40 / + oocytes, chromatin length was observed to vary from 2.4–5.8 μm. Thus, in oocytes heterozygous for the α tub67C P40 mutation, the chromatin mass fails to elongate properly. To determine whether or not the observed defect in chromatin stretching was a consequence of the slightly shorter spindles found in FM7/X; α tub67C P40 / + oocytes , we calculated the axial ratio (length over width) of the chromatin and plotted this value against spindle length. This metric of axial ratios allowed us to evaluate the stretching of the chromosomes in a manner independent of spindle length. For FM7/X; +/+ oocytes, the axial ratios range from one to ten, whereas in oocytes obtained from FM7/X; α tub67C P40 / + females, they range from one to two . Thus, even when differences in spindle length are taken into account, oocytes from FM7/X; α tub67C P40 / + females still display an obvious defect in chromatin stretching. Even in those FM7/X; α tub67C P40 / + oocytes with the longest spindles, little or no stretching of the chromatin mass is observed and the chromatin remains almost spherical. Taken together, these data reveal a defect in the stretching of chromosomes during prometaphase in FM7/X; α tub67C P40 / + oocytes, which is clearly distinct from an effect on spindle lengthening. Lengthening of the spindle and chromatin during meiotic prometaphase is paralleled by the coalescence of a Drosophila centromere-resident protein, MEI-S332, at the most poleward ends of the chromatin mass . We examined the distribution of MEI-S332 protein in prometaphase oocytes bearing the α tub67C P40 mutation by immunolocalization. In wild-type spindles, the MEI-S332 protein is distributed over the surface of the chromatin in discrete foci during early prometaphase, and coalesces symmetrically at the extreme poleward tips during spindle assembly and elongation . As expected, MEI-S332 protein was found on the poleward edges of the chromatin masses in FM7/X; +/+ spindles . In contrast, the MEI-S332 localization pattern from oocytes derived from FM7/X; α tub67C P40 / + females indicated that the centromeres were positioned abnormally . In no case (0/15) was the MEI-S332 protein properly positioned at the distal tips of the elongating chromatin mass, as is observed in wild-type oocytes . Rather, MEI-S332 either failed to be completely localized at opposite poles of the main chromosome mass or was found entirely on one side of the main chromatin mass . These cytological studies demonstrate that heterozygosity for the α tub67C P40 mutation leads to a defect in both chromatin stretching and centromere positioning. The genetic studies of the α tub67C P40 mutation were carried out in two types of females: females that were heterozygous for a normal sequence X chromosome and an FM7 balancer chromosome, denoted FM7/X , and females carrying two normal sequence X chromosomes, denoted X/X . In X/X females the X chromosomes recombine at least once in >90% of these oocytes, while in FM7/X females, the presence of the multiply inverted balancer chromosome reduces the frequency of X chromosomal exchange to <1% of normal . A comparison of the frequency of errors of meiotic segregation in FM7/X and X/X females allowed us to differentially assess the effects of mutations such as α tub67C P40 on the chiasmate and achiasmate segregation systems. As shown in Fig. 4 , FM7/X; α tub67C P40 / + females display 20-fold higher levels of X chromosome nondisjunction than do FM7/X; +/+ control females, suggesting that most, if not all, of these cases of X chromosome nondisjunction are due to a failure of achiasmate segregation. Indeed, even those few nondisjunction events that were observed in X/X; α tub67C P40 / + females occurred in the 5–8% of the oocytes in which the two X chromosomes failed to undergo exchange (data not shown). An additional mutant allele, derived from α tub67C P40 and denoted α tub67C P40 Δ , displayed an enhancement of meiotic chromosome missegregation relative to the original P element allele . The α tub67C P40 Δ allele differs from the original P element insertion only in that a substantial internal portion of the P element has been removed. Heterozygosity for α tub67C P40 Δ , as assayed in FM7/X females, leads to high levels of achiasmate nondisjunction; however, the α tub67C P40 Δ mutant has little or no effect on the segregation of chiasmate X chromosomes (in X/X females). For both of these alleles, the observed chromosome missegregation must primarily be due to nondisjunction rather than loss, as the frequency of diplo- X exceptions always equals or slightly exceeds that of nullo- X exceptions (data not shown). FM7/X; α tub67C P40 / + and FM7/X; α tub67C P40 Δ / + females also display elevated levels of 4 th chromosome nondisjunction (again with an approximate equality of diplo- 4 and nullo- 4 exceptions). In both cases, the effect on 4 th chromosome missegregation was less than half of the effect on X chromosome segregation. Two lines of evidence suggest that most of the observed 4 th chromosome nondisjunction events are a secondary consequence of failures of X chromosome missegregation and not a direct effect of heterozygosity for the α tub67C mutations. First, 4 th chromosome nondisjunction is rarely observed in females bearing structurally normal (i.e., recombining) X chromosomes, despite the fact that the 4 th chromosomes themselves are always achiasmate. Second, most of the 4 th chromosome nondisjunction occurs in oocytes that were simultaneously nondisjunctional for the X chromosomes, and reflects cases where the two 4 th chromosomes segregate away from the two nondisjoining X chromosomes (data not shown). We also analyzed the meiotic effects of four other female-sterile recessive alleles of the α tub67C gene obtained by Matthews et al. 1993 . When heterozygous, these alleles induce only low levels of X or 4 th chromosome nondisjunction in females bearing two normal sequence X chromosomes . However, in the presence of the FM7 balancer chromosome, heterozygosity for the α tub67C 2 allele increases X nondisjunction to levels comparable to those observed in FM7/X; α tub67C P40 / + females, whereas the α tub67C 1 , α tub67C 3 , and α tub67C 4 alleles exhibited intermediate levels (7.1–8.2%) of X chromosome (data not shown). These data demonstrate that disruption of achiasmate segregation is a property of many alleles of the α tub67C locus. The dominant effects of the α tub67C alleles appear to be antimorphic, since heterozygosity for a deficiency of the α tub67C locus ( Df(3L)AC1 ) had no effects on achiasmate segregation (data not shown). The Nod kinesin-like protein is specifically required for achiasmate segregation in Drosophila . Homozygosity for loss-of-function nod mutations specifically causes achiasmate chromosomes to nondisjoin or to be lost at high frequencies . Cytological studies of prometaphase oocytes from homozygous nod females reveal that achiasmate chromosomes are found precociously at the poles of the developing meiotic spindle and frequently dissociate from the spindle . During prometaphase, Nod protein is localized along the entire lengths of oocyte chromosomes . Here, it acts to retard the poleward migration of achiasmate chromosomes and serves, in essence, as a stabilizing plateward force. As shown in Fig. 5 A, heterozygosity for a recessive loss-of-function mutation of nod ( nod b17 ) strongly suppressed the meiotic effects of the α tub67C P40 and α tub67C P40 Δ alleles. All four of the α tub67C mutations isolated by Matthews et al. 1993 are also suppressed by heterozygosity for a loss-of-function nod allele (data not shown). This effect is not allele-specific with respect to nod , because all three nod alleles tested, including a deficiency ( Df(1)nod ), exhibit a similar capability to suppress the phenotype of the α tub67C P40 allele. These data are consistent with the view that the relative abundance of functional αtub67C and Nod proteins is critical to the faithful segregation of achiasmate chromosomes. To test this hypothesis further, we measured chromosome segregation in oocytes with various levels of nod + and α tub67C . We kept tubulin levels constant, and tested the effect of increasing the dose of nod + . Using a duplication of nod + , we showed that three wild-type copies of nod + in an otherwise wild-type background increased chromosome missegregation . The frequencies of X and 4 th missegregation were lower than, but qualitatively similar to, the effect on achiasmate chromosome nondisjunction observed in oocytes from females heterozygous for α tub67C P40 . We also compared the effect of three copies of nod + in the presence of different levels of wild-type α tub67C . X and 4 th chromosome nondisjunction levels were substantially elevated in FM7/X; α tub67C P40 / + females carrying the nod + duplication, compared with flies with the nod duplication and two wild-type copies of α tub67C . Finally, we determined the effect of varying the number of copies of nod + in the presence of constant levels of wild-type α tub67C , i.e., in α tub67C P40 / + oocytes. Again, in the presence of comparable α tub67C P40 levels, the levels of chromosome missegregation showed an almost linear relationship with the levels of nod . Reducing the copy number of nod + suppresses the chromosome missegregation in oocytes from FM7/X ; α tub67C P40 / + females. Therefore, we determined what effect a loss-of-function allele of nod ( nod b17 ) has on the cytological defects observed in FM7/X; α tub67C P40 / + oocytes. We measured chromatin mass and spindle lengths in oocytes derived from FM7, nod b17 /X; α tub67C P40 / + females , and found that chromatin mass length in FM7, nod b17 /X; α tub67C P40 / + oocytes paralleled those of wild-type and ranged in length from 3–12.5 μm, with no chromatin mass shorter than 3 μm . Plotting of the chromatin length or axial ratio versus the spindle length demonstrated a parallel elongation of chromatin and spindles in nod -suppressed oocytes . Heterozygosity for nod with two copies of wild-type α tub67C has no effect on chromatin mass elongation (data not shown). Moreover, in FM7, nod b17 /X; α tub67C P40 / + oocytes, MEI-S332 protein is once again normally localized . Reducing Nod levels restores the ability of chromatin to be elongated in mature spindles and restores proper centromere positioning. However, the effect of the α tub67C P40 mutation on decreasing overall spindle length (average = 12.2 μm) in oocytes from α tub67C P40 / + females was not suppressed by reducing the dosage of nod + (average spindle length = 11.1 μm). Thus, reducing the dose of nod + can suppress the chromosome missegregation phenotype, the defect in chromatin elongation, and centromere mispositioning, but does not suppress the reduction in spindle length created by α tub67C P40 . Altering Nod dosage in oocytes bearing the α tub67C mutations indicates that Nod plays a role in centromere positioning; this effect would have been impossible to detect using loss-of-function nod alleles alone. Our data demonstrate that the fidelity of achiasmate chromosome segregation is sensitive to the relative levels of functional αtub67C and Nod proteins. The consequences of altering the level of one of these two proteins are ameliorated or exacerbated by changes in the level of the other. We propose that when the level of wild-type αtub67C protein is reduced, the poleward force(s) are compromised, and this leads to a failure of chromatin elongation and centromere positioning. We can imagine four mechanisms to explain the disruption of poleward forces by α tub67C mutations. The first proposes that the presence of the aberrant αtub67C P40 subunits results in reduced production of poleward force from a minus end directed microtubule-based motor: a kinesin or a dynein . Failure of these motors to generate poleward force could prevent the migration of kinetochores and/or the elongation of chromosome arms. The second hypothesis suggests reduced coupling between a plus end directed kinesin at the kinetochore and kinetochore microtubules . This could lead to poor coupling of kinetochore microtubules to the kinetochore, resulting in a failure of the mechanism that actually provides force for poleward kinetochore motility. A third possibility is that a plus end directed motor responsible for sliding antiparallel microtubules is compromised and this prevents sliding of microtubules bound to the chromatin. Finally, the observed impairment in poleward movement may reflect reduced binding of a microtubule-associated protein(s) or kinesin that regulates poleward flux or microtubule dynamics . Nod could also play a role in microtubule dynamics, and thereby regulate poleward and antipoleward forces. As noted above, cytological and genetic studies are consistent with a model in which Nod functions in wild-type spindles to provide both a plateward force and a function which is important for centromere positioning. When poleward forces and chromatin stretching are reduced by heterozygosity for α tub67C P40 , decreasing the dose of nod + ameliorates this defect. We speculate that this suppression occurs because the reduced plateward forces now more closely equal the impaired poleward forces. Similarly, increasing the amount of Nod in both wild-type and in α tub67C P40 / + oocytes should, and more importantly does, increase the frequency of meiotic errors in oocytes. These observations are consistent with a model in which Nod protein serves as a stabilizing plateward force for the segregation of achiasmate chromosomes, and that this force is balanced by poleward forces which are dependent on the level of functional αtub67C protein. The model presented in the preceding paragraph does not require the physical interaction of the αtub67C and Nod proteins, only their separate roles in creating opposing forces. It is, however, at least possible that the two proteins do indeed physically interact. In this case, the observed defects in chromosome and centromere movements in oocytes carrying mutations in α tub67C gene might be the result of poisonous or rigor-like interactions between Nod protein bound to the chromatin and the mutant αtub67C protein. The observation that the effects of these α-tubulin mutations is restricted to achiasmate chromosomes is puzzling in terms of the more global defects observed cytologically. We can only surmise that the presence of chiasmata is prophylactic to the kinds of errors created by these tubulin mutations. Perhaps the types of kinetochore orientation mechanisms that successfully ensure the segregation of chiasmate bivalents are to some degree more fail-safe than those ensuring the segregation of achiasmate homologues. | Study | biomedical | en | 0.999995 |
10601330 | Spinal cords (C3) and sciatic nerves were harvested from 30-d-old CGT +/+ and CGT −/− mice that were perfused through the heart with either 4 or 1% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.3. Spinal cords were postfixed for 1 h in the same fixative, cryopreserved in 30% sucrose in PB for 48 h at 4°C and sectioned at 10 μm. Sciatic nerves were teased apart to yield single fiber preparations. Spinal cord sections and sciatic teased fibers were blocked for 1 h in PB containing 10% normal goat serum, 5% fish gelatin, and 0.1% Triton X-100 and incubated in the primary antibody overnight at 4°C. The primary antibodies used in this study were directed against ankyrin G (mouse monoclonal, Zymed and gift from Dr. Van Bennett, Duke University, Durham, NC) , neurofascin (rabbit polyclonal, gift from Dr. Van Bennett) , paranodin (rabbit polyclonal) , potassium channel Kv1.1 (rabbit polyclonal, gift from Dr. Bruce Tempel, University of Washington School of Medicine, Seattle, WA; mouse monoclonal, Upstate Biotechnology) , sodium channels (rabbit polyclonal, gift from Dr. Rock Levinson, Health Sciences Center, Denver, CO) , and myelin basic protein (mouse monoclonal, Sternberger Monoclonal, Inc.; rabbit polyclonal, Dako, Inc.). After primary antibody incubation, the tissue was thoroughly rinsed in PB, blocked for 1 h as described previously, and incubated in the appropriate secondary antibody conjugated to biotin or Texas red for 2 h. After rinsing the tissue four times for 5 min, the tissue incubated in the secondary antibody conjugated to biotin was incubated in streptavidin fluorescein (diluted 1:200) for 30 min and thoroughly rinsed. The tissue was coverslipped using Vectashield (Vector Labs, Inc.). For double-labeling, the above procedure was repeated using a different species-specific antibody and the appropriate secondary antibody conjugated to the proper fluorochrome. When myelin basic protein was the second antigen to be labeled, the tissue was postfixed in 4% paraformaldehyde in PB for 10 min at −20°C, rinsed, and permeabilized in −20°C acetone for 10 min. All labeled slides were analyzed by both conventional fluorescent and scanning confocal microscopy. Images were generated on a Leica TCS-NT Laser Scanning microscope using a 40× oil objective and a pinhole size of 1.0 Airy disk units. Spinal cord and sciatic nerve tissues were harvested from 30-d-old galactolipid-deficient and wild-type mice, diced into small pieces, homogenized in 1% SDS in PB, and placed in a boiling water bath for 10 min. Insoluble material was removed by centrifugation at 5,000 g for 10 min. Protein concentrations were determined using a modified Lowry assay (Bio-Rad, Inc.). 100 μg of protein was separated using a precast 4–15% gradient polyacrylamide gel (Jule, Inc.), transferred to nitrocellulose, and stained with Ponceau S. The nitrocellulose was blocked for 1 h in 5% milk solids, 5% normal goat serum, and 0.1% Triton X-100 in PB, incubated in antiparanodin (1:3,000) for 1 h at room temperature, rinsed in PB, blocked, incubated in goat anti–rabbit secondary antibody conjugated to HRP, rinsed in PB, and visualized using ECL™ according to the manufacturer's instructions (Amersham Pharmacia Biotech). To better understand the consequences that altered axo-glial interactions of the CNS and PNS have on the establishment of ion channel domains, we analyzed the distribution of the nodally clustered voltage-gated sodium channels and the paranodal/juxtaparanodal Kv1.1 potassium channels in the CGT-deficient mice. In both the mutant and wild-type mice, CNS and PNS sodium channels were concentrated in small regions that were presumptive nodes of Ranvier . Similar results have been reported previously regarding sodium channel distribution in the PNS of galactolipid-deficient mice . Since we reported previously that CNS nodal length is increased in the galactolipid-deficient mice , we measured the length and the width of the sodium channel domain and report node length as a function of axon caliber. In the CNS, the length to width ratio was significantly greater in the mutant (1.17 ± 0.05; n = 3 mice and 36 nodes; P < 0.02 by t test) compared with littermate wild-type (0.75 ± 0.13; n = 3 mice and 35 nodes) mice, indicating that the sodium channel domains, which likely corresponds to nodal length, were increased in the galactolipid-deficient animals. In contrast, potassium channel distribution was dramatically altered in the CNS of the CGT-deficient mice. In the CNS and PNS of wild-type mice, intense labeling was commonly observed in the juxtaparanodal region . The juxtaparanode was easily distinguished from the paranodal and nodal regions, since its diameter was conspicuously larger. Potassium channel antibody reactivity was occasionally observed in the paranodal region; however, the labeling intensity was greatly reduced. In the CNS of the mutant animals, fewer juxtaparanodal regions were immunolabeled with the Kv1.1 antibody , whereas diffuse labeling over long stretches of axons was occasionally observed . When paranodal/juxtaparanodal potassium channel accumulations were present, the width of the labeling pattern did not change, indicating that the diameter of these axons does not change at the interface between the paranode and juxtaparanode in the galactolipid-deficient mice. In the PNS, paranodal/juxtaparanodal regions were typically labeled, although the labeling was frequently less intense and more diffuse as compared with the wild-type sciatic nerve fibers . To verify the location of the sodium and potassium channel accumulations, we double-labeled CNS and PNS tissues with antibodies directed against the ion channels . The findings from these double-labeling experiments supported the single immunolabeling experimental observations that the sodium channel distribution was not altered in the mutants, since these channels maintained a nodal localization in both the CNS and PNS. Using the sodium channel clustering as an indicator of node position, we have quantitatively demonstrated that the distribution of potassium channels is dramatically altered in both the CNS and PNS ( Table ). In wild-type mice, potassium channels were primarily restricted to the juxtaparanodal regions with a minority of axons exhibiting paranodal distribution. In the galactolipid-deficient mice, the channels were rarely limited to the juxtaparanode. Instead, the potassium channels in the CNS were frequently clustered in the paranodal region, diffusely distributed along the internode, or were not detected, whereas in the PNS they were primarily observed only in the paranodes. In addition to facilitating the potassium channel quantitative analysis, the double-labeling approach also revealed that the prominent separation between the ion channel domains in the wild-type tissue is frequently absent in the mutant. In the mutant mice the potassium and sodium channel domains occasionally overlapped . The structural abnormalities at the node of the galactolipid-deficient mice appear to be related to compromised axo-glial interactions, such that we have analyzed the distribution of two potential neuronal adhesion molecules: paranodin and neurofascin. In addition, we have determined the distribution of the cytoskeleton-associated molecule ankyrin G . Using a combination of immunocytochemical techniques and confocal microscopy, we demonstrated the complete absence of paranodin accumulation in the paranodal regions of the myelinated fibers of the spinal cord in the CGT −/− mouse . In the galactolipid mutants, paranodin appeared to be diffusely distributed along the axon , resembling the expression pattern of unmyelinated fibers . In the sciatic nerve, paranodin was localized to the paranodal region; however, the staining intensity was reduced and the border between the paranode and the juxtaparanode was not as clearly defined as in the wild-type sciatic nerve . Paranodin was not detected in the paranode of any of the CNS fibers examined and reduced accumulations of paranodin were always observed in the paranode of the PNS fibers observed. Western blot analysis revealed no difference in the level of paranodin expression between the galactolipid mutant and wild-type animals for either the spinal cord or the sciatic nerve , indicating that the diminished immunoreactivity was a result of abnormal paranodal accumulations. In contrast, the distribution of the nodal proteins neurofascin and ankyrin G (data not shown) did not appear altered in either the spinal cord or the sciatic nerve of the mutant. We reported previously that mice incapable of synthesizing the myelin galactolipids GalC and sulfatide exhibit structural abnormalities of the nodal and paranodal regions that are likely due to compromised axo-glial interactions . In this study, we demonstrate that in the CNS, the distribution of the Shaker- type Kv1.1 potassium channels is altered, whereas the clustering of the voltage-gated sodium channels is only mildly affected. Furthermore, we show a complete disruption in the axolemmal organization of the potential axo-glial adhesion molecule paranodin. In the PNS, we demonstrate similar trends with regard to ion channel organization and paranodin distribution; however, the abnormalities are less dramatic. The regional differences correlate well with the structural data, since the morphological abnormalities in the PNS are less severe. Taken together, we propose that the disruption in the distribution of paranodin is further evidence that axo-glial interactions are disrupted in the galactolipid-deficient mice, and that these aberrant interactions impair appropriate ion channel segregation. The pattern of potassium channel distribution that we report for the galactolipid-deficient mice is consistent with a previous report of shiverer mice , which also display compromised axo-glial interactions in the CNS . In both shiverer and galactolipid-deficient mice, potassium channels frequently do not cluster in the paranodal/juxtaparanodal region but are diffusely distributed throughout the internodal regions. Furthermore, the sciatic nerve of shiverer mice display elongated paranodal/juxtaparanodal potassium channel labeling . This alteration in ion channel distribution correlates well with the mild disruption in paranodal Schwann cell–axon interactions . Likewise, potassium channel distribution is altered in the PNS of the CGT −/− mice coinciding with compromised axo-glial interactions and reduced paranodal accumulation of paranodin. Consistent with abnormal potassium channel distribution, particularly in conjunction with aberrant paranode structure, action potential duration is increased in the CNS of the galactolipid-deficient mice . In addition, action potential amplitude is decreased in the CNS , and to a lesser degree in the PNS . Furthermore, the addition of 4-aminopyridine, an inhibitor of potassium channels, results in little or no change in PNS amplitude, whereas CNS amplitude increased 25%. This difference likely reflects the greater alteration of potassium channel distribution in the CNS compared with the PNS in these mutants. Axo-glial interactions do not only influence potassium channel distribution. Clustering of sodium channels in the PNS also appears dependent upon the appropriate association of the Schwann cell with the axon . In the CNS, Kaplan et al. 1997 reported that oligodendrocyte contact is not required for initial sodium channel clustering in vitro, but a recent report demonstrates that axo-glial contact, as indicated by paranodin and myelin-associated glycoprotein labeling, is required for the sodium channel accumulation in vivo . In the galactolipid-deficient mice, sodium channels are concentrated in nodal regions. This finding demonstrates that normal paranodal axo-glial contacts are not essential for nodal clustering of sodium channels. Although gross clustering of sodium channels to the nodal gap is not dependent upon the formation of the paranodal septate-like junctions, these axo-glial junctions may be important in establishing and maintaining the interface between the sodium and potassium domains, since these domains occasionally overlap in both the CNS and the PNS of the galactolipid-deficient mutant. Using ultrastructural analysis, we have shown previously that axo-glial interactions are disrupted in the galactolipid-deficient mice . Here we provide evidence that these interactions are also disrupted at the molecular level, since paranodin, a known component of the septate-like junctions that form between the myelin sheath and the axolemma , does not appropriately accumulate in the paranodal region. Presently, the mechanism responsible for proper axolemmal distribution of paranodin is not known. Since paranodin has multiple potential cell adhesion domains including an extracellular lectin-binding domain , an attractive model is that GalC and/or sulfatide directly bind paranodin and facilitate its accumulation in the paranodal axolemma. Nevertheless, there is no evidence to suggest that the galactolipids accumulate in the paranodal region. Another possibility centers on the role that the galactolipids play in detergent-insoluble-complex (DIGs) formation and trafficking. In oligodendrocytes, DIGs, which are raft-like microdomains composed of the myelin galactolipids and proteins, are thought to be responsible for the molecular organization of the myelin sheath . Therefore, in mice that lack GalC and sulfatide, an as yet unidentified paranodin ligand may be abnormally distributed in the oligodendrocyte of CGT −/− mice, resulting in the disruption of paranodin intracellular targeting. The rearrangement of axolemmal proteins in the CGT −/− mice appears to be specific to the paranodal region, since the membrane arrangement of ankyrin G and neurofascin is not grossly affected. The clustering of neurofascin precedes myelination , therefore it is not surprising that its distribution is not affected by a myelin gene mutation. In contrast, the clustering of ankyrin G and voltage-dependent sodium channels is temporally associated with the elaboration of myelin-associated glycoprotein-positive myelin-forming processes . Therefore, if the distribution of sodium channels and ankyrin G is dependent on myelin, the mechanism by which these proteins are spatially organized apparently does not require the myelin galactolipids and is distinct from the process that regulates potassium channel and paranodin distribution. In summary, mice that are incapable of producing the myelin galactolipids have compromised axo-glial interactions as evidenced by CNS and PNS structural abnormalities . Furthermore, the distribution of paranodin, a prominent component of the paranodal septate-like junctions , is dramatically altered with no paranodal accumulation in the CNS. The disruption in axo-glial interactions leads to the abnormal distribution of the Shaker -type Kv1.1 potassium channels in both the CNS and the PNS. Therefore, our data indicate that axo-glial interactions are essential not only for the proper myelin formation but also axolemmal organization. | Study | biomedical | en | 0.999996 |
10601331 | HeLa and L929 cells were grown in 9-cm diam glass petri dishes (Falcon) in DME supplemented with 10% FCS, 10 μg/ml antibiotics (penicillin and streptomycin), and 2 mM l -glutamine (GIBCO BRL) at 37°C in an atmosphere of 5% CO 2 . For synchronization, HeLa cells grown to ∼50% confluence were arrested in early S phase with 2 mM hydroxyurea (Sigma Chemical Co.) for 16 h, then released by several washes in PBS before a final wash in fresh medium. FACS analysis showed that these cells remained in S phase for 10 h before entering G2 (3 h), and finally mitosis (1 h). For late S phase samples, cells were routinely collected 7.5–8 h after release. For immunofluorescence, cells were grown on glass coverslips in culture dishes. For Trichostatin A (TSA; Sigma Chemical Co.) treatment, exponentially growing cells were incubated in the presence of TSA (50 ng/ml, a dose compatible with cell growth) in complete medium for a chosen time. 5-bromo-2′-deoxyuridine (BrdU; Sigma Chemical Co.) incorporation was performed by incubating exponentially growing cells in the presence of 40 μM BrdU for 10 min. Cells were then washed in PBS and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. For pulse–chase experiments, cells were washed twice with prewarmed PBS and once in prewarmed medium after the BrdU pulse, and then cultured for different time periods. The cells were further processed for isolation of nuclei or fixed with 2% paraformaldehyde in PBS. Biotin-16-deoxyuridine (BiodU) incorporation on isolated nuclei was performed essentially as described by Krude et al. 1997 . In brief, after nuclei isolation in vitro run-on was initiated by adjusting the nuclear preparation to final concentrations: 40 mM K-Hepes, pH 7.8; 7 mM MgCl 2 ; 3 mM ATP; 0.1 mM each of GTP, CTP, and UTP; 0.1 mM each of dATP, dGTP, and dCTP; 40 μM bio-dUTP; 20 mM creatine phosphate; 0.5 mM DTT; and 2.5 μg phosphocreatine kinase (all Boehringer Mannheim Corp.). After a 10-min incubation at 37°C, reactions were stopped by a 25× dilution in PBS and immediately fixed in 2% paraformaldehyde. Nuclei were then deposited onto polylysine-coated coverslips by gentle centrifugation through a 30% cushion of glycerol in PBS, rinsed twice with PBS, and permeabilized in 0.2% Triton X-100 in PBS for 5 min. For the BiodU detection FITC-conjugated streptavidin (Enzo) was used, and fluorescence analysis carried out as described. Paraformaldehyde-fixed cells or nuclei were washed twice in PBS and then incubated in 0.2% Triton X-100 in PBS for 5 min and blocked in 5% BSA, 0.1% Tween 20 in PBS (blocking buffer) for 10 min. Specific antibodies were then added at the appropriate dilution in blocking buffer. Incubation was carried out for 45 min at room temperature. To visualize the staining, FITC or Texas red-conjugated goat anti–rabbit or anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) were used. During the final washing steps with 0.1% Tween 20 in PBS, 0.25 μg/ml, 4,6-diamino-2-phenylindole (DAPI; Sigma Chemical Co.) was added for DNA staining. In experiments involving BrdU immunodetection, a denaturation step in 4 N HCl for 10 min at room temperature was incorporated, after the permeabilization treatment, to render BrdU incorporated into DNA accessible. Neutralization was achieved by washes in PBS, and the slides were further processed as described above. Since detection of the nuclear proteins studied here is sensitive to HCl extraction, a specific protocol was used for double staining with BrdU. Proteins were first revealed (first and second antibodies) on paraformaldehyde-fixed cells as described. After 3 washes in PBS and 0.1% Tween 20, a second fixation step was included using either 2% paraformaldehyde in PBS for 15 min or 5 mM ethylene glycol-bis(succinimidylsuccinate; Pierce Chemical Co.) in PBS for 30 min at 37°C, followed by several washes in 0.3 M glycine in PBS. After this step, DNA was denatured with 4 N HCl, before BrdU revelation. The coverslips were mounted in Vectashield (Vector Laboratories Inc.). Despite postfixation, some loss of signal could still occur. Incorporation of BiodU (on isolated nuclei) into DNA was used instead of BrdU as an alternative (not shown). This allowed us to avoid the HCl denaturation since the biotinyl residue is accessible in the double helix of DNA. Rabbit polyclonal antibodies (pAbs) against the different acetylated isoforms of histones H4 and H2A have been characterized in detail . They recognize H4 isoforms acetylated on one of the four lysines (K) acetylated in vivo or H2A acetylated at lysine 5 (H2A Ac 5). They were used at final dilutions of 1:500 for R101/12 (K12), R14/16 (K16), and R123 (H2A Ac 5), and 1:1,000 for R232/8 (K8) and R41/5 (K5). mAbs against HP1α (2HP1H5) and HP1β (IMOD-1A97), kindly provided by Dr. R. Losson and Dr. P. Chambon (CNRS, Strasbourg, France), were used at final dilutions of 1/400. The mAb against human CAF-1 p150 (mAb1) was kindly provided by Dr. B. Stillman . The rabbit pAb1, directed against the human CAF-1 p60, was kindly provided by Dr. Marheineke . The specificities of the antibodies were tested by Western analysis on all sources of material studied. Monoclonal rat anti-BrdU was purchased from Harlan; Sera-Labo, Texas red goat anti–rabbit immunoglobulin (Ig), FITC goat anti–rabbit Ig, and Texas red goat anti–rabbit Ig antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. Image acquisition was performed using a Leica TCS-4D confocal scanning microscope, equipped with an Acousto-Optical Tuneable Filter (AOTF), with a 100× NA 1.4 plan-apochromat oil immersion objective. Single optical sections are presented. FITC and Texas red were excited by the argon-krypton laser at 488 and 568 nm, respectively. Red and green fluorescence were separated by a 580-nm long-pass dichroic beam-splitter. A 520-nm band-pass filter and a 590-nm long-pass filter were used to minimize cross-talk and stop laser scattered light. DAPI staining, shown in Fig. 1 , was acquired with the UV laser. Before acquiring a double staining z series, the intensity of excitation wavelengths and the power of photodetectors were adjusted to avoid cross-talk. The fluorescence signals from both fluorochromes were recorded simultaneously in one scan, and saved separately on two channels to be processed independently. Three-dimensional, two color images of doubly stained nuclei were recorded using the same equipment. The voxel dimensions (x, y, z) of each recorded stack was adjusted to 0.05 × 0.05 × 0.25 μm to verify the Shannon sampling criteria . The averaging parameter was adjusted to have the best signal/noise ratio without bleaching the sample during the acquisition process. For H4Ac and H2AAc staining we could not reach this criteria without bleaching, leading us to perform the quantitative analysis on single optical sections (2D analysis). The same setup was used for all sample acquisition to facilitate comparison between data. Numerical data were stored in 8-bit tiff format series for each color and transferred for processing and analysis. Noise reduction and image quantification was developed in house. Metamorph software (Universal Imaging) was used as a support for executing our own Dynamic Link Libraries (DLL) written in Visual Basic and C languages for displaying results and basic treatments. A nonlinear rank-adaptive denoising filter was applied to better distinguish fluorescent structures from background fluorescence without loss of resolution in transition regions. Red and green volumes were thresholded separately to avoid background contributions in the quantitative analysis. To make the measurements reproducible and comparable, red and green thresholds, designated Tr and Tg, respectively, remained unchanged for each volume corresponding to each series. Voxels are sorted in four categories: C, colocalized; R, red only; G, green only; and N, noise. In N, voxels displaying green and red signal intensities below both thresholds Tr and Tg were counted. In C, voxels displaying green and red signal intensities above both thresholds Tr and Tg were counted. In G, voxels displaying green signal intensity above Tg and red signal intensity below Tr were counted. In R, voxels displaying red signal intensity above Tr and green signal intensity below Tg were counted. Data presented in Fig. 2 and Fig. 6 were computed similarly in 2D, using pixels instead of voxels. A control experiment, in which replication sites pulse-labeled with BrdU were revealed by a balanced mix of Texas red and FITC secondary antibodies, gave 90% of colocalizing BrdU within the total population of BrdU [(C) / (C+R) ratio], using the same parameters as in Fig. 4 . This 90% approaches the 100% normally anticipated. The difference is due to the definition of our thresholds, which eliminates noise, but also leads to systematic underestimation of our values. In addition, we find that these values are dependent on the shape of the objects revealed in each color and this has to be taken into account for interpretation of the data. In all the graphs, for each point several experimental measurements were collected and the average value has been plotted in arbitrary units. For each couple of markers, the plots represent the red signals colocalizing with the green signals in arbitrary units [(C) / (C + R) ratio] normalized to the maximal value obtained in all the experiments involving this couple of markers. For comparison of the relative enrichment of the various acetylated isoforms of histones at either CAF-1 or BrdU sites, all histone signals (in green, which were similar in shape) were considered as one marker. Normalization was thus achieved using the common maximal value obtained for each set of experiments involving either CAF-1 or BrdU independently. To investigate possible disorganization on a large scale that could occur during replication of heterochromatin domains, we followed the distribution of HP1α and HP1β, two heterochromatin variants of HP1 . Using an mAb, we examined the relationship between HP1α or -β and replication foci by immunofluorescence in mouse (L929) and human (HeLa) cells. The fluorescence signals from both fluorochromes were recorded simultaneously in a single scan under conditions that ensure that the illumination and detection were free of lateral shifts, a precondition for secure recognition of colocalization in the specimen. Different stages of S phase were identified by the distinct BrdU incorporation patterns characteristic of early, mid, or late replicating foci . Typical results obtained with early or late replication profiles are presented in Fig. 1 . Our confocal analysis revealed, for both HP1α and HP1β, intensely labeled areas in dots corresponding to large blocks of pericentric heterochromatin identified by dense DAPI staining in mouse cells at all stages of S phase. In addition, we reproducibly observed a general diffuse nuclear staining, the significance of which remains to be established. Hp1α and -β dots were never associated with replication foci in early S phase. Remarkably, in cells characterized by a late replication pattern, a significant proportion of BrdU incorporation sites could be found within HP1 (α or β) dots. We further confirmed these observations in HeLa cells . We conclude that, for two different species, the existence of Hp1α and -β domains can coincide at this scale, with regions of DNA undergoing replication. From mammals to yeast, it has been demonstrated that H4 underacetylation is a characteristic feature of heterochromatin . To examine the state of H4 acetylation at both early and late replication foci in HeLa cells, we combined pulse labeling with BrdU and immunodetection using antibodies specific for H4 acetylated at lys 5, 8, 12, or 16 . An antibody to acetylated H2A was used as a control for an acetylation group unrelated to de novo chromatin assembly. Confocal analysis showed that the different H4 and H2A antibodies displayed a general punctate nuclear staining (enriched in some regions and excluded from the nucleolus). In early S cells, replication foci appeared equivalently associated with the four acetylated isoforms of H4. In contrast, in late S cells, we distinguished two different staining patterns : H4 isoforms acetylated at lys 5 or 12 were specifically enriched at late replication foci, whereas isoforms acetylated at lys 8 or 16 were excluded from these particular regions as is acetylated H2A. Similar results were obtained using mouse L929 or Xenopus A6 cells (not shown). This was further confirmed using a less destructive method (not shown, see Materials and Methods) that reveals the replication foci by in vitro run-on in the presence of Bio-16-dUTP . To quantify these observations, we developed a program to estimate the degree of colocalization between two markers in sets of confocal optical sections. This program and experimental controls to validate its application are described in Materials and Methods. Thus, we could analyze the average behavior of several hundred foci, providing a more accurate estimation of the statistical significance of our observations . From these data, we conclude that H4 acetylated at lys 5 or 12 is specifically enriched at late replicating foci, whereas forms acetylated at lys 8 or 16 are excluded. Since acetylation at lys 5 and 12 is specific to newly synthesized histone H4, this enrichment probably corresponds to the deposition of H4 on newly replicated heterochromatin regions. It is remarkable, however, that this specific enrichment could only be detected at late replication foci. The absence of specific enrichment of H4Ac 5 and 12 at early replication foci could be due to the multiple acetylation/deacetylation events occurring concurrently in euchromatin regions that may mask changes associated specifically with replication. Alternatively, the difference between the early and late replication profile could reflect a different mechanism for H4 deposition. CAF-1 previously had been reported to be generally located at replication foci . However, a strict comparison between early and late replication foci was not available. Using an mAb directed against p150-CAF-1 , a general localization of this subunit at both late and early replication foci was observed by confocal analysis . Quantification of these data in sets of confocal sections, demonstrated that newly replicated regions are equally associated with CAF-1 in either early or late replication foci . Importantly, we observed that not all the p150-CAF-1 foci were associated with replication foci (green spots), prompting us to analyze the temporal association of CAF-1 with DNA synthesis. In early S phase cells , double staining patterns revealing each specific acetylated isoform of H4, together with the p150-CAF-1 subunit, were similar. In contrast, in late S phase p150-CAF-1 could be found associated with H4Ac 5 or 12, but never with H4Ac 16 or 8. Since H4Ac 8, in addition to H4Ac 5 and 12, were found in a complex with CAF-1 in human nuclear extracts , these findings suggest that the CAF-1 complex including H4Ac 8 is not involved at late replication foci. The association of CAF-1 with early and late replication foci argues for a general chromatin assembly mechanism throughout S phase. Remarkably, H4Ac 5 and 12 association with CAF-1 reproduces the pattern of H4 acetylation associated with DNA replication in heterochromatin regions . We used the BrdU/BiodU double labeling approach to relate a temporal link between two DNA synthesis events to the spatial overlap of their corresponding signals. Synchronized cells were allowed to progress to late S phase, pulsed first with BrdU (see Materials and Methods) and chased for various times in vivo before a second pulse labeling was performed on isolated nuclei by in vitro run-on in the presence of Bio-16-dUTP. We compared the localization of the BrdU-labeled patches with sites of DNA synthesis after various times, by merging both signals acquired by confocal microscopy . In nuclei labeled immediately after the first pulse, both in vivo and in vitro replication sites clearly overlapped. Indeed, individual foci appeared mainly yellow, as shown in Fig. 4 B, even though the colocalization was not perfect, as expected for two consecutive events. Within 15 min, a partial separation of the two signals could already be observed. In the same domain we could visualize red (in vivo pulse with BrdU), green (in vitro pulse with BiodU), and yellow signals (both pulses). Although we cannot exclude that some red, green, and yellow domains could correspond to closely linked clusters of separate smaller foci, the frequency of this arrangement diminishing with time is in favor of a vast majority being the same foci. Foci in which only BiodU (green) or BrdU (red) was incorporated were likely to be those that had been switched on or off (respectively) during the chase. Consistent with this interpretation, we observed that the number of the latter increased with time, reflecting the progressive extinction of foci, whereas only a few new foci appeared during the chase, presumably due to the ending of S phase. In the same way, within double-labeled domains, separation between both signals increased with time and the respective BiodU and BrdU signals could be perfectly distinguished after 30 min. One hour after BrdU pulse, only a few BrdU foci were still associated with BiodU foci. In summary, the overlap between the two signals decreased as a function of time, due to both extinction and firing of replicating foci during the chase and to the progressive separation of clusters corresponding to DNA synthesized during the two pulses within one focus. Quantification of BrdU-labeled regions associated with BiodU-labeled regions in sets of confocal sections showed a decrease in their spatial association, consistent with the visual observation . These results are in agreement with previous in vivo studies in rodent cells using double pulse labeling in vivo and different methods of quantification . The consistency of these data further supports the validity of our method. In this analysis, patches of synthesis occurring at 15 min intervals (corresponding approximately to the synthesis of 50 Kb between both pulses) can be distinguished. Thus, this analysis could be used as a reference to study the large scale relationships between factors of interest and DNA synthesis. We investigated how the association of recently replicated DNA with CAF-1 evolved as a function of time in a pulse–chase experiment. Synchronized cells were allowed to progress to late S phase, pulsed with BrdU (see Materials and Methods), and chased for various times in vivo. After the chase, CAF-1 and sites of BrdU incorporation were revealed, and their localization compared by merging both signals acquired by confocal microscopy. Immediately after the pulse, BrdU incorporation sites widely overlapped with CAF-1 sites , although labeled sites were not exclusively yellow, as shown in Fig. 5 B. The signals corresponding to CAF-1 and BrdU had not separated within 20 min. This contrasts with the signals corresponding to two successive DNA synthesis events, which were separated at 15 min after BrdU pulse . After 50 min, CAF-1 and BrdU signals had begun to separate, and reached complete separation at 80 min after pulse. As in the previous experiment, few foci with a single color appeared as chase time increased. Hence, CAF-1 association with BrdU incorporation sites appears related to DNA synthesis, but does not strictly follow the same dynamics. Considering the association of BrdU signal with CAF-1, our quantitative analysis showed no significant change within the first 20 min after BrdU pulse . After this time, following the extinction of replication foci, the percentage of colocalization decreased as a function of time. Furthermore, comparing the quantitative analysis of BrdU/CAF-1 and BrdU/BiodU pulse–chase experiments, a time difference of 20 min could be seen between release of CAF-1 and cessation of DNA synthesis. It is remarkable that after 80 min post-BrdU pulse, whereas replication had stopped within foci labeled at the time of the pulse, CAF-1 could still be found associated with BrdU incorporation sites. Further supporting these findings, we found that the two largest subunits of CAF-1, p60 and p150, detected respectively using a pAb and an mAb, behaved similarly in this assay. A similar analysis performed on early replicating foci gave comparable results (not shown), further suggesting a general behavior of CAF-1 throughout S phase. This behavior of CAF-1 is consistent with the rapid association of this assembly factor with newly replicated regions of DNA and its retention for at least 20 min at these locations. We then investigated how the association of H4Ac 5 with recently replicated regions of the genome evolved as a function of time in a pulse–chase experiment carried out as described above. Considering data in Fig. 2 and Fig. 3 showing that replication specific acetylated isoform of H4 (H4Ac 5 and 12) could not be detected enriched at early replication foci, we concentrated our study on late replication foci. Immediately after the pulse, BrdU incorporation sites were widely stained with H4Ac 5 antibody , although labeled sites were not exclusively yellow, as shown in Fig. 6 . The signals corresponding to BrdU were still associated with H4Ac 5 after a 20-min chase. Thus, clusters of newly synthesized DNA can been found associated with H4Ac 5 for at least 20 min. After 50 min, BrdU incorporation sites appeared partially dissociated from H4Ac 5 and both signals were completely distinct in G2 phase (after a 3-h chase). BrdU signal associated with H4Ac 5 was determined after different chase times on several optical sections from different nuclei to establish the statistical significance of our observations . Similar data were obtained for H4Ac 12 in both human and Xenopus (not shown). Thus, H4 acetylation at lys 5 and 12 appeared to be transiently associated with late replicated sequences in both mammals and amphibians. This result contrasts with the maintenance of lys 12 acetylation reported for heterochromatin from both yeast and Drosophila . The loss of H4Ac 5 and 12 enrichment at late replicated sequences is likely to result from an enzyme catalyzed deacetylation or a loss of epitope accessibility. Preliminary immunolabeling experiments have failed to detect any specific enrichment of deacetylase catalytic subunits at late replication foci (not shown). This may be because the specific enzymes involved still have to be identified, though we cannot exclude the possibility that the deacetylase activity at replication foci is locally regulated by a cofactor or a modification, rather than by varying the amount of enzyme present. Alternatively, without any specific enrichment of HDAC, the lack of HAT in these regions could be sufficient to account for H4 deacetylation following replication as supported by Fig. 7 . Most importantly, these data strongly support the existence of a time window of ∼20 min, during which heterochromatin regions are enriched in acetylated H4, followed by a deacetylation event. An asynchronous population of HeLa cells was pulsed with BrdU and chased for 90 min either in the presence or absence of TSA, a histone deacetylase inhibitor . After a 90-min chase in the absence of TSA, all four acetylated isoforms of H4 were reproducibly excluded from late replicated regions . This is consistent with data in Fig. 6 showing that H4Ac 5 was no longer associated with late replicated foci after 80 min chase. In the presence of TSA, however, association of H4Ac 5 and 12 was detected, whereas H4Ac 8 and 16 staining were excluded from BrdU-stained regions. These data demonstrate that the loss of H4Ac 5 and 12 after 90 min chase is due to a deacetylation event. Under these conditions, an enhancement of H4Ac 5, 8, and 12 staining at the nuclear periphery was observed in all cells of an asynchronous population, indicating that it is unrelated to the replication process . Although the significance of this observation is unclear, it provided a useful marker for the TSA effect. We next investigated the dynamics of H4 acetylation in heterochromatin regions outside S phase. To ensure that cells were out of S phase, they were cultured 14 h after the BrdU pulse, allowing late replicated cells to exit mitosis. At this time, cells were either mock-treated or treated with TSA (90 min, 50 ng/ml). In mock-treated cells, BrdU staining was excluded from staining with any of the four acetylated isoforms, consistent with the general underacetylated state of H4 in heterochromatin regions. In TSA-treated cells, despite an increase in H4Ac 5, 8, and 12 at the nuclear periphery, the BrdU staining remained excluded from all acetylated isoforms of H4 . Thus, none of the acetylated forms of H4 could be stabilized by TSA treatment under these conditions. Taken together, these results indicate that the dynamics of histone H4 acetylation in heterochromatin regions is strictly limited to lys 5 and 12 and restricted to the time of DNA replication. Immunofluorescence analysis of heterochromatin domains at late replicating foci shows that these domains contain specific acetylated forms of histone H4, together with the largest subunit of CAF-1. The enrichment of the histone H4 acetylated at lys 5 and/or 12 was in striking contrast with the exclusion of H4Ac 8 and 16. We followed the large scale dynamics of this domain through the temporal analysis of DNA synthesis, the presence of CAF-1, acetylated H4, Hp1α, and Hp1β. Thus, we can distinguish properties of heterochromatin domains that are either stable (Hp1α and -β concentration) or transient (H4Ac 5 and 12, and CAF-1). Comparison with early replicating foci/euchromatin regions shows that CAF-1 is also associated with DNA synthesis in these regions. In contrast, we could not reveal any specific enrichment in H4Ac 5 and 12, compared with 8 and 16. We discuss how the replication-associated cycle of acetylation/deacetylation may have a specific importance in heterochromatin regions. The existence of local concentrations of specific proteins has been proposed to compartmentalize chromatin regions, creating an index system for the nucleus . The local concentration of specific proteins, such as Hp1α and -β, could also be critical for defining domains that should be repressed. In our study, no major changes in the distribution of Hp1α and -β were observed during DNA replication of heterochromatin regions . This was surprising at first, since the presence of Hp1α and -β may reflect a high level of DNA condensation that may not necessarily be compatible with the replication process. We thus conclude that, if the replication fork is destabilizing the nucleosome , this is a local phenomenon that does not affect the stability of the larger scale organization in a detectable manner. The maintenance of the local concentration of Hp1α and -β as part of nucleoprotein complexes, either directly interacting or simply in close proximity with DNA, may ensure the rapid reassociation of the replicated regions into heterochromatin. Furthermore, the ability of HP1 proteins to oligomerize has led to the hypothesis that they can cross-link chromatin domains by the formation of concatenated multiprotein complexes . Thus, Hp1α and -β appear to be components that favor the maintenance of a heterochromatin region. Interestingly, in mouse cells, association of some genes with an HP1β domain has been shown to correlate with their transcriptional repression . One would thus predict that in vertebrates, overexpression of HP1β could result in a spreading or expansion of these domains, possibly producing phenotypes resembling PEV . Additional modification adding to stability in heterochromatin, such as methylation of DNA in vertebrates , should also be incorporated into the overall picture. Future experiments will provide insights into these issues. Analysis of the distribution of CAF-1 and H4Ac 5, compared with DNA synthesis at heterochromatin regions, demonstrated a dynamic behavior that was not strictly parallel to DNA synthesis . No major differences were observed between the location of CAF-1 and patches of DNA corresponding to individual replication foci observed within 20 min after their labeling in HeLa cells. For this chase time, we established that two DNA synthesis events are already clearly separated. The maintenance of CAF-1 association with these patches of DNA after their synthesis is further emphasized by the detection of CAF-1 foci beyond the DNA replication time. This CAF-1 enrichment at the location of newly synthesized DNA soon after its synthesis, and its persistence, exceeds the time involved in synthesizing the DNA length corresponding to a nucleosome and depositing one set of H3 and H4 at the replication fork . This extended time period is comparable to the postreplicative phase required for proper chromatin maturation . An attractive hypothesis is that it could define the time needed to process a unit of a size between 50 and 200 Kb, reminiscent of the chromatin loops. This would be compatible with the recent report on CAF-1–coupled inheritance of chromatin acting postreplicatively in vitro . Similarly, we determined that newly synthesized DNA sequences were associated with H4Ac 5 for ∼20 min in late replication foci. The existence of a pool of acetylated histone H4 concentrated in the region of heterochromatin at the time of replication constitutes a strong argument for believing that these histones are used to form the new nucleosomes at these foci. It is striking though, that, at a large scale level, this replication-associated acetylated isoform could not be specifically detected at early replication foci although CAF-1 was still found . Since the biochemical approach has indicated a general usage of H4Ac 5 and 12 throughout the genome, however, multiple acetylation/deacetylation events occurring concurrently in these regions may mask changes associated specifically with replication. In heterochromatin, remarkably, the acetylation level of H4 associated with histone deposition at the replication fork appears as a dominant event. The parallel between CAF-1 and H4Ac 5 and 12 in heterochromatin raises the possibility that CAF-1 association with acetylated histones helps to coordinate loading of acetylated histone H4 with a deacetylation event. This latter event could be promoted through the interaction of the smallest subunit of CAF-1 with a histone deacetylase previously detected in human cell extracts . The heterochromatin specific effect of CAC deletions in yeast strains leading to the redistribution of the telomeric protein Rap1p, together with a loss of silencing , could be explained by defects in such a coordination when replicating heterochromatin regions. Future experiments should address whether perturbation of CAF-1 in higher eucaryotes similarly affects heterochromatin function. This may also relate to the possible role of CAF-1 associated with DNA repair . By facilitating the deacetylation step during the chromatin dynamics associated with DNA repair, CAF-1 may help to avoid the persistence of inappropriate chromatin organization. Our data are consistent with the existence of a general mechanism for nucleosome assembly employed throughout the genome. In this pathway, histone deposition would make use of acetylated histones chaperoned by CAF-1 that would facilitate deacetylation. Depending on the local environment, particularly the presence of HATs/HDACs and their regulators, it may be possible to generate distinct acetylated chromatin configurations at a steady state. Inhibition of histone deacetylase by TSA showed that the dynamics of acetylation of histone H4 in heterochromatin is restricted to K5 and K12, and to the time of the DNA replication . Prolonged exposure to the drug led to the disruption of heterochromatin domains (HP1 staining, not shown, Taddei, A., and G. Almouzni unpublished results). These observations are reminiscent of those obtained in Saccharomyces pombe in which a TSA treatment disrupted centromeric organization and delocalized the swi6 protein . Further investigations are necessary to determine the exact basis of this phenomenon. It is intriguing that H4 acetylation seems to not be required for general nucleosome formation, per se, either in vitro or in vivo . It has been proposed that the so-called futile cycles of enzymatic metabolism may be needed for rapid response to changes in the environment and for evolution . It is interesting to consider the possibility that the acetylation/deacetylation cycle chaperoned by CAF-1 serves the same purpose. In that respect, it is noteworthy that mutations in histone tails affecting all the acetylatable sites in histone H4 gave rise in yeast to a checkpoint phenotype . The importance of the alternative states generated by the futile cycle could be envisaged at two different levels. At a cellular level, this could help to monitor S phase progression, DNA damage processing, and perhaps determine how S phase events are coordinated. At the level of a population, a certain degree of adaptability can be advantageous during evolution, as was discussed for the epigenetic inheritance of centromeres . This is exemplified in yeast with the switching mechanism at mating type loci that depends on replication . We studied here how a preestablished structure could be propagated, the next challenge will be to understand how these structures can be formed de novo, which will be critical during the development of an organism. | Study | biomedical | en | 0.999997 |
10601332 | Nuclear isolation buffer (buffer N) consisted of 10 mM Hepes, pH 7.5, 2 mM MgCl 2 , 250 mM sucrose, 25 mM KCl, 1 mM DTT, 1 mM PMSF, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin A. Cell lysis buffer consisted of 20 mM Hepes, pH 8.2, 5 mM MgCl 2 , 10 mM EDTA, 1 mM DTT, and 20 μg/ml cytochalasin B and protease inhibitors. A GST-AKAP95 fragment covering amino acids 387–692 and including the RII-binding domain of human AKAP95 (designated GST-AKAP95Δ1-386) was described previously . A synthetic peptide derived from the AKAP Ht31 was described previously and contained the amphipathic helix structure of Ht31 required for RII binding ; thus, Ht31 was used as a specific inhibitor of AKAP–RII interaction. Control Ht31-P peptides corresponded to Ht31 with two isoleucine residues mutated to prolines, disrupting the amphipathic helix structure of AKAPs . Affinity-purified rabbit polyclonal antibodies against AKAP95 (Upstate Biotechnology Inc.) were described elsewhere . Two novel mAbs against human AKAP95 (mAb36 and mAb47) were developed in conjunction with Transduction Laboratories. Each mAb was produced by immunizing mice with the GST-AKAP95Δ1-386 fusion peptide. Anti–human RIIα mAbs (Transduction Laboratories) were described earlier . Affinity-purified rabbit antibodies against human lamin B receptor (LBR) or heterochromatin protein 1α, rabbit antiserum against a peptide of human lamin B, and human anti–lamin B autoantibodies (gifts from J.-C. Courvalin, Institut Jacques Monod, Paris, France) were described previously . Anti–human Eg7 polyclonal antibodies were generated by immunizing rabbits with a peptide comprising the last 15 amino acids of human Eg7 (KTTPILRASARRHRS) . HeLa cells were grown in EMEM medium (GIBCO BRL) as described in Eide et al. 1998 . For microinjections, cells were synchronized in S phase by a double thymidine (2.5 mM) block . Cells were synchronized in M phase by subsequent exposure to 1 μM nocodazole for 18 h . Mitotic indexes were typically ∼95%. For nuclear microinjections, HeLa cells were plated onto glass coverslips at 10 6 cells per 28-cm 2 dish and synchronized in S phase by double thymidine block. Nuclei were microinjected with 25–50 pl of solution using a Narishige micromanipulator and picoinjector. Injection solution consisted of EMEM containing 10 μg/ml of a 150-kD FITC-dextran (Sigma Chemical Co.) to visualize the injections. Nuclei were injected with either 2–5 pg affinity-purified anti–AKAP95 polyclonal antibodies (at ∼0.1 mg/ml IgG), mAb36 (diluted 1/10), 2–5 pg preimmune IgGs (at ∼0.1 mg/ml), or ∼250 pg GST-AKAP95Δ1-386 peptide together with anti–AKAP95 antibodies. Inhibitors, antagonists, or catalytic peptides were injected at concentrations indicated in Results. Successful nuclear injections were assessed by retention of the FITC-dextran within the nucleus 1 h after injection (see Results). After a resting period of 6 h, injected cells were synchronized in M phase with 1 μM nocodazole. Mitotic cells were microinjected as described above while arrested in M phase with nocodazole. Injected cells remained in nocodazole for up to 2 h after injection during the period of observation. Mitotic HeLa cells were washed once in ice-cold PBS, once in 20 vol of ice-cold lysis buffer, and packed at 800 g for 10 min. The cell pellet was resuspended in 1 vol of lysis buffer and incubated for 30 min on ice. Cells were homogenized by a 2× 2-min sonication on ice and the lysate was centrifuged at 10,000 g for 15 min at 4°C. The supernatant was cleared at 200,000 g for 3 h at 4°C in a Beckman SW55 rotor. The clear supernatant (mitotic cytosolic extract) was aliquoted, frozen in liquid nitrogen, and stored at −80°C. Protein concentration of the extract was usually ∼15 mg/ml. Interphase cytosolic extracts were prepared as above from unsynchronized HeLa cells (95–98% in interphase) except that EDTA was omitted from the cell lysis buffer. Confluent unsynchronized HeLa cells were harvested, washed in PBS, sedimented at 400 g , and resuspended in 20 vol of ice-cold buffer N containing 10 μg/ml cytochalasin B. After a 30-min incubation on ice, cells were homogenized on ice by 140–170 strokes of a tight fitting (B-type) glass pestle in a 7-ml homogenizer. Nuclei were sedimented at 400 g for 10 min at 4°C and washed twice by resuspending in buffer N and centrifuging as above. Essentially, all nuclei recovered were morphologically intact, as judged by phase-contrast microscopy and labeling with 10 μg/ml FITC-conjugated ConA (data not shown) . Nuclei were used fresh or frozen at −80°C in buffer N containing 70% glycerol. High salt (2 M NaCl)–extracted nuclear matrices were prepared from purified nuclei as described previously . Matrices were solubilized in 8 M urea/10 mM Tris-HCl, pH 8.0. To prepare interphase chromatin, 10 8 HeLa nuclei suspended in 200 μl buffer N containing 1% Triton X-100 were incubated with 5 U of micrococcal nuclease (Sigma Chemical Co.) at 37°C for 5 min. Digestion was terminated by adding EDTA to 5 mM and the mixture was centrifuged at 10,000 g for 5 min. The supernatant (S1) was collected and the pellet was resuspended in 2 mM EDTA and incubated for 15 min at 4°C. After sedimentation as above, the supernatant (S2) was combined with S1 to yield the chromatin fraction, proteins were precipitated with 10% TCA and dissolved in SDS sample buffer. To solubilize mitotic condensed chromatin, mitotic cell lysates were centrifuged at 10,000 g for 10 min. The pellet was resuspended in 500 μl buffer and sedimented at 1,000 g for 20 min in a swingout rotor (Eppendorf) through a 1-M sucrose cushion made in buffer N. Mitotic chromosomes were recovered from the pellet. This pellet was extracted with 1% Triton X-100 at 4°C for 30 min, sedimented at 15,000 g for 15 min, and the detergent-insoluble fraction was digested with 5 U/ml micrococcal nuclease at 37°C for 5 min. After sedimentation as above, proteins of the supernatant (soluble chromatin) were precipitated in 10% TCA and dissolved in SDS-sample buffer. Purified HeLa cell nuclei (2,000 nuclei/μl) were permeabilized in 500 μl buffer N containing 0.75 μg/ml lysolecithin for 15 min at room temperature. Excess lysolecithin was quenched by adding 1 ml of 3% BSA made in buffer N and incubating for 5 min on ice, before sedimenting the nuclei and washing once in buffer N. Nuclei were resuspended at 2,000 nuclei/μl in 100 μl buffer N containing affinity-purified anti–AKAP95 antibodies (1:40 dilution; 2.5 μg) or 2.5 μg of preimmune rabbit IgGs. After a 1-h incubation on ice with gentle agitation, nuclei were sedimented at 500 g through 1 M sucrose for 20 min and held in buffer N on ice until use. Antibody loading of nuclei was verified by immunofluorescence. AKAP95 was not proteolyzed during permeabilization and antibody loading procedures, and lysolecithin-treatment of nuclei did not affect nuclear envelope disassembly or chromatin condensation in mitotic extract (data not shown). A nuclear disassembly/chromatin condensation reaction consisted of 20 μl mitotic extract, 1 μl nuclear suspension (∼2 × 10 4 nuclei), and 0.6 μl of an ATP-generating system . The reaction was started by addition of the ATP-generating system and allowed to proceed at 30°C for up to 2 h. The extract supported nuclear envelope disassembly and chromatin condensation without apoptotic proteolysis of nuclear envelope proteins or DNA degradation . Chromatin condensation was monitored by after staining samples with 0.1 μg/ml Hoechst 33342. Chromatin was considered to condense when it acquired an irregular and compact morphology sometimes with distinct chromosomes or chromosome fragments. In some instances, extracts were preincubated with inhibitors for 30 min on ice before adding nuclei and the ATP-generating system. Percent chromatin condensation was calculated as the ratio of condensed chromatin masses per number of nuclei examined. For biochemical analyses, condensed chromatin was sedimented at 1,000 g through 1 M sucrose as described above and recovered from the pellet. HeLa chromatin condensed in mitotic extract was recovered by sedimentation at 1,000 g through 1 M sucrose, washed by resuspension and sedimentation in lysis buffer, and incubated for up to 2 h in fresh mitotic extract, either intact or immunodepleted of AKAP95 or RIIα. In some experiments, this extract contained antibodies or inhibitors. DNA was labeled with Hoechst and examined as above. Premature chromatin decondensation (PCD) referred to swelling of the chromatin into ovoid or spherical objects, with no discernible chromosomes. Kinetics of PCD was evaluated by measurement of the proportion of chromatin masses undergoing decondensation at regular time intervals. 5 μl of HeLa cytosolic extract was mixed with 5 μl of a 2× histone kinase buffer (160 mM β-glycerophosphate, 20 mM EGTA, 30 mM MgCl 2 , 2 mM DTT, 20 μg/ml each of aprotinin, leupeptin, and pepstatin A, and 100 nM PKI). The kinase reaction was initiated by addition of 10 μl of a cocktail containing 2.5 mg/ml histone H1, 0.7 mM ATP, and 50 μCi of γ-[ 32 P]ATP. The reaction was carried out at room temperature for 15 min and stopped by addition of 15 μl of 2× SDS loading buffer and boiling. Proteins were resolved by 15% SDS-PAGE, the gel was dried, and phosphorylation of histone H1 was visualized by autoradiography. Immunoblotting of nuclei, chromatin, and reaction supernatants was performed as described using the following antibodies: rabbit anti-AKAP95 (1:250 dilution), rabbit anti-lamin B (1,000), anti-LBR (1:500), RIIα mAb (1:250), anti-Eg7 (1:500), and HRP-conjugated secondary antibodies. Blots were revealed with chloronaphtol/hydrogen peroxide or by enhanced chemiluminescence (Amersham). For immunoprecipitations, whole mitotic or interphase cells (as indicated) were sonicated twice for 2 min on ice in immunoprecipitation (IP) buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1% Triton X-100, and protease inhibitors), and the lysate was centrifuged at 15,000 g for 15 min. The supernatant was precleared with protein A/G agarose (1:25 dilution) at 4°C for 1 h. Immunoprecipitations were carried out with relevant antibodies (anti–RIIα mAb, anti–AKAP95 polyclonal, and anti-Eg7, each at a 1:50 dilution) from the supernatant at room temperature for 2.5 h, followed by incubation with protein A/G agarose (1:25 dilution) for 1.5 h and centrifugation at 4,000 g for 10 min. Immune complexes were washed three times in IP buffer and proteins were eluted in 2× SDS boiling sample buffer. RIIα was also immunoprecipitated from mitotic extract, or chromatin, or reaction supernatant fractions diluted 10× in IP buffer and sonicated (chromatin fraction only). AKAP95 or RIIα was also immunodepleted from undiluted cytosolic extracts using affinity-purified anti–AKAP95 antibodies or anti–RIIα mAbs at a 1:50 dilution. For immunofluorescence analysis , cells, nuclei, or condensed chromatin masses were settled onto poly- l -lysine–coated coverslips, fixed with 3% paraformaldehyde, permeabilized with 0.5% (cells) or 0.1% (nuclei) Triton X-100, and proteins were blocked with 2% BSA in PBS/0.01% Tween 20 (PBST). Primary antibodies (anti–AKAP95 polyclonal, anti-LBR, anti–RIIα mAb, anti–lamin B human autoantibody) and secondary antibodies were used at a 1:100 dilution. DNA was stained with 0.1 μg/ml Hoechst 33342. Observations were made on an Olympus AX70 epifluorescence microscope using a 100× objective, and photographs were taken with a Photonic Science CCD camera and OpenLab software (Improvision Co.). The subcellular localization of human AKAP95 was examined throughout the HeLa cell cycle. Immunofluorescence analysis using an affinity-purified anti–AKAP95 antibody confirmed that AKAP95 was exclusively nuclear in interphase, colocalized with DNA, and was excluded from nucleoli . As early as prometaphase and until telophase, AKAP95 staining colocalized with chromosomes over a diffuse cytoplasmic labeling . In late telophase/interphase, AKAP95 labeling became excluded from the cytoplasm and restricted to the daughter nuclei . Similar results were obtained with two newly developed anti–AKAP95 mAbs, mAb36 and mAb47, and in human 293T fibroblasts with all three antibodies (data not shown). Immunoblotting analysis of AKAP95 revealed similar levels of AKAP95 in whole interphase and mitotic cell lysates . Fractionation of interphase and mitotic cells confirmed that AKAP95 was exclusively nuclear in interphase . At mitosis, ∼75% of AKAP95 cofractionated with chromatin, whereas ∼25% was detected in a 200,000- g supernatant fraction . No AKAP95 was detected in membrane fractions of interphase or mitotic cells . Furthermore, fractionation of purified HeLa nuclei into chromatin and high salt–extracted nuclear matrices revealed that ∼80% of AKAP95 partitioned with the matrix, whereas ∼20% cofractionated with chromatin . Thus, AKAP95 is restricted to the nuclei of interphase cells, where it associates primarily with the nuclear matrix. At mitosis, AKAP95 redistributes into a major chromatin-associated fraction and a minor soluble pool. Upon nuclear reassembly at the end of mitosis, AKAP95 is sequestered into the daughter nuclei where it re-acquires an interphase distribution. Association of AKAP95 with RIIα in interphase and mitosis was investigated in dual immunofluorescence and immunoprecipitation experiments. In interphase, AKAP95 labeling was clearly distinct from the cytoplasmic staining of RIIα . At mitosis, RIIα exhibited a more uniform cytoplasmic labeling that mostly overlapped with that of AKAP95 . Although both AKAP95 and RIIα were detected by immunoblotting in interphase whole cell lysates , immunoprecipitation of RIIα from such lysates did not coprecipitate AKAP95 in interphase, but did coprecipitate AKAP95 at mitosis . Immunoprecipitation of RIIα also coprecipitated AKAP95 from both a mitotic cytosolic fraction and from mitotic chromatin solubilized by micrococcal nuclease digestion . This illustrates the assembly of both soluble and chromatin-associated AKAP95–RIIα complexes at mitosis. The dynamics of formation of the chromatin-associated mitotic AKAP95–RIIα complex was investigated using a cytosolic extract prepared from mitotic HeLa cells. The extract supports disassembly of exogenous purified interphase HeLa nuclei, including nuclear envelope breakdown and chromatin condensation (see Materials and Methods). Immunofluorescence and immunoblotting analysis of input nuclei and condensed chromatin showed that AKAP95 was associated with the condensing chromatin . As in vivo, a minor portion of AKAP95 was also released into the cytosol during chromatin condensation (data not shown). Concomitantly, RIIα, undetected in input interphase nuclei, was recruited from the extract onto the chromatin surface where it colocalized and cofractionated with AKAP95 . Furthermore, immunoprecipitation of RIIα from condensed chromatin coprecipitated AKAP95 , illustrating the formation of a chromatin-associated AKAP95–RIIα complex during chromatin condensation in vitro. It is noteworthy that association of AKAP95 with chromatin also occurred in mitotic extract immunodepleted of RIIα (data not shown), indicating that mitotic redistribution of AKAP95 is independent of RIIα. Altogether, these results indicate that in mitotic extract, nuclear AKAP95 is redistributed and primarily associates with condensing chromosomes in an RIIα-independent manner. Concomitantly, AKAP95 recruits RIIα from a cytosolic pool onto chromatin. Thus, one function of chromatin-associated AKAP95 at mitosis is to target PKA, via RIIα, to condensing chromosomes. The functional significance of the chromatin-associated AKAP95–RIIα complex was first investigated using the mitotic extract. To this end, we attempted to block the function of nuclear AKAP95 by introducing affinity-purified anti–AKAP95 antibodies (or control preimmune rabbit IgGs) into purified HeLa nuclei as described in Materials and Methods. Antibody-loaded nuclei were exposed to mitotic extract and nuclear disassembly assessed after 2 h by DNA staining and immunofluorescence analysis using anti–lamin B and anti–LBR antibodies. Fig. 4 A shows that anti–AKAP95 antibodies, but not preimmune IgGs, inhibited chromatin condensation although nuclear envelope disassembly took place as judged by the absence of lamin B or LBR labeling. Inhibition of chromatin condensation occurred with most (∼90%) chromatin masses examined and was reproduced by mAb36, whereas mAb47 was little effective . Inhibition of chromatin condensation was specific for anti–AKAP95 antibodies since it did not occur with the following: anti–AKAP95 antibodies introduced into nuclei with 0.1 mg/ml of the GST-AKAP95Δ1-386 peptide used to generate the antibodies ; and affinity-purified polyclonal antibodies against heterochromatin proteins HP1α , HP1β, or HP1γ (not shown) . Preincubation of anti–AKAP95 antibodies in the extract or immunodepletion of soluble AKAP95 did not inhibit condensation . This illustrates a role for nuclear rather than cytosolic AKAP95 in chromatin condensation. Additionally, loading nuclei with anti–AKAP95 antibodies did not block the recruitment of RIIα onto the chromatin, as judged by immunofluorescence and immunoblotting analyses of condensed chromatin (data not shown). This argues that inhibition of chromatin condensation with anti–AKAP95 antibodies does not result from inhibition of the PKA-anchoring activity of AKAP95. To determine whether inhibiting nuclear AKAP95 function in vivo affected mitotic chromosome condensation, nuclei of HeLa cells synchronized in S phase by a double thymidine block were microinjected with ∼2–5 pg of anti–AKAP95 polyclonal antibodies or mAb36, each with or without 250 pg of competitor GST-AKAP95Δ1-386 peptide, or with preimmune IgGs. Successful nuclear injections were verified by coinjection of a 150-kD FITC-dextran and scored by retention of the dextran within the nuclei 1 h after injection . Injected cells were released from the thymidine block, exposed to 1 μM nocodazole, and the proportion of cells arrested at mitosis was determined after 17 h. A fraction of injected cells was also analyzed by immunofluorescence using antibodies against LBR, a marker of the inner nuclear membrane. Fig. 5 shows that, after nocodazole treatment, cells injected with anti–AKAP95 antibodies did not undergo chromosome condensation, as shown by the absence of detectable metaphase chromosomes under bright field microscopy and after DNA staining in most cells , and by mitotic indexes of 15–20% . In contrast, preimmune IgGs or anti–AKAP95 antibodies injected with GST-AKAP95Δ1-386 did not prevent chromosomes from forming a metaphase plate . In addition, despite the lack of chromosome condensation in antibody-injected cells, the release of FITC-dextran from the nucleus into the cytoplasm of these cells after nocodazole treatment argued that the nuclear envelope was disassembled. This was verified by immunofluorescence analysis of LBR . Breakdown of the nuclear envelope implies that immunoblocking of nuclear AKAP95 does not prevent entry into mitosis per se, but rather affects chromosome structure at this stage of the cell cycle. However, it should be noted that microinjection of a 25-fold higher concentration of anti–AKAP95 antibodies blocked nuclear envelope breakdown in the majority of injected cells (data not shown). The recruitment of RIIα by AKAP95 onto condensing chromatin led us to determine whether the putative role of AKAP95 in chromatin condensation required association with RIIα and anchoring of PKA. To this end, the RII-anchoring inhibitor peptide Ht31 (500 nM) was preincubated in mitotic extract before adding nuclei. Chromatin condensed to the same extent with Ht31 and control Ht31-P peptides that do not compete binding of RII to AKAPs (data not shown). Thus, the role of AKAP95 in chromosome condensation in vitro is independent of its RII-anchoring function. Furthermore, neither chromatin condensation nor nuclear envelope disassembly was affected by inhibiting PKA activity in the extract with 1 μM of the PKA inhibitor PKI, or by downregulating cAMP signaling with 100 μM of the cAMP antagonists Rp-8-Br-cAMPS or Rp-8-CPT-cAMPS (data not shown). Thus, chromosome condensation in mitotic extract does not require cAMP signaling or PKA activity. The effects of disrupting AKAP-RII anchoring and downregulating cAMP/PKA signaling on chromosome condensation at mitosis were investigated by microinjecting nuclei of interphase HeLa cells with 50 nM Ht31, 50 nM Ht31-P, 10 nM PKI, or 10 μM Rp-8-Br-cAMPS, and assessing mitotic indexes after nocodazole treatment as done previously after immunoblocking of AKAP95. As shown in Table , neither reagent inhibited nuclear envelope breakdown (data not shown) nor chromosome condensation, as judged by mitotic indexes of 79–90%. Therefore, disruption of AKAP95-RII anchoring or cAMP/PKA inhibition does not impair mitotic chromosome condensation. Although chromatin condensation occurred normally in the mitotic extract containing anti–AKAP95 antibodies, we consistently observed a phase of decondensation of the chromatin upon prolonged (>4 h) incubation in the extract (data not shown). This raised the hypothesis that inhibition of AKAP95 might affect the morphology of the condensed chromatin. To test this possibility, chromatin was allowed to condense for 2 h in mitotic extract, anti–AKAP95 antibodies were added (1:50 dilution) and chromatin morphology was examined by DNA staining after another 2 h of incubation. Chromatin decondensation was observed in extract containing anti–AKAP95 antibodies , but not in the extract containing preimmune IgGs or anti–AKAP95 antibodies together with 0.1 mg/ml of GST-AKAP95Δ1-386 competitor peptide . Thus, immunoblocking AKAP95 in the extract prevented the chromatin from remaining in a condensed form. To determine whether immunodepletion of AKAP95 had a similar effect, chromatin condensed in mitotic extract was purified by sedimentation and further incubated in a fresh extract immunodepleted of AKAP95. Chromatin exposed to AKAP95-depleted, but not mock-depleted, extract underwent decondensation within 2 h , indicating that AKAP95 is implicated in maintaining chromatin in a condensed form. Moreover, the extent of chromatin decondensation after immunoblocking or immunodepletion of AKAP95 was more limited than decondensation of mitotic chromatin exposed to an interphase extract , suggesting the involvement of distinct decondensation pathways. Consequently, chromatin decondensation in mitotic extract was referred to as premature chromatin decondensation (PCD). As PCD might be interpreted as a consequence of premature exit of the extract from the M phase, we tested this possibility by measuring the level of histone H1 kinase activity in the mitotic extract incubated for 2 h without or with anti–AKAP95 antibodies and in AKAP95-depleted extract. Fig. 6 B shows that elevated (mitotic) levels of H1 kinase activity were maintained in antibody-containing and immunodepleted extracts (compare with basal H1 kinase activity in interphase HeLa cell extract). This result indicates that immunoblocking or immunodepleting AKAP95 did not release the extract from the M phase, and lends further support to the hypothesis that PCD and interphase chromatin decondensation involve distinct processes. Conclusive evidence that AKAP95 was required for maintenance of condensed chromatin in vitro was provided in a rescue experiment. Purified condensed chromatin was exposed to mitotic extract immunodepleted of AKAP95. Increasing concentrations of GST-AKAP95Δ1-386 were added and after 2 h proportions of PCD were determined by DNA staining. GST-AKAP95Δ1-386 dramatically inhibited PCD in a concentration-dependent manner . We determined whether GST-AKAP95Δ1-386 was also able to promote recondensation of prematurely decondensed chromatin. Purified condensed chromatin was allowed to undergo PCD for 2 h in AKAP95-depleted mitotic extract. Subsequent addition of GST-AKAP95Δ1-386 to the extract restored chromatin condensation also in a concentration-dependent manner . Thus, GST-AKAP95Δ1-386 was capable of inhibiting PCD and recondensing prematurely decondensed chromatin in AKAP95-depleted extract, indicating that AKAP95 is required for maintaining chromatin in a condensed form. Furthermore, since anti–AKAP95 antibodies do not block binding of RIIα to AKAP95, the data also argue that AKAP95 has an intrinsic function in the maintenance of condensed chromatin. The relevance of these in vitro observations on chromosome structure during mitosis was investigated. HeLa cells arrested in the M phase with 1 μM nocodazole were injected with affinity-purified anti–AKAP95 antibodies and, after 1 h, chromatin morphology was assessed by phase-contrast and DNA staining while cells remained in nocodazole. Anti–AKAP95 antibodies readily induced decondensation of chromosomes that could no longer be distinguished by phase-contrast . Furthermore, no nuclear envelope was detected, arguing that decondensation was reminiscent of PCD observed previously in vitro and distinct from interphase nuclear reformation. In contrast, coinjection of anti–AKAP95 antibodies with 250 pg GST-AKAP95Δ1-386 did not alter the state of chromosome condensation . These results indicate that chromosome decondensation is not an artefactual consequence of exposure of cells to nocodazole. Rather, they show that as in in vitro, immunoblocking AKAP95 at mitosis induces PCD, indicating that functional AKAP95 is needed for maintaining chromosomes condensed throughout mitosis. Whether AKAP95–RIIα association and cAMP signaling through PKA were required for maintenance of condensed chromatin during M phase was examined. Purified condensed chromatin was added to a mitotic extract preincubated with 500 nM Ht31 (or control Ht31-P) peptides to disrupt AKAP95–RII interactions (data not shown) and PCD was assessed after 1 h by DNA staining. Fig. 8 A shows that while chromatin remained condensed with Ht31-P, Ht31 induced PCD, indicating a requirement for AKAP95–RII interaction to maintain chromatin condensed. Whether PKA activity and cAMP signaling were involved in this process was determined using PKA inhibitor PKI (1 μM) and the cAMP antagonist Rp-8-Br-cAMPS (100 μM). Both reagents induced PCD, whereas control activation of PKA with 1 μM cAMP had no effect . Adding free C subunits together with anti–AKAP95 antibodies also induced PCD , suggesting that only PKA bound to chromatin-associated AKAP95 is implicated in maintaining condensed chromatin. Altogether, these results indicate that maintenance of condensed chromatin in mitotic extract requires functional AKAP95, cAMP signaling events mediated by PKA and anchoring of PKA (via RIIα) to chromosomes by AKAP95. The requirement for RIIα binding to AKAP95 and PKA activity for maintenance of condensed chromatin was tested further by assessing chromatin morphology after immunodepleting RIIα from the extract. Up to 60% of condensed chromatin masses incubated in the mitotic extract depleted of RIIα underwent PCD over a 2-h period . Moreover, immunoblotting analysis of chromatin at the beginning (0 h) and at the end (120 min) of incubation in an RIIα-depleted extract showed that whereas input chromatin harbored RIIα, most RIIα was solubilized by 120 min . In contrast, no obvious change in the amount of AKAP95 bound to chromatin was detected under these conditions . Together with our previous observation that RIIα is recruited from the cytosol to the chromatin via AKAP95, these results reflect a dissociation of RIIα from the chromatin and suggest that, as with AKAP95, keeping RIIα on condensed chromatin involves an equilibrium between soluble and chromatin-associated pools of PKA. The requirements for AKAP–RII interaction and cAMP/PKA signaling events to maintain chromosomes condensed at mitosis were investigated by microinjecting M phase–arrested HeLa cells with either 50 nM Ht31 or Ht31-P, 10 nM PKI, 10 μM Rp-8-Br-cAMPS, ∼1 ng C, or 2–5 pg anti-AKAP95 antibodies together with ∼1 ng C. The cells remained exposed to nocodazole for 1 h after injections and were examined by phase-contrast microscopy and DNA staining. Data of Fig. 9 show that Ht31 (but not Ht31-P), PKI and Rp-8-Br-cAMPS induced PCD, indicating that PKA-AKAP anchoring, PKA activity, and cAMP signaling are required for maintenance of condensed chromatin throughout mitosis. Coinjection of anti–AKAP95 antibodies with C also promoted PCD , arguing that functional inhibition of AKAP95 promotes chromatin decondensation even in the presence of unbound PKA. Several proteins have been shown to be required for mitotic chromosome condensation, including pEg7 , a component of the 13S condensin complex . To determine whether AKAP95 was part of the condensin complex or interacted with the complex, immunoprecipitations were carried out first from lysates of interphase or mitotic HeLa cells. Immunoblotting analysis of human Eg7 using a polyclonal antibody against the 15–carboxy-terminal amino acids of human Eg7 revealed that interphase and mitotic cells harbored similar levels of Eg7 . Immunoprecipitation data showed that AKAP95 and Eg7 did not coprecipitate from interphase cell lysates, indicating that these proteins do not interact at this stage of the cell cycle . In contrast, in mitotic cell lysates, AKAP95 and Eg7 coprecipitated regardless of the antibody used , suggesting that AKAP95 and Eg7 interact at mitosis. Since AKAP95 was found to be associated with chromatin at mitosis , we tested the possibility that the AKAP95–Eg7 interaction was mediated by DNA. To this end, mitotic chromatin was isolated, solubilized with micrococcal nuclease, and the DNA was digested with 400 μg/ml DNase I in the presence of protease inhibitors. Immunoprecipitation of AKAP95 or Eg7 from the DNase-treated fraction revealed that both proteins also coprecipitated , suggesting that the mitotic AKAP95–Eg7 interaction is not mediated by DNA and is probably direct. pEg7, the Xenopus homologue of human Eg7, has been previously shown to be recruited onto condensing chromosomes at mitosis . Results from our laboratory have shown that chromatin condensation in the mitotic extract is accompanied by association of Eg7 with chromatin (Landsverk, H., K.L. Guellec, and P. Collas, unpublished observation). Since pEg7 is required for chromosome condensation, we determined whether immunoblocking nuclear AKAP95 would affect the recruitment of Eg7 to chromatin in vitro. Nuclei loaded with preimmune IgGs (−α-AKAP95) or anti–AKAP95 antibodies (+α-AKAP95) were allowed to condense for 2 h in mitotic extract. Chromatin masses were sedimented and immunoblotted using anti–Eg7 antibodies. As expected from our previous results anti–AKAP95 antibodies blocked chromatin condensation. Remarkably, the antibodies also prevented the recruitment of Eg7 to chromatin that normally occurs in extract either in the absence of antibodies (not shown) or with nuclei loaded with preimmune IgGs . Loading nuclei with anti–HP1α antibodies did not impede association of Eg7 with chromatin , indicating that inhibition of Eg7 recruitment by anti–AKAP95 antibodies is not a mere consequence of steric hindrance imposed by the antibody. Altogether, these results suggest that functional inhibition of AKAP95 prevents the association of Eg7 to chromatin required for condensation. This paper provides evidence that AKAP95 is a multivalent protein with a role in chromosome condensation at mitosis. AKAP95 acts as an anchor for a PKA-signaling complex onto mitotic chromosomes, which is required for maintenance of chromosomes in a condensed form throughout mitosis. AKAP95 also appears essential for the recruitment onto chromosomes of Eg7, a component of the condensin complex. The data also provide insights on the significance of the rise in cAMP level and PKA activity during mitosis. Several lines of evidence illustrate a function of AKAP95 in chromosome condensation and maintenance of chromosomes in a condensed form during mitosis. First, immunoblocking intranuclear AKAP95 inhibits chromatin condensation in vitro and in vivo. Second, immunoblocking AKAP95 during mitosis or in vitro, or immunodepletion of soluble AKAP95 from the mitotic extract, induces PCD. Inhibition of chromatin condensation with anti–AKAP95 antibodies is unlikely to result from a nonspecific steric effect of the antibodies since antibodies against all variants of the heterochromatin protein HP1 are ineffective. Clearly, AKAP95 is implicated in chromatin condensation before PKA associates with chromatin and interacts with AKAP95. This is consistent with the established downregulation of PKA required for mitotic nuclear envelope breakdown and with the absence of PKA in interphase nuclei . Thus, in principle, AKAP95 and RII cannot interact until the nuclear envelope has broken down. However, once nuclear envelope disassembly has occurred, the role of AKAP95 in maintaining condensed chromosomes requires cAMP/PKA signaling and AKAP95–PKA interaction. A third observation supporting the involvement of AKAP95 in regulating chromosome structure at mitosis is the finding that a recombinant AKAP95 fragment (GST-AKAP95Δ1-386) prevents PCD in AKAP95-depleted extract, and restores condensation of prematurely decondensed chromatin, both in a dose-dependent manner. Thus, the domain(s) of AKAP95 required for this activity and for interaction of AKAP95 with chromatin reside(s) in the carboxy-terminal 306 amino acids (amino acids 387–692) of the protein. As expected from the RII binding requirement for maintenance of condensed chromatin, this AKAP95 fragment also contains the RII binding motif . The consequences of immunoblocking AKAP95 may be accounted for by the hypothesis that AKAP95 function may be disrupted by antibodies only when they bind AKAP95 that is not associated with chromatin. Indeed, the antibodies block condensation when incorporated into interphase nuclei before association of AKAP95 with chromatin (Landsverk, H., and P. Collas, unpublished results). Thus, it is possible that anti–AKAP95 antibodies incubated in mitotic cytosol fail to inhibit chromatin condensation because by the time the nuclear envelope breaks down, nuclear AKAP95 has already associated with chromosomes. Similarly, induction of PCD in cytosol immunodepleted of AKAP95 suggests that the establishment of an equilibrium between chromosome-associated and soluble AKAP95 is critical for maintenance of condensation. By binding AKAP95 when it dissociates from chromosomes as well as soluble AKAP95, the antibodies may sharply decrease the on-rate of AKAP95 to chromosomes, causing decondensation of the chromatin. Since it associates with chromatin early during mitotic nuclear disassembly, AKAP95 could conceivably either directly affect chromatin structure, or facilitate the recruitment of factors controlling mitotic chromosome condensation . Sequence analysis of human AKAP95 reveals that AKAP95 is unlikely to belong to the family of SMC proteins, factors shown to affect chromosome structure at mitosis and during development . Unlike SMC proteins, AKAP95 displays no coiled-coil domains, no amino-terminal ATP-binding sites and no DA box, a signature motif of SMCs . Likewise, AKAP95 displays no consensus site for topoisomerase II ; thus, unlike SMCs or topoisomerase II, AKAP95 is probably unlikely to directly impose a conformational change on chromatin inducing condensation. Rather, our data suggest that a function of AKAP95 is to allow the recruitment of components of the condensin complex. First, immunoblocking of AKAP95 inhibits association of Eg7 with chromatin in vitro. Eg7 is the human homologue of Xenopus pEg7, a protein associated with the 13S condensin complex , suggesting that anti–AKAP95 antibodies prevent binding of the condensin complex onto chromosomes. Second, AKAP95 can be coprecipitated with Eg7 from solubilized, DNase-digested mitotic chromatin, suggestive of an interaction between AKAP95 and Eg7 that is not mediated by DNA. Collectively, these findings raise the attractive possibility that one function of AKAP95 is to facilitate recruitment and/or anchoring of condensins to chromatin at mitosis. The multivalent nature of AKAP95 is not only evidenced by its role in chromatin condensation, but also by the observation that AKAP95 maintains a crucial PKA-anchoring function during mitosis. Whereas AKAP95 and RII are found in a different compartment in interphase, a chromosome-associated AKAP95–PKA signaling complex is established at mitosis. Induction of PCD after immunodepletion of RIIα from mitotic cytosol reflects an equilibrium between chromatin-associated (via AKAP95) and soluble RIIα, and a requirement for a soluble pool of RIIα to maintain the chromatin fraction of the AKAP95–RIIα complex saturated and functional. Disruption of PKA anchoring with Ht31 peptides induces PCD in vitro and in vivo, indicating that the AKAP95–PKA interaction is essential for the maintenance of condensed chromatin. Several physiological functions implicating AKAP-anchored PKA have been reported using similar disruption approaches . Formation of the AKAP95–PKA complex early during chromosome condensation may be critical to mediate the increasing cAMP signal as mitosis progresses . It is conceivable that maintenance of a condensed chromatin structure results from PKA-dependent phosphorylation of chromatin substrates such as histones or other DNA-related structural regulators such as topoisomerases . Our results provide some significance for the rising level of cAMP and increasing PKA activity during mitosis . Implications of PKA downregulation in entry into mitosis have been addressed previously . It has been reported that cAMP levels and PKA(−type II) activity are significant or even rise during mitosis. We propose that rising cAMP levels during mitosis activate chromatin-associated PKA, which together with AKAP95, is required to ensure proper chromosome condensation throughout mitosis. This view is supported by the induction of PCD by cAMP antagonists or PKI. Moreover, anti–AKAP95 antibodies added to cytosol in vitro or in vivo together with free PKA C subunit also promote PCD. Since cytosolic anti–AKAP95 antibodies presumably inhibit binding of soluble AKAP95 to chromatin, the latter observation argues that only C bound to chromosomes is relevant for maintenance of condensed chromatin, whereas free cytosolic C is ineffective. Higher PKA activity at the end of mitosis facilitates chromatin decondensation and nuclear reassembly . This scenario implies the induction of antagonistic effects of cAMP/PKA signaling on chromosome structure at mitosis that are temporally distinct. It also implicates the existence of a molecular switch upstream of the cAMP/PKA pathway, that controls the dual effect of cAMP/PKA activity at mitosis. A likely candidate might be M phase–promoting factor, as a threshold level of this activity has been shown to be required to initiate activation of the cAMP/PKA pathway during mitosis . The dual effect of PKA during mitosis and at the exit of mitosis could also be due to the redistribution of AKAP95, which dissociates from PKA at mitosis exit. Although the structural determinants mediating AKAP–RII interactions in general are being characterized , whether additional processes modulate these associations is unknown. Circumstantial evidence suggests the involvement of posttranslational modification of RII in such regulation. First, RIIα is primarily localized in the centrosome–Golgi area in interphase HeLa cells, presumably via a centrosomal AKAP . Release of RIIα from centrosomes at mitosis correlates with CDK1-mediated phosphorylation of RIIα . Second, CDK1-mediated phosphorylation of RIIα in vitro is sufficient to induce partial solubilization of RIIα from a Triton X-100 insoluble pool . Third, association of AKAP95 with RIIα is mitosis-specific. Finally, decondensation of in vitro–condensed chromatin in an interphase extract is associated with release of RIIα from chromatin-associated AKAP95 (Collas, P., data not shown). This argues that binding of RIIα to AKAP95 at mitosis is not a mere consequence of both proteins being in the same subcellular compartment; rather, a modification of either protein seems to affect their interaction. Together with the data of Keryer et al. 1998 , our results argue for a cell cycle–regulated redistribution of RIIα, suggesting an additional mechanism regulating PKA type II subcellular localization. An emerging feature of AKAPs is their ability to anchor entire signaling complexes other than PKA to specific substrates. To date, AKAP79, AKAP220, gravin, yotiao/AKAP450/CG-NAP, and ezrin have been shown to act as polyvalent anchoring proteins for signaling units involving PKA or protein kinase C together with protein phosphatases . Additional binding partners of AKAP95 besides PKA at mitosis have yet to be identified. Nevertheless, our results extend the concept that AKAP95 is likely to be a multivalent targeting molecule anchoring signaling complexes as well as chromatin remodeling factors in a cell cycle–regulated manner. It will be exciting to determine whether additional AKAPs also harbor specific intrinsic cellular functions. | Study | biomedical | en | 0.999998 |
10601333 | The p105 protein was coimmunoprecipitated with anti-SMN mAb 2B1 and the band was excised from a single one-dimensional Coomassie stained polyacrylamide gel and in-gel digested with trypsin (unmodified, sequencing grade; Boehringer Mannheim Corp.) as described in Shevchenko et al. 1996 . Tryptic peptides were recovered from gel pieces by extraction with 5% formic acid and acetonitrile. The combined extracts were pooled together, dried in a speed vac, redissolved in 5% formic acid, and analyzed by nanoelectrospray tandem mass spectrometry (nano-ES MS/MS) as described in Wilm et al. 1996 . Nano-ES MS/MS was performed on a API III triple quadrupole instrument (PE Sciex) equipped with a nano-ES ion source developed in EMBL . Comprehensive protein and expressed sequence tag (EST) databases were searched using PeptideSearch v. 3.0 software developed by M. Mann and P. Mortensen (University of Southern Denmark, Odense, Denmark). No limitations on protein molecular weight and species of origin were imposed. Several peptides of the p105 band analyzed by MS identified a human EST sequence using the peptide sequence tag algorithm. The EST clone was assumed to be incomplete since it lacked a start codon and encoded for a putative protein of only 456 amino acids. The cloning of the full-length Gemin3 cDNA was achieved by hybridization screening of a human leukemia 5′-STRETCH PLUS cDNA library (from CLONETECH) using a subfragment (EcoR1–EcoR1) of the EST clone #AA303940 as a probe. The EST EcoR1 fragment contained a region of the Gemin3 open reading frame (ORF) encoding amino acids 368–548. 12 independent partial cDNA clones with insert sizes ranging from 1–2.5 kb were isolated, all of which contained overlapping regions of the same ORF that encoded for a putative protein of 824 amino acids. Since the clone lacked an in frame stop codon upstream of the start codon, we performed two successive rounds of rapid amplification of 5′-cDNA ends by PCR (5′-RACE PCR) to extend further upstream the cloning of the Gemin3 cDNA. In brief, total mRNA from HeLa-S3 cells was prepared using the TRIZOL reagent (Life Technologies, Inc.). For the generation of cDNA, 2 μg of mRNA was reverse-transcribed using Superscript™II reverse transcriptase (Life Technologies, Inc.). The reaction was primed with Gemin3 cDNA-specific primers, GSPA (5′-TTCCACTTCCAGGCCG) or GSPC (5′-TCTTGGGGCTTTCCTCAGG), in the first or second round of RACE, respectively. The cDNAs were then purified using a GlassMAX spin cartridge, tailed with dCTP and TdT, and amplified by PCR using the Gemin3 specific primer GSP2 (5′-TCTTTCCTCTCCTCCC) and the Universal Amplification primer (UAP) from Life Technologies, Inc. (5′-CUACUACUACUAGGCCACGCTTCGACTAGTAGTAC). PCR products were cloned into the pCR2.1 vector by TA cloning (Invitrogen) and sequenced. The two independent 5′-RACE experiments yielded products terminating at the same 5′ position. The extended 5′ Gemin3 cDNA contained an in frame stop codon upstream of the start codon. By in vitro transcription–translation, we confirmed that the cloned cDNA encoded the full-length Gemin3 protein (see below). The [ 35 S]methionine-labeled proteins were produced by an in vitro coupled transcription–translation reaction (Promega Biotech) in the presence of [ 35 S]methionine (Nycomed Amersham, Inc.). 6His-Gemin3 and 6His-SMN fusion protein was expressed from a pET bacterial expression system in the Escherichia coli strain BL21(DE3) and purified using nickel chelation chromatography using Novagen His-bind buffer. GST–Gemin3 fusion protein was expressed from a GST expression vector pGEX-5X-3 (Pharmacia Biotech, Inc.) in the E. coli strain BL21 and purified using glutathione-Sepharose (Pharmacia Biotech, Inc.) according the manufacturer's protocol. Anti-Gemin3 antibodies 11G9 and 12H12 were prepared by immunizing Balb/C mice with 6His-tag COOH-terminal domain of Gemin3 (from amino acids 368–548) purified from nickel chelation chromatography using Novagen His-Bind buffer kit. Hybridoma production, screening, and ascites fluid production were performed as previously described . Immunoprecipitations of in vitro translated proteins were carried out in the presence of 1% Empigen BB buffer as previously described . Immunoprecipitations of SMN, the Sm proteins, and Gemin3 from cells were carried out using total HeLa lysate in the presence of 1% Empigen BB buffer as previously described . Immunoprecipitations and purifications of the SMN, Gemin2, Sm, and Gemin3 complexes were carried out using total HeLa lysate in the presence of 0.5% Triton X-100 as previously described . For immunoblotting, proteins were resolved on 12.5% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher and Schuell, Inc.) using a BioTrans Model B Transblot apparatus (Gelman Science) according to the manufacturer's instructions. The membranes were then incubated in blocking solution (PBS 5% nonfat milk) for at least 1 h at room temperature, rinsed with cold PBS, and then incubated in blocking solution with primary antibody for at least 1 h at room temperature. The membranes were subsequently washed three times in PBS containing 0.05% NP-40, and bound antibodies were detected using the peroxidase-conjugated goat anti–mouse IgG plus IgM (Jackson ImmunoResearch Laboratories). The antibody-decorated protein bands were visualized by an ECL Western blotting kit (Nycomed Amersham, Inc.) after washing three additional times with PBS containing 0.05% NP-40. HeLa cells were cultured in DME supplemented with 10% FBS (both from GIBCO BRL). HeLa cells, plated on glass coverslips, were transfected by the standard calcium phosphate method. Following overnight incubation with DNA, cells were washed and fresh medium was added. Transfected cells were fixed and processed by immunofluorescence staining after an additional 24–36 h of incubation. Immunofluorescence staining was carried out essentially as previously described . Double-label immunofluorescence experiments were performed by separate sequential incubations of each primary antibody diluted in PBS containing 3% BSA, followed by the specific secondary antibody coupled to either FITC or Texas red. All incubations were carried out at room temperature for 1 h. Laser confocal fluorescence microscopy was performed with a Leica TCS 4D confocal microscope. Images from each channel were recorded separately and, where indicated, the files were merged. Antibodies used in these experiments were as follows: mouse IgG1 monoclonal anti-Gemin3 (11G9 and 12H12; this work); mouse IgG1 monoclonal anti-SMN ; rabbit polyserum anti-p80 coilin ; mouse IgG3 monoclonal anti-Sm ; and SP2/O, a nonimmunoglobulin chains secreting mouse hybridoma (ATTC). The rabbit affinity-purified anti-Exon7 antibody was made against the polypetide encoded by the SMN exon7 . Purified GST or GST fusion proteins (2 μg) bound to 25 μl of glutathione-Sepharose beads were incubated with 10 6 cpm of the in vitro translated protein mix in 1 ml of binding buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM EDTA, 0.1% NP-40, 2 μg/ml leupeptin and pepstatin A, and 0.5% aprotinin). After incubation for 1 h at 4°C, the resin was washed five times with 1 ml of binding buffer. The bound fraction was eluted by boiling in SDS-PAGE sample buffer, and run on SDS-PAGE. The gels were fixed for 30 min and the signal enhanced by treatment with Amplify solution (Nycomed Amersham, Inc.). For direct in vitro binding , purified GST or GST–Gemin3 proteins (2 μg) bound to 25 μl of glutathione-Sepharose beads were incubated with 5 μg of purified 6His-SMN or 6His-mB in 1 ml of binding buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl, 2 mM EDTA, 0.05% NP-40, 2 μg/ml leupeptin and pepstatin A, and 0.5% aprotinin). After incubation for 1 h at 4°C, the resin was washed five times with 1 ml of binding buffer. The bound fraction was eluted by boiling in SDS-PAGE sample buffer, and analyzed by SDS-PAGE and Western blot using a rabbit polyclonal anti-His-tag antibody (Santa Cruz Biotech). HeLa cells were fractionated according to Dignam et al. 1983 . S100 fractions (400 μl of ∼20 mg/ml protein) in buffer F (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, 500 mM KCl) were loaded on a Superose 6 HR 10/30 column (Pharmacia Biotech, Inc.). The column was then washed with buffer A (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5% glycerol). Fractions (0.5 ml) were collected, and 30 μl of each fraction was resolved on SDS-PAGE, followed by Western blotting. The Gemin3 GenBank/EMBL/DDBJ accession number is AF171063. Immunoprecipitations from [ 35 S]methionine-labeled HeLa cell lysates with anti-SMN and anti-SIP1 mAbs revealed the presence of several protein components in the SMN–SIP1 complex . Among the proteins that can be coimmunopurified with anti-SMN and anti-SIP1 antibodies, only some of the major low molecular mass proteins, identified as the Sm proteins, have been characterized so far . In addition to SMN, SIP1, and the Sm proteins, there is a doublet at ∼97 kD, and additional bands at 175, 95, 60, and 50 kD that coimmunopurified with the anti-SMN antibody. The two proteins of the 97-kD doublet were eluted from the gel, digested with trypsin, and the resulting peptides sequenced by nano-ES MS as described previously . In this paper, we describe the molecular cloning and characterization of the high molecular weight protein of this doublet (p105). Several peptides from this band identified a human EST sequence using the peptide sequence tag algorithm . Several additional cDNA clones were obtained by hybridization screening of a human leukemia 5′-STRETCH PLUS cDNA library, using this EST clone as a probe. We isolated 12 independent partial cDNA clones with insert sizes ranging from 1–2.5 kb, all of which contained overlapping regions of the same ORF. 5′ RACE PCR was used to extend this cDNA further upstream. A cDNA clone containing the longest ORF was constructed and conceptual translation of its nucleotide sequence revealed a potential initiator methionine, preceded by an in frame stop codon. This cDNA encodes a putative protein of 824 amino acids with a calculated molecular mass of 92.2 kD and a pI of 6.5. We then determined that this cDNA encodes the p105 protein that coimmunoprecipitates with SMN (see Gemin3 and SMN Colocalize in Gems). Thus, this is a full-length cDNA clone for a novel component of the SMN complex that we termed Gemin3, for component of gems number 3 (see below). Because of the existence of several unrelated proteins called SIP1 , we tentatively rename the SMN-interacting SIP1 Gemin2, for component of gems number 2 . Gemin3 has high amino acid sequence similarities with the RNA helicase core region of the human eukaryotic initiation factor 4A-II (eIF4A-II). eIF4A-II is a DEAD box RNA helicase that belongs to the SFII superfamily of helicase . A scheme depicting the modular structure of Gemin3 and the predicted amino acid sequence of Gemin3 aligned with the sequence of eIF4A-II are presented in Fig. 3 . This alignment showed the presence of seven motifs in the Gemin3 protein, which are characteristic of the RNA helicase core region. Database searches with the COOH-terminal nonconserved region did not reveal significant homology to any other protein or any recognizable motifs. To investigate the interaction of Gemin3 with SMN and to characterize Gemin3 further, we generated mAbs to it by immunizing mice with a purified bacterially produced recombinant 6His-Gemin3 fragment (amino acids 368–548). Two hybridomas, 11G9 and 12H12, were selected for additional studies. Several lines of evidence demonstrate that these hybridomas indeed produce mAbs that recognize Gemin3 specifically. First, both 11G9 and 12H12 specifically immunoprecipitate Gemin3 produced by in vitro transcription and translation from the Gemin3 cDNA, but do not immunoprecipitate similarly produced hnRNP A1 or SMN proteins . Second, the mAb 11G9 efficiently recognized purified 6His-Gemin3 on Western blots, but did not recognize similarly produced and purified 6His-Gemin2 . Finally, on an immunoblot of total HeLa lysate, both 11G9 and 12H12 recognized a single protein of ∼105 kD . mAbs 11G9 or 12H12 did not recognized a specific protein on a Western blot with total mouse 3T3 cell lysate or Xenopus laevis XL-177 cell lysate. However, 11G9 specifically immunoprecipitated a single protein of ∼105 kD from these cell lysates (data not shown), suggesting that Gemin3, like SMN, is conserved in vertebrates. Indirect laser confocal immunofluorescence microscopy using antibodies 11G9 and 12H12 was performed on HeLa cells to determine the subcellular localization of Gemin3. Fig. 5 A shows that Gemin3 is found throughout the cytoplasm and also displays intense staining of prominent discrete nuclear bodies that are readily discernible by differential interference contrast . This pattern is similar to that seen for SMN and Gemin2 , except that the nucleoplasmic staining of Gemin3 is stronger. To determine if the nuclear structures stained by 11G9 are gems or coiled bodies, we performed double-label immunofluorescence experiments using antibodies against Gemin3 and either p80 coilin as a marker of coiled bodies or SMN as a marker of gems . In many cell lines, gems and coiled bodies entirely overlap by antibody staining, however, in the HeLa PV strain used here, these two bodies are frequently found separate from each other . Therefore, we used HeLa PV cells to examine whether Gemin3 is located in gems or in coiled bodies. As can be seen in Fig. 5C and Fig. D , the nuclear structures that contain Gemin3 are clearly distinct from coiled bodies, but Gemin3 completely colocalized with SMN in gems . The colocalization of Gemin3 with SMN strongly supports the conclusion that these two proteins exist as a complex in the cell. Gemin3 is thus the third constituent of gems described so far. Compared with visual observation, the confocal photographs in Fig. 5 , slightly overestimates the nuclear staining and underestimates the cytoplasmic staining for Fig. 5C and Fig. D . To characterize further the Gemin3 complex, immunoprecipitations using anti-Gemin3 mAbs and [ 35 S]methionine-labeled HeLa cells were carried out in the presence of either Triton X-100 or the more stringent detergent, Empigen BB . The immunoprecipitated proteins were then analyzed by SDS-PAGE. As references for these immunoprecipitations, we also included an immunoprecipitation with the anti-Sm mAb Y12 and an immunoprecipitation with the anti-SMN mAb 2B1. As shown in Fig. 6 A, several proteins can be coimmunoprecipitated with Gemin3 and the pattern of immunoprecipitated proteins is very similar to that obtained with the anti-SMN antibody. In addition to Gemin3, SMN, and Gemin2, there are several prominent bands at 175, 95, and 50 kD. The two groups of proteins at 28 and 15 kD have been identified previously as the Sm B/B′, D1-3, E, F, and G proteins of snRNPs . In addition, there are bands that coimmunoprecipitate only with anti-SMN (at 60 kD) or anti-Gemin3 (at 115 kD) mAbs. As further evidence for the specificity of the antibodies used, the immunoprecipitations were performed in the presence of Empigen BB. Under these conditions, anti-Gemin3 and anti-SMN antibodies immunoprecipitate Gemin3 and SMN proteins respectively . Interestingly, a protein of 95 kD is still present under these conditions in both of these immunoprecipitations, but not in the control SP2/O immunoprecipitation, suggesting that this unidentified protein interacts tightly with both Gemin3 and SMN. To confirm the coimmunopurification results, we tested for interaction of Gemin3 with SMN, Gemin2, and the Sm proteins in HeLa cells in vivo by immunoprecipitations and Western blot experiments. The anti-Gemin3 mAb 11G9 was used for immunoprecipitation from total HeLa cell extracts, and these were then resolved by SDS-PAGE and an immunoblot was probed with the anti-SMN antibody . As shown in Fig. 6 C (lane 11G9 IP), 2B1 readily detects SMN in the 11G9 immunoprecipitates, indicating that SMN is coimmunoprecipitated with Gemin3. Because SMN is associated with Gemin2 to form a stable complex in vivo and in vitro , we also investigated whether Gemin3 could be coimmunoprecipitated with Gemin2. As shown in Fig. 6 C, the anti-Gemin2 mAb 2S7 clearly detects Gemin2 in the anti-Gemin3 11G9 immunoprecipitates . In a reciprocal experiment, the Gemin3 protein could also be coimmunoprecipitated by the anti-SMN mAb 2B1 and the anti-Gemin2 mAb 2S7 . Because SMN and Gemin2 are found in a complex with the Sm proteins, we asked whether Gemin3 can be coimmunoprecipitated with the spliceosomal snRNP Sm core proteins as well. Fig. 6 D shows that Gemin3 is present in the anti-Sm mAb Y12 immunoprecipitates like SMN and Gemin2 . No Gemin3, SMN, Gemin2, or Sm proteins were detected in a SP2/O immunoprecipitate (data not shown). These results demonstrate that Gemin3, SMN, Gemin2, and the Sm proteins are associated in vivo in a complex that can be immunoprecipitated by either anti-SMN, anti-Gemin2, anti-Sm, or anti-Gemin3 antibodies. Further support for the existence in vivo of a complex that contains SMN, Gemin2, and Gemin3 was obtained from gel filtration experiments. HeLa cytoplasmic S100 extract was fractionated on a Superose 6 HR 10/30 high performance gel filtration column and each fraction was subjected to SDS-PAGE, followed by Western blot with anti-Gemin3, anti-SMN, and anti-Gemin2 mAbs. Gemin3, SMN, and Gemin2 comigrated and showed a peak at ∼800 kD, demonstrating that they are components of a large macromolecular complex . Interestingly, a second pool of SMN-Gemin2, lacking Gemin3, is observed in a lower molecular weight complex that peaks at ∼150 kD, suggesting that at least two different SMN–Gemin2 subcomplexes exist in vivo. However, we cannot exclude the possibility that the 150-kD subcomplex corresponds to a fraction of SMN-Gemin2 that dissociates from Gemin3 during cell fractionation and/or chromatography. We reported previously that a SMN–Gemin2 complex migrates at >300 kD after filtration of a cytoplasmic S100 extract on a TSK-GEL G3000-SW column . The Superose 6 HR 10/30 gel filtration column used here allowed us to obtain a better resolution of the cytoplasmic SMN complex and to better estimate its size as ∼800 kD. To further analyze the Gemin3 complex, we performed in vitro protein-binding assay between Gemin3 and several components of the SMN complex. For in vitro binding assays, Gemin3 was produced as a fusion protein with glutathione S-transferase (GST), and SMN and Gemin2 were produced and labeled with [ 35 S]methionine by in vitro transcription and translation in rabbit reticulocyte lysate. Purified GST or GST–Gemin3 fusion immobilized on gluthatione-Sepharose were incubated with labeled SMN or Gemin2 proteins. After extensive washing, bound proteins were eluted by boiling in SDS-containing sample buffer and the eluted material was analyzed by SDS-PAGE and detected by fluorography. Full-length SMN, but not Gemin2, bound specifically to immobilized GST–Gemin3 , but not to GST alone (data not shown). To investigate whether Gemin3 interacts with Sm proteins, purified GST or GST–Gemin3 recombinant proteins were used for binding assays with in vitro [ 35 S]methionine-labeled Sm proteins B, D1, D2, D3, E, F, and G . The results, shown in Fig. 7 B, demonstrate that the Sm proteins B and D3 bind to GST–Gemin3, whereas there is no detectable binding to GST alone (data not shown). D2 binds Gemin3 only weakly and we note that the profiles of Sm protein binding to SMN and Gemin3 are not identical . For example, SMN binds to D1 whereas Gemin3 does not . To address the possibility that some component of the rabbit reticulocyte lysate mediates these interactions, wild-type full-length SMN and SmB were produced as recombinant 6His-tagged proteins and incubated with GST or GST–Gemin3. After several rounds of washing, bound proteins were solubilized by boiling in SDS sample buffer, resolved by SDS-PAGE, immunoblotted, and probed with a rabbit polyclonal antibody specific to the 6His-tag. As shown in Fig. 7 C, SMN and SmB bind specifically to Gemin3, but not to GST alone. We conclude that both SMN and SmB interact directly with Gemin3. To further characterize the interaction between Gemin3 and SMN, we first tested whether SMN carrying two well-characterized mutations found in SMA patients, the Y272C point mutant (SMNY272C) and the exon7 deletion mutant (SMNΔEx7), the major product of the SMN2 gene , are able to interact with Gemin3. SMN wild-type and mutants were produced and labeled with [ 35 S]methionine by in vitro transcription and translation in rabbit reticulocyte lysate. Full-length wild-type SMN bound specifically to immobilized GST–Gemin3 . However, SMNY272C and SMNΔEx7 are severely defective in their ability to bind GST–Gemin3. No detectable binding was observed to GST alone. Similar results were observed using purified recombinant 6His-SMN wild-type and mutant proteins instead of in vitro translated products (data not shown). SMN oligomerization and Sm binding are not mutually exclusive, and in fact, Sm binding is strongly enhanced by SMN oligomerization . To determine whether SMN self-association enhances Gemin3 interaction, GST–SMN, or GST as a control, was preincubated with a molar excess of recombinant 6His-tag SMN to form SMN oligomers. After removing the unbound 6His-SMN by washing, in vitro translated [ 35 S]methionine-labeled Gemin3 and SmB were added and assayed for binding . As expected, SmB binding was strongly enhanced by SMN oligomerization , however, Gemin3 binding was not affected. The unwinding activity of DEAD box RNA helicases may not be sequence specific. The target specificity of these proteins is, at least in some cases, provided by their interaction with specific proteins of the RNP substrate. These interactions appear to be mediated via the unique auxiliary domain that each RNA helicase contains . Therefore, we investigated the role of the unique COOH-terminal domain of Gemin3 (amino acids 430–825) in the interaction and cellular localization of this novel DEAD box RNA helicase. To do so, we constructed three deletion mutants of Gemin3 and first tested their binding to GST–SMN. Wild-type and mutant myc-Gemin3 constructs were transcribed and translated in rabbit reticulocyte lysate in the presence of [ 35 S]methionine, and the resultant translated products were assayed for binding to GST–SMN as described above. As Fig. 8 B indicates, the wild-type myc-Gemin3 protein and myc-ΔN368C277Gemin3 mutant proteins interact specifically with GST–SMN, but not with GST alone. The myc-ΔC328Gemin3 and myc-ΔN548 Gemin3 mutant proteins clearly do not interact with GST–SMN. Thus, the COOH-terminal domain of Gemin3 (amino acids 456–547) mediates the interaction of SMN with Gemin3. We then monitored the expression and cellular localization of the myc-tagged mutants in transfected HeLa cells. Because gems represent a marker of a concentrated pool of the SMN complex in the nucleus, it is likely that the incorporation of a myc-tagged Gemin3 protein into gems results from its interaction with SMN in vivo. Double-label immunofluorescence microscopy, using anti-myc tag antibodies to detect either the transfected wild-type myc-Gemin3 or the myc-ΔGemin3 mutants, and the anti-SMN mAb 2β1 antibody, showed accumulation of the wild-type (data not shown) and myc-ΔN368C277Gemin3 mutants into gems . However, the myc-ΔC328Gemin3 and myc-ΔN548 (data not shown) Gemin3 mutant proteins accumulate essentially in the cytoplasm without any gems or nucleoplasm localization. This strongly suggests that the COOH-terminal region of Gemin3 (amino acids 456–547) is responsible for the localization of Gemin3 into gems. The molecular characterization of the spinal muscular atrophy gene product, SMN, demonstrated that it is concentrated in novel nuclear structures called gems . Coiled bodies and gems represent nuclear structures that appear to be involved in RNA metabolism, and in many of the cell lines studied, these two bodies are often found in association . SMN is also found in the cytoplasm, where, together with its tightly associated partner, Gemin2, it functions in the assembly of snRNP particles . In the nucleus, SMN is required for pre-mRNA splicing, and likely serves to assemble and maintain the splicing machinery in an active form . To perform these functions, SMN must either have an intrinsic activity or recruit other proteins that can actively affect structural transitions to the complex in certain RNP targets. Several factors that have the capacity to serve in such functions, including assembly and disassembly of components of the splicing machinery, have been described. Many of these factors are DEAD/DEAH box RNA helicases that are essential for splicing . Prp43, for instance, is required for the disassembly of the snRNP–intron lariat complex , Prp22 is needed to release the mature mRNA from the spliceosome , and Prp24 acts as a recycling factor for U4 and U6 snRNP . Using a biochemical approach to characterize new components of the SMN complex, we have identified a novel DEAD box RNA helicase termed Gemin3. Gemin3 forms a stable complex with SMN in vivo and in vitro, and it colocalizes with SMN in nuclear gems. Several lines of evidence suggest that Gemin3 and SMN function as a complex in vivo. SMN and Gemin3 can be coimmunoprecipitated and both are present in a large (∼800 kD) complex that also contains Gemin2. Anti-SMN, anti-Gemin2, or anti-Gemin3 mAbs immunoprecipitate the spliceosomal snRNP core Sm proteins, as well as several other unidentified proteins. Gemin3 interacts directly with SMN and with several snRNP Sm core proteins, including B/B′, D2, and D3. In addition, Gemin3 is uniformly distributed in the cytoplasm, where snRNP assembly takes place, and it can be specifically coimmunoprecipitated with the cytoplasmic pool of Sm proteins (data not shown). Together, these findings suggest that Gemin3 may play an important role in spliceosomal snRNP biogenesis. DEAD box proteins have been found to be involved in many aspects of RNA metabolism, including pre-mRNA splicing, translation, snRNP–snRNP interactions, mRNA degradation, and mRNA transport in eukaryotes and prokaryotes . One of the major questions about the function of each DEAD/DEAH box RNA helicase is the identification of the specific RNA target for it. Some of the enzymes of this family can unwind generic RNA substrates in vitro . For these enzymes, the specificity towards particular RNAs, therefore, appears to be determined by factors that interact with their unique auxiliary domains. For example, the DEAH box RNA helicase Prp16 is recruited to the spliceosome via its unique NH 2 -terminal domain . The specific substrate for Gemin3 has not yet been identified and this remains a central question of interest. In a series of preliminary experiments, we have not been able to detect RNA helicase or RNA-dependent ATPase activity for recombinant Gemin3, so far. It is possible that such activity will only manifest itself when Gemin3 is associated with other proteins as part of a complex or that it will be detectable once a specific RNA or RNP target is found. The interaction of Gemin3 with SMN is direct, and we found that amino acids 456–547 of Gemin3 mediate this interaction and, likely as a consequence of this, the localization of Gemin3 to the gems. Thus, we propose that Gemin3 provides the enzymatic activity of the SMN complex to affect structural transitions in its RNA targets. The SMN protein is capable of forming an oligomer of >400 kD in vitro and we show here that SMN comigrates with an ∼800-kD complex that also contains Gemin2 and Gemin3. It is likely that SMN oligomerization is critical for the nucleation of this large complex. In addition to Gemin3 and Gemin2, several Sm proteins interact with SMN, and we therefore propose that SMN forms a docking platform to bring together, in the appropriate spatial arrangement, the multiple proteins that are involved in the de novo assembly and regeneration of its RNP (e.g., snRNP) substrates. Interestingly, the interaction of SMN with Gemin3 is severely reduced by mutations found in SMA patients, such as the point mutant SMNY272C or the exon 7 deletion. Thus, the formation of the SMN platform seems critical for SMN function because SMA affects both the capacity of SMN to oligomerize, as well as to interact with several Sm proteins and Gemin3. As a likely consequence of these defective interactions, the function of SMN in the regeneration of the splicing machinery is abolished . Coiled bodies contain the highest local concentration of p80 coilin and are enriched in components of three major RNA processing pathways: pre-mRNA splicing; histone mRNA 3′ maturation; and pre-mRNA processing . Gems contain the highest local concentration of SMN, Gemin2, and Gemin3 and are often found associated with coiled bodies . Although the definitive function of these two nuclear bodies has not been completely elucidated, the characterization of their protein and RNA contents represents an important step toward the understanding of their functions. Further studies of Gemin3, a novel DEAD box containing protein and component of gems, should shed light on the functions of the SMN complex and gems. | Study | biomedical | en | 0.999996 |
10601334 | Astrocytes were isolated from the cerebral cortex of E18 rat embryos using a minor modification of previously described methods . In brief, cerebral hemispheres were obtained from E18 brains and the meninges were carefully removed. Brain tissue was digested with papain (Worthington Biochemical Corp.) at 37°C for 15 min and plated in 175-cm 2 culture flasks (two brains/flask). Cells were grown in MEM with 10% FCS for 10 d and agitated strongly on a shaking platform to separate astrocytes from microglia and oligodendroglia. Cells were then replated into 150-mm diam dishes and grown for an additional 7 d. Cultures used for experiments were comprised of >95% astrocytes, based on the morphological (fibroblast-like appearance with the formation of a cobblestone cell layer) and immunohistochemical (detection of glial fibrillary acidic protein [GFAP] with anti-GFAP antibody; Sigma Chemical Co.) criteria. When cells achieved confluence, the medium was replaced with serum-free MEM and cultures were subjected to H for the indicated times (up to 22 h) using an incubator equipped with an H chamber (Coy Laboratory Products) as described . Using this chamber, the ambient oxygen tension in culture medium bathing the cells was ∼8–10 Torr . In some experiments, cells were returned to the ambient atmosphere after H and incubated for 4 h (R). In other experiments, cells were maintained in normoxia and exposed to either calcium ionophore A23187 (1 μM for 8 h) (Sigma Chemical Co.), tunicamycin (1 μg/ml for 8 h) (Sigma Chemical Co.), or hydrogen peroxide (80 μM for 4 h) (Wako Chemicals). Alternatively, cultures were subjected to heat shock at 42°C for 2 h and then returned to 37°C and incubated for an additional 2 h. 293 cells and BHK cells were cultured in DMEM and transfected with the indicated expression vectors as described below. Total RNA was extracted from cultured astrocytes subjected to normoxia or H for 20 h using Qiagen RNeasy kit, and differential display was performed as described . In brief, RT-PCR with avian myeloblastosis virus reverse transcriptase (Amersham Pharmacia Biotech) and ∼300 random primers (12–21 oligonucleotides) were used to prepare cDNAs. PCR products were separated by acrylamide gel (6%) electrophoresis, and cDNA bands of interests were cut out from the gel. After cloning the cDNAs into pGEM-T vector (Promega), DNA sequencing was performed using a DNA sequencer (model 377; Applied Biosystems). Differential expression of candidate genes in H versus normoxia was confirmed by Northern blotting using 32 P-radiolabeled cDNAs as probes. One of the cDNA fragments obtained by differential display, termed T41, was used to screen an adult cDNA library (lambda ZapII cDNA library; Stratagene). A cDNA of 2.3 kb was obtained spanning the entire open reading frame (ORF) and polyadenylation signal, and both strands were sequenced. Sequence searches and comparisons were carried out using several databases, including the National Center for Biotechnology Information, FASTA and BLAST molecular analysis systems, and EMBL/ GenBank/DDBJ. The human SERP1/RAMP4 cDNA, which included the complete ORF of rat SERP1/RAMP4, was obtained from a human expression sequence tag (EST) clone through the IMAGE consortium. DNA sequencing was performed on both strands for each clone. Total RNA (10–15 μg), isolated from cultured astrocytes or rat tissues, was separated on agarose/formaldehyde (1%) gels and transferred onto Immobilon N membranes (Millipore). A cDNA fragment of SERP1/RAMP4 was labeled with 32 P by the random hexamer procedure (specific activity 0.5–3 × 10 9 cpm/μg DNA) and was used to probe membranes with immobilized RNA. After washing in 2× SSC and 0.5× SDS for 1 h, membranes were subjected to autoradiography. Northern blots were also performed with rat Sec61α, human Sec61β (see below), rat GRP78 (cloned by PCR with specific primers), rat HSP72 , human ORP150 , and human oligosaccaryltransferase (OST; cloned by PCR with specific primers) cDNA fragments as probes. Unilateral middle cerebral artery (MCA) occlusion was performed in male Sprague-Dawley rats (250 g) as described . After 12 h of ischemia, rats were killed and brains were frozen at −80°C. Serial coronal sections were cut and SERP1/RAMP4 mRNA was detected by in situ hybridization using previously described techniques . In brief, sense and antisense riboprobes for SERP1/RAMP4 were in vitro transcribed from the rat SERP1/RAMP4 cDNA inserted into the pGEM T vector. After linearizing the vector with NcoI (for the sense probe) or SpeI (for the antisense probe), reaction mixtures were incubated with [ 35 S]UTP (NEG-039H; Dupont-NEN) and SP6 or T7 RNA polymerase (Promega). Brain sections were then hybridized with either sense or antisense probes and washed, dried, and subjected to autoradiography. 2 d later, films were developed and brain images were examined. For some sections, slides were covered with photographic emulsion (Eastman Kodak Co.) for 2 wk, and were then developed and analyzed by dark-field microscopy. SERP1/RAMP4 antigen was also detected by immunoprecipitation followed by the Western blotting with anti–SERP1/RAMP4 antibody as described below. SERP1/RAMP4 cDNA encoding the complete ORF was amplified by PCR using primers tagged with FLAG epitope at the NH 2 terminus and cloned into pcDNA3 (Invitrogen). The rat Sec61α and human Sec61β cDNAs were cloned as described and hemagglutinin (HA)-tagged, amplified by PCR, and ligated into pcDNA3. All constructs were sequenced before transfection studies (see below). Anti–human Sec61β antibody was generously provided by Dr. Tom A. Rapoport (Harvard University, Boston, MA). Reagents to detect receptor for advanced glycation endproducts (RAGE) at the protein level were prepared as described . Antibodies and cDNA for CD8 were kindly provided by Dr. Kazunori Imaizumi (Osaka University Medical School, Osaka, Japan). To obtain antibody reactive with SERP1/RAMP4, a peptide with the sequence CTQRGNVAKTSRNAPEEK (containing an extra cysteine residue at the NH 2 terminus), was synthesized and conjugated to keyhole limpet hemocyanin. Rabbits were immunized by conventional methods. Once high titer antibody was obtained, the antiserum was affinity-purified using a column with immobilized synthetic peptide (Proton Kit 1; Multiple Peptide Systems). For detecting SERP1/RAMP4 protein, cultured astrocytes or 293 cells transfected with pcDNA3/FLAG-tagged SERP1/RAMP4 were lysed in 1% NP-40/10 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 10 μg/ml aprotinin, and immunoprecipitation or Western blotting was performed using rabbit anti–SERP1/RAMP4 antibody or mouse anti-FLAG antibody. Sites of primary antibody binding were determined by enhanced chemiluminescence method (Amersham Pharmacia Biotech), or using alkaline phosphatase–conjugated secondary antibodies. Immunoblotting for other molecules used specific antibodies for each epitope, and the same general procedure as that for SERP1/RAMP4 was used. To assess the subcellular localization of SERP1/RAMP4, BHK cells transiently transfected to overexpress FLAG-tagged SERP1/RAMP4 or YSY6p, were subjected to double-immunostaining with mouse anti-FLAG (Sigma Chemical Co.) and rabbit anti–protein disulfide isomerase (PDI) antibodies (the latter kindly provided from Dr. Ryuichi Masaki, Kansai Medical University, Osaka, Japan) . Sites of primary antibody binding were visualized using TRITC-conjugated anti–mouse antibody (Sigma Chemical Co.) and FITC-conjugated anti–rabbit antibody (Sigma Chemical Co.). In other studies, RAGE was detected using rabbit anti-RAGE IgG followed by FITC-conjugated anti–rabbit antibody. 293 or BHK cells were transiently transfected with expression constructs for RAGE or CD8 (2 μg DNA/ml) alone or with pcDNA/SERP1/RAMP4 (2 μg DNA/ml) using lipofectamine (Life Technologies, Inc.). Where indicated, ER stress was induced by addition of the calcium ionophore A23187 (1 μg/ml) or tunicamycin (1 μM). After overnight incubation, stabilization of membrane proteins (RAGE or CD8) was analyzed by immunoblotting or by immunoprecipitation, the latter after pulse–chase labeling. Metabolic labeling was accomplished by addition of [ 35 S]methionine (American Radiolabeled Chemicals) to the culture medium (0.25 mCi/ml), followed by incubation for 30 min in methionine-free DMEM, and a 1-h chase period in complete medium (i.e., without [ 35 S]methionine). As a control experiment, stabilization of IκBα under stress conditions was also studied using anti-IκBα antibody (Santa Cruz Biotechnology, Inc.). Antiubiquitin antibody (StressGen Biotechnologies Corp.) was used to characterize the smear RAGE antigen under stress condition. In some experiments, cells were released from the stress after overnight incubation with tunicamycin (1 μM) (i.e., the medium was replaced with tunicamycin-free medium) and glycosylation of RAGE was analyzed by metabolic labeling with [ 35 S]methionine (0.25 mCi/ml) for 3 h followed by immunoprecipitation with anti-RAGE antibody. 293 cells (5 × 10 6 cells) were transfected with expression constructs for FLAG-tagged SERP1/RAMP4, or the membrane protein RAGE (see above). After overnight incubation, cultures were lysed in 1% NP-40 or 2% deoxycholate (the latter followed by DNase I treatment)/10 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 10 μg/ml aprotinin, and immunoprecipitation was performed with various antibodies, such as anti-FLAG (Sigma Chemical Co.), anti-Sec61α, anti-Sec61β, anti-RAGE , anti-GRP78 (StressGen Biotechnologies Corp.), anti-ORP150, or anticalnexin (StressGen Biotechnologies Corp.) antibodies. Immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with indicated antibodies. Cross-linking with disuccinimidyl suberate (DSS) (Pierce) was performed on membrane fractions isolated from 293 cells cotransfected with expression constructs for SERP1/RAMP4 and HA-tagged Sec61β as described . In brief, 10 7 cells were homogenized with a Dounce homogenizer in 0.25 M sucrose, 10 mM acetic acid, 10 mM triethanolamine, 1 mM EDTA, pH 7.4, and 1 mM PMSF, and then centrifuged at 1,000 g for 10 min. Supernatant was spun again at 10,000 g for 30 min and supernatant from the second spinning was subjected to final centrifugation at 100,000 g for 60 min to clarify a cytosolic fraction. Cross-linking was carried out by incubating membrane fractions with 1 mM DSS in PBS for 1 h. Coimmunoprecipitation (co-IP) was then performed as described above. Laser densitometric analysis was performed to standardize the results of Western and Northern blotting with Quality One software (Pdi, Inc.) as described previously . Cultured rat astrocytes were exposed to H, and after RT-PCR, a differentially expressed amplicon of 400 bp termed T41 was identified. Northern analysis using this cDNA as a probe and total RNA harvested from hypoxic astrocytes confirmed selective upregulation compared with normoxia (see below), and led us to clone the full-length cDNA. A rat brain cDNA library was screened, and a 2.3-kb cDNA clone was obtained that contained only a single ORF and polyadenylation signal. The cDNA encoded a protein of 66 amino acids, termed SERP1, including a putative transmembrane-spanning domain at the COOH terminus . Based on sequence homology database searches, SERP1 displayed 29% identity at the amino acid level with the yeast protein YSY6p, which was identified as a high-copy suppressor of SecY/Sec61 mutant . Human and Caenorhabditis elegans homologues of SERP1 were encoded in EST clones, and showed 100 and 53% identities, respectively. We further found that SERP1 is identical to a ribosome-associated membrane protein termed RAMP4 which was copurified with Sec61 complex . Rat, human, and C. elegans SERP1 cDNA sequences can be obtained from EMBL/GenBank/DDBJ under accession nos. AB018546, AB022427, and Z81095, respectively. Northern blotting of total RNA harvested from rat tissues demonstrated similar levels of SERP1/RAMP4 transcripts in a range of normal organs, whereas heart, lung, and muscle displayed lower levels of mRNA . Cultured astrocytes exposed to H showed a time-dependent increase in SERP1/RAMP4 transcripts, although this occurred after relatively long-term oxygen deprivation . When cultures were returned to the ambient environment after hypoxia (R), an increase in SERP1/RAMP4 mRNA was kept for at least 4 h . Levels of SERP1/RAMP4 mRNA under H and H/R were then compared with those of Sec61 complex (Sec61α and Sec61β) and the molecular chaperons in ER (GRP78, ORP150), the latter known to be induced in H or other types of ER stress . Transcripts of Sec61α and Sec61β increased in a manner paralleling SERP1/RAMP4, whereas GRP78 and ORP150 displayed enhanced expression at earlier times and began to decline soon after replacement into normoxia. Quantification of Northern analysis demonstrated that SERP1/RAMP4 and GRP78 mRNAs increased by ∼5.1- and 6.0-fold in H, compared with normoxia, respectively . Treatment of normoxic cultures with tunicamycin or A23187, both of which result in accumulation of unfolded polypeptides within the ER , caused a ∼4.2- and 5.8-fold increase in SERP1/RAMP4 transcripts, respectively, whereas hydrogen peroxide and HSP were without effect . Accompanying H/R- and ER stress–induced elevation of SERP1/RAMP4 mRNA, there was a five- to sixfold increase in SERP1/RAMP4 antigen (∼10 kD) as shown using immunoprecipitation followed by immunoblotting with anti-SERP1 antibody . Immunostaining of BHK cells transiently transfected with FLAG-tagged SERP1/RAMP4 displayed an overlapping distribution of SERP1/RAMP4 antigen and the ER marker PDI . Although SERP1/RAMP4 has a putative n -glycosylation site at its NH 2 terminus, treatment of SERP1/RAMP4-transfected cells with tunicamycin or incubation of SERP1/RAMP4 protein itself with endoglycosidase H revealed no change in SERP1/RAMP4 (with respect to either its subcellular distribution or migration on SDS-PAGE), suggesting that glycosylation had not occurred (data not shown). To extend our studies of SERP1/RAMP4 expression in response to oxygen deprivation in vivo, distribution of SERP1/RAMP4 transcripts and the expression of SERP1/RAMP4 antigen were assessed in rat brain after MCA occlusion. In situ hybridization showed increased SERP1/RAMP4 transcripts, especially in the periischemic penumbral region, compared with lower levels of expression in nonischemic areas . Microautoradiography suggested that induction of SERP1/RAMP4 transcripts occurred in both neurons and astrocytes in the ischemic hemisphere . Immunoprecipitation followed by Western blotting with anti-SERP1/RAMP4 antibody confirmed ∼5.7-fold induction of SERP1/RAMP4 antigen in rat ischemic brain . The effect of SERP1/RAMP4 on processing of two integral membrane glycoproteins, RAGE and CD8, was studied by cotransfecting 293 cells with expression vectors for each along with pcDNA/SERP1/RAMP4. Transient transfection of cultures with pcDNA3/RAGE followed by treatment of cells with tunicamycin or A23187 (data not shown) resulted in trapping of RAGE in the ER followed by degradation. Similar results were observed when CD8 was overexpressed and cultures were treated with these drugs. Intracellular levels of RAGE were analyzed by immunoblotting and metabolic labeling with [ 35 S]methionine followed by immunoprecipitation . 293 cells overexpressing RAGE and exposed to tunicamycin or A23187 showed a decrease in the amount of RAGE antigen (∼58 and 36%, respectively, compared with quiescent cultures; M r ∼55 kD in glycosylated form and 52 kD in unglycosylated form), as well as a poorly defined smear of RAGE immunoreactivity in the upper portion of the membrane, the latter likely due to aggregation of RAGE , and/or several more rapidly migrating immunoreactive bands, consistent with the occurrence of degradation . Immunoprecipitation of cell lysates with anti-RAGE antibody followed by Western blotting with antiubiquitin antibody showed that the smear of RAGE immunoreactivity includes polyubiquitinated RAGE (data not shown). Although cotransfection of quiescent 293 cells with pcDNA3/RAGE and pcDNA3/SERP1/RAMP4 had no apparent effect on RAGE antigen , overexpression of SERP1/RAMP4 in tunicamycin- or A23187-treated cultures increased the intensity of the major RAGE band to ∼80 and 86% of the intensity in untreated controls, respectively. Furthermore, both higher and lower molecular weight immunoreactive RAGE bands disappeared in cotransfectants overexpressing RAGE and SERP1/RAMP4 . In contrast, overexpression of Sec61β instead of SERP1/RAMP4 did not have a similar effect on RAGE and overexpression of SERP1/RAMP4 did not stabilize cytosolic proteins, such as IκBα, which also undergoes degradation in response to stress conditions under study (data not shown). 293 cells transfected with pcDNA3/RAGE were labeled with [ 35 S]methionine for 30 min after treatment of cell cultures with tunicamycin or A23187. Immunoprecipitation of RAGE resulted in the appearance of the expected immunoreactive species . However, RAGE present after 60 min into the chase period was substantially reduced in tunicamycin or A23187-treated cells (32 and 5% of untreated control, respectively), presumably due to degradation . Cotransfection with pcDNA/SERP1/RAMP4, along with pcDNA3/RAGE, maintained, at least in part, levels of RAGE in cells exposed to tunicamycin (∼73% of untreated control) or A23187 . Similar results were obtained when pcDNA3/CD8 was transfected instead of pcDNA3/RAGE . In view of the enhanced expression of SERP1/RAMP4 in both H and R, and its homology with the yeast protein YSY6p , further studies were performed to assess the contribution of SERP1/RAMP4 to protein refolding (glycosylation) after stress. SERP1/RAMP4 overexpression had no detectable effect on the glycosylation of RAGE in the presence of tunicamycin . However, metabolic labeling with [ 35 S]methionine for 3 h using pcDNA3/RAGE-transfected 293 cells after quenching tunicamycin treatment demonstrated that SERP1/RAMP4 overexpression not only stabilized unglycosylated RAGE but also facilitated, at least in part, the glycosylation of RAGE . These effects of SERP1/RAMP4 overexpression during and after the stress were not due to induction of the OST complex, the enzymes that are required for (N-linked) glycosylation (data not shown). To analyze the mechanism underlying the chaperon-like functions of SERP1/RAMP4 (by stabilizing and facilitating glycosylation of membrane proteins) under and after ER stress, we first studied the interaction of SERP1/RAMP4 with integral membrane protein RAGE. However, co-IP of 293 cells transfected with pcDNA3/RAGE and pcDNA3/SERP1/RAMP4 did not show a complex comprised of both molecules under either normal and stress conditions. Next, the binding of SERP1/RAMP4 to Sec61 complex was examined, since RAMP4 was originally copurified with Sec61 complex . Co-IP study using whole cell lysates in NP-40–containing buffer or co-IP after cross-linking of membrane fractions revealed that SERP1/RAMP4 formed a stable complex with Sec61β both in transfected cells and under endogenous conditions. In Fig. 6 b, lane 4, the two major bands at ∼42 and 27 kD may represent the heterotrimer and heterodimer of Sec61β and SERP1/RAMP4 complex based on the molecular masses of both molecules. It should be noted that transfection of cells with pcDNA3/SERP1/RAMP4 resulted in at least 10 times higher expression of proteins, based on immunoprecipitation and Western blotting (data not shown). To rule out the possibility that Sec61β formed a complex with SERP1/RAMP4 after its dissociation from Sec61 complex in NP-40–containing buffer, similar co-IP studies were performed with cell lysates made in the presence of deoxycholate . Under these conditions, not only Sec61β but also Sec61α was coimmunoprecipitated with SERP1/RAMP4. Formation of a complex between RAGE and Sec61 complex was also studied by co-IP; Sec61β–RAGE complex was detected in response to stress in both NP-40– and deoxycholate-containing buffer, although Sec61α–RAGE binding was detected only in the presence of deoxycholate . SERP1/RAMP4 also became associated with calnexin, which is a membrane protein and a molecular chaperon in ER . Such an interaction occurred under control (endogenous) conditions and after overexpression of SERP1/RAMP4. In these co-IP studies, ∼10 and 15% of Sec61β was coimmunoprecipitated with SERP1 and unglycosylated RAGE, respectively (data not shown). A key facet of the cellular response to the environmental stress imposed by oxygen deprivation appears to be induction of molecular chaperons. In this context, previous studies from our and other laboratories have demonstrated induction of molecular chaperons in ER, GRP78, GRP94, and a novel 150-kD polypeptide termed ORP150 in hypoxic and ischemic conditions. Suppression of ORP150 transcripts in oxygen-deprived cultures of human embryonic kidney cells increased their vulnerability to H-induced apoptosis , suggesting that a stress response in ER might have a important role in adaptation to ischemic challenge. The current studies describe another polypeptide, SERP1, whose expression is increased by H/R although the induction is preceded by those of molecular chaperons in ER, and which participates in the ER stress response due to the accumulation of unfolded proteins in the ER. SERP1 encodes a polypeptide localized to the ER and comprised of only 66 amino acids, which was identical to RAMP4. RAMP4 was originally copurified with the core component of the protein-translocation machinery of the ER, the Sec61 complex, and it was recently reported that RAMP4 controls the glycosylation of major histocompatbility complex class II–associated invariant chain . Sec61 is a complex composed of α, β, and γ subunits, which assemble into a channel through which nascent proteins enter the ER . A putative transmembrane-spanning domain at the COOH terminus probably anchors SERP1/RAMP4 in the ER membrane. The orientation of SERP1/RAMP4 in the ER is likely to present the NH 2 terminus to the cytosol, as predicted by previous studies of other similar proteins , and consistent with the apparent lack of glycosylation of the putative N-glycosylation site at the SERP1/RAMP4 NH 2 terminus. SERP1/RAMP4 displays homology with yeast YSY6p, identified based on its capacity to suppress the defect in protein export of a SecY (Sec61 homologue in bacteria) mutant . The identity between SERP1 and RAMP4, and the homology between SERP1 and YSY6, as well as induction of SERP1 in response to ER stress at mRNA and protein levels, suggested the hypothesis that SERP1 might participate in the biosynthesis or degradation of secretory and membrane proteins. To evaluate the effect of SERP1/RAMP4 on protein processing, experiments were performed to address whether SERP1/RAMP4 has any effect on stabilization and refolding of membrane proteins during stress conditions (i.e., ER stress). Induction of ER stress with tunicamycin or A23187 in 293 cells promoted aggregation and degradation of two integral membrane proteins, RAGE and CD8, members of the Ig superfamily of cell surface molecules with a single transmembrane-spanning domain. Cotransfection of 293 cells to overexpress SERP1/RAMP4 prevented accelerated aggregation and degradation of RAGE and CD8 in the setting of ER stress . Although it is not yet clear whether these effects of SERP1/RAMP4 occurred by directly preventing the degradation of membrane proteins or by promoting their refolding in the ER, our results support the latter concept, because SERP1/RAMP4 did facilitate, at least in part, glycosylation of integral membrane proteins in the ER after ER stress was relieved without induction of the OST. Co-IP and cross-linking studies revealed that SERP1/RAMP4 interacts with the Sec61α and Sec61β subunits, which bind to RAGE (and by analogy, other membrane proteins) in response to cellular perturbation . It is important to note that SERP1/RAMP4 is likely to interact with other ER proteins in addition to Sec61α and Sec61β, as indicated by the presence of several bands after immunoblotting of SERP1/RAMP4 after cross-linking . However, in a previous cross-linking study, RAMP4 was not detected in a complex with Sec61β . We speculate that the reason for this difference with our results is likely to derive from the use of different cross-linking agents. Kalies et al. 1998 used bis-maleimidohexane, which depends on optimal juxtaposition of cysteines, whereas our study used DSS, which does not interact with sulfhydryl groups. SERP1/RAMP4 is coprecipitated with calnexin , a membrane protein and a molecular chaperon in ER known to associate with folding intermediates of glycoproteins (monomeric glycoproteins) and believed to play a major role for the quality control apparatus in ER . Together these results suggest that the stabilization of membrane proteins during and after ER stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, components of the translocon such as Sec61α and Sec61β, and ER chaperons, such as calnexin. Increased expression of components of the Sec61 complex begins relatively late during H (>12 h) and is sustained during R. This suggests that induction of the Sec61 complex probably has an integral role for increased biosynthesis of luminal and membrane proteins in replacement of those damaged during stress. It is noteworthy that expression of SERP1/RAMP4 parallels that of components of the Sec61 complex and not that of luminal ER chaperons, which declines during R. This may indicate that the functions of the Sec61 complex and SERP1/RAMP4 during de novo protein synthesis are tightly coupled. These considerations underline the importance of further studies to determine the detailed function of SERP1/RAMP4 in the context of cell stress, and the contribution of SERP1/RAMP4 to cellular processing of native polypeptides or disease-related proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR) or amyloid-beta precursor protein (APP) . | Study | biomedical | en | 0.999997 |
10601335 | Rabbit polyclonal antibodies against p115 were generated by immunization with purified rat p115 . For affinity purification of antibodies, purified GST-p115 fusion protein was run on 7.5% acrylamide gels, and transferred to nitrocellulose. Strips of nitrocellulose containing p115 were incubated with immune serum in PBS, 5% dried milk, and 0.1% Tween 20 for 3 h at room temperature. Bound antibodies were eluted with 0.1 M glycine, pH 3.0, and then neutralized with 1/10 vol 1 M phosphate buffer, pH 7.4. These anti–p115 antibodies were either used in transport assays or cross-linked to protein A–Sepharose 4 FF (Pharmacia Biotech, Inc.) with dimethyl pimelimidate (Pierce Chemical Co.) according to the manufacturer's protocol, and subsequently used for p115 immunodepletion experiments. Mouse polyclonal antibodies against GM130 were generated by immunization with inclusion bodies from bacteria expressing full-length GM130 . Anti-giantin monoclonal G1/133 and anti–ERGIC-53 monoclonal G1/93 were provided by Dr. Hans-Peter Hauri (University of Basel, Basel, Switzerland). Polyclonal antibodies against Mann II were kindly provided by Dr. Marilyn Farquhar (University of California, San Diego, CA). mAbs against Mann II were from Sigma Chemical Co. Anti–rab1 antibodies were a gift of Dr. Mark McNiven (Mayo Foundation, Rochester, MN). Polyclonal antibodies against Mann I were provided by Dr. Kelley Moreman (University of Georgia, Athens, GA). Monoclonal anti–VSV-G protein (P5D4) was provided by Dr. Kathryn Howell (University of Colorado, Denver, CO). Goat anti–rat and anti–mouse antibodies conjugated with FITC or rhodamine were purchased from Jackson ImmunoResearch. Full-length p115 was cloned into the BamHI-NotI restriction sites of the pGEX-6P-2 GST vector (Pharmacia Biotech, Inc.). p115 was obtained by PCR using as a template a pBluescript-II-KS plasmid (Stratagene) encoding rat p115 cDNA previously described . GST-p115 fusion protein expression and purification were performed according to the manufacturer's protocol. p115 was affinity-purified from rat liver cytosol using affinity-purified polyclonal antibodies raised against p115 cross-linked to beads as previously described . The p115 preparation was judged homogenous by Coomassie blue staining. Ascites raised against p115 (3A10) were concentrated by centrifugation in a Microcon 30 (Pierce Chemical Co.) and dialyzed into MI buffer (48 mM K 2 HPO 4 , 140 mM NaH 2 PO 4 , 4.5 mM KH 2 PO 4 , pH 7.2). Denaturation was by boiling to 100°C for 3 min. Before injection, solutions were centrifuged at 15,800 g for 20 min, and then mixed with 1/10 vol of Texas red–dextran (TR-dextran, 10,000 D, 50 mg/ml) and 10× MI buffer. Injections into the cytoplasm of WIF-B cells were performed with an Eppendorf transjector 5246 and micromanipulator 5171 attached to a Zeiss Axiovert 100 microscope. The pressure was 50 hectoPascal for 0.1–0.2 s using an Eppendorf femtotip needle. After injection, cells were incubated with Ham's F12 medium (Gibco Laboratories) at 37°C for 2–3 h, fixed, and processed for immunofluorescence. In some experiments, 2.5 μg/ml BFA was used. Fluorescent images were collected with the MCID analysis system (Zeiss Axiovert 100 microscope) or with a Zeiss confocal microscope (LSM410). Digitized images were cropped, assembled, and labeled in Adobe Photoshop. The ER to Golgi transport assay was performed as described previously . In brief, normal rat kidney (NRK) cells, or LEC-1 (a CHO-derived cell line deficient in NAGT-1 and analogous to CHO15B) cells were grown on 10-cm petri dishes to 80–90% of confluence and infected with the temperature-sensitive strain of the vesicular stomatitis virus, VSVtsO45 at 32°C for 3–4 h . The cells were pulse-labeled with 35 S-trans label (200 mCi/ml; ICN) at the restrictive temperature (42°C) for 10 min, chased with complete medium for 5 min, and perforated by hypotonic swelling and scraping to make semi-intact cells. A transport reaction was performed in a final total volume of 40 μl in a buffer that contained 25 mM Hepes-KOH, pH 7.2, 75 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM EGTA, 1.8 mM CaCl 2 , 1 mM N -acetylglucosamine, ATP regeneration system (1 mM ATP, 5 mM creatine phosphate, and 0.2 IU rabbit muscle creatine phosphokinase), 5 μl rat liver cytosol, 5 μl of semi-intact cells in 50 mM Hepes-KOH, pH 7.2, and 90 mM potassium acetate. Transport was initiated by transfer of cells to 32°C. After 90 min of incubation, cells were pelleted, resuspended in appropriated buffer, and digested with endoglycosidase H (endo-H) or endoglycosidase D (endo-D) as described previously . The samples were analyzed on 8% SDS-PAGE and fluorography. The transport was quantified using GS-700 imaging densitometer (Bio-Rad Laboratories). For antibody inhibition of transport assay, affinity-purified p115 antibodies were added into the complete assay cocktail and incubated on ice for 30 min before use. Staging experiments were performed as previously described . Rat liver cytosol was prepared as previously described . Immunopurified anti–p115 antibodies cross-linked to protein A–Sepharose were incubated (2 h at 4°C) with rat liver cytosol, and the level of p115 depletion was tested by SDS-PAGE and immunoblotting. This analysis was performed as described previously . In brief, NRK cells plated on coverslips were infected with VSVtsO45 at 32°C for 30 min, followed by an incubation at 42°C for 3 h, and then shifted to ice and permeabilized with digitonin (20 mg/ml). Coverslips were incubated in various transport cocktails at 32°C for 90 min. For antibody inhibition of the transport assay, affinity-purified p115 antibodies were added into the complete transport mix and incubated on ice for 30 min followed by an incubation at 32°C for 90 min. Transport was determined by transferring coverslips to ice and fixing them in 3% formaldehyde/PBS for 10 min. The coverslips were processed for double label immunofluorescence. Cells grown on glass coverslips were washed three times in PBS and fixed in 3% paraformaldehyde in PBS for 10 min at room temperature. Paraformaldehyde was quenched with 10 mM ammonium chloride, and cells were permeabilized with PBS, 0.1% Triton X-100 for 7 min at room temperature. The coverslips were washed (three times, 2 min per wash) with PBS, and blocked in PBS, 0.4% fish skin gelatin, 0.2% Tween 20 for 5 min, followed by blocking in PBS, 2.5% goat serum, 0.2% Tween for 5 min. Cells were incubated with primary antibody diluted in PBS, 0.4% fish skin gelatin, and 0.2% Tween 20 for 45 min at 37°C. Coverslips were washed (five times, 5 min per wash) with PBS and 0.2% Tween 20. Secondary antibodies coupled to FITC or rhodamine were diluted in 2.5% goat serum and incubated on coverslips for 30 min at 37°C. Coverslips were washed with PBS and 0.2% Tween 20 as above, and mounted on slides in 9:1 glycerol/PBS with 0.1% q-phenylenediamine. Fluorescence patterns were visualized with an Olympus IX70 epifluorescence microscope. Optical sections were captured with a CCD high resolution camera equipped with a camera/computer interface. Images were analyzed with a power Mac 9500/132 computer using IPLab Spectrum software (Scanalytics Inc.). The requirement for p115 has been previously studied only in in vitro–reconstituted assays . To examine the function of p115 in vivo, monoclonal anti–p115 antibodies were microinjected into WIF-B cells , and their effect on Golgi structure was monitored by the distribution of Mann II. As shown in Fig. 1 , injection of anti–p115 antibodies had an effect on Golgi structure. A field of cells is shown by phase-contrast (A), and the injected cells are identified by their content of coinjected TR-dextran (B). Within 2 h after injection, the Golgi complexes in all injected cells were disassembled, and Mann II was present in relatively large punctate structures dispersed throughout the cells (C). The structures appeared superficially similar to those observed in cells transfected with mutant forms of p115 lacking portions of the globular head or the coiled-coil tail . Injection of control antibodies against the bile canalicular plasma membrane protein 5′-nucleotidase did not perturb Golgi structure (D–F). ER to Golgi traffic can be disrupted by brefeldin A (BFA), which inhibits a guanine nucleotide exchange factor for ADP ribosylation factor; thus, preventing assembly of COP I coats, and ultimately blocking normal traffic between ER and the Golgi . This results in COP I–independent tubulation of the Golgi and the relocation of Golgi proteins into the ER . BFA-induced membrane redistribution has given valuable insights into components and mechanisms involved in regulation of the secretory pathway , and we combined BFA treatments with microinjection of anti–p115 antibodies to characterize the effect of anti–p115 antibodies on ER–Golgi traffic. The redistribution of Mann II from the Golgi into the ER in control WIF-B cells or in cells injected with anti–p115 antibodies was examined first. As shown in Fig. 2 , Mann II was detected in a normal perinuclear Golgi pattern in uninjected cells or in injected cells immediately after the antibody injection and before BFA treatment (A). The injected cells were identified by the presence of anti–p115 IgG (B). When an analogous field of cells was treated with BFA for 30 min, Mann II relocated from the Golgi to the ER in uninjected and injected cells (C). Only a single time point was analyzed, and it remains possible that the kinetics of Mann II redistribution may have varied in injected and uninjected cells. Some Golgi staining was still evident in uninjected and injected cells after the BFA treatment (C, arrowheads). After 30 min, the majority of anti–p115 IgG was associated with membranes, presumably representing p115 localization (D), as shown previously . The results suggest that anti–p115 antibodies do not block the Golgi to ER redistribution of Mann II induced by BFA treatment. To examine if the antibodies inhibit the movement of Mann II from the ER, WIF-B cells were first treated with BFA for 30 min to relocate Mann II from the Golgi to the ER, and then injected with anti–p115 antibodies. The temporal sequence of Golgi recovery after BFA wash-out was followed. As shown in Fig. 3 A, punctate structures dispersed throughout the cell were detected after a 10-min BFA wash-out in both, uninjected and injected cells. The size and distribution of the elements were comparable, suggesting that the antibodies do not block early stages of BFA recovery. A difference was seen after 30 min of BFA wash-out, when defined Golgi structures could be seen in uninjected cells (C, arrows), while significantly smaller, dispersed structures were present in the injected cells (C, arrowheads). The effect was also evident after 120 min of BFA wash-out, when compact Golgi structures were seen in uninjected cells (E, arrows), whereas dispersed punctate structures persisted in the injected cells (E, arrowheads). In some injected cells, a more Golgi-like Mann II pattern was seen (E, asterisk), but the structure appeared less compact and organized. These results suggest that anti–p115 antibodies prevent the reassembly of normal Golgi complexes. The inhibitory effects of anti–p115 antibodies on the maintenance and reassembly of normal Golgi structure, coupled with the previous finding that p115 is present on VTCs and cycles between the Golgi and earlier secretory compartments , suggested a potential role for p115 in ER to Golgi transport. To analyze if p115 is present on functional VTCs transporting cargo from the ER to the Golgi, we used a temperature-sensitive strain of the vesicular stomatitis virus (VSVtsO45) as a transport marker . The viral VSV-G protein fails to exit the ER at 42°C, the nonpermissive temperature, but after shifting the cells to the permissive temperature of 32°C, a wave of VSV-G protein enters the secretory pathway, and its movement from the ER to the Golgi can be monitored morphologically . NRK cells infected with the virus and cultured at the nonpermissive temperature for 3 h contain VSV-G protein in the ER, whereas p115 is predominantly detected in the Golgi region . When infected cells were subsequently shifted from 42 to 15°C and incubated at 15°C for 3 h, VSV-G protein movement to the Golgi was arrested in peripheral VTCs that also contained p115 . We have shown previously that p115 colocalizes with the VTC marker, ERGIC-53 when VTCs are preferentially accumulated during low (15°C) temperature incubation . Bonafide Golgi proteins such as galactosyl-transferase or Mann II do not redistribute to peripheral VTCs after low temperature treatment . When infected cells were shifted from 42 to 32°C for 1 h, VSV-G protein and p115 colocalized in the Golgi . These results indicate that p115 is a component of VTCs that move VSV-G protein from the ER to the Golgi, and raise the possibility that p115 could be involved in VTC dynamics. Although p115 has been identified as a cis- to medial-Golgi transport factor , its yeast homologue, Uso1p, has been shown to act in ER to Golgi traffic . To examine directly p115 participation in ER to Golgi traffic, we used a previously developed semi-intact cell transport assay . NRK cells were infected with VSVtsO45 and radiolabeled at 42°C. Cells were permeabilized to remove endogenous cytosol, supplemented with exogenous transport cocktails, and shifted to 32°C to initiate VSV-G protein transport. Delivery of VSV-G protein to the Golgi was assessed by its carbohydrate processing, as defined by endo-H resistance. VSV-G protein oligosaccharide chains are processed during transport by the sequential actions of enzymes localized throughout the Golgi stack. The processing involves the sequential function of Mann I and N -acetylglucosamine transferase I (NAGT-1), both considered cis-Golgi enzymes , and of Mann II, localized in the medial/trans Golgi stack . After processing by Mann I, VSV-G acquires endo-D sensitivity, whereas subsequent processing by NAGT-1 and Mann II confers endo-H resistance. As shown in Fig. 5 A, when complete transport cocktail was added to permeabilized cells, ∼50% of VSV-G protein was processed to an endo-H–resistant form (top band, lane 2), and this is set as 100% processing. The percent processing is analogous to the level of processing reported previously . In contrast, when transport was analyzed with an ATP-depleting system, VSV-G protein remained sensitive to endo-H (bottom band, lane 1), and this is set as 0% processing. The addition of increasing amounts of affinity-purified anti–p115 antibodies (from 0.1 to 0.8 μg) led to a dose-dependent inhibition of VSV-G protein processing to the endo-H–resistant form (lanes 3–6). To provide quantitative data on VSV-G processing, analogous data from repeated experiments ( n = 3) were evaluated by densitometry, and the average of relative percent is presented in the accompanying bar graph. The relative processing was reduced by 15% in the presence of 0.1 μg of antibody with >80% inhibition when 0.4 μg of anti–p115 antibodies were added. When preimmune antibodies were added to the transport assay, normal processing of VSV-G protein was observed (data not shown). To ensure that the inhibitory effects of the anti–p115 antibodies were due to an interaction with p115, the antibodies were preincubated either with GST-p115 fusion protein or GST transferred to nitrocellulose strips. The nonbound fractions were tested for inhibitory activity in the semi-intact transport assay. As shown in Fig. 5 B, lane 3, preincubation of the antibody with GST-p115 nitrocellulose strips efficiently neutralized its inhibitory effect on traffic, and processing of VSV-G protein to the endo-H–resistant form was comparable to the control situation with complete cytosol (lane 2). In contrast, inhibition was still apparent with antibodies incubated with GST strips (lane 4), and processing of VSV-G protein to the endo-H–resistant form was comparable to that in the absence of ATP (lane 1). These results suggest that anti–p115 antibodies block ER to Golgi transport through a specific interaction with p115. To extend these findings, VSV-G protein transport in the presence of limiting amounts of p115 was analyzed. In the semi-intact cell transport assay, exogenous rat liver cytosol must be added to provide cytosolic and peripheral membrane proteins released during permeabilization . p115 is peripherally associated with membranes, and during cell disruption, is largely released into the cytosol . Rat liver cytosol contains high amounts of p115 (∼0.5 μg/mg). To examine if such exogenously added p115 is required for transport, p115 was removed from the cytosol by immunodepletion. The extent of immunodepletion was analyzed by immunoblotting . The immunoblot was evaluated by densitometry, and the relative amount of p115 present is shown in the accompanying bar graph. Up to 60% of cytosolic p115 was removed in lane 4. When such p115-depleted cytosol was used in the transport assay, VSV-G protein processing to the endo-H–resistant form was inhibited by ∼90%. Immunodepletion of cytosol with nonspecific antibodies (affinity-purified rabbit anti–goat IgG, lane 3) or preimmune serum (data not shown) had no effect on VSV-G protein transport. Reactions containing complete transport cocktail (lane 2) or cocktail containing an ATP-depleting system (lane 1) were analyzed as positive and negative controls and defined as 100 and 0% VSV-G protein processing, respectively. To examine if addition of purified p115 could rescue transport, p115-depleted cytosol (analogous to that in lane 4) was supplemented with increasing amounts of purified p115. p115 was purified from rat liver cytosol on affinity-purified anti–p115 antibodies cross-linked to a protein A–Sepharose column. As shown in Fig. 5 D, the material eluted from the anti–p115 IgG column (lane 1) contains a band of ∼110 kD, whereas no such band was eluted from a control preimmune IgG column (lane 2). To ensure that the 110-kD band was p115, the 110-kD region was excised from a gel, subjected to tryptic digestion and the peptides were separated and characterized by MALDI mass spectrometry combined with sequence database searching. The peptide mass map of 10 major peptides match with p115, and no other rat protein in that molecular mass range was found in the search. Analogous purified p115 was added to the p115 depleted cytosol. As shown in Fig. 5 C, p115 panel, lanes 5–7, and bar graph, the supplemented cytosol contained 110, ∼140, and ∼160% of p115 in untreated cytosol, respectively. When such supplemented cytosols were used in the transport assay, VSV-G protein was processed to the endo-H–resistant form with ∼40, ∼50, and ∼100% processing efficiency, respectively (VSV-G panel, lanes 5–7, and bar graph). Taken together, these results indicate that p115 is essential for VSV-G protein delivery from the ER to the Golgi. Previous work suggested that p115 functions to tether donor and acceptor membranes before membrane fusion , but did not place the p115 requirement relative to requirements for other known transport factors. Studies on Uso1p suggested that its function is regulated by Ypt1p , and Ypt1p and its mammalian homologue rab1 are essential for ER to Golgi transport . To order the sequence of rab1- and p115-requiring transport steps in mammalian cells, staging experiments were performed. In the first stage, p115-depleted cytosol was added to the semi-intact cell transport assay, and the cells were incubated at the permissive temperature for 60 min to allow VSV-G protein to accumulate in the p115-arrested compartment. The cells were collected, and in the second stage, incubated with either p115-depleted cytosol, complete cytosol, rab1-depleted cytosol, or complete cytosol supplemented with anti–rab1 antibodies. Rab1-depleted cytosol contained <5% of the rab1 found in untreated cytosol (data not shown), and 3 μg of anti–rab antibodies were used, an amount similar to that shown previously to be inhibitory in the semi-intact cell transport assay . As shown in Fig. 6 A (gel and bar graph, lane 1), when p115-depleted cytosol was used in both stages of the transport assay, <10% of VSV-G protein was processed to the endo-H–resistant form. This represents the background of the assay and is set as 0% relative processing. When complete cytosol (lane 2) was added in the second stage, ∼35% of VSV-G protein was transported to the Golgi and acquired endo-H resistance (set as 100% relative processing), the same extent as when completed cytosol was used in both stages (data not shown). Significantly, when rab1-depleted cytosol (lane 3), or complete cytosol containing anti–rab1 antibodies (lane 4) were used, comparable ∼90% level of relative processing was observed, indicating that progression of VSV-G protein from the p115–depletion blocked compartment to the Golgi does not require rab1. In agreement with previous results showing partial inhibition after rab1 depletion , when stages I and II were performed with rab1-depleted cytosol, VSV-G transport was blocked by ∼60% (lane 5). When complete cytosol was added in the second stage (lane 6), VSV-G protein processing was >80% of that in lane 2. In contrast, when p115-depleted cytosol was added in the second stage (lane 7), VSV-G protein processing was inhibited by ∼60%, the same extent as when rab1-depleted cytosol was used in both stages (lane 5). This suggests that movement of VSV-G protein from the rab1–depletion blocked compartment to the Golgi requires p115. Together, the data indicate that rab1 is required before the p115-requiring step of VSV-G protein transport. Using the same ER to Golgi transport assay, it has been shown that Ca 2+ is required at a last transport step before membrane fusion . To determine if addition of anti–p115 antibodies or p115 depletion inhibits ER to Golgi transport at a stage before or after the Ca 2+ requirement, staging experiments were performed. In the first stage, 10 mM EGTA was added to the semi-intact cell transport assay, and the cells were incubated at the permissive temperature for 60 min to allow VSV-G protein to accumulate in the EGTA-arrested compartment. The cells were collected, and in the second stage, incubated with either cytosol containing 10 mM EGTA, complete cytosol, p115-depleted cytosol, or complete cytosol supplemented with anti–p115 antibodies. As shown in Fig. 6 B (gel and bar graph, lane 1), when EGTA was added to both stages of the transport assay, ∼3% of VSV-G protein acquired endo-H resistance. This represents the background of the assay and is set as 0% relative transport. When complete cytosol (lane 2) was added in the second stage, ∼35% of VSV-G protein was processed, and this represents 100% relative processing. The ∼35% processing is analogous to the percent processing observed when complete cytosol is added to both stages of transport (data not shown). Significantly, when p115-depleted cytosol (lane 3), or complete cytosol containing p115 antibodies (lane 4) was added in the second stage, ∼100% relative processing was observed, suggesting that progression of VSV-G protein from the Ca 2+ -arrested compartment to the Golgi does not require p115. This was confirmed by reverse staging. As shown in Fig. 6 B (lanes 5 and 6), addition of p115-depleted cytosol to both stages of the transport assay resulted in ∼15% of VSV-G protein acquiring endo-H resistance (∼40% relative processing). (In these experiments, p115 depletion was incomplete [data not shown] and accounts for the incomplete inhibition of VSV-G protein transport.) When complete cytosol was added in the second stage (lane 7), ∼30% of VSV-G protein was processed (∼90% relative processing). When cytosol containing EGTA was added in the second stage, ∼5% of the VSV-G protein was transported to the Golgi and acquired endo-H resistance (∼10% relative processing) (lane 8). This suggests that movement of VSV-G protein from the p115–depletion blocked compartment to the Golgi stack requires Ca 2+ . Together, the results indicate that p115 is required before the Ca 2+ -requiring stage of VSV-G protein transport. Morphological examination of VSV-G protein localization in the presence of EGTA suggests that Ca 2+ is required for VTC delivery to the Golgi stack . Our finding that p115 is required at a stage of transport before the Ca 2+ requirement suggested that p115 might also be required at that stage. To define where anti–p115 antibodies and p115 depletion blocked transport of VSV-G protein, morphological transport assays were performed. Addition of anti–p115 antibodies to the morphological transport assay prevented VSV-G protein from moving to the Golgi . A representative experiment (from >10 analyses) is presented. VSV-G protein was not detected in the ER, indicating that anti–p115 antibodies had no effect on VSV-G protein exit from the ER and its delivery to post-ER transport intermediates, but prevented delivery of such intermediates to the Golgi. This resulted in accumulation of VSV-G protein in peripheral VTCs, most of which were labeled with anti–p115 antibodies (C, arrowheads). Anti–p115 antibodies were also detected in the Golgi (C, arrows), perhaps because of incomplete removal of p115 from membranes during permeabilization. Alternatively, anti–p115 antibodies might act by trapping p115 on Golgi membranes through the formation of inactive complexes. Addition of equivalent amounts of preimmune antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi, where it colocalized with p115 (D–F). The same pattern was observed when monoclonal anti–Mann II antibodies were added to the reaction (data not shown). When p115-depleted cytosol was used in the morphological transport assay, VSV-G protein was also not transported to the Golgi, as evidenced by its lack of colocalization with Mann II . Two representative cells from two different experiments are shown in these panels. VSV-G protein localized predominantly to peripheral VTCs, indistinguishable from those seen in the presence of anti–p115 antibodies . VSV-G protein was almost exclusively in peripheral VTCs, and was not present to any significant extent in the ER, confirming that p115 is not involved in ER exit and delivery of cargo to more distal transport intermediates. VSV-G protein was efficiently transported to the Golgi when complete cytosol was used . Taken together, these results indicate that p115 function is required after the formation of post-ER VTCs but before their delivery to the Golgi stack. This defines a novel function for p115, and identifies the first step of membrane transport in which p115 participates. Since addition of anti–p115 antibodies or p115 depletion inhibits VSV-G protein delivery to the Golgi and causes its accumulation in peripheral VTCs, we expected that the arrested VSV-G protein will not be processed by any of the Golgi glycosyl-modifying enzymes. In agreement, our data show that the arrested VSV-G protein remains endo-H sensitive, indicating a lack of processing by NAGT-1 and Mann II. To examine whether the arrested VSV-G protein is processed by the cis-Golgi enzyme Mann I, endo-D resistance/sensitivity was analyzed in LEC-1 and NRK cells. Mutant LEC-1 cells that lack NAGT-1 were used first because in these cells oligosaccharide modifications stop after Mann I processing . VSV-G protein is endo-D resistant while in the ER and becomes endo-D sensitive after being transported and processed by Mann I. When infected cells were permeabilized and used in the semi-intact cell transport assay in the presence of an ATP-depleting system, VSV-G protein was endo-D resistant . When complete transport cocktail was used, a proportion (∼60%) of VSV-G protein became endo-D sensitive, and this is taken as the standard to which other reactions are compared . Unexpectedly, VSV-G protein was processed to the endo-D–sensitive form when anti–p115 antibodies were added to the transport assay in an amount analogous to that found to block acquisition of endo-H resistance in NRK cells . Addition of control antibodies had no effect on VSV-G protein processing . Similarly, when p115-depleted cytosol was used in the transport assay, VSV-G protein was processed to the endo-D–sensitive form . Even a significant depletion of p115 (>80% of p115 was depleted in lanes 5 and 6 as compared with lane 2) had no effect on the amount of VSV-G protein sensitive to endo-D. The level of p115 (∼20% of control) that supported the processing to the endo-D–sensitive form in LEC-1 cells was unable to support processing to the endo-H–resistant form in NRK cells . The level of endo-D processing in reactions containing cytosol immunodepleted with anti–p115 antibodies was similar to that when control or preimmune antibodies were used for immunodepletion. To test VSV-G protein processing by Mann I while in pre-Golgi transport intermediates, we arrested VSV-G protein transport by incubating the LEC-1 cells at 15°C . As shown in Fig. 9 C, VSV-G protein is resistant to endo-D when cells are incubated at 42°C (lanes 1 and 4) and becomes endo-D sensitive when incubated at 32°C (lanes 2 and 5). When cells were incubated at 15°C, ∼70% of VSV-G protein was endo-D sensitive (lanes 3 and 6), suggesting that the VSV-G protein was processed by Mann I. Analogous experiments were performed in NRK cells, but both endo-H and endo-D resistance/sensitivity of VSV-G protein arrested by various treatments were tested. These digestions were done in parallel since only endo-D sensitivity of an endo-H–sensitive form of VSV-G will define whether VSV-G protein is processed by Mann I but not NAGT-1 and Mann II. As shown in Fig. 9 D, when transport was performed in the presence of an ATP-depleted transport cocktail, VSV-G protein is endo-H sensitive (lane 1) and endo-D resistant (lane 5). When transport was performed in the presence of a complete transport cocktail, VSV-G becomes endo-H resistant (lane 2) and is endo-D resistant (lane 6). When affinity-purified anti–p115 antibodies were added to the complete transport assay, VSV-G protein is endo-H sensitive (lane 3) and endo-D resistant (lane 7). These results differ from the results of analogous experiments performed in LEC-1 cells , in which addition of affinity-purified anti–p115 antibodies leads to VSV-G protein that is endo-D sensitive. In an attempt to clarify this difference, we analyzed the endo-H and endo-D sensitivity of VSV-G protein arrested in peripheral VTCs when NRK cells are incubated at 15°C. As shown in Fig. 9 D, lanes 4 and 8, VSV-G protein was endo-H sensitive and endo-D resistant. This result differs from that in LEC-1 cells, where an endo-D–sensitive form was observed after 15°C incubation. Together, the data show that endo-D–sensitive VSV-G protein is seen in LEC-1 cells in the presence of anti–p115 antibodies or when cells are incubated at 15°C, whereas the same treatments of NRK cells result in a VSV-G protein that is endo-D resistant. A likely explanation for this difference is that the lack of p115 requirement in LEC-1 cells is due to incomplete inactivation/removal of endogenous p115 from those cells as opposed to NRK cells. Alternatively, since we observe a difference in VSV-G protein endo-D sensitivity in intact LEC-1 and NRK incubated at 15°C, oligosaccharide processing of VSV-G protein might be different in the two cell lines. Currently, we have no explanation for the different results in LEC-1 and NRK cells, and cannot define whether p115 is required for transport of VSV-G protein into a Mann I–containing compartment. The differences in the biochemical results in LEC-1 and NRK cells prompted us to examine Mann I localization in traffic-arrested NRK cells since all previous morphological analyses have been done in NRK cells. Previous EM immunoperoxidase studies indicated that in NRK cells, Mann II is localized in the medial-cisternae, whereas Mann I is localized in the trans-cisternae and the TGN, and at the light immunofluorescence level, both Mann II and Mann I are concentrated in the Golgi region . In agreement, Mann I and II colocalized in the Golgi region in cells incubated at 37°C, although Mann I was also present in a more diffuse non-Golgi pattern . Significantly, Mann I redistributed from the Golgi to peripheral punctate structures when cells were incubated at 15°C, whereas Mann II did not show such redistribution . To characterize these pre-Golgi elements, VSVtsO45-infected cells were shifted from 40 to 15°C for 3 h, and then processed to localize Mann I and VSV-G protein. As shown in Fig. 10G–I , Mann I redistributed from the Golgi to peripheral structures, some of which (arrowheads, I) contained VSV-G protein. In contrast, Mann II did not show redistribution . To determine whether Mann I can redistribute to pre-Golgi VTCs arrested by p115 depletion, semi-intact cells incubated with transport assays supplemented with complete cytosol or with p115-depleted cytosol were processed for double label immunofluorescence to localize VSV-G protein and Mann I. As shown in Fig. 10M–O , when transport was performed with complete cytosol, Mann I was detected in a typical perinuclear Golgi structure, where it colocalized with VSV-G protein. In contrast, when transport was performed with p115-depleted cytosol, Mann I was found colocalizing with peripheral VTCs . As already shown in Fig. 8 , Mann II did not show such relocation. Together, these results indicate that Mann I can cycle to pre-Golgi compartments during cargo transit from the ER to the Golgi. Furthermore, our data show that p115 is not required for events before and including the recycling of Mann I from the Golgi to peripheral compartments, but is essential for the subsequent events leading to delivery of cargo and Mann I to the Golgi. Functional p115 appears to be required for normal Golgi morphology in intact cells since injection of anti–p115 antibodies into WIF-B cells resulted in the appearance of Golgi fragments scattered throughout the injected cells. This antibody effect appears specific since microinjection of anti–5′-nucleotidase antibodies does not lead to changes in Golgi morphology. Normal Golgi architecture varies in different cells, but seems to be dependent on the balance of incoming and outgoing membrane traffic through the structure. Perturbation of the anterograde or retrograde traffic by various treatments of cells leads to Golgi disruption. Specifically, changes in Golgi morphology were observed in cells overexpressing ADP-ribosylation factor , injected with rab1a mutant , or anti–β-COP antibodies . Similarly, treatment of cells with BFA , nocodazole , or okadaic acid has a dramatic effect on Golgi integrity. The exact mechanism of the disruptive action of anti–p115 antibodies on Golgi structure is currently unknown, but it is possible that the antibodies perturb transport pathways between the ER and the Golgi. Specifically, the antibodies could inhibit p115 activity in vivo by blocking p115 interactions with GM130 , giantin , or another, as yet unidentified, protein. Alternatively, anti–p115 antibodies might act by trapping p115 on the membranes through the formation of inactive complexes. Presence of anti–p115 antibodies in cells did not prevent the redistribution of Mann II to the ER after BFA treatment, but the anti–p115 antibodies prevented the reassembly of Golgi complexes during subsequent BFA wash-out. The requirement for functional p115 manifested late, after exit of Golgi enzymes from the ER and delivery to peripherally scattered punctate structures. Significantly, these structures did not move towards the MTOC and did not coalesce into a centrally located Golgi complex. The effect of anti–p115 antibodies was similar to that of anti–EAGE (anti–β-COP) antibodies, which also interfere with the proper reassembly of compact Golgi complexes in a juxtanuclear region during a BFA wash-out . Although our findings place the functional requirement for p115 in ER to Golgi traffic between the VTCs and the Golgi, it remains to be determined which step of the anterograde or retrograde transport is blocked by anti–p115 antibodies in vivo. The previous finding that p115 is abundant on VTCs was extended in this study to show that p115-containing VTCs are functional and carry cargo VSV-G protein from the ER to the Golgi. The possible involvement of p115 in this part of the secretory pathway was examined using previously established ER to Golgi transport assays that measure biochemically or morphologically the movement of VSV-G protein from the ER to the Golgi in semi-intact cells. Our biochemical results show that anti–p115 antibodies or p115 depletion specifically block VSV-G protein transport before acquisition of endo-H resistance, believed to represent the delivery of VSV-G protein to the cis/medial-cisternae of the Golgi stack. Unexpectedly, morphological examination showed that in the presence of anti–p115 antibodies or p115 depletion, VSV-G protein was not delivered to the Golgi stack, but instead was arrested in pre-Golgi peripheral VTCs. These findings indicate that p115 is not required for VSV-G proteins to efficiently exit the ER and be delivered to morphologically normal peripheral VTCs, but is essential for the subsequent delivery of such VTCs to the Golgi stack. What is the role of p115 in ER to Golgi traffic? Because p115 is predominantly associated with VTCs and p115 depletion arrests transport in VTCs, we favor the hypothesis that p115 functions while on VTCs. A possible role for p115 could be to mediate delivery of essential components from the Golgi to VTCs, by tethering Golgi-derived recycling vesicles to VTCs before fusion. This hypothesis is suggested by the finding that a subpopulation of Golgi-derived COP I vesicles (albeit produced under the nonphysiological conditions of GTPγS treatment) is enriched in giantin, a protein known to bind p115 . Giantin is an extended coiled-coil transmembrane protein and the interaction between p115 on VTCs and giantin on COP I vesicles could theoretically span the distance of >400 nm, raising the possibility that p115 on VTCs might act to catch and tether giantin-containing Golgi-derived COP I vesicles. Fusion of such vesicles would deliver components essential for VTC maturation and subsequent delivery to the Golgi stack. Alternatively, p115 might function to mediate VTC motility on microtubules. This is suggested by the fact that the p115-induced block in ER to Golgi traffic is biochemically and morphologically similar to that caused by 15°C incubation, when dynein/dynactin–mediated movement of VTCs on microtubules is inhibited . Furthermore, VSV-G protein is arrested in peripheral VTCs in cells overexpressing the dynamitin subunit of the dynactin complex , and their pattern is indistinguishable from VTCs arrested by anti–p115 antibody addition or p115 depletion. Overexpression of dynamitin disrupts the dynactin complex, separating the Arp-1 filament from the dynein-binding arm of p150 Glued and prevents microtubule-mediated motility. Interestingly, a link between microtubule movement and rab proteins has been established by the finding that the Golgi-localized rab6 interacts with rabkinesin, a Golgi-associated kinesin-like motor protein that binds to microtubules and is likely to mediate membrane movement along microtubules . Whether rab1 might also have motor proteins (perhaps of the dynein/dynactin family) as one of its effectors remains to be determined. However, it is interesting that p115 structure is similar to that of kinesin (a dimer with globular heads, a central coiled-coil tail, and a tail domain), although p115 does not contain an ATP-binding/hydrolysis domain and does not interact with microtubules (data not shown). It is equally possible that p115 is required to recruit or activate other uncharacterized factors that will ultimately allow VTC transport and fusion. We do not consider it likely that p115 function in VTC dynamics involves interaction with GM130, since GM130 is not detected on VTCs or Golgi-derived COP I vesicles , and appears restricted to the Golgi stack. It is more probable that p115–GM130 interactions occur after the VTCs are delivered to the Golgi stack. Functional p115 was shown to be required in ER to Golgi traffic after the rab1-dependent step of transport. Rab1 has been shown previously to be required after COP II vesicle budding and after the assembly of peripheral VTCs . In semi-intact cells supplemented with the rab1a (N124I) mutant that is defective for guanine nucleotide binding, VSV-G protein has been shown at the ultrastructural level to accumulate in tubular structures with associated coated vesicular profiles , but the morphology of the p115-arrested transport intermediates remains to be analyzed. The behavior of p115 and rab1 in mammalian cells is analogous to that of Uso1p and Ypt1 in yeast, since Ypt1p appears to act upstream of Uso1p, and might be indirectly involved in recruiting Uso1p to the membranes . Whether rab1 performs a similar function for p115 is uncertain since significant differences in the dynamics of transport intermediates (e.g., yeast do not appear to have extensive VTCs) are evident. p115 was found to act before the Ca 2+ -requiring step, which is considered to be the last stage of transport before fusion . SNARE pairing precedes Ca 2+ requirement , and our data are consistent with the reported requirement for Uso1p before SNARE complex formation . This report documents the first instance of a morphologically observed relocation of Mann I from the Golgi stack to peripheral pre-Golgi structures. The relocation is apparent in cells in which ER to Golgi transport is blocked by 15°C incubation or p115 depletion, suggesting that Mann I (like other Golgi proteins) cycles between the Golgi and the ER. The mechanism of Golgi protein recycling is largely undefined. Recycling might occur directly from the Golgi to the ER, since direct delivery via tubular structures is observed during BFA-induced relocation of Golgi proteins, and at least one Golgi glycosylating enzyme, GalT, can be detected in similar tubules under physiological conditions . The BFA-induced tubular relocation of Golgi proteins is COP I–independent . Alternatively, recycling might occur from the Golgi to VTCs. This pathway would be consistent with the biochemical data showing that VSV-G protein arrested in pre-Golgi transport intermediates can be processed by NAGT-1, which recycles via small vesicles from the Golgi to the pre-Golgi intermediates . The recent results showing that NAGT-1 binds coatomer in vitro , and NAGT-1, as well Mann II and Gal-T, bud from the Golgi in COP I–coated vesicles suggest that COP I–coated vesicles might transport Golgi-derived glycosyl transferases to pre-Golgi transport intermediates. Based on the structural similarity of Mann I to other glycosyltransferases (a type II transmembrane protein), it is possible that it also recycles via Golgi-derived COP I–coated vesicles. Previous work has defined the kinetics with which Golgi resident proteins cycle between the Golgi and the ER. Several Golgi proteins such as KDEL-R, TGN38 , and gp27 , and glycosylating enzymes such as N -acetylgalactosaminyltransferase-2 (GalNAc-T2) and GalT have been shown to cycle with half lives between 20 min and 2–3 h . Based on our observation that the majority of Mann I relocates from the Golgi and appears in blocked VTCs during a 90-min incubation at 37°C (the time course of the semi-intact transport assay), it appears that Mann I cycles with relatively fast kinetics. Although the semi-intact cell system used in our studies is not designed to analyze recycling pathways, the results showing relocation of Mann I, but not of Mann II to peripheral VTCs suggests that Mann II cycles with significantly slower kinetics. The recycling kinetics of Mann I seem more similar to those of the ERGIC proteins, KDEL-R and ERGIC53, which cycle rapidly at 37°C. These proteins also relocate to peripheral VTCs during longer (usually ∼3 h) incubations at 15°C. However, Mann I does not appear to be a bonafide component of the ERGIC since EM analysis showed it to be localized throughout the Golgi stack, with highest concentration in the trans-cisternae and the TGN, rather than the cis-face of the stack . This suggests that rapid cycling is not restricted to residents of the ERGIC, and that proteins with steady state distribution in more distal Golgi compartments including the TGN, also contain signals for rapid cycling. Whether the recycling mechanisms are the same and involve COP I vesicles, or are distinct remains to be elucidated. Our morphological results have direct bearing on the controversy regarding the mode of cargo transport through the Golgi. The finding that Mann I efficiently relocates from the Golgi stack to colocalize with peripheral cargo-containing VTCs is less consistent with the anterograde vesicular traffic model than with the maturation model for exocytic traffic. Our results are not consistent with the anterograde vesicular traffic model, in which glycosyl-transferases remain relatively stationary in distinct Golgi cisternae while the cargo is shuttled from proximal to distal cisterna in small vesicles. The observed relocation supports the maturation model, in which cargo progresses through the secretory pathway in a compartment that is remodeled by the sequential recycling of processing enzymes from later Golgi compartments. | Study | biomedical | en | 0.999999 |
10601336 | Yeast cells were grown in standard rich medium (YPD), sporulation, or SD minimal media containing required supplements and 0.2% yeast extract where indicated . Strains used were SEY6210 , 6210 drs2Δ , 6210 arf1Δ , 6210 chc1-ts , PRY6222 ( MATa leu2-3,112 ura3-52 his3- Δ200 trp1-Δ901 ade 2-101 suc2-Δ9 drs2Δ::TRP1 ), GPY1103 , EGY101-16D , LSY93.1-10A , EGY1211-6B , TBY103 ( MATa leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 sec23-1 ), TGY144 ( MAT α leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801 sec1-1 ), TGY1906 ( MATa leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 pan1-20 ), TGY1912 ( MATa leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801 suc2-Δ9 end4-1 ), SEY5185 ( MATa leu2-3,112 ura3-52 sec18-1 ), BHY161 , BHY163 , 6210 vps33 Δ , and 6210 vps35 Δ . To clone SWA3 , strain CCY2808 was transformed with a genomic library and Leu + transformants were selected on synthetic dextrose-Leu media containing 4 μg/ml adenine. After 7–10 d of incubation at 30°C, 50 colonies that appeared to sector (out of 21,000 transformants) were streaked on 5′-fluoroorotic acid (FOA) plates. Library plasmids were rescued from the four strains that grew on FOA and were retransformed into CCY2808. Only the transformants harboring pPR10 were able to sector on YPD at 30°C and were able to grow at 20°C. Partial sequencing indicated that the other three plasmids contained ARF2 . pPR10 contained a fragment of chromosome I from coordinates 94916–104338. Deletion of the BamHI fragment contained solely in DRS2 (to produce pPR10ΔBamHI) destroyed the complementing activity of pPR10. The full-length DRS2 gene contained on a SpeI-SnaBI fragment was subcloned into SpeI-SnaBI digested pRS315 or pRS425 to produce pRS315-DRS2 and pRS425-DRS2. Both plasmids complemented the cold-sensitive growth defect of CCY2808. A drs2 deletion plasmid (pGCR1) was constructed by replacing a BamHI-SnaBI fragment in pPR10 with an ∼1.1-kb BamHI-PvuII fragment containing TRP1 from pJJ280 . The plasmid pGCR1 was linearized with SacI and HpaI and transformed into SEY6210 to produce 6210 drs2Δ . The correct integration event was confirmed by PCR. Site-directed mutagenesis of DRS2 was performed by the megaprimer PCR method . To generate megaprimers coding for either a D to N or D to E mutation at amino acid position 560, reverse primers TCCTGTCTTGTTACTGAATATAT and TCCTGTCTTTTCACTGAATATAT, respectively, were combined with forward primer TTTGTCACCGTTGAATTAATC into a PCR reaction mixture containing pRS315-DRS2. The gel purified ∼150-bp product was added to a second PCR reaction containing the reverse primer TTCATCATCCAATCTTTCCAG and pRS315-DRS2. The resulting ∼650-bp products were digested with BspEI and BglII, and then ligated into BspEI/BglII cut pRS315-DRS2 to produce pDRS2(D560N) and pDRS2(D560E). Sequencing of the resulting plasmids indicated that the specific mutations had been introduced with no additional mutations. The 2-μm KEX2HA plasmid was constructed by subcloning a SalI/EagI digestion fragment of pSN218 , containing the KEX2-HA sequence, into SalI/EagI-digested pRS426 to produce p426KH. The HA.11 mouse monoclonal antibody (Covance) was used at a 1:250 dilution to detect Kex2-HA by immunofluorescence. Cell labeling, immunoprecipitation , FM4-64 uptake , and Ste3p turnover experiments were performed as previously described. Ste3p-myc was detected with 9E10 c-myc antibody (Oncogene Research Products) and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) followed by the enhanced chemiluminescence detection (Amersham Corp.). Subcellular fractionation was performed as previously described , except that cells were converted to spheroplasts and washed twice in buffer containing 0.8 M sorbitol before lysing with a Dounce homogenizer in buffer A containing 0.1 mg/ml RNase A. The cells were preincubated for 1 h at 15° or 30°C, lysed, and subjected to centrifugation at 13,000 g and the resulting supernatant was then centrifuged at 100,000 g . Immunoblots bearing 20 μg of protein from each sample were probed with mouse antibodies to Chc1p , Aps1 and Aps2 , and ARF1 . Clathrin-coated vesicles (CCVs) were prepared from SEY6210 and 6210 drs2Δ as previously described . For 15°C-treated samples, cells from a 3-liter culture grown to 6–8 OD were collected, resuspended into 1 liter of pre-chilled fresh YPD, and incubated at 15°C for 1 h before further processing. After gel filtration chromatography, 1.2 ml of every other fraction (total 5 ml per fraction) from fractions 15–35 were precipitated with TCA (10% final concentration). The precipitates were resuspended in 50 μl Laemmli sample buffer, and one third of each sample was subjected to immunoblotting and probed with antibodies to Chc1p, Mnn1p , and Pep12p . Spheroplasts were prepared for electron microscopy as previously described except that the initial glutaraldehyde fixation was overnight at 4°C. 50–60-nm sections were viewed on an electron microscope (CM12; Philips). Vesicle fractions were similarly prepared, but included the following modifications: 5-ml fractions were fixed overnight at 4°C by addition of 700 μl of 25% glutaraldehyde (3% final concentration). Fixed vesicles were pelleted at 100,000 g for 70 min. The vesicle pellet was washed in 100 mM sodium cacodylate, pH 6.8, 5 mM CaCl 2 , and then stained identically to the cell blocks, with one exception. A 30-min room temperature incubation with 1% tannic acid in 100 mM sodium cacodylate, pH 7.4, was included before en bloc staining with uranyl acetate. Dehydration, embedding, and section poststaining were performed as for the cell samples. A TrpE-DRS2 fusion construct was prepared by ligating the BglII/HindIII cut DRS2 fragment (1,180 bp) from pPR10 into the BamHI/HindIII-digested pATH2 vector . The resulting plasmid contained an in-frame fusion between TrpE and a region of Drs2p (amino acids 528–920) carrying ATPase motifs and predicted to be a cytoplasmic loop. This plasmid was transformed into JM101 cells and the resulting fusion protein was expressed and purified by the method of Koerner et al. 1991 , except that the procedure was scaled up 10-fold to increase yield. Also, cracking buffer (10 mM sodium phosphate, pH 7.2, 1% β-mercaptoethanol, 1% SDS, 6 M urea) was used together with sonication to resuspend the insoluble portion of the total cell lysate before separation by preparative SDS-PAGE. The band from the preparative gel corresponding to the TrpE/Drs2 fusion protein was cut out and electroeluted using an Elutrap (Schleicher & Schuell, Inc.). Polyclonal rabbit antiserum to the Drs2-TrpE fusion protein was prepared at Scantibodies Laboratory. Drs2p antibodies were affinity purified twice as described using 500 μg of gel purified TrpE/Drs2 fusion protein bound to an Immobilon (Millipore Corp.) strip by capillary action. Affinity-purified anti–Drs2p antibody was used at a concentration of 1:1,000 for immunofluorescence and 1:2,000 for Western blots. Cell lysates for Western blots were prepared by vortexing cells with glass beads in SDS-urea sample buffer (40 mM Tris-HCl, pH 6.8, 8 M urea, 0.1 mM EDTA, 1% β-mercaptoethanol, 5% SDS) and heating at 50°C for 5 min. Boiling of samples caused aggregation of Drs2p such that the protein would remain in the stacking gel during SDS-PAGE. To clone the SWA3 gene, a yeast genomic library ( CEN, LEU2 ) was used to transform the swa3-1 mutant, and the transformants were screened for complementation of the cold-sensitive growth and arf1Δ synthetic lethal phenotypes. A single library plasmid was isolated that was able to complement both mutant phenotypes and further subcloning analyses revealed that a 5-kb SpeI-SnaBI genomic fragment containing the full-length DRS2 gene (and no other open reading frame, ORF) retained the complementing activity (see Materials and Methods). A drs2 null strain was generated by replacing most of the DRS2 coding sequences with TRP1 . Consistent with the phenotype previously reported for drs2Δ strains and that exhibited by the swa3 mutants , the drs2Δ null mutant was unable to grow at 20°C or below, but grew well at temperatures above 23°C. Linkage analysis indicated that SWA3 is allelic to DRS2, and synthetic lethality between arf1Δ and drs2Δ was confirmed by crossing the single mutants and characterizing the progeny by tetrad analysis . This genetic interaction suggested that Drs2p may be involved in an ARF-dependent vesicle-mediated protein transport event(s). To determine if drs2Δ would exhibit synthetic lethality with other mutations that perturb transport vesicle formation, we crossed drs2Δ with strains harboring ts mutations in subunits of COPI ( ret1-1 , sec21-1 , and sec27-1 encoding α, γ, β′-COP, respectively), the ER transport vesicle coat COPII ( sec12-1 and sec23-1 ), and the clathrin heavy chain ( chc1-5, chc1- Δ57, chc1-521 ), and analyzed the progeny by tetrad dissection. All of the sec drs2Δ and the ret1 drs2Δ double mutants were obtained at the expected frequency (approximately one fourth of the progeny) and most grew well at 26.5°C . The exception was sec21-1 drs2Δ mutants, which grew somewhat more slowly at this temperature than either single mutant. Among those growing well at 26.5°C, double mutants harboring drs2Δ and sec12-1 or sec23-1 also grew at 20°C, which is a nonpermissive temperature for drs2Δ single mutants. This suggests that sec12 and sec23 are able to partially suppress the cold-sensitive growth defect of drs2Δ , and that COPII may act upstream of Drs2p. In stark contrast, chc1-ts drs2Δ double mutants were nearly always inviable. All of the spores that failed to grow in the tetrad analysis shown in Fig. 1 B were predicted to be chc1-5 drs2Δ double mutants based on the genotype of the viable spores from each tetrad. The drs2Δ and chc1-5 single mutants grew nearly as well as wild-type progeny at this temperature . Microscopic examination of the plates indicated that the drs2Δ chc1-5 spores germinated and divided a few times to form microcolonies, indicating that mitotic growth, rather than germination, was impaired. In addition, this genetic interaction was not allele-specific since all three of the chc1 ts alleles tested were synthetically lethal with drs2Δ . To further address the specificity of the synthetic lethal interaction between drs2Δ , arf1Δ , and chc1-ts alleles, we crossed drs2Δ with several mutants that exhibit a defect in protein transport through the secretory or endocytic pathways. Double mutants combining drs2Δ with sec1-1, sec7-1, sec14-1, sec18-1, vps15Δ , vps21 Δ , vps33 Δ , vps35 Δ , ypt7Δ , or end4-1 were obtained at the expected frequency, although the end4-1 drs2Δ , vps33Δ drs2Δ , and sec18-1 drs2Δ mutants exhibited a slow growth phenotype. Interestingly, a mutant allele of the yeast Eps15 homologue, pan1-20 , was also synthetically lethal with drs2Δ . Eps15 is a cytoplasmic protein that localizes to clathrin-coated pits and may have an adaptor-like function in clathrin coated vesicle formation . Of particular note was the lack of genetic interactions with sec7 and sec14 , which both perturb protein transport through the yeast Golgi complex. One of the more striking phenotypes of yeast clathrin mutants is the mislocalization of several late Golgi (TGN) proteins that are required for pro–α-factor proteolytic processing. This results in the secretion of fully glycosylated pro–α-factor rather than the mature peptide . If clathrin function at the TGN is perturbed in the drs2Δ mutant, we would expect this mutant to secrete pro–α-factor. To test this, wild-type, drs2Δ , and clc1Δ (clathrin light chain null) strains were grown at 30°C, and after shifting to 20°C for 1 h, were metabolically labeled and chased at this nonpermissive temperature for drs2Δ . Aliquots of cells were removed at the chase times indicated in Fig. 2 , converted to spheroplasts, and then centrifuged to separate intracellular (I) from extracellular (E) fractions. α-Factor was then recovered from each sample by immunoprecipitation. At the beginning of the chase, labeled α-factor was present throughout the secretory pathway of the cells, as indicated by the presence of the core glycosylated ER proform, Golgi-modified hyperglycosylated proforms and the mature form . In wild-type cells, complete proteolytic processing of pro–α-factor occurred within 15 min, and most of the mature α-factor was secreted and degraded in the extracellular space. However, the clc1Δ cells were clearly deficient in the processing of pro–α-factor, and most of the hyperglycosylated precursor was secreted into the extracellular space within 15 min, as previously reported . Similarly, the drs2Δ mutant secreted the hyperglycosylated pro–α-factor and partially processed α-factor forms into the extracellular space . The drs2Δ mutant also exhibited a modest defect in Golgi-specific glycosylation since the hyperglycosylated pro–α-factor secreted from drs2Δ cells showed a slightly faster mobility within the SDS-polyacrylamide gel relative to that from the clc1 Δ strains. The kinetics of pro–α-factor secretion at this temperature (20°C) was nearly equivalent to that of clc1Δ cells. These results indicate that late Golgi function is specifically perturbed in the drs2Δ cells and suggest a loss of clathrin function at the TGN at the nonpermissive temperature. In pulse–chase experiments performed at 30°C, most of pro–α-factor was processed and secreted as the mature form from drs2Δ cells (data not shown), indicating that this defect was temperature conditional. Moreover, the pro–α-factor processing defect was observed within 15 min of preincubation at 20°C, suggesting a rapid onset of the temperature conditional phenotype (data not shown). To examine protein transport through the endocytic pathway of drs2Δ cells, we followed the turnover of the α-factor receptor, Ste3p, which is constitutively endocytosed and delivered to the yeast vacuole where it is degraded . A c-myc tagged Ste3p expressed from a galactose-regulated promoter was used so new Ste3p synthesis can be shut off by shifting the cells to glucose. Wild-type, drs2Δ , and arf1Δ cells were grown in galactose at 30°C to induce expression of the construct and populate the plasma membrane with the Ste3-myc protein. Glucose was added to the culture, which was then shifted to 15°C, and the disappearance of Ste3-myc was followed over time by immunoblotting. Even though the arf1Δ mutant displays an abnormal endosome morphology , the kinetics of Ste3p transport to the vacuole as measured by Ste3-myc degradation was very similar to that of wild-type cells . In contrast, the rate of Ste3-myc turnover was three- to fivefold slower in the drs2Δ cells compared with wild type, suggesting a defect in endocytosis. To control for recovery of protein in each sample, the same blots were probed for carboxypeptidase Y (CPY), which was recovered equally in each sample (data not shown). drs2Δ cells cultured in glucose did not express Ste3p-myc, indicating that glucose repression of the GAL promoter was not perturbed (data not shown). Again, this phenotype was temperature conditional since the rate of Ste3-myc turnover at 30°C in the drs2Δ strain was only slightly (1.4-fold) slower than the wild-type strain (data not shown). Clathrin mutants display a similar defect in Ste3p turnover caused by inefficient internalization of the receptor from the plasma membrane . However, examination of drs2Δ cells by immunofluorescence localization of Ste3p-myc at each time point after glucose was added indicated an accumulation of Ste3-myc in intracellular structures with very little plasma membrane staining (data not shown). This result suggested that transport of Ste3p from the endosome to the vacuole, rather than internalization from the plasma membrane, was perturbed. To more specifically test whether uptake from the plasma membrane or transport from endosomes to the vacuole was defective in drs2Δ cells at the nonpermissive temperature, we stained cells with the fluorescent endocytic marker FM4-64 on ice, and then shifted the cultures to 15°C to initiate endocytosis of the dye . The FM4-64 was rapidly cleared from the plasma membrane of both wild-type and drs2Δ cells (data not shown), and in wild-type cells it was delivered to the vacuole membrane in 30–60 min (WT, 1 h). In contrast, the FM4-64 accumulated in endosomes of drs2Δ cells, which appear as punctate fluorescent spots in Fig. 3 B ( drs2Δ , 1 h). Even after 4 h at 15°C, most of the FM4-64 was not in the vacuoles and stained what appeared to be clusters of smaller structures ( drs2Δ , 4 h). As the drs2Δ cells warmed up on the microscope stage, the FM4-64 was rapidly delivered to the vacuoles at all times tested, indicating that the defect was fully reversible (data not shown). In addition, vacuoles in drs2Δ cells that were stained with FM4-64 at 30°C did not appreciably fragment when the cells were shifted to 15°C (data not shown), so the structures stained at 15°C were indeed endosomes and not fragmented vacuoles. These studies indicated that the endocytic defect observed in drs2Δ cells was attributable to a defect in the endosome-to-vacuole pathway rather than clathrin-dependent endocytosis from the plasma membrane. If so, CPY transport should be affected as well since this protein follows a TGN to endosome to vacuole delivery route. To test this, we examined the transport of CPY to the vacuole in the drs2Δ mutant. CPY is synthesized in the ER as the p1 precursor form and is modified on N-linked oligosaccharides by the Golgi α1,3 mannosyltransferase (Mnn1p) to form the p2 precursor. p2 CPY is sorted from secreted proteins in the TGN and ultimately processed to the mature form in the vacuole . Wild-type, drs2Δ , and arf1Δ cells were pulse-labeled and chased at either the permissive or nonpermissive temperature of drs2Δ . Aliquots of cells were removed at the chase times indicated and CPY was recovered by immunoprecipitation. As shown in Fig. 4 , drs2Δ cells displayed near wild-type CPY transport kinetics at 30°C, while at 15°C the transport of CPY in drs2Δ mutants was significantly delayed relative to that in the wild-type cells, and was similar to the defect observed in arf1Δ cells at either temperature (approximately threefold slower transport kinetics). The partial glycosylation defect observed in drs2Δ (and arf1Δ cells) prevented the formation of a p2 CPY form that could be resolved from the p1 form in SDS-polyacrylamide gels. Thus, the kinetics of ER-to-Golgi transport could not be assessed for CPY. However, the ER-to-Golgi transport kinetics for α-factor and invertase in drs2Δ cells was found to be nearly wild type at the nonpermissive temperature, as scored by disappearance of the ER core form . Therefore, it is unlikely that ER-to-Golgi transport for CPY is disturbed in drs2Δ cells. These data are most consistent with the interpretation that protein transport from the late Golgi or endosomes to the vacuole is perturbed by the drs2Δ mutation. Interestingly, the chc1-5 allele isolated in the arf1Δ synthetic lethal screen also exhibits a partial glycosylation defect and an approximately threefold slower transport kinetics for CPY . Most of the sec mutants that exhibit a temperature-conditional block in protein transport also accumulate an organelle or vesicular intermediate of the secretory pathway at the nonpermissive temperature. For example, the sec7 and sec14 mutations block protein transport out of the Golgi complex and accumulate Golgi structures called Berkeley bodies . The Golgi cisternae in these mutants are sometimes stacked and appear to adopt a deep, cup-shaped morphology that in electron micrographs of thin sections present double-membrane ring or crescent shaped structures depending on the section plane. Clathrin mutants also exhibit aberrant membrane structures similar to Berkeley bodies, although these structures are rarely stacked . Morphological studies by electron microscopy revealed that drs2Δ cells accumulated aberrant double-membrane ring and crescent-shaped structures at both 15° and 30°C . The double-membrane rings in the drs2Δ cells measured 200–250 nm in diameter (average, 240 nm) and often presented a significant gap between the concentric membranes, which should be equivalent to the luminal space of the cisternae. Representative double-membrane ring structures are marked with white arrowheads in Fig. 5 A. Very similar ring structures also accumulated in the chc1 Δ mutant . These types of structures were never observed in wild-type cells; however, ring structures could be found that were more highly fenestrated and appeared to be breaking down into tubules or vesicles . In fact, similar fenestrated ring structures accumulate dramatically in arf1Δ cells . Some of the double-membrane rings in the drs2Δ cells were also modestly fenestrated . To quantitate the effect of temperature on the accumulation of ring structures in wild-type and drs2Δ cells, these cells were grown at 26.5°C and shifted to the temperature indicated in Fig. 5 E for 2 h before fixation. Regions containing 23–25 cell sections were randomly selected and the number of crescent-shaped structures, double-membrane rings, and vesicles (50–100-nm spheres) were counted and expressed as the number of structures per cell section. Relative to the wild-type strain, the drs2Δ mutant accumulated four- to eightfold more crescent and ring structures at the temperatures examined . In this analysis, structures similar to that shown in Fig. 5 D were counted as rings in the wild-type cells. There was also a greater accumulation of these structures in the drs2Δ cells at the colder temperatures, which was particularly evident for the double-membrane rings. The number of vesicles in the drs2Δ cells dropped modestly at the colder temperatures, although within the range of values observed for the wild-type cells. Golgi membranes that accumulate at the nonpermissive temperature in the sec7 mutant have been reported to become more extensively stacked in low glucose medium . Some structures appear stacked in the drs2Δ mutant grown in 2% glucose , but no significant difference was observed in the morphology of the membranes accumulating in drs2Δ cells incubated in low glucose (0.1%) medium for 2 h at the nonpermissive temperature. However, the effect of low glucose on Berkeley body structure in a sec7 mutant in our strain background was modest (data not shown). Plasma membrane invaginations have been proposed to represent endocytic intermediates in yeast and are extended in some mutants that exhibit a defect in endocytosis [for example, sjl1 sjl2 and pan1 ]. However, no difference in the depth of plasma membrane invaginations between wild-type and drs2Δ cells was observed under any of the growth conditions described above. A mammalian cytosolic ARF guanine nucleotide exchange factor requires both PS and phosphatidylinositol-4,5-bisphosphate for optimal membrane binding and subsequent activation of ARF . This observation suggested a requirement for Drs2p (a potential PS translocase) to recruit ARF and clathrin to membranes. To test whether Drs2p plays a role in regulating ARF activation and therefore association of ARF and clathrin with membranes, we fractionated both wild-type and drs2Δ cellular lysates by differential centrifugation to compare the amounts of ARF, clathrin, and adaptin subunits recovered in the 13,000- g pellets, and the 100,000- g pellets and supernatants. A small increase in the amount of clathrin heavy chain found in the 100,000- g supernatant fraction was occasionally observed in the mutant samples relative to the wild-type samples at both 15° and 30°C; otherwise, the fractionation pattern for clathrin was very similar between the two strains (data not shown). In addition, the fractionation pattern for ARF and the two adaptin small subunits (Aps1p for AP-1 and Aps2p for AP-2) was nearly identical between drs2Δ and wild-type samples. Therefore, the association of ARF and clathrin with bulk cellular membranes seemed to be unaffected in the drs2Δ mutant, although we cannot rule out the possibility that the distribution of ARF between specific organelles is perturbed. To further assess clathrin function in the drs2Δ mutants, we asked whether CCVs could be purified from this mutant. CCV preparations were generated from drs2Δ and wild-type cells with or without a 1-h shift to 15°C (see Materials and Methods). Cell lysates were centrifuged at 21,000 g for 30 min to pellet large organellar membranes (e.g., plasma membrane, vacuolar membrane, ER, and mitochondria) and the resulting supernatant was centrifuged at 100,000 g to pellet vesicles. The 100,000- g pellet was then applied to a Sephacryl S-1000 gel filtration column , and the fractions were probed for the clathrin heavy chain by immunoblotting. As shown in Fig. 6 A, the clathrin heavy chain was highly enriched in fractions 23 to 27 for both wild-type and drs2Δ samples from cells shifted to 15°C or 30°C . In addition, from Coomassie blue–stained gels, we estimated that the recovery of clathrin heavy chain in these fractions was comparable for both strains at both temperatures (data not shown). However, a clear difference between wild-type and mutant samples was evident when fractions highly enriched for the clathrin heavy chain were examined by EM. Peak clathrin fractions obtained from wild-type cells were substantially enriched in CCVs with a clear lipid bilayer present in a substantial number of the vesicle profiles . In contrast, most of the vesicle profiles in the drs2Δ peak clathrin fractions contained an electron dense center, but no lipid bilayer within the clathrin basket . In addition, the drs2Δ fractions contained what appeared to be partially assembled clathrin lattices that were rarely observed in wild-type fractions. CCVs with a lipid bilayer were occasionally observed in the drs2Δ fractions, but were found at <1/10 of the frequency per section as compared with the wild-type samples (multiple sections from three different preparations were compared). This was a temperature-conditional phenomenon as CCV preparations from drs2Δ cells maintained at a permissive temperature were indistinguishable from wild-type samples (data not shown). It is well established that clathrin triskelions can self assemble into baskets in slightly acidic, low ionic strength buffers . The method we used for isolating CCVs from yeast applies such buffer conditions to retain assembled coats on vesicles. In addition, the lysate is incubated for 30 min at 30°C with RNase to reduce the amount of ribonucleoprotein complexes in the P100 sample. These conditions are likely to induce assembly of free clathrin triskelions into baskets and lattices within the drs2Δ cell lysate. It appears that these clathrin structures pellet at 100,000 g and elute from the S-1000 column in similar fractions as bona fide CCVs. The simplest interpretation of these data is that the drs2Δ cells are deficient in producing CCVs at the nonpermissive temperature. It is also possible that clathrin dissociates more readily from CCVs produced in drs2Δ cells and reassembles into baskets during purification, thus preventing the isolation of bona fide CCVs. In either case, there is a marked difference between drs2Δ and wild-type cells either in the ability to produce CCVs or in the physical properties of CCVs from these strains. Even though most large membranes pellet at 21,000 g , we had noticed by EM that the 100,000- g pellet from drs2Δ cells contained a significant number of aberrant membrane structures that were similar in appearance and size to those observed in cell sections . Because drs2Δ cells exhibited both Golgi and endosome-associated defects, it was possible that both organelles would accumulate in the drs2Δ mutant. Therefore, we examined the S1000 column fractions from the CCV preparations for membranes containing a late Golgi protein, α1,3-mannosyltransferase (Mnn1p), and an endosomal t-SNARE, Pep12p . In fractions from wild-type cells, Mnn1p eluted slightly later than clathrin, which likely represents small vesicles or small Golgi fragments. However, a substantial change in the elution profile of Mnn1p-containing membranes was observed in fractions from drs2Δ cells. A peak of Mnn1p eluted just after the void volume and spread from fractions 16–31. This change in Mnn1p distribution was observed with samples prepared from drs2Δ cells incubated at both 15°C and 30°C (data not shown), and correlated with the large membrane structures observed by EM in these fractions . Membranes of any sort were difficult to find by EM in fraction 16 from wild-type samples (data not shown). Relative to Mnn1p, a wider distribution of Pep12p-containing membranes was found in the S1000 fractions from wild-type cells. However, the Pep12p elution profile was not significantly different between the wild-type and drs2Δ fractions . These data suggest that the abnormal membrane structures that accumulate in the drs2Δ cells are Golgi membranes and not endosomal membranes. To analyze the intracellular localization of Drs2p, we prepared a rabbit polyclonal antiserum against a bacterially expressed fragment of Drs2p (amino acid residues 528–920). Affinity-purified anti–Drs2p antibodies recognized an ∼150-kD protein on an immunoblot of a wild-type cell lysate , which is in close agreement to the predicted mass of 154 kD. This protein was not present in a drs2Δ cell lysate and was found in greater abundance in wild-type strain carrying DRS2 on a multicopy plasmid . Thus, the 150-kD protein is equivalent to Drs2p. Drs2p was recently suggested to localize to the plasma membrane based on cofractionation of an epitope-tagged Drs2p with the plasma membrane ATPase, Pma1p, in sucrose gradient fractions from a crude cell lysate . However, we found that membranes containing Drs2p could be quantitatively separated from membranes containing Pma1p by differential centrifugation as described above for the CCV preparations. Equal amounts of protein from the lysate (L), pellet (P21), and supernatant (S21) of 21,000 g and pellet (P100) and supernatant (S100) of 100,000 g were probed for Drs2p and Pma1p. Nearly all of Drs2p remained in the supernatant after centrifuging at 21,000 g (S21) while most of Pma1p was found in the pellet (P21) . All of the Drs2p in the S21 was pelleted during the subsequent 100,000- g centrifugation step and was found in the P100 fraction. The fractionation profile for Drs2p shown in Fig. 7 B is very similar to how CCV and late Golgi proteins such as Kex2p fractionate. To further characterize the localization of Drs2p, a P100 fraction was applied to the bottom of a sucrose gradient and centrifuged to equilibrium . Fractions were collected from the top and probed for Drs2p and Mnn1p and assayed for GDPase and Kex2p . GDPase marks early Golgi compartments, Kex2p the late Golgi (or TGN), and Mnn1p is typically split between these two regions of the gradient. The fractionation pattern for Drs2p clearly matches that of Kex2p . In similar gradients, we found that the endosomal marker Pep12p was broadly distributed throughout the gradient (data not shown), although others have observed a defined peak in fractions significantly lighter than that of Kex2p . In no case did the Pep12p fractionation pattern match with that of Drs2p. In addition, the S1000 column elution profile of membranes containing Drs2p (from wild-type cells grown at 30°C) matched that of membranes containing Mnn1p . The Drs2p elution profile overlapped that of CCVs , raising the possibility that some Drs2p is present within the CCVs. This possibility is currently being tested; however, it appeared that the bulk of Drs2p resides in Golgi membranes. To determine whether Drs2p and Kex2p reside in the same membrane structures, wild-type cells overexpressing a hemagglutinin (HA)-tagged Kex2p were stained simultaneously with antibodies to Drs2p and HA. Localization of Drs2p by immunofluorescence produced a punctate staining pattern that is typical for the yeast Golgi complex . Although the signal was weak, it was clearly above the background staining observed for the drs2Δ strain . The HA antibody also produced a punctate staining pattern and, importantly, most of the structures that were positive for Drs2p also stained for Kex2-HA. As expected, very little overlap was observed between Drs2p and the early Golgi marker Och1-HA (data not shown). We could not detect plasma membrane staining with the Drs2p antibodies, which is inconsistent with a previous report suggesting that Drs2p resides in the plasma membrane . Overexpression of Drs2p from a multicopy plasmid resulted in an ER staining pattern , so this method could not be used to enhance the signal. To determine whether the lack of plasma membrane staining with Drs2p antibodies was due to a technical limitation, we tested whether we could detect plasma membrane staining for an antigen that is present in the Golgi, endosomes, and plasma membrane. Wild-type spheroplasts were stained with antibodies to α1,3-linked mannose residues that are found on many glycoproteins. For these cells, staining of the plasma membrane is clearly observed as a stained rim around the cells . Thus, by both immunofluorescence and subcellular fractionation studies, it appears that most, if not all, of Drs2p is found in late Golgi membranes. Drs2p is predicted to be a P-type ATPase based on the presence of five well-conserved ATPase motifs involved in the binding and hydrolysis of ATP . For P-type ATPases, an aspartic acid within the second motif forms an aspartyl-phosphate catalytic intermediate that is essential for ATP hydrolysis . Sequence alignments between Drs2p and other P-type ATPases predict that aspartic acid 560 (D560) in Drs2p would form the aspartyl-phosphate intermediate . To test whether this amino acid is critical for Drs2p function, we mutated D560 to either an asparagine (D560N) or a glutamic acid (D560E) residue and examined the ability of the mutants to complement the cold-sensitive growth defect of the drs2Δ strain. These mutations would be expected to cause minimal structural changes in Drs2p, but should abolish the presumed ATPase activity of this protein. The drs2Δ strain carrying either the wild-type DRS2 gene (Wild-type), an empty vector ( drs2Δ ), or two independent isolates of each mutant (D560N and D560E) were tested for growth at 30° and 20°C . As previously reported, the drs2Δ strain failed to grow at 20°C, but grew well at 30°C. None of the strains carrying the D560 point mutations were able to grow at 20°C , even though each of the strains expressed a wild-type level of Drs2p (data not shown). These data support the assignment of Drs2p as a P-type ATPase and suggest that the ATPase activity of Drs2p is essential for its function in vivo. In this report, we present several lines of evidence that strongly implicate the integral membrane P-type ATPase Drs2p in late Golgi function, and suggest a link between Drs2p and CCV formation from Golgi membranes. (a) A specific synthetic lethal interaction was found between drs2Δ , arf1Δ , and chc1-ts alleles. (b) The drs2Δ mutant exhibits a cold-sensitive defect in the proteolytic processing of pro–α-factor in the yeast TGN that is comparable with the defect shown by clathrin mutants. (c) The drs2Δ mutant accumulates aberrant Golgi structures that are morphologically comparable to the membrane structures that accumulate in clathrin mutants. (d) We observed a substantial decrease in the yield of CCVs that could be isolated from the drs2Δ mutant preincubated at the nonpermissive temperature. (e) Drs2p localizes to late Golgi membranes containing Kex2p. Our rationale for performing the arf1Δ synthetic lethal screen was that it could provide an unbiased, in vivo approach to identify proteins that regulate ARF function, or participate with ARF in CCV or COPI vesicle formation. This was the first yeast genetic screen we are aware of that has uncovered a mutant allele of the clathrin heavy chain gene . While mutations at many loci can influence the viability of a chc1 Δ strain , there are no other reports of mutations that are lethal in combination with chc1-ts alleles other than arf1Δ . This provides genetic support for the substantial biochemical evidence in the literature implicating ARF in clathrin recruitment to Golgi membranes , and also provides validation for the arf1Δ synthetic lethal approach. We were initially concerned about the specificity of the drs2Δ – arf1Δ synthetic lethal interaction, even though four alleles of the SWA3/DRS2 gene were recovered in this genetic screen. However, from crosses with 19 different mutants that perturb the secretory or endocytic pathways, we found a specific interaction between pairwise combinations of arf1Δ , drs2Δ , and chc1-ts alleles. The drs2Δ allele was also synthetically lethal with pan1-20 , an eps15-related protein that interacts with yAP180 (yeast homologue of clathrin assembly protein AP180) in a yeast two-hybrid assay . Importantly, there was no genetic interaction between drs2Δ and mutant ts alleles encoding three different COPI subunits, even though combination of arf1Δ with these COPI alleles produces a synthetic growth defect . These results suggest that Drs2p is specifically involved in clathrin, but not COPI, function. This interpretation is supported by the finding that membranes containing Drs2p could be separated from early Golgi membranes where COPI would be expected to function, and cofractionated with late Golgi membranes where clathrin function is required. These genetic analyses prompted us to examine the drs2Δ mutant for defects in the secretory and endocytic pathways. The secretion kinetics and Golgi-specific glycosylation of pro–α-factor and invertase were only modestly perturbed. However, a substantial cold-sensitive defect in the Kex2p-dependent processing of pro–α-factor was observed. This resulted in secretion of the pro–α-factor precursor, a phenotype also exhibited by clathrin mutants . Kinetic defects were also observed for CPY transport to the vacuole and the turnover of the Ste3p pheromone receptor within the vacuole. These phenotypes are likely caused by a delay in endosome-to-vacuole transport that was clearly observed using the fluorescent endocytic tracer FM4-64. Thus, the drs2Δ mutation seems to specifically perturb late Golgi (TGN) and endosome function. As visualized by EM, the drs2Δ mutant accumulates abnormal membrane-bound structures that were morphologically equivalent to structures that accumulate in clathrin mutants. The abnormal membrane structures that were isolated from drs2Δ cells by differential centrifugation and gel exclusion chromatography were enriched for a late Golgi protein, but not for an endosomal marker protein. Since the localization studies suggest that Drs2p is a late Golgi (TGN) resident, these data strongly suggest that Drs2p acts in the late Golgi to maintain normal structure and function of this compartment. It is possible that Drs2p has some function in the endosome as well, or the effect on endosome function could be a secondary consequence of perturbing late Golgi function. Perhaps the most compelling evidence that Drs2p plays a role in clathrin function is the striking difference in the appearance of CCV preparations between drs2Δ and wild-type cells. Very few bona fide CCVs could be isolated from drs2Δ cells preincubated at the nonpermissive temperature. These preparations contained clathrin baskets and lattices with no associated membrane. In contrast, CCV preparations from drs2Δ cells maintained at 30°C were indistinguishable from wild-type samples. These results suggest that Drs2p is required to form clathrin-coated vesicles from Golgi membranes at temperatures below 23°C. However, it is also possible that the association of clathrin coats with vesicle membranes from drs2Δ cells is less stable and the coat dissociates during cell lysis. In either case, loss of Drs2p clearly perturbs CCVs. At this time, it is not possible to distinguish whether the effect of Drs2p on clathrin function is direct or a secondary consequence of the abnormal TGN structure. However, this effect is specific since the TGN of drs2Δ cells functions normally in the ability to sort vacuolar proteins and in protein secretion, despite the abnormal morphology. This suggests that late secretory vesicles bud normally from the TGN in drs2Δ cells. The genetic interactions also showed a high degree of specificity between drs2Δ and clathrin mutations. Therefore, there is not a wholesale loss of Golgi function in drs2Δ cells at the nonpermissive temperature. Indeed, most of the specific defects observed can be explained by a loss of clathrin function. Even the accumulation of abnormal membrane at the permissive temperature could be the result of inefficient CCV budding. The temperature-conditional defects observed for a strain carrying a complete loss of function allele is somewhat unusual. This suggests that Drs2p is required to overcome an inherently cold-sensitive process in the cell (perhaps CCV budding), such that the growth defect is caused by the combination of low temperature and loss of Drs2p. However, this cold-sensitive process is not at the extreme of the normal growth range for yeast; drs2Δ cells fail to grow at room temperature (20°C) and the mutant phenotypes described here are observed at this temperature. In addition, some of the drs2Δ phenotypes, such as arf1Δ synthetic lethality and abnormal Golgi morphology, are also observed at 30°C. Thus, it appears that Drs2p plays a role in Golgi function at all temperatures examined but is only essential below 23°C. Alternatively, it is possible that Drs2p function is essential at all temperatures, but loss of Drs2p is compensated by one or more of the Drs2p-related P-type ATPases at higher temperatures. In either case, Drs2p clearly plays a critical role for organisms such as yeast since the ability to adapt to daily fluctuations in temperature is essential for their survival. What is the biochemical function of Drs2p? Many P-type ATPases use the energy of ATP hydrolysis to pump cations such as Ca 2+ , H + , Na + , or heavy metals across a membrane against their electrochemical gradient. These transporting ATPases contain signature motifs that allowed the identification of 16 P-type ATPases in the yeast genome that can be phylogenetically grouped into six distinct families . Four of the families correspond to the cation transporters just described, one family contains two ORFs of unknown function, and the last family are potential aminophospholipid transporters, which includes Drs2p and four other uncharacterized ORFs. Drs2p is 28–35% identical to its four other yeast family members, but is 47% identical to bovine or murine ATPase II from chromaffin granules (FASTA comparisons), arguing that Drs2p and ATPase II are orthologues. The closest related P-type ATPases to the Drs2p family are the Ca 2+ transporters (21% identity between Drs2p and Pmc1p). However, Drs2p and ATPase II are missing negatively charged amino acids in transmembrane domains 4 and 6 that are essential for cation transport and have hydrophobic residues in their place. This is consistent with the proposed role of ATPase II and Drs2p in transporting aminophospholipids and makes it less likely that Drs2p transports cations. The drs2Δ mutant has been reported to exhibit a defect in the translocation of a fluorescent PS derivative across the plasma membrane . However, Siegmund et al. 1998 have recently challenged this observation and have failed to detect a difference between wild-type and drs2Δ cells in the translocation of fluorescent lipid derivatives across the plasma membrane. This group also found no difference in the amount of PE exposed on the outer leaflet of the plasma membrane as detected by trinitrobenzene sulfonic acid labeling. These latter observations could suggest that Drs2p is not an aminophospholipid translocase, but it is more likely that the Drs2p protein is simply not present at the plasma membrane and therefore is not required to maintain an asymmetric distribution of aminophospholipids in this membrane. In fact, we cannot detect Drs2p at the plasma membrane by immunofluorescence localization or subcellular fractionation . Measurement of translocase activity with Golgi membrane fractions is more complicated because the direction of flip is expected to be from the luminal to the cytoplasmic leaflet and thus requires incorporation of the probe into the luminal leaflet. Thus, further work is required to determine if Drs2p is an aminophospholipid translocase, as suggested by its homology to ATPase II. Since an aminophospholipid translocase activity is the only biochemical function attributed to ATPase II and Drs2p in the literature, it is relevant to speculate on how membrane asymmetry may affect Golgi structure and perhaps clathrin function. The bilayer couple hypothesis of Sheetz and Singer 1974 proposes that asymmetric changes in the two leaflets of a bilayer should induce conformational changes in the membrane. In fact, induced bilayer asymmetry can cause conversion of spherical liposomes into tubular and interconnected vesicular structures . In this regard, it is possible that changes in the asymmetric distribution of PS or PE could influence the formation of tubules or fenestrated regions of Golgi cisternae to produce a structure on which clathrin can assemble more productively. This is consistent with the morphological defect observed for Golgi membranes in the drs2Δ mutant, which are notable for their lack of fenestration or tubular regions. Particularly in comparison to the arf1Δ mutant in which the Golgi is highly fenestrated or tubular in appearance . Others have proposed that the transbilayer movement of lipid could induce the bending of membranes to facilitate vesicle budding . Zha et al. 1998 have reported that changes in the composition of the plasma membrane outer leaflet caused by sphingomyelinase treatment can induce an energy-independent budding of functional endocytic vesicles. In addition, Farge et al. 1999 have recently reported that exogenous PS incorporated into the external leaflet of the plasma membrane was pumped to the inner leaflet and markedly enhanced the level of bulk endocytosis. Thus, it is feasible that Drs2p actively participates in CCV budding from Golgi membranes, and becomes essential for this process as the temperature drops below a specific threshold where a decreased fluidity of the membrane may prevent clathrin from performing this function alone. A third possibility is that an increase in the PE or PS concentration of the cytoplasmic leaflet may influence the recruitment or activity of peripherally associated proteins. For example, the association of an ARF guanine nucleotide exchange factor with Golgi membranes might be influenced by the PS concentration . In addition, clathrin can be recruited on chemically defined liposomes, but requires an unusually high concentration of PE (40% of total lipid) to produce well-defined clathrin-coated buds on the membranes . One might expect that an enzyme that pumps PE to the cytoplasmic membrane could produce a high local concentration of this lipid, particularly if the coat proteins restrict lateral diffusion of PE. Purification and reconstitution of Drs2p in chemically defined liposomes will help define the biochemical function of this protein and perhaps shed light on how Drs2p influences clathrin function in vivo. | Study | biomedical | en | 0.999996 |
10601337 | Polyclonal antibodies have been generated in rabbit against the cytoplasmic domain of the chicken kidney AE1-4 anion exchanger that was expressed as a bacterial fusion protein in E. coli . In brief, the region of AE1-4 encompassing amino acids 1–359 was subcloned into the pFLAG-ATS prokaryotic expression vector (IBI) downstream of the eight–amino acid FLAG epitope sequence. The expression of this bacterial fusion protein is driven by the inducible tac promoter. After induction with IPTG, the fusion protein was purified from bacterial extracts by immunoaffinity chromatography using a monoclonal antibody to the FLAG epitope. The purified fusion protein was used as a source of antigen to inject rabbits. Madin-Darby canine kidney (MDCK) cells were maintained in DME supplemented with 5% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO 2 . Cells were transfected with various AE1 anion exchanger cDNAs subcloned into the pcDNA3 eukaryotic expression vector (Invitrogen). Media was changed 24 h after transfection, and 48 h after transfection cells were analyzed for AE1 expression. Alternatively, at 48 h after transfection cells were split 1:10 in complete media containing 600 μM G418. Cells that could grow in the presence of G418 were selected and used for various assays. Sense primers complementary to nucleotides 89–103, 137–151, or 185–198 of the chicken AE1-4 anion exchanger cDNA were generated. Sense primers were flanked at their 5′ ends with a HindIII restriction site, which represents a unique site at the 5′ end of the AE1-4 cDNA in the pcDNA3 polylinker, followed by a Kozak sequence , and an AUG codon. Each sense primer was used for a polymerase chain reaction (PCR) in combination with an antisense primer corresponding to nucleotides 199–219 of the AE1-4 cDNA. This antisense primer encompassed a unique BamHI restriction site in the AE1-4 cDNA. PCR reactions using the full-length AE1-4 cDNA as a template were performed using Pfu polymerase (Stratagene). PCR products were digested with HindIII and BamHI restriction enzymes, and purified on low melting point agarose gels. The AE1-4Δ21, AE1-4Δ37, and AE1-4Δ53 constructs were generated by using these PCR products to replace the 5′ end of the AE1-4 cDNA in pcDNA3 that had been removed by digestion with HindIII and BamHI. Point mutants were constructed using the Altered Sites II site-directed mutagenesis kit (Promega). In brief, the AE1-4 cDNA was subcloned into the pALTER-1 vector. After alkaline denaturation, a mutagenic oligonucleotide was annealed to the full-length AE1-4 cDNA template. The sequences of the mutagenic oligonucleotides are as follows: Y44A (5′-TCCACGTAGCCCTCA GC GGTGTCCCTGTGGGC-3′), Y47A (5′-TCG-TGCAGCTCCACG GC GCCCTCATAGGTGTC-3′), Y44A,Y47A (5′-TCGTGCAGCTCCACG GC GCCCTCA GC GGTGTCCCTGTGGGC-3′), and N638T (5′-CCGCGGGCGGTGCCG G TGGTCACCTCCAGCCC-3′). The mutant strand was elongated and ligated using T4 DNA polymerase and T4 DNA ligase, and cotransformed into a repair-minus E . coli strain along with helper phage DNA. All mutants were confirmed by sequencing using the dideoxy method. The mutant cDNAs were subcloned into pcDNA3 for transfection. MDCK cells transiently or stably expressing wild-type or mutant AE1 anion exchangers were harvested and detergent lysed by incubation in 150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM MgCl 2 , 2 mM EGTA, 6 mM β-mercaptoethanol, and 1% (vol/vol) Triton X-100 for 5 min on ice. In some instances, the cells were treated with 25 μg/ml of the actin depolymerizing drug, latrunculin B, for 1 h before lysis. Immunolocalization analyses have shown that phalloidin-stained microfilaments are absent in MDCK cells treated with this drug (data not shown). The lysate from treated or untreated cells was microcentrifuged for 5 min to separate the detergent soluble and insoluble fractions. The detergent insoluble pellet was resuspended in immunoprecipitation buffer containing 170 mM NaCl, 20 mM Tris (pH 7.5), 5 mM EGTA, 5 mM EDTA, 0.1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, and 1% (wt/vol) sodium deoxycholate and sonicated three times. After sonication, the insoluble fraction was microcentrifuged for 5 min and the pellet discarded. The detergent soluble and insoluble fractions were then electrophoresed on a 6% SDS polyacrylamide gel, and the proteins were electrophoretically transferred to nitrocellulose. The filters were blocked with 5% powdered milk and incubated overnight with a 1:30,000 dilution of a chicken AE1-specific peptide antibody . After washing, the filters were incubated with goat anti–rabbit (GAR) IgG conjugated to horseradish peroxidase, and immunoreactive species were detected on Kodak Biomax MR film using enhanced chemiluminescence. Cells expressing the various anion exchanger polypeptides were grown in 100-mm culture dishes or on 24-mm polycarbonate filters (Costar). The cells were washed once with methionine-free DME and incubated with this media for 15 min at 37°C. At this time, 65 μCi/ml 35 S-Translabel™ (ICN) was added to the cells and they were incubated an additional 15 min at 37°C. After washing, the cells were incubated in DME containing 5% fetal calf serum for times ranging from 0 to 48 h. At each time point, the cells were detergent extracted as described above and separated into detergent soluble and insoluble fractions. In some instances, total cell lysates were prepared by directly lysing cells in immunoprecipitation buffer. Protein A–agarose beads that had been preloaded with rabbit polyclonal antibodies directed against amino acids 1–359 of chicken AE1-4 were added to these fractions and incubated at 4°C overnight. The protein A–agarose beads were then washed three times with immunoprecipitation buffer, and the immune complexes were released by incubation in SDS sample buffer. Immunoprecipitates were resolved on a 6% SDS polyacrylamide gel. The gels were treated with PPO in DMSO, dried, and exposed to Kodak Biomax MR film at −80°C. MDCK cells stably expressing the various chicken AE1 anion exchanger polypeptides were pulsed with 35 S-Translabel, chased for 0, 1, or 4 h, and AE1 immunoprecipitates were prepared as described above. After washing of the protein A–agarose beads with immunoprecipitation buffer, the beads were washed with 10 mM Tris (pH 7.4), and 150 mM NaCl (TBS). The beads were then resuspended and digested either with 2.5 mU endoglycosidase H (endo H), or 0.1 U N-glycosidase F as described previously . After digestion, the beads were washed one time in TBS and immune complexes were released by incubation in SDS sample buffer and analyzed on a 6% SDS polyacrylamide gel. The observed digestion patterns were the same when digests were carried out at 37°C. Three different labeling schemes were used to label MDCK cells stably expressing the chicken AE1-4 anion exchanger. (a) The cells were pulsed with 35 S-Translabel™ in methionine-free DME for 15 min, followed by 15 min of chase in methionine-free DME. The cells were then chased an additional 45 min in methionine-free DME in the absence or presence of 1.5 mg/ml chymotrypsin. (b) The cells were incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35 S-Translabel™, and a 15-min chase (a total of 45 min). (c) The cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin. For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates as described above. A fraction of the total cell lysate was analyzed by immunoblotting analysis using a polyclonal antibody raised against chicken erythroid α-spectrin (Sigma) that recognizes α-fodrin or a chicken AE1-specific peptide antibody . MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35 S-Translabel™ as described above. After a 45-min chase, either 10 μg/ml brefeldin A (BFA), 25 mM ammonium chloride, or 0.4 M sucrose was added to the media, and the cells were incubated until a total chase period of 4 h had elapsed. At each time point, AE1 immunoprecipitates were prepared from total cell lysates as described above. Immunoprecipitates were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography. X-ray films from the immunoblotting analyses and the immunoprecipitation experiments were scanned using DeskScan II 2.1 software, and quantitative densitometry was performed using NIH Image. Each value presented in the text represents the average from three independent experiments in which the exposures of individual bands were not yet saturating. MDCK cells transiently or stably expressing the various chicken AE1 anion exchanger polypeptides were either grown on coverslips or on polycarbonate filters (Costar). The cells were washed in PBS, and fixed by incubating with 1% paraformaldehyde in PBS for 10 min at room temperature. The cells were then permeabilized in PBS containing 0.5% Triton X-100 or with acetone. After permeabilization, cells were incubated with a chicken AE1-specific peptide antibody, followed by incubation with donkey anti–rabbit (DAR) IgG conjugated to lissamine. After washing, immunoreactive polypeptides were visualized using a Zeiss Axiophot microscope or a BioRad laser scanning confocal microscope. Background levels of staining were observed in control experiments in which the peptide antigen used to generate the AE1-specific peptide antibody were included as a competitor. In some instances, actin microfilaments were stained by incubating cells with 1 μg/ml phalloidin conjugated to fluorescein isothiocyanate (FITC; Sigma). Additional assays also examined whether the localization of AE1 anion exchangers was affected by pre-extracting MDCK cells with PBS containing 0.5% Triton X-100 before fixation in paraformaldehyde. The kidney from a 17-d-old chicken embryo was isolated and fixed by incubation for 30 min in 2% paraformaldehyde in PBS at 4°C. After fixation, the tissue was rinsed in PBS and incubated overnight at 4°C in 0.5 M sucrose in PBS. The tissue was then frozen in embedding media and 3-μm tissue sections were cut using a cryotome. The tissue sections were postfixed in 1% paraformaldehyde in PBS for 5 min, and extracted in PBS containing 0.5% Triton X-100. The sections were then incubated with a chicken AE1-specific peptide antibody and a monoclonal antibody directed against the H + -ATPase (the kind gift of Dr. S. Gluck). After washing, sections were incubated with donkey anti–rabbit (DAR) IgG conjugated to lissamine and donkey anti–mouse (DAM) IgG conjugated to fluorescein. Immunoreactive polypeptides were visualized using a BioRad laser scanning confocal microscope. Previous studies have indicated that three variant AE1 anion exchanger transcripts, AE1-3 , AE1-4 , and AE1-5 , are expressed in chicken kidney . The AE1-3 and AE1-5 transcripts initiate translation at the same AUG and encode identical predicted polypeptides of ∼94 kD that lack the 78 NH 2 -terminal amino acids of the chicken erythroid AE1-1b anion exchanger . The AE1-4 transcript contains an in-frame AUG in its unique 5′ sequence, and encodes a predicted polypeptide of ∼101 kD that contains 63 amino acids at its NH 2 terminus that are absent in AE1-3. To determine the localization of these variant anion exchangers in the cells of the kidney collecting duct, tissue sections prepared from a 17-d-old chicken embryo were probed with an AE1-specific peptide antibody, and with a monoclonal antibody directed against the H + -ATPase. Analysis of these tissue sections by confocal microscopy revealed that most (>95%) AE1 expressing cells in the collecting duct exhibited a basolateral pattern of localization for AE1, and an apical pattern of localization for the H + -ATPase , typical of acid-secreting, α-intercalated cells . However, a small percentage of the AE1 anion exchanger expressing cells did not exhibit a polarized distribution for AE1. In the cell marked by the arrow in Fig. 1 , AE1 accumulated in both the basolateral and apical membrane, while the H + -ATPase was restricted to the apical membrane. The cells that exhibited a nonpolarized distribution for AE1 typically lacked the highly elaborated apical membrane observed in cells where AE1 exclusively accumulated in the basolateral membrane. Whether these cells represent α-intercalated cells that have not yet fully differentiated is not known. The AE1-specific peptide antibodies used in these immunolocalization studies recognized a sequence present in both AE1-3 and AE1-4. Therefore, we were unable to determine whether the alternative NH 2 termini of the chicken kidney AE1 variants affect their localization in the cells of the kidney collecting duct. To investigate whether the variant cytoplasmic domains of these electroneutral transporters may be involved in directing their intracellular localization in kidney epithelial cells, the AE1-3 and AE1-4 anion exchangers were transiently expressed in MDCK cells. After the establishment of a polarized epithelial phenotype, transfected cells were fixed and stained with AE1-specific antibodies, and immunoreactive polypeptides were visualized by confocal microscopy. This analysis revealed that AE1-4 primarily accumulates in the basolateral membrane of transfected MDCK cells . In contrast, AE1-3 accumulates in or near the apical membrane where it exhibits a diffuse pattern of localization . Although AE1-3 is primarily apical in the cells shown in Fig. 2 A, this variant transporter also accumulated in an undefined intracellular compartment in some of the transfected cells (data not shown). These data suggest that the alternative NH 2 -terminal cytoplasmic domains of AE1-3 and AE1-4 serve as signals that direct these variant transporters to opposite membrane domains in this polarized epithelial cell type. Fractionation studies have examined whether the differences in intracellular localization of the AE1-3 and AE1-4 anion exchangers correlated with differences in other properties of the variant transporters. Confluent MDCK cells transiently expressing AE1-3, AE1-4, or a point mutant of AE1-4 that eliminates its single N-linked glycosylation site, AE1-4N638T, were lysed with isotonic buffer containing 1% Triton X-100, and separated into soluble and insoluble fractions by centrifugation. Immunoblotting analysis of these fractions with AE1-specific antibodies detected a discrete polypeptide of ∼80 kD in AE1-3 transfected cells that was primarily detergent soluble . In contrast to AE1-3 expressing cells, several polypeptides ranging in size from ∼97 to ∼115 kD were detected in both the detergent soluble and insoluble fractions of cells transfected with AE1-4 . The polypeptides detected in AE1-4 expressing cells are similar in size to the array of AE1 anion exchangers detected in chicken kidney membrane preparations . Quantitation of several fractionation experiments identical to those described above has indicated that ∼5% of AE1-3 is detergent insoluble, while ∼45% of AE1-4 is detergent insoluble. Although AE1-4N638T exhibited fractionation properties similar to AE1-4, a single species of ∼95 kD accumulated in cells transfected with this construct . This suggests the complex array of polypeptides observed in AE1-4 transfected cells is due to heterogeneity in the N-linked modifications acquired by this AE1 variant. The size of the polypeptides detected for both AE1-3 and AE1-4 in this analysis is smaller than their predicted molecular masses. The basis for this discrepancy is currently unknown. Pulse–chase analyses have investigated the mechanisms involved in generating the steady state profile of anion exchangers detected in AE1-3 and AE1-4 transfected cells. MDCK cells stably transfected with AE1-3 or AE1-4 were pulsed with 35 S-Translabel™ for 15 min, and chased for 1 or 4 h. At each time point, the cells were lysed and immunoprecipitates prepared using AE1-specific peptide antibodies were either undigested, digested with endo H, or digested with N-glycosidase. These studies revealed an AE1-3 polypeptide of ∼80 kD accumulated in MDCK cells at the end of a 15-min pulse . This species was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼78 kD. The ∼80-kD AE1-3 polypeptide underwent no further modification, and was completely turned over by the end of a 4-h chase. Analysis of AE1-4 transfected cells revealed a discrete AE1-4 species of ∼97 kD accumulated in MDCK cells after the 15-min pulse . This polypeptide was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼95 kD. Quantitation of three independent experiments has shown that ∼50% of the newly synthesized AE1-4 polypeptides are turned over during a 4-h chase. The remaining AE1-4 polypeptides acquired additional modifications that eventually resulted in a diffuse array of polypeptides ranging in size from ∼105 to ∼112 kD . The ∼105- to ∼112-kD polypeptides were still sensitive to digestion with N-glycosidase yielding a polypeptide of ∼95 kD. However, the N-linked sugar modifications of the ∼105- to ∼112-kD polypeptides were insensitive to digestion with endo H . This suggests that AE1-4 initially receives high mannose or biantennary hybrid sugars on its single N-linked site. By 1 h after synthesis, ∼10% of these modifications are converted to endo H–resistant complex sugars, and by 4 h after synthesis the bulk of the newly synthesized AE1-4 polypeptides have received these more complex endo H-resistant sugar modifications. The addition of these complex sugars to AE1-4 requires the activity of α-mannosidase II and N-acetylglucosamine transferase, markers of the medial compartment of the Golgi. This suggests that AE1-4 passes through the medial compartment of the Golgi between 1 and 4 h after synthesis. The acquisition of mature N-linked sugars by chicken erythroid AE1 anion exchangers occurs via recycling of newly synthesized polypeptides from the plasma membrane to the Golgi . To determine whether a similar mechanism is responsible for the slow acquisition of endo H-resistant sugars by AE1-4, pulse–chase studies have examined the time after synthesis that newly synthesized AE1-4 becomes susceptible to digestion with extracellular chymotrypsin. This analysis revealed that the immunoprecipitable AE1-4 species of ∼97 kD was not susceptible to digestion with extracellular chymotrypsin that was present in the media during the pulse and for the first 15 min of chase . However, when chymotrypsin was included in the media from 15 min to 60 min of chase, all of the immature AE1-4 species of ∼97 kD was susceptible to digestion . Immunoblotting analysis revealed that the steady state population of AE1-4 was digested to the same extent regardless of the time when chymotrypsin was added . Additional controls revealed that α-fodrin was not susceptible to chymotrypsin digestion at either of the time points indicating the susceptibility of newly synthesized AE1-4 to chymotrypsin was not due to the cells becoming leaky during digestion. These results indicate that essentially all of the newly synthesized polypeptides arrive at the plasma membrane by 1 h after synthesis. Furthermore, these data suggest that the acquisition of endo H–resistant N-linked sugars by AE1-4 between 1 and 4 h of chase primarily occurs via recycling of cell surface polypeptides to the Golgi. Interestingly, when cells were shifted to 4°C after 1 h of chase to block vesicular trafficking and incubated with chymotrypsin for an additional 45 min at 4°C, newly synthesized AE1-4 was resistant to chymotrypsin digestion . This result together with the fact that newly synthesized polypeptides have undergone cell surface delivery by 1 h of chase suggests that newly synthesized AE1-4 is rapidly internalized after surface delivery. Previous studies have shown that recycling of chicken erythroid AE1 anion exchangers from the cell surface to the Golgi is sensitive to ammonium chloride , BFA , and 0.4 M sucrose , reagents known to affect different steps in the endocytic pathway. Pulse–chase analyses have investigated whether the recycling of AE1-4 from the cell surface to the Golgi in MDCK cells exhibits a similar sensitivity to these reagents. Each reagent was added to MDCK cells expressing AE1-4 after a 15-min pulse and a 45-min chase, and the effect of the reagent on the acquisition of complex N-linked sugars by AE1-4 was monitored during a subsequent chase. These studies revealed that 0.4 M sucrose, which can inhibit clathrin-dependent endocytosis by dissociating clathrin from the plasma membrane , completely blocked the acquisition of mature N-linked sugars by newly synthesized AE1-4 . This result is consistent with the hypothesis that after delivery to the plasma membrane, the rapid internalization of AE1-4 occurs in a clathrin-dependent fashion. In contrast to 0.4 M sucrose, the posttranslational processing of AE1-4 in the presence of ammonium chloride or BFA was similar to that observed in control cells . The observation that AE1-4 recycling is insensitive to ammonium chloride and BFA suggests that the Golgi recycling pathway for AE1 in MDCK cells is distinct from the recycling pathway previously described for AE1 in chicken embryonic erythroid cells . During the establishment of epithelial polarity, the actin cytoskeleton of MDCK cells undergoes a dramatic reorganization. Cells grown in subconfluent monolayers possess discrete actin stress fibers in the basal membrane. During polarization, these stress fibers become much less prevalent as actin is redistributed for the most part to sites of cell–cell contact. To assess the potential role of the actin cytoskeleton in directing the intracellular localization of AE1-3 and AE1-4 in MDCK cells, subconfluent monolayers of cells transfected with these variant transporters were double stained with AE1-specific antibodies and FITC-phalloidin. These studies revealed that the localization of AE1-4 in transfected cells substantially overlapped the distribution of both phalloidin-stained stress fibers in the basal membrane and cortical actin at sites of cell–cell contact . In contrast, there was minimal if any overlap of AE1-3 with phalloidin-stained microfilaments in transfected cells (data not shown). To determine whether association with filamentous actin could account for the detergent solubility properties of AE1-4 , transfected cells were extracted in situ with 0.5% Triton X-100 before fixation and staining with AE1-specific antibodies and phalloidin. This analysis revealed that AE1-4 still colocalized with phalloidin-stained stress fibers and filamentous actin at cell–cell junctions in these preextracted cells suggesting that AE1-4 is associated with the actin cytoskeleton. In contrast to AE1-4, there was essentially no detectable fluorescence in AE1-3 expressing cells that were detergent extracted before fixation and staining with AE1 antibodies (data not shown). Pulse–chase studies have investigated the kinetics with which newly synthesized AE1-4 associates with the detergent insoluble fraction of MDCK cells. Subconfluent MDCK cells stably transfected with AE1-4 were pulsed for 15 min and chased for times ranging from 1 to 48 h . At each time point, the cells were detergent fractionated and immunoprecipitates were prepared from the detergent soluble and insoluble fractions using AE1-specific antibodies. Quantitation of three independent experiments identical to the one shown in Fig. 7 A has revealed that newly synthesized AE1-4 was almost entirely (>95%) detergent soluble after 1 h of chase. By 2 h of chase, ∼50% of the AE1-4 polypeptides have acquired complex N-linked sugars, and ∼15% of the polypeptides have become detergent insoluble. The pool of polypeptides with complex N-linked sugars does not increase between 2 and 4 h of chase. However, there is a substantial reduction in the amount of the ∼97-kD polypeptide during this time period suggesting that most of the polypeptides that have not acquired complex N-linked sugars by 2 h of chase are turned over. In addition, there is a gradual shift of the remaining polypeptides from the detergent soluble to the insoluble pool such that by 8 h of chase ∼45% of the polypeptides are detergent insoluble. By 12 h of chase, ∼90% of the newly synthesized polypeptides have turned over, and immunoprecipitable AE1-4 was almost undetectable after 48 h of chase. These pulse–chase studies suggest that association of newly synthesized AE1-4 with the detergent insoluble fraction does not provide a mechanism for preferentially stabilizing AE1-4 in MDCK cells, since detergent soluble and insoluble AE1-4 turned over at a similar rate at the later time points of the analysis. Similar studies with MDCK cells that had been grown on permeable supports 5 d after cell–cell contact yielded identical results (data not shown) indicating the detergent fractionation properties and turnover rate of AE1-4 are not affected by the state of polarity of these epithelial cells. The in situ extraction studies described above suggest that the acquisition of detergent insolubility by newly synthesized AE1-4 is at least in part due to its association with the insoluble actin cytoskeleton. To directly assess the role of the actin cytoskeleton in the maintenance of AE1-4 insolubility, transfected cells grown in subconfluent monolayers were treated with the actin-depolymerizing drug, latrunculin B, for 1 h before detergent lysis. Immunoblotting analysis revealed that the detergent insoluble pool of AE1-4 was dramatically reduced in cells treated with this reagent . Interestingly, similar studies with cells grown in confluent monolayers revealed that latrunculin B treatment only had a small but reproducible effect on the detergent insolubility of AE1-4 . These results indicate that the maintenance of AE1-4 insolubility is almost entirely dependent upon an intact actin cytoskeleton in subconfluent cells. However, as cells develop a more polarized phenotype, alternative mechanisms are primarily responsible for the insolubility of AE1-4. Other investigators have identified dominant cytoplasmic sorting signals that direct the basolateral sorting of integral membrane polypeptides in epithelial cells . The studies described above have indicated that the alternative NH 2 -terminal cytoplasmic sequences of the chicken AE1-3 and AE1-4 anion exchangers direct these variant transporters to the apical and basolateral membrane, respectively, of polarized MDCK cells. To determine the sequences at the NH 2 terminus of AE1-4 that direct its basolateral accumulation, a variety of mutants have been generated . NH 2 -terminal truncation mutants that deleted the NH 2 -terminal 21 (AE1-4Δ21) or 37 (AE1-4Δ37) amino acids of AE1-4 primarily accumulated in the basolateral membrane of stably transfected MDCK cells . In contrast, AE1-4Δ53, which lacks the NH 2 -terminal 53 amino acids of AE1-4, exhibited a diffuse pattern of localization in or near the apical membrane, and accumulated in an undefined intracellular compartment of transfected cells . This indicated that sequences between amino acids 37 and 53 of AE1-4 are required for its basolateral accumulation in MDCK cells. The region between amino acids 37 and 53 of AE1-4 contains two tyrosine residues, at positions 44 and 47. Other investigators have shown that tyrosine residues are critical for the basolateral sorting of several type I integral membrane proteins in MDCK cells . To investigate the role of these residues in directing the intracellular localization of AE1-4, point mutants were generated in which an alanine was substituted for each of the tyrosine residues separately or together. Substituting alanine for either of these residues separately, AE1-4(Y44A) or AE1-4(Y47A), had no effect on the basolateral accumulation of AE1-4 . In contrast, the AE1-4(Y44A,Y47A) double mutant primarily accumulated in the apical membrane and in an undefined intracellular compartment of transfected MDCK cells . In a small percentage of the cells, the AE1-4(Y44A,Y47A) mutant also accumulated in the basolateral membrane. These data suggest that efficient basolateral targeting of AE1-4 in MDCK cells is dependent upon these cytoplasmic tyrosine residues. Interestingly, either one of the tyrosines at amino acids 44 or 47 is sufficient to direct the basolateral accumulation of this variant transporter. To determine whether the basolateral accumulation of the AE1-4 mutants correlated with their ability to colocalize with the actin cytoskeleton, subconfluent monolayers of MDCK cells expressing the mutant transporters were double stained with AE1-specific antibodies and FITC-phalloidin. This analysis revealed that only those mutants that were efficiently targeted to the basolateral membrane in confluent monolayers, including AE1-4Δ21, AE1-4Δ37, AE1-4(Y44A), and AE1-4(Y47A), colocalized both with stress fibers in the basal membrane of subconfluent MDCK cells, and with cortical actin at sites of cell-cell contact . In addition to colocalizing with stress fibers in the basal membrane of cells, AE1-4Δ37 often accumulated in clusters in the basal membrane that were also stained by FITC-phalloidin . These actin-containing clusters were rarely observed with the other mutant constructs, and they were negative for staining with talin antibodies, a marker for focal adhesions (data not shown). Although AE1-4(Y44A,Y47A) colocalized with cortical actin at sites of cell-cell contact in a small percentage of transfected cells , this mutant transporter was never observed to colocalize with stress fibers in the basal membrane of cells. These results suggest that association of AE1-4 with specific elements of the actin cytoskeleton, like efficient basolateral targeting, requires at least one of the tyrosine residues at amino acids 44 and 47 of the polypeptide. At this time, we can not distinguish whether the association of AE1-4 with the detergent insoluble actin cytoskeleton is a prerequisite for or a consequence of the basolateral sorting of this variant transporter in MDCK cells. To determine whether the NH 2 -terminal truncation and point mutants of AE1-4 affected the detergent solubility properties and posttranslational modifications of this variant transporter, confluent MDCK cells stably transfected with each construct were detergent lysed, and separated into soluble and insoluble fractions by centrifugation. Immunoblotting analysis of these fractions with AE1-specific antibodies revealed that the AE1-4 mutants could be grouped into three classes. Quantitation of three independent experiments identical to the one shown in Fig. 10 has revealed that the first class of mutants, AE1-4Δ21 and AE1-4(Y44A), was similar to wild-type AE1-4 in terms of the percent of the steady state population that was detergent insoluble (∼40%), and the percent of the molecules that acquired complex N-linked sugars (∼95%). The second class of mutants, AE1-4Δ37 and AE1-4(Y47A), exhibited partial defects in their posttranslational processing . The percentage of AE1-4(Y47A) polypeptides that acquired complex N-linked sugars (∼45%) was reduced relative to that observed with AE1-4, and both AE1-4Δ37 and AE1-4(Y47A) were substantially reduced in their ability to associate with the detergent insoluble fraction of MDCK cells (∼5% of total protein). Interestingly, unlike AE1-4, most of the AE1-4(Y47A) polypeptides that associated with the detergent insoluble fraction possessed only simple N-linked sugars suggesting that AE1-4(Y47A) may acquire detergent insolubility by a mechanism distinct from that used by AE1-4. Finally, AE1-4Δ53 failed to acquire complex N-linked sugars and was entirely detergent soluble . A similar phenotype was observed for AE1-4(Y44A,Y47A) suggesting that Golgi recycling and the acquisition of detergent insolubility, like efficient basolateral targeting, requires at least one of the tyrosines at amino acids 44 and 47 of AE1-4. Pulse–chase analyses have examined whether mutations that alter the posttranslational processing of AE1-4 affect the kinetics with which these polypeptides acquire complex N-linked sugars as well as their stability. These studies revealed that each mutant acquired N-linked modifications that were sensitive to endo H during a 15-min pulse . During the chase period, AE1-4Δ37 and AE1-4(Y47A) acquired additional sugar modifications that were resistant to endo H. However, the percentage of newly synthesized AE1-4Δ37 and AE1-4(Y47A) polypeptides that acquired complex N-linked sugars by 4 h of chase was substantially lower than that observed for AE1-4 . These data indicate that tyrosine 47 and sequences in the NH 2 -terminal 37 amino acids of AE1-4 are both necessary for efficient Golgi recycling of AE1-4. The inefficient recycling of AE1-4Δ37 and AE1-4(Y47A) may account for the reduced ability of these mutant transporters to associate with the detergent insoluble fraction of MDCK cells. Similar analyses with AE1-4Δ53 and AE1-4(Y44A,Y47A) indicated that these mutants acquired no additional modifications after the pulse, and like AE1-3, they were almost completely turned over by 4 h of chase . The pulse–chase and immunoblotting studies indicate that Golgi recycling requires at least one of the tyrosine residues at amino acids 44 and 47 of AE1-4. Furthermore, these data suggest that the stable accumulation of newly synthesized AE1 in this kidney epithelial cell type is completely dependent upon its ability to undergo recycling from the plasma membrane to the Golgi. The alternative NH 2 -terminal sequences of the variant chicken kidney AE1 anion exchangers direct the intracellular trafficking of these membrane transporters in transfected MDCK cells. The AE1-4 variant accumulates in the basolateral membrane of transfected cells, while the AE1-3 variant, which lacks the 63 NH 2 -terminal cytoplasmic amino acids of AE1-4, primarily accumulates in the apical membrane. In addition to targeting AE1-4 to the basolateral membrane of transfected cells, the NH 2 -terminal 63 amino acids of AE1-4 are also required for recycling of this membrane transporter from the plasma membrane to the Golgi. Previous studies have shown that the association of chicken erythroid AE1 anion exchangers with cytoskeletal ankyrin occurs during recycling of newly synthesized polypeptides from the plasma membrane to the Golgi . The data described here have shown that association with the detergent insoluble cytoskeleton and the stable accumulation of the chicken kidney AE1 variants in MDCK cells is also linked to their ability to undergo Golgi recycling. The sorting of type I membrane proteins to the basolateral membrane of kidney epithelial cells has been shown to be dependent upon dominant cytoplasmic sorting signals that direct the vectorial transport of newly synthesized proteins from the TGN to the basolateral membrane . Many of these basolateral sorting signals are tyrosine-dependent . The accumulation of variant chicken AE1 anion exchangers in the basolateral membrane of transfected MDCK cells is also dependent upon cytoplasmic tyrosine residues. Furthermore, the NH 2 -terminal cytoplasmic sequence of AE1-4 contains two tyrosine residues at amino acids 44 and 47, either one of which is sufficient to direct the basolateral accumulation of this variant transporter. A similar result has previously been observed for the low density lipoprotein receptor, which contains two tyrosine-dependent cytoplasmic sorting signals, either one of which is sufficient to direct the basolateral sorting of this type I membrane protein . Interestingly, the same tyrosine residues that are involved in the basolateral accumulation of variant AE1 anion exchangers in MDCK cells are also required for the recycling of AE1 from the plasma membrane to the Golgi. Substitution of alanine for tyrosine 44 and 47 of AE1-4 results in a polypeptide that is almost completely deficient in Golgi recycling, and like AE1-3, the AE1-4(Y44A,Y47A) mutant is rapidly degraded. Either of the tyrosine residues at amino acids 44 and 47 of AE1-4 is sufficient to direct the basolateral accumulation of this type III membrane protein. However, there are differential requirements for these tyrosine residues for Golgi recycling. Substitution of an alanine for tyrosine 47 reduces the efficiency of Golgi recycling of AE1-4 by ∼50%, while a similar substitution at tyrosine 44 has no significant effect on recycling. Tyrosine 47 resides within the sequence, YVEL, that is conserved among all characterized AE1 anion exchangers except for chicken AE1-3 , and the mammalian kidney AE1 variants . This tetrapeptide matches the sequence motif, YXXΦ, where X is any amino acid and Φ is a hydrophobic residue. This sequence motif has been shown to associate with the μ subunits of the AP1, AP2, and AP3 adaptor complexes , and function as an endocytic and trans Golgi network recycling signal for several type I membrane proteins. The hydrophobic residue in the YXXΦ motif is critical for the ability of this sequence to serve as a sorting signal , and for its ability to associate with adaptins . Future studies will address whether additional residues in the YVEL tetrapeptide of AE1-4 are involved in directing the intracellular trafficking of this variant transporter in MDCK cells. The molecular basis of detergent insolubility of AE1 in MDCK cells varies as a function of polarity. Immunolocalization analyses coupled with studies using the actin-depolymerizing drug, latrunculin B, have indicated that the detergent insolubility of AE1-4 in subconfluent, nonpolarized MDCK cells is almost entirely dependent upon an intact actin cytoskeleton. Although the exact role of the actin cytoskeleton in directing the intracellular trafficking of AE1-4 is unclear, the ability of mutant AE1 constructs to accumulate in the basolateral membrane of this cell type correlated with their ability to colocalize with phalloidin-stained stress fibers. Immunolocalization studies with AE1-4Δ37 have further suggested that the organization of the actin cytoskeleton is modified as a consequence of associating with this AE1-4 mutant. Filamentous actin colocalizes with AE1-4Δ37 in large clusters in the basal membrane of subconfluent MDCK cells. This reorganization of actin, which was not observed with wild-type AE1-4 or the other mutant constructs, may contribute to the slow rate at which AE1-4Δ37 recycles to the Golgi and acquires mature N-linked sugars. As cells establish a more polarized epithelial phenotype, the maintenance of detergent insolubility of AE1-4 is only partially dependent upon the actin cytoskeleton. The alternative interactions responsible for the maintenance of AE1-4 insolubility in polarized MDCK cells are not known. However, the fact that chicken erythroid AE1 anion exchangers acquire detergent insolubility in part through their association with cytoskeletal ankyrin suggests that the detergent insolubility of AE1-4 in confluent MDCK cells could arise as a result of their association with the epithelial ankyrin 3 isoform . Previous investigators have postulated that the stable accumulation of the Na + ,K + -ATPase in the basolateral membrane of polarized MDCK cells is dependent upon its association with the detergent insoluble cytoskeleton via its interaction with ankyrin and fodrin . Regardless of the specific interaction(s) that accounts for the detergent insolubility of AE1-4 in MDCK cells, the stability of this variant transporter is not dependent upon its continued association with the detergent insoluble fraction, since detergent soluble and insoluble AE1-4 turn over at a similar rate. Other investigators have hypothesized that phosphorylation of the tyrosine residue in the YVEL tetrapeptide in the cytoplasmic domain of skate erythroid AE1 may be involved in regulating its association with elements of the cytoskeleton, such as ankyrin . Along these lines it is interesting to note that substitution of an alanine for the tyrosine in the Y 47 VEL sequence of AE1-4 only partially blocks Golgi recycling, while it almost entirely inhibits the ability of this polypeptide to associate with the detergent insoluble fraction of confluent MDCK cells. Since AE1-4(Y47A) appears to be unimpaired in its ability to associate with the actin cytoskeleton, these results suggest the possibility that tyrosine 47 may be directly involved in mediating the association of AE1-4 with alternative cytoskeletal receptors, such as ankyrin 3. Our analyses have suggested that a subset of the newly synthesized AE1-3 anion exchanger is directed to the apical membrane of transfected MDCK cells. However, the rapid turnover of this polypeptide in this epithelial cell type prevents high levels of this variant transporter from accumulating in the apical membrane domain. This rapid rate of turnover may account for the fact that polypeptides with the predicted molecular mass of AE1-3 are not detected in chicken kidney membrane preparations by immunoblotting analysis . Although it is possible that AE1-3 accumulates in the apical membrane of a subset of cells in the kidney collecting duct, AE1-3 may also function by modulating AE1-4 activity through heterodimer formation. Future studies will determine whether AE1-3 and AE1-4 have the capacity to heterodimerize in vivo and the consequences of heterodimerization on AE1-4 localization and stability in MDCK kidney epithelial cells. | Study | biomedical | en | 0.999997 |
10601338 | Purified TeNT (2 × 10 7 mouse lethal doses/mg of protein) was a gift from Dr. William Habig (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). Except as noted, purified BoNT A was purchased from List Biological Laboratories, Inc. and BoNT C was from the Centre for Applied Microbiology and Research (5.2 × 10 7 and 1.0 × 10 7 mouse LD 50 /mg protein, respectively). BoNT A (9.4 × 10 6 mouse LD 50 /mg protein) from Calbiochem-Novabiochem Corp. was used for the experiments presented in Fig. 5 , a–c, and Fig. 8 . BoNT A provided by Drs. Eric Johnson and Michael Goodenough (University of Wisconsin, Madison, WI) was used for the experiments in Fig. 4 and Fig. 5 . Concentration of BoNT A was adjusted based on the IC 50 for blockade of glycine release; the effects of each preparation on exo- and endocytosis were tested and found to be identical. BoNT B (1.26 × 10 6 LD 50 /mg protein) was purchased from Calbiochem-Novabiochem Corp. [ 3 H]Glycine (sp act 12.2 Ci/mmol) was purchased from Amersham Corp. Affinity-purified rabbit polyclonal antisynapsin 1a was obtained from Chemicon International, Inc. Affinity-purified rabbit polyclonal antibodies against amino acids 1–32 of the variable domain of vesicle-associated membrane protein (VAMP) 1 and against the COOH-terminal 12 amino acids of synaptosomal-associated protein of 25 kD (SNAP-25) were a gift of Dr. Cesare Montecucco (University of Padova, Padova, Italy). FM1-43 was obtained from Molecular Probes and HRP Type VI from Sigma Chemical Co. Timed pregnant C57Bl/6NCR mice were obtained from the Frederick Cancer Research and Development Center. Neuronal cell cultures were prepared from mouse fetuses, 13 d in gestation as described previously . Dissociated spinal cord cells were plated at 1 × 10 6 cells per 35-mm plastic culture dish coated with Vitrogen-100 ® (Collagen Corp.). For immunohistochemistry and FM1-43 uptake, spinal cord cells were plated at 2.5 × 10 5 cells per dish onto a confluent glial feeder layer . Cultures were maintained at 35°C in a humidified atmosphere containing 10% CO 2 , fed twice weekly by partial medium replacement, and were used between the third and fifth week after plating. TeNT (10–100 ng/ml) or BoNT A (10–300 ng/ml) was added to the cultures in serum-free medium for 16–26 h before experiments (unless noted otherwise). Immunohistochemistry for synapsin, VAMP, and SNAP-25 was performed as described in detail , using antisynapsin at 1:1,000, anti-VAMP at 1:400, and anti–SNAP-25 at 1:400. K + -evoked, Ca 2+ -dependent release of glycine was assayed as described previously . In brief, cultures were labeled with [ 3 H]glycine, rinsed, and incubated in Ca 2+ -free solutions containing 3 mM K + with 0.5 mM EGTA, followed by 56 mM K + (no EGTA), and finally in depolarizing medium containing 2 mM Ca 2+ and 56 mM K + . For this study, 20 μl of incubation medium was withdrawn either at 1-min or 20-s intervals as noted for scintillation spectrometry. Nystatin-perforated whole cell patch clamp experiments were performed on control and toxin-treated neurons. Patch electrodes were fire-polished; electrode resistance was ∼2 MΩ. Resting membrane potentials were measured and spontaneous postsynaptic currents (PSCs) were continually recorded with an Axopatch-1B amplifier, and analyzed with an ITC-16 computer interface, a Macintosh computer, and Synapse software developed by Instrutech Corporation and Synergistic Research Systems. For recording, the cultures were bathed in 1 ml of a Hepes-buffered salt solution (HBSS) containing 136 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, 10 mM glucose, and 0.1% BSA at pH 7.25 with the osmolality adjusted with sucrose to 325 ± 5 mmol/kg . The pipette medium contained 145 mM KCl, 5 mM NaCl, 2 mM MgCl 2 , 5 mM Hepes, 1 mM EGTA, and 100 nM free Ca 2+ , pH 7.3. After baseline recordings were obtained, 1 ml of HBSS containing 56–90 mM KCl (with NaCl adjusted) was applied over the course of 8–18 s to the surface of the patched neuron under direct microscopic observation. With addition of the high K + solution, resting membrane potentials were depolarized (average −60.7 ± 4.1 mV to −16.7 ± 1.7 mV; n = 9). PSC amplitude and duration, beginning within 1 s of the full application of K + , were integrated as PSC area with Synapse software. For FM1-43 loading, control and toxin-exposed cultures were rinsed in HBSS and depolarized for 5 min at 35°C with 56 mM KCl in isosmotic HBSS containing 2 mM CaCl 2 and 2 μM FM1-43 . Cultures were rinsed several times over 20–30 min in HBSS either containing 1 μM tetrodotoxin, or without Ca 2+ and containing 0.5 mM EGTA, to clear surface membranes of the dye while preventing spontaneous network activity and consequent loss of FM1-43 from within labeled terminals. Living cultures were photographed in this rinse solution; some cultures underwent a second round of depolarization (in the absence of FM1-43) to destain synaptic terminals. FM1-43 labeled cultures were photographed using a Zeiss Photomicroscope II with a 40× plan-neofluar lens and T-MAX 3200 film. For uptake of HRP, incubations were identical to the above except that the stimulation medium contained 8–10 mg/ml HRP and the cultures were depolarized for 2–5 min. HRP was visualized after glutaraldehyde fixation (see below) by a 15-min incubation in 3,3′-diaminobenzidine tetrahydrochloride (0.75 mg/ml) in 0.05 M Tris-HCl, pH 7.6, containing 0.01% hydrogen peroxide. Cultures were prepared for electron microscopy as described in detail in Neale et al. 1978 . In brief, fixation in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.3, was followed by postfixation in 1% osmium tetroxide, en bloc staining with uranyl acetate, rapid dehydration in an ethanol series and embedding in the culture dish. After curing, the Epon disc was separated from the plastic dish and fields of interest were selected, removed, and mounted on Epon blanks for ultrathin sectioning. Stained ultrathin sections were examined in a JEM-1010 (JEOL USA Inc.) electron microscope fitted with a goniometer stage. Synaptic terminals were located and the specimens were tilted if necessary to obtain a cross-sectional view of the apposing synaptic membranes. Terminals were photographed at 40,000×. Electron micrographs were printed at 100,000×. The distance between individual synaptic vesicles and the presynaptic membrane was measured manually, and those vesicles lying within 10 nm were scored as morphologically docked. The distance of 10 nm appears reasonable in light of the estimated size of the coiled-coiled core complex proteins linking the synaptic vesicle to the presynaptic membrane . Micrographs were digitized and NIH Image software was used to measure the length of active zones. Vesicle counts were obtained directly from micrographs. Unpaired t tests were used to determine differences between samples. Spinal cord cultures attain electrophysiologic maturity 3 wk after plating when the frequency of spontaneous postsynaptic potentials approaches a maximum . Morphologic development also proceeds rapidly after plating. Fig. 1 illustrates the morphology of neurons (a) and the number and distribution of synaptic terminals (b) in a 3-wk-old culture. Synaptic boutons contain one or more active zones and a complement of either round or flattened small electron lucent vesicles, and occasional larger dense core vesicles . Neurotransmitter release has been assayed by preloading control and toxin-exposed cultures with [ 3 H]glycine or [ 3 H]glutamine and determining the level of radioactivity released into the culture medium after 5 min of depolarization with 56 mM KCl and 2 mM CaCl 2 . In cultures exposed for 20 h to either TeNT or BoNT A (0.06 nM or 10 ng/ml), K + -evoked release of both glycine and glutamate appears completely arrested . High extracellular concentrations of Ca 2+ (5–10 mM) have been shown to partially overcome a BoNT A–induced block in exocytosis . K + depolarization sustained for several minutes might induce an influx of Ca 2+ sufficient to trigger rapid and/or transient synaptic vesicle exocytosis that is not obvious in a 5-min assay. To rule out the possibility that, in BoNT A–treated cultures, there is a bolus of neurotransmitter released within seconds of depolarization that might not be detected by this assay, we examined cultures for glycine release at 20-s intervals after the addition of depolarizing medium . Radioactivity released from cultures in 56 mM K + without Ca 2+ is taken as the baseline; values above this are considered a result of Ca 2+ -dependent K + -induced release. The rate of release from control cultures increases with age in vitro; variability among controls is due most likely to differences in number and maturity of synaptic terminals. In six experiments from three dissections assayed between 23 and 30 d after plating, loss of radiolabeled glycine from TeNT-treated cells is similar in high K + medium with or without Ca 2+ , even at times shortly after exposure to Ca 2+ . The same absence of Ca 2+ -dependent glycine release is seen in sister cultures treated with BoNT A. Assays terminated after 20 s in high K + plus Ca 2+ similarly showed no significant difference between counts released from TeNT- and BoNT A–treated cultures. Experiments repeated on cultures of spinal cord neurons grown on a feeder layer of cortical astrocytes yielded similar results (data not shown). We attempted to induce transmitter release by a medium change either from 0 to 2 mM Ca 2+ (both with 56 mM K + , as shown) or from 3 mM to 56 mM K + (both with Ca 2+ , not shown). In all cases (eight experiments), results are the same; radioactivity lost from BoNT A–treated cultures is very nearly identical to that lost from sister cultures treated with TeNT, and does not exceed that lost in high K + medium without Ca 2+ . In addition, whole cell patch clamp recordings were obtained from neurons before and after depolarization. In untreated control cultures, application of 56–90 mM K + with 2 mM Ca 2+ produces synaptic currents . When baseline recordings are obtained from similar cultures maintained in 1 μM tetrodotoxin (TTX), K + -evoked synaptic currents are observed at levels similar to controls . Synaptic currents are not detected in cultures exposed for 16–20 h to either TeNT or BoNT A , at toxin concentrations 35–50% as high as those used for the morphologic studies. The observations in cultures treated with BoNT A are indistinguishable from those in TeNT, with levels of activity at least 20-fold less than in controls . These data were obtained within 1 s after application of high K + medium; manual analysis of records during the application of high K + provided no evidence of synaptic events in either TeNT- or BoNT A–treated cultures. Control and toxin-blocked cultures were stained immunohistochemically for the synaptic proteins known to be substrates for proteolysis by these toxins. In control cultures, antibodies against VAMP yield a staining pattern almost identical to that seen with antisynapsin . However, VAMP is not detectable in cultures when neurotransmitter release is arrested by TeNT . Similarly, the COOH terminus of SNAP-25 is evident in untreated cultures , and is not observed in BoNT A–blocked cultures . Synaptic fine structure was examined in sister cultures at the time of synaptic blockade, with and without K + depolarization . The length of individual active zones was measured and docked synaptic vesicles (within 10 nm of the presynaptic membrane) were counted ( Table ). In toxin-blocked cultures, the average length of the active zones is not significantly different from controls (i.e., 0.45–0.50 μm). In nonstimulated but spontaneously active control preparations , ∼7.8 ± 4.7 (SD) vesicles/μm of active zone appear docked. In toxin-blocked cultures, the number of docked synaptic vesicles is almost twice as high as in control cultures . In controls, ∼25% of active zones have >10 docked vesicles/μm, whereas in cultures exposed to TeNT or BoNT A, ∼80% of active zones have >10 vesicles/μm. Experiments with BoNT B (not shown) show identical results. However, when control cultures are stimulated with 56 mM KCl and 2 mM CaCl 2 before fixation , there is a reduction in the number of vesicles at the active zone with a lower percentage (16%) of active zones showing >10 vesicles/μm. In both TeNT- and BoNT A–blocked cultures, K + depolarization fails to elicit neurotransmitter release and does not reduce the number of docked vesicles, with 78% of the active zones having >10 docked vesicles/μm for TeNT, and 89% for BoNT A. Thus, relative to nonstimulated controls, TeNT- and BoNT A–blocked terminals have more synaptic vesicles apposed to the presynaptic membrane of the active zone, and K + stimulation does not reduce this number, as it does in controls. Thus, functional assays of toxin action are corroborated by morphologic evidence for proteolysis of toxin substrates and for impedance of synaptic vesicle exocytosis. To demonstrate further the toxin-induced electrical quiescence, we used the styryl dye FM1-43 for the optical detection of synaptic activity . Depolarization of control cultures in the presence of FM1-43 results in a pattern of fluorescence consonant with the labeling of synaptic terminals . Uptake of FM1-43 is not observed with depolarization in the absence of Ca 2+ and is markedly reduced without depolarization . As expected, cultures treated for 22 h with 10 ng/ml TeNT , 10–100 ng/ml BoNT C , or BoNTs B or D (10 ng/ml; not shown), fail to release neurotransmitter with K + depolarization, and also fail to take up FM1-43. In marked contrast, cultures blocked with 10 ng/ml of BoNT A, which exhibit no K + -evoked release of neurotransmitter show intense K + -stimulated uptake of FM1-43. Increasing the concentration of BoNT A to 200 ng/ml or 300 ng/ml (not shown) failed to abolish FM1-43 uptake. As in controls, FM1-43 loading of BoNT A–treated cultures requires both extracellular Ca 2+ and elevated K + . These results indicate that BoNT A blockade of neurotransmitter release does not prevent vesicle membrane reuptake by endocytosis, and that membrane retrieval is dependent on Ca 2+ . Evidence that FM1-43 labeling is associated with recycled synaptic vesicles is provided by the ability to destain a labeled preparation by further stimulation in the absence of dye. Indeed, K + stimulation of control cultures preloaded with FM1-43 results in dye loss . When dye-loaded cultures are stimulated in the absence of Ca 2+ , exocytosis of labeled vesicles is prevented and fluorescence is retained . However, in BoNT A–blocked cultures that have been loaded with FM1-43, subsequent depolarization (in the presence of Ca 2+ ) does not induce destaining . This provides further evidence that synaptic vesicles in BoNT A–blocked cultures are unable to undergo exocytosis. To strengthen the hypothesis that FM1-43 staining of BoNT A–blocked terminals is related to vesicle membrane endocytosis, we examined the fine structure of synaptic terminals. In spontaneously active control cultures , clathrin-coated pits and vesicles are seen only occasionally, although empty clathrin baskets are not uncommon. When control cultures are stimulated , small coated pits and vesicles (arrows) are observed with increased frequency and empty clathrin baskets with decreased frequency. In cultures blocked with TeNT or with BoNT A , synaptic vesicles accumulate at the presynaptic membrane, empty clathrin baskets are typical, and coated pits and vesicles are extremely rare. When TeNT-blocked cultures are stimulated with K + , these synaptic features remain unchanged, suggesting a lack of membrane movement. When BoNT A–blocked cultures are depolarized , the number of docked synaptic vesicles remains elevated, although clathrin-coated pits are found with increased frequency and empty clathrin baskets have disappeared. Thus, stimulated BoNT A cultures take on those features which suggest active retrieval of synaptic vesicle membrane. To confirm that synaptic terminals incapable of vesicle exocytosis can be stimulated to retrieve vesicle membrane, the vesicle-loading experiments were repeated with HRP substituted for FM1-43 in the stimulation medium. Control and toxin-blocked cultures were depolarized by K + with Ca 2+ for 5 min in the presence of 10 mg/ml HRP, rinsed for 30 min in medium containing TTX, and then fixed. A large proportion of synaptic vesicles in control cultures contain HRP reaction product. As expected, HRP-labeled vesicles are rare in TeNT-blocked cultures . Depolarization of BoNT A–blocked cultures, however, results in the labeling of a much greater proportion of small vesicles . When BoNT A–blocked cultures are fixed almost immediately after depolarization (i.e., without an extended rinse), clathrin-coated vesicles of the appropriate size are labeled with HRP (insets). These experiments lend further credence to the proposal that, in BoNT A–blocked neurons, synaptic vesicles can be endocytosed in the absence of temporally associated vesicle exocytosis, and indicate that the recycling process involves the clathrin-coated membrane organelles observed in control preparations. To confirm that HRP-labeled vesicles in control cultures are competent for exocytosis, we loaded terminals by stimulating for 2 min in the presence of 8 mg/ml HRP. HRP-loaded terminals were subjected to a second interval of K + depolarization and the change in percentage of labeled vesicles was determined . After HRP-loading of the control cultures, ∼10% of total synaptic vesicles contain HRP . In cultures stimulated after loading, labeled vesicles decrease to 2.5% of total vesicles . Without Ca 2+ in the stimulation medium, the percentage of labeled vesicles remains high (10%; not shown). Thus, the loss of HRP is consistent with synaptic vesicle exocytosis. In BoNT A–blocked cultures, HRP loading (as above) labels ∼5% of the synaptic vesicles . This percentage is not decreased by subsequent depolarization , confirming that exocytosis is not stimulated in the presence of BoNT A. When loading of either BoNT A–blocked or control cultures is attempted in the absence of extracellular Ca 2+ , after a rinse in Ca 2+ -free buffer, there is no HRP in synaptic vesicles ( Table ), and there are no HRP-labeled cisternae. In control cultures, failure to load can be taken as a consequence of the zero Ca 2+ -induced block in exocytosis. The absence of vesicle recycling in BoNT A–blocked terminals under these conditions is not due to a failure of vesicle exocytosis since the endocytosis of HRP in blocked cultures occurs in the absence of measurable exocytosis. Therefore, it must be concluded that the endocytic retrieval of synaptic vesicle membrane requires Ca 2+ . In another set of cultures (data not shown), we mock-loaded BoNT A–blocked cultures (2 min in high K + , with Ca 2+ but no HRP) from one to four times before adding HRP to the stimulation medium. The number of vesicles labeled with HRP decreases with each stimulation interval. When HRP is added for a fifth stimulation, labeled vesicles are rare. Thus, there appears to be a finite source of surface membrane that can be retrieved and recycled as synaptic vesicles. In spinal cord cell cultures, biochemical, electrophysiologic, and morphologic assays provide evidence for ample exo- and endocytosis of synaptic vesicles. Treatment of cultures with TeNT results in the cessation of both neurotransmitter release by synaptic vesicle exocytosis and retrieval of synaptic vesicle membrane by endocytosis. In contrast, in BoNT A–blocked cultures, substantial endocytosis occurs in the absence of evidence for exocytosis as measured by these assays. The data as a whole imply that a reservoir of synaptic vesicle membrane exists on the plasma membrane of the nerve terminal, that calcium is required to initiate the uptake of this membrane for recycling, and that BoNT A, unlike TeNT, does not interfere with the process of synaptic vesicle membrane retrieval. Atypical accumulation of docked synaptic vesicles is associated with tetanus intoxication and with disruption of the function of the N -ethylmaleimide–sensitive factor . This morphology suggests an inability of synaptic vesicles to fuse with the presynaptic membrane and is entirely compatible with the demonstration that TeNT is a zinc endopeptidase that cleaves VAMP , a homologue of a protein critical for transport vesicle docking and fusion at the yeast and mammalian Golgi apparatus. TeNT and BoNTs A and C, each acting on a different protein of the ternary core complex , induce blockade of neurotransmitter release, which correlates with toxin cleavage of its specific substrate . We report here that BoNTs A and B also cause an accumulation of synaptic vesicles at the active zone at a time when neurotransmitter release is arrested. These observations support the requirement of the ternary complex (VAMP, syntaxin, and SNAP-25) for vesicle fusion but not for vesicle docking. The occurrence of docked vesicles in toxin-treated preparations has been explained by the ability of synaptotagmin, a synaptic vesicle integral membrane protein, to bind to SNAP-25 on the presynaptic membrane . This binding is not dependent on Ca 2+ or on the presence of syntaxin or VAMP, and occurs even with BoNT A- or E-cleaved SNAP-25. Functional docking (i.e., leading to vesicle fusion), however, requires that the proteins of the core complex be intact . This is consistent with our observation that K + -induced depolarization of control cultures reduces the number of vesicles at the active zone, whereas depolarization of toxin-blocked cultures is not similarly effective. Thus, the clostridial neurotoxins block neurotransmitter release by preventing synaptic vesicle fusion. The synaptic blockade induced by BoNT A in this study is at variance with several reports of the failure of BoNT A to produce a complete block in transmitter release. The latter studies introduced toxin into normally nonreceptive PC12 or chromaffin cells by mechanical cracking or digitonin permeabilization of the cells, or intracellular injection of recombinant BoNT A light chain . It remains unclear whether this difference in the extent of blockade reflects the underlying biology of toxin internalization into intact neurons or differences in assay conditions. It might be argued that, under the experimental conditions described here, BoNT A blocks the release of glycine and glutamate , but fails to block the release of other transmitters. If so, we would expect to see some nerve terminals with, and others without, evidence of vesicle recycling. In fact, HRP-labeled vesicles are seen in every synaptic terminal. Alternatively, if BoNT A had caused the loss of stored transmitter from synaptic vesicles, depolarization would induce the exocytosis of empty vesicles. However, there is considerable evidence that BoNT A does not interfere with the synthesis and/or storage of acetylcholine nor TeNT with that of GABA . Finally, vesicle recycling in BoNT A–blocked cultures may have occurred as a result of rapid or low level K + -evoked exocytosis that was below the sensitivity or time resolution of our assays. Both glycine release and synaptic currents are maintained in high K + for several minutes in control cultures, and about half the number of vesicles are recycled in BoNT A–blocked cultures as in untreated controls. It seems unlikely that both the neurotransmitter release assays and whole cell patch clamp analysis would fail to detect exocytic responses of this magnitude. While it may not be possible to prove that absolutely no exocytosis occurs with BoNT A, there remains a striking disproportion between the level of exocytosis that could be measured and what might be expected to account for the endocytosis that is observed. We have used the dissociation of synaptic vesicle exo- from endocytosis to show that vesicle endocytosis requires Ca 2+ . Our results contrast with those of Ryan et al. 1993 who reported endocytosis of synaptic vesicle membrane, in cultures of hippocampal neurons, in the absence of extracellular Ca 2+ . The time course of the decrease in intraterminal Ca 2+ , elevated by the initial membrane depolarization, suggests that superfusion with FM1-43 was begun before the free Ca 2+ concentration had returned to the baseline. This residual elevation may have been sufficient, even in the absence of either extracellular Ca 2+ or simultaneous depolarization, to initiate membrane retrieval. Ca 2+ is required to initiate vesicle endocytosis after vesicle depletion induced by α-latrotoxin at the frog neuromuscular junction (NMJ) or by prolonged high frequency action potential stimulation in lamprey giant reticulospinal synapses . In the latter preparation, an extracellular Ca 2+ concentration of 11 μM was sufficient to induce clathrin-mediated synaptic vesicle endocytosis . Synaptic vesicle recycling studied in synaptosomes also supports a Ca 2+ requirement for endocytosis at a concentration lower than that required for maximal exocytosis . In that study, Ca 2+ entry acts as the trigger by activating calcineurin which dephosphorylates a number of proteins, including amphiphysin and dynamin, implicated in clathrin-mediated endocytosis . Low extracellular Ca 2+ allowed near maximal endocytosis after very submaximal exocytosis, leading to the speculation that the synaptic terminal membrane might always contain a pool of recoverable membrane with “exocytosis adding to this pool and endocytosis drawing from it” . Experiments at the NMJ of Drosophila shibire ts1 mutants suggest that any Ca 2+ -requiring step in endocytosis occurs early, i.e., before the accumulation of endocytic membrane invaginations . Secretory vesicle membrane retrieval by rapid endocytosis in adrenal chromaffin cells and melanotrophs also appears to be Ca 2+ -dependent although it is not mediated by clathrin-coated vesicles . In this study, endocytosed FM1-43 and HRP in control cultures are associated with recycled synaptic vesicles that are exocytosis-competent. Although tracer exocytosis is not possible in BoNT A–blocked terminals, HRP-labeled vesicles are observed docked . Further studies are required to confirm that the recycled vesicle membrane contains the appropriate complement of proteins and that the vesicles themselves contain neurotransmitter. Nonetheless, the present work stands as a direct demonstration of a Ca 2+ requirement for endocytosis. In neuroendocrine cells, endocytosis does not always strictly follow exocytosis and, under certain circumstances, exocytosed membrane may reside in the plasma membrane until specific conditions drive retrieval . However, immunofluorescence fails to detect synaptic vesicle proteins on the terminal plasmalemma of the NMJ under resting conditions . Similar studies in rat hippocampal cell cultures, using antibodies directed against the lumenal NH 2 terminus of synaptotagmin I (Syt lum -Abs) also demonstrated a lack of surface membrane signal, even after depolarization for several minutes . However, radioimmunoassays using [ 125 I]Syt lum -Abs provide quantitative data demonstrating a significant pool of synaptotagmin I at the cell surface even at steady state . This pool comprises about half the synaptotagmin measured after K + depolarization for 3 min. Given that antibody binding reflects the presence of synaptic vesicle membrane, it could be inferred that a sizable membrane reserve exists in the surface membrane of the synaptic terminal. This membrane reserve, then, could serve as the source of membrane internalized in BoNT A–treated neurons. The present results suggest that TeNT may disrupt a component of the reuptake mechanism that is unaffected by BoNT A. VAMP-2 is recognized by AP3 for the formation of synaptic vesicles from endosomes of PC12 cells. Tetanus toxin inhibited the formation of synaptic vesicles and their coating with AP3 in vitro, whereas BoNTs C and E had no effect . If VAMPs were similarly a sorting signal for synaptic vesicle endocytosis in the nerve terminal, TeNT might prevent the retrieval of vesicle membrane, whereas BoNT A would not. The synaptic vesicle recycling pathway described at the NMJ involves vesicle collapse into the plasmalemma and membrane retrieval, away from the active zone, by clathrin-coated pits and vesicles, the latter fusing to form intermediate cisternal compartments that bud new synaptic vesicles . The formation of cisternae by the coalescence of small endocytic vesicles , implies a homotypic fusion between recycling, but not mature, vesicles that might involve SNARE proteins or their equivalent that are not affected by BoNT A. Alternatively, the cisternae may be sections of tubular invaginations of the plasma membrane formed by bulk endocytosis and not the products of vesicle fusion at all . Other aspects of this recycling pathway have been challenged with evidence suggesting that membrane retrieval at the NMJ by clathrin-coated vesicles occurs after only high frequency stimulation, and that the physiologic pathway for exo-endocytosis involves the formation of a fusion pore and rapid retraction of the vesicle . Electron microscopy of giant synapses in which endocytosis has been blocked by compromising dynamin function demonstrate the accumulation of clathrin-coated pits after either a low or high frequency stimulation . Thus, it appears that clathrin plays a role in a single process that operates to recycle synaptic vesicles. Furthermore, recycling kinetics are similar after high and low frequencies and appear sufficiently slow to allow for vesicle collapse and membrane retrieval and sorting through an intermediate endosomal compartment , although involvement of this intermediate compartment remains in controversy . Vesicle recycling studies using FM1-43 at the NMJ under the influence of staurosporine have been reconciled with the fusion pore model of exocytosis. Despite the similarity of some of the staurosporine data with those obtained using BoNT A, there are a number of important differences. First, there is no evidence for neurotransmitter release; i.e., a membrane fusion event, from BoNT A–exposed motor endplates or spinal cord cultures . Second, depolarization of BoNT A–blocked neuronal terminals results in labeling of synaptic vesicles with HRP; and third, repeated intervals of stimulation before HRP incubation result in a run-down of endocytosis, suggesting a finite source of retrievable surface membrane. Our observation of HRP labeling of coated vesicles and membrane cisternae in BoNT A–treated neurons, after K + -induced depolarization in the presence of extracellular Ca 2+ , is entirely consistent with the collapse of synaptic vesicles into the plasma membrane and subsequent membrane retrieval by endocytic mechanisms. That synaptic vesicle recycling occurs as unitary quantal events, which are temporally linked to single action potentials , is indisputable evidence that the coupling between vesicle exo- and endocytosis is highly regulated. Membrane retrieval appears to be driven by something other than membrane incorporation . Further studies aimed at understanding how synaptic vesicle endocytosis occurs during BoNT A, and not during TeNT, blockade will undoubtedly provide insight into this basic mechanism and may lead to the identity of the trigger and the cascade of events it initiates. | Study | biomedical | en | 0.999996 |
10601339 | Dictyostelium -specific dynein antibodies used were: M4, a mouse polyclonal antibody against dynein IC; IC144, a rat polyclonal antibody against IC; and NW127, a rabbit polyclonal antibody against dynein heavy chain (HC). Antibody generation will be described in Results. M4 polyclonal antibody was used to screen a λgt11 cDNA expression library made from Dictyostelium cells developed for 4 h (Clontech Laboratories, Inc.). 10 immunoreactive phage clones were isolated, 3 of which were positive by epitope selection. The longest of these, IC10, had an open reading frame of 1,956 nucleotides. The other two clones were partial sequences contained within the IC10 sequence . The full-length clone IC10 was used as a PCR template to amplify various IC truncation mutants. 33-nucleotide extensions were added to the 3′ PCR primers (5′-TTA TAA ATC TTC TTC ACT AAT TAA TTT TTG TTC-3′) to produce the COOH-terminal myc epitope tags. BamH1 sites were added at the 5′ ends of all PCR primers to facilitate subsequent cloning. PCR products were cloned into the BamH1 site of pVEII , downstream of a discoidin I-γ promoter, whose activity can be repressed by including folate in the medium and induced by withdrawing folate. AX3 wild-type Dictyostelium cells were transformed by electroporation with 10 μg plasmid DNA as described previously . Transformants were cloned in 96-well plates in HL5 medium with 50 μg/ml G418. Folate (1 mM) was added to the medium during selection and expansion of the clones. Several independent clones of each class were analyzed in each experiment to control for possible mutations caused by clonal variation. For immunoprecipitations (IPs), 4 × 10 7 cells were collected and washed twice with 15 mM Na-KPO 4 buffer, pH 6.5. After resuspension in 1 ml IP buffer (50 mM Pipes, pH 6.8, 5 mM EDTA, 100 mM NaF, 25 mM Na pyrophosphate, 2.5 mM DTT, 1 mM PMSF, 50 μg/ml leupeptin, 50 μg/ml pepstatin, 1 mM ATP) cells were lysed by sonication. The cell lysate was cleared by centrifugation at 38,000 g for 30 min at 2°C. Protein A–Sepharose preincubated with the IP antibody was added to the cell lysate and the mixture incubated with rocking for at least 2 h at 4°C. Dynein HC antibody NW127 was used to immunoprecipitate dynein complex, whereas affinity-purified capping protein β antibody R18 (a generous gift from Dr. John Cooper, Washington University, St. Louis, MO) was used to immunoprecipitate the dynactin complex. Sepharose bead–bound immune complexes were collected by centrifugation and washed four times with IP buffer. The final pellets were resuspended in 30 μl 2× SDS sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol), boiled for 5 min, centrifuged, and the supernatant collected. For Western blots, samples were separated on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked in 5% nonfat milk and incubated with primary antibody followed by HRP-conjugated secondary antibody. Blots were developed in Renaissance enhanced chemiluminescence reagent (NEN Life Science Products) and exposed to X-ray film. For each gradient, 2 × 10 8 cells were washed twice with 15 mM Na-KPO 4 buffer, pH 6.5, resuspended in 0.4 ml of lysis buffer (100 mM Pipes, pH 6.8, 1 mM EDTA, 5 mM EGTA, 5 mM MgSO 4 , 2.5 mM DTT, 1 mM PMSF, 50 μg/ml leupeptin, 50 μg/ml pepstatin) and lysed by sonication. After centrifugation at 38,000 g for 30 min at 2°C, the lysates were loaded on an 11-ml 5–20% continuous sucrose density gradient made in lysis buffer without protease inhibitors. The gradients were centrifuged in a SW41 rotor (Beckman Instruments Inc.) at 31,500 rpm for 16 h (4°C). Fractions (0.8 ml) were collected from the bottom of the gradient and protein samples prepared for Western blot analysis. Cells grown on sterile coverslips were fixed and indirect immunofluorescence performed with various antibodies followed by 4,6-diamidino-2-phenylindole (DAPI) staining. To stain the Golgi complex, cells were fixed in −15°C methanol for 5 min and stained with a comitin mAb (a gift from Dr. Angelika A. Noegel, University of Cologne, Cologne, Germany). For tubulin staining, cells were fixed in 1.85% formaldehyde in 15 mM Na-KPO 4 , pH 6.5, for 5 min at room temperature, then extracted in −15°C methanol for 5 min, and stained with a rat anti–α-tubulin mAb (Serotech, Ltd.). For centrosome staining, cells were fixed with either of the two methods described above and stained with polyclonal γ-tubulin antibody and several different mAbs specific for Dictyostelium centrosomes (NAB350, 4/148, and 2/165) . Conventional immunofluorescence microscopy was carried out on an Axioskop microscope (Carl Zeiss, Inc.) equipped with a 100×, 1.4 NA oil immersion objective. Images were taken with a cooled CCD camera (Hamamatsu Photonic Systems) controlled by MetaMorph 3.6 imaging system (Universal Imaging Corp.) and were processed with MetaMorph and Adobe Photoshop 5.0 (Adobe Systems, Inc.). Dictyostelium cells were washed in 15 mM Na-K PO 4 buffer, fixed in ice-cold 70% ethanol, and stained with 50 μg/ml propidium iodide. RNase was included in the staining solution to remove double-stranded RNA. The nuclear DNA content of the stained samples was analyzed using FACS-Calibur (Beckton Dickinson). Cells grown on coverslips were fixed in 4% glutaraldehyde in 0.1 M Sorensens phosphate buffer, pH 7.4, rinsed, and postfixed in 1% osmium tetroxide in 0.1 M Sorensens phosphate buffer, pH 7.4 for 1 h. After dehydration in ethanol and propylene oxide, they were embedded in POLY/BED 812 (Polysciences Inc.). After polymerization at 60°C for 48 h, coverslips were removed, and serial sections were cut about 90-nm thick on a Reichert-Jung Ultracut E ultramicrotome (Leica, Inc.). After staining in 4% uranyl acetate followed by Reynolds lead, sections were viewed and photographed on a JEOL JEM 1220 transmission electron microscope at 15,000× to 40,000× magnification. Cytoplasmic dynein was isolated from vegetatively growing Dictyostelium cells . Dictyostelium dynein contains a 530-kD dynein HC, an 83-kD IC, and a 58-kD light IC . The 83-kD IC, purified from SDS-PAGE gels, was used to generate polyclonal antisera in mice. One of these antisera, M4, reacted specifically with an 83-kD protein on Western blots of Dictyostelium whole cell extract and with the IC of purified dynein . M4 was used to screen a Dictyostelium cDNA expression library. A full-length clone, IC10, encodes a protein of 651–amino acids, predicted to have an M r of 72 kD. For subsequent experiments, we generated additional Dictyostelium dynein antibodies: polyclonal rat antibody IC144 was raised against bacterially expressed IC , and polyclonal rabbit antiserum NW127 was generated against SDS gel–purified dynein HC . Dictyostelium dynein IC is 39% identical and 56% similar to the rat IC, and 24% identical and 48% similar to the axonemal dynein IC sequence. In vitro binding studies have mapped the p150/Glued binding domain to the NH 2 -terminal 123 amino acids of rat dynein IC . Although the primary sequences of Dictyostelium and rat dynein IC are very divergent (26% identical) at the NH 2 terminus, their predicted secondary structures are similar. Both have a coiled–coil domain near the NH 2 terminus followed by a serine-rich region. The structural similarity suggests that this region of the Dictyostelium IC is likely the dynactin binding domain. In contrast to the NH 2 -terminal domain, the COOH-terminal half of Dictyostelium and rat IC show 71% identity, suggesting that this domain may be involved in a conserved dynein IC function such as binding to other dynein subunits. Our hypothesis was that the NH 2 -terminal domain of IC is important for targeting dynein activity such as binding to dynactin, whereas the COOH terminus is crucial for dynein HC binding. To functionally define the domains of dynein IC and to generate cell lines with defective dynein function, we overexpressed different IC domains in wild-type Dictyostelium . We designed several dynein IC truncation mutants that deleted or disrupted the hypothetical HC binding domain or the predicted dynactin binding domain . ICΔC lacks the COOH-terminal 373 amino acids, whereas ICΔN47 and ICΔN106 lack the NH 2 -terminal 47 or 106 residues, respectively. Mutant IC expression was controlled by a discoidin promoter, whose activity can be repressed by addition of folate to the medium . This system makes it possible to conditionally express potentially deleterious mutants in Dictyostelium . Mutant protein expression was assessed using Western blots of whole cell lysates from cells induced for 3 d with IC-specific IC144 antibody . For all three mutants, the mutant expression level was >10 times that of the endogenous wild-type IC. In vitro studies indicated that dynein IC can associate directly with dynactin complex and thus could serve as a link between the two large complexes . To test the IC truncation mutants for binding to either complex, we immunoprecipitated dynein with a dynein HC antibody (NW127) and dynactin using an affinity-purified capping protein β antibody (R18) . From wild-type Dictyostelium lysates, the endogenous IC coprecipitated with both NW127 and R18 antibody , suggesting that wild-type IC associates with both dynein and dynactin complexes. However, each of the IC truncations was deficient in association with one of the two complexes. Although a significant amount of ICΔC was detected in the capping protein IP, very little was detectable in dynein HC IP . This suggests that the COOH-terminal deletion abolished the ability of IC to bind to dynein HC while preserving its dynactin binding activity. In contrast, both ICΔN106 and ICΔN47 mutants bound dynein HC well but associated poorly with dynactin. The ratio of ICΔN to endogenous IC associated with dynactin is greatly reduced when compared with their ratio in cell lysates . This suggests that the NH 2 -terminal IC region is crucial for dynactin association and is consistent with previous studies that mapped the p150 binding activity to the NH 2 -terminal region . ICΔN47 showed better HC binding than ICΔN106, indicating that whereas the COOH-terminal conserved region of IC is required for dynein HC association, the binding is stabilized by residues that extend into the NH 2 -terminal domain. To further investigate the dynein complex in IC mutant cells, cell lysates were fractionated on a 5–20% sucrose density gradient. Wild-type dynein migrated as a 20S complex containing both the HC and IC . In ICΔC cells, ICΔC protein did not cosediment with the dynein HC and wild-type IC , providing independent evidence that this mutant failed to associate with dynein. Also, since wild-type IC comigrated with HC, dynein complex was not affected by ICΔC expression. For both ICΔN47 and ICΔN106 cells, a significant amount of the mutant IC cosedimented with HC at the normal position for dynein. The ratio of mutant IC to wild-type IC in the dynein fractions was consistent with that in dynein IPs, with the majority of dynein complexes containing the truncated IC mutant. In ICΔN106, a large amount of the mutant IC migrated near the top of the gradient, most likely due to its high expression level. These results demonstrate that the IC truncation mutants are defective in their ability to mediate dynein–dynactin association, since they efficiently bind to only one partner but not the other. Therefore, overexpression of these truncated ICs would compete with endogenous wild-type IC for binding to one of the two partners, thereby disrupting the dynein–dynactin association. Consistent with this, there was less wild-type IC bound to HC in ICΔN-expressing cells, and greatly reduced wild-type IC in capping protein IPs in ICΔC expressing cells . We analyzed the three different IC mutant cell lines to determine the consequence of mutant IC expression. Interestingly, all three mutants produced similar phenotypes by affecting a range of dynein-dependent functions. By phase-contrast microscopy, induced mutant cells appeared larger and flatter than control or uninduced mutant cells . To control for nonspecific effects due to high level exogenous protein expression, we also expressed full-length myc-tagged IC at levels similar to the mutants. None of the abnormalities seen in the mutants were observed (data not shown). Several studies have highlighted the importance of cytoplasmic dynein in Golgi apparatus positioning . Therefore, Golgi complex localization is a good in vivo indicator of cytoplasmic dynein and dynactin function. Localization of the Golgi complex was detected by indirect immunofluorescence using a mAb against comitin . In wild-type Dictyostelium cells, the Golgi complex appears as a compact perinuclear cluster whose center coincides with the MT organizing center (MTOC) . In contrast, in all three IC mutants the Golgi complex was dispersed throughout the cytoplasm , supporting the idea that ICΔN and ICΔC expression disrupted dynein function. Consistent with the change in cell morphology in the IC mutants, we observed dramatic changes in the organization of the MT network. Cells induced for 3 d were analyzed by indirect immunofluorescence with tubulin antibody and DAPI staining of DNA. In wild-type cells , interphase MTs formed extended radial arrays originating from the MTOC . However, in both ICΔC and ICΔN cells, the interphase MTs were profoundly disorganized. The most striking phenotype was that the majority of the mutant cells showed the MT network collapsed into a bundle . The MTOC of the bundles were often displaced to the cell periphery. Some cells showed a large yet relatively normal looking MT network . The MTOC in both groups of cells were larger than normal, appearing as a large ring with a hollow center. As discussed below, the centrosomes in these cells were also abnormally large. We also observed other MT abnormalities in a small number of mutant cells. Some cells exhibited multiple cytoplasmic MT asters , some of which were nucleus-associated, whereas many were not. Occasionally we observed cells lacking obvious MT organization , in which individual MTs were randomly distributed instead of focusing to an organizing center. Mutant cells with MT abnormalities often showed big irregular interphase nuclei with abnormal DNA distributions. We performed FACS ® analysis to characterize the total DNA content in individual cells . The vast majority of wild-type cells contained 2 N DNA, with a minor peak at 4 N . In contrast, the majority of ICΔC cells contains >2 N DNA. Furthermore, the major peak of the mutant cells fell somewhere between 2 N and 4 N, and the rest had a wide range of DNA content that was not a multiple of the normal. This result suggests that the mutant cells were aneuploid, probably due to defects in chromosome segregation. The abnormal nuclear DNA content and decreased viability (discussed below) of IC mutants suggested that the IC truncations caused defects in mitosis. Yet as judged by tubulin and DAPI staining, there was no increase in the mitotic index after induction. Like control cells, <2% of the induced IC mutant cells were in mitosis. Of the small numbers of mitotic spindles observed, some seemed normal, and others were monopolar or multipolar. In general, cells expressing IC truncations did not accumulate in mitosis, although there was clearly a mitotic defect. Because of the profound MT disorganization, we next focused our attention on the MTOC or centrosome. We examined the centrosomes by indirect immunofluorescence using antibodies specific for several Dictyostelium centrosomal components (NAB350, 4/148, 2/165, and γ-tubulin) . All four antibodies have been shown to stain the corona of Dictyostelium centrosomes, which appear as dots by immunofluorescence. Over 99% of wild-type cells showed a single centrosome of uniform shape and size associated with the periphery of each nucleus . However, all three IC truncation mutants showed a variety of centrosome abnormalities, including alterations in size, shape, number, and position. The centrosome abnormalities were rare in repressed cells, but dramatically increased upon induction. The most frequent abnormality in the IC mutants was an enlarged centrosome associated with an enlarged nucleus , or less frequently, with multiple nuclei . Interestingly, the increased size and intensity of the centrosomes almost always correlated with increased cell size and nuclear DNA content. Occasionally elongated or dumbbell-shaped centrosomes were observed, which seemed to be two centrosomes closely adjacent to each other , a pattern not observed in wild-type interphase cells. This strongly suggests that the abnormally large centrosomes may result from failed centrosome separation after duplication. We also observed a small number of IC mutant cells with multiple centrosomes and only one or two nuclei , especially late in the induction course. Whereas some of these centrosomes were nucleus-associated, many were not. To examine whether the centrosome and MT abnormalities were related, we double-labeled the centrosome and tubulin . Except in cells with no apparent MT organization, centrosomal components always colocalized with the centers of MT asters. The abnormal size and number of centrosomes correlated with the abnormal size and number of MT networks. To better understand the effect of IC mutations on MT organization and centrosome morphology, we determined MT and centrosome morphology at various timepoints after induction . The phenotypes fell into one of five categories: (1) normal MT network; (2) bundled MTs; (3) large centrosomes; (4) multiple MT asters; and (5) disorganized MT without an obvious organizing center. Before induction (day 0), >90% of the ICΔC cells had normal MT networks and centrosomes . 1 d after induction, the MT network was bundled in >50% of the cells, although the sizes of the centrosomes were normal. As induction proceeded, the cells, their MT networks, and their centrosomes became enlarged. On day 3 after induction, ∼60% of the cells had large MT bundles and ∼70% showed large centrosomes. The large MT networks were usually accompanied by large centrosomes and large nuclei. Less than 10% of the cells showed relatively normal MT networks. However after 4 d, the number of cells with normal MT networks began to increase. By day 6, 73% of the population had normal MTs and centrosomes. This time course established two primary phenotypes: MT bundling and unusually large centrosomes. MT bundling became apparent very early after induction and peaked by 3 d. The large centrosomes appeared on day 2 and peaked on day 3. Whereas disruption of the MT network occurred once the level of mutant IC became high enough to disrupt dynein function, the effect on centrosomes occurred only after mitosis. Peak expression of the phenotypic defects correlates with the level of mutant IC . Accordingly, after day 4, ICΔC expression decreased, leading to increased number of cells with normal MT networks. A similar pattern was observed in ICΔN47 cells . MT bundling and large centrosomes appeared and peaked around the same time as in ICΔC cells. ICΔC and ICΔN mutants had similar effects on dynein function, further suggesting that the major function of the IC is to mediate dynein–dynactin interaction, and that most dynein functions require dynactin association. The return of normal MT organization along with decreasing mutant expression could occur because the mutant phenotype was reversible, or because the severely affected cells died and were eventually outgrown by cells that no longer expressed the IC truncation. Therefore, we determined cell viability after induction by measuring the plating efficiency. In contrast to 91% for wild-type, ICΔC cells induced for 3 d had a plating efficiency of 12%, indicating that mutant cells had significantly decreased viability. The number of viable mutant cells was comparable to the number with normal MT networks (9%), consistent with the idea that cells with MT or centrosome abnormalities may be nonviable, most likely due to mitotic defects. One striking phenotype of IC mutants was the presence of apparently larger centrosomes. Apparently large centrosomes in the light microscope could result from closely positioned, morphologically normal centrosomes produced by defective centrosome duplication or separation, or from the abnormal accumulation of centrosomal material. To distinguish these possibilities, we examined centrosome morphology at the ultrastructural level. The interphase Dictyostelium centrosome is a nucleus-associated body consisting of a rectangular, electron-dense core surrounded by an amorphous matrix or corona from which MTs radiate . The core is a tripartite structure of ∼280 × 220 × 130 nm in size . As expected, the length of wild-type centrosome cores averaged 285 ± 45 nm, whereas the ICΔC mutants were substantially larger, averaging 387 ± 104 nm in length, with some nearly twice the length of wild-type . The tripartite organization of the core and the corona of these long centrosomes appeared similar to wild-type. The observation of centrosomes twice the normal length in interphase cells suggests they may arise from failure of the centrosome replication cycle which normally doubles the length of centrosomes during prophase and then separates the two halves longitudinally to produce the spindle poles . We also observed paired centrosomes in interphase ICΔC cells . In these pairs, two centrosomes were <300 nm apart, a configuration not seen in wild-type cells. The large centrosomes detected by fluorescence microscopy represented both longer and closely paired centrosomes. These abnormalities suggest that dynein mutants are defective in centrosome replication or separation. In this study we tested the hypothesis that IC-mediated dynein–dynactin interaction is required for dynein-dependent functions in vivo by expressing IC truncation mutants in wild-type Dictyostelium cells. The NH 2 -terminal domain bound only dynactin, whereas the COOH-terminal domain associated with dynein. When overexpressed, both NH 2 -terminal and COOH-terminal mutants resulted in dispersion of the Golgi complex, as expected for defective dynein function . We also found a variety of additional phenotypes, including dramatic alteration in the MT network, changes in centrosome size and number, and altered nuclear DNA content. These results demonstrate the essential role of the IC in mediating dynein–dynactin interaction in vivo, and suggest that this interaction is important for most dynein-dependent cellular functions. In addition, this study revealed a novel role for dynein in centrosome replication and separation. The ability of ICΔN47 and ICΔN106 to associate with dynein, together with ICΔC's failure to bind, shows that the COOH-terminal domain of the IC is required for HC association. ICΔN47 bound the HC more efficiently than did ICΔN106, whereas a more extended NH 2 -terminal truncation, ICΔN278, associated only weakly with the HC (data not shown). The IC is a WD-repeat–containing protein . Analysis of the Dictyostelium IC predicts presence of six WD domains located in the COOH-terminal half of the molecule. Since ICΔC, with the WD-repeats deleted, was unable to bind to HC, WD domains seem to be required for HC association. However, even the largest NH 2 -terminal truncation, ICΔN278, which has intact WD repeats, failed to efficiently bind the HC, suggesting that the WD repeats are not sufficient for HC binding. The prediction of six potential β-propeller structures suggests that the IC adopts a structure similar to the G protein β subunit . In this model the loops that connect the β-propellers on one face of the IC would bind the HC, leaving the loops on the opposite side of the propeller available for another association. However, as is the case for Gβ association with Gγ, an extended region (residues 107–278) containing a predicted coiled–coil domain appears important for stable IC–HC association. The IC could contribute to dynein function by: (a) facilitating dynein–dynactin interaction, thus targeting dynein to specific cargos via dynactin; (b) regulating dynein enzymatic activity; or (c) mediating the association of other subunits with the dynein complex. In vitro studies showed that IC interacts with p150 subunit of dynactin, providing a link between dynein and dynactin . Our results provide direct evidence for dynein–dynactin interaction in vivo by showing that the IC associated with both complexes in immunoprecipitates of the cell lysates. In ICΔN cells, most dynein molecules carry truncated IC subunits defective in dynactin binding. In contrast, in ICΔC cells, the dynein complex was intact and only its association with dynactin was blocked. Since the ICΔN and ICΔC mutants produced indistinguishable phenotypes , it is likely that a primary role of the IC is to mediate dynein–dynactin interaction. However, this does not exclude the possibility that the IC may regulate dynein by additional means. As a first approach to study IC function, we attempted to generate Dictyostelium cells lines with a disrupted IC gene but never obtained IC-null mutants. Combined with the failure to generate dynein HC-null lines in Dictyostelium , this strongly suggests that dynein function is required for the viability of Dictyostelium cells. Therefore, we expressed IC truncation mutants controlled by an inducible promoter to disrupt dynein function in a regulated fashion. When repressed, cells carrying the mutant expression construct grew well. However, upon induction, viability decreased as abnormal phenotypes appeared. Judged by plaque formation on bacterial lawns, only 12% of the cells were viable 3 d after induction, further confirming that dynein function is essential for Dictyostelium viability. Previous studies on cytoplasmic dynein have revealed different requirements for dynein in other organisms. Dynein function is not essential for yeast and filamentous fungi, including Neurospora and Aspergillus , whereas dynein is required for the viability of Drosophila and mammalian cells . In this regard, Dicytostelium 's dynein requirement is similar to that of higher eukaryotic systems. How does dynein affect cell viability? The centrosome and nuclear abnormalities in the IC mutants indicate mitotic defects. Large centrosomes likely result from failure of centrosome separation after its duplication in prophase. Subsequent failure of spindle formation or separation would affect chromosome separation producing the aneuploidy we observed. This idea is supported by the observation of large centrosomes and increased nuclear DNA content only when induction exceeded one cell cycle. Cells unable to accomplish mitosis might be expected to arrest in mitosis due to a mitotic checkpoint mechanism. However, we did not see an increase in mitotic index in IC mutants. Cells with large centrosomes and abnormal nuclei appeared to be in interphase. This suggests that Dictyostelium cells can bypass the mitotic checkpoint. Consistent with this, Dictyostelium cells treated with MT-disrupting drugs such as nocodazole showed only a transient increase in mitotic index that then returned to normal . However such cells were inviable, consistent with the decreased viability we observed. Expression of IC truncation mutants in wild-type Dictyostelium cells resulted in dramatic changes in the interphase MT network. The most dominant phenotype was the collapse of normally radial MT arrays into bundles. This indicates a role for dynein–dynactin interaction in the organization of interphase MTs. The transition of well-extended radial MT arrays to bundles when dynein function was disrupted suggests a change in the balance of the forces responsible for the normal extended morphology of the MT network. A similar MT bundling phenotype has been observed in Dictyostelium cells overexpressing the head domain of the HC . Based on this, dynein was proposed to provide a traction force to keep the MTs extended as a radial array . Our results support this model. As a minus end–directed MT-based motor, dynein has the proper polarity to generate a traction force on the MTs, provided it has an anchoring point. Dynein and dynactin have been localized on both membranous organelles and the cell cortex . Dynein–dynactin anchored at the cell cortex could provide a point of attachment acting as a tension generating element that holds the plus-ends of MTs in place. Similarly, dynein–dynactin anchored on cellular organelles such as the nucleus and vesicles could also provide a traction force along the sides of MTs. Release of this connection by disrupting the dynein–dynactin interaction could collapse the MT network. Perhaps the most striking phenotype of the IC truncation mutants was the abnormal centrosome morphology, suggesting defects in centrosome replication and separation. Centrosomes duplicate once each cell cycle. Daughter centrosomes separate early in mitosis to form the spindle poles, and after mitosis, become the MTOCs for the daughter cells. In Dictyostelium , the interphase centrosome has a box-shaped, multilayered core. In prophase the three-layered core doubles in length and then splits longitudinally to expose an inner face that nucleates polar and kinetochore MTs in prometaphase. The spindle then elongates to separate the chromosomes in anaphase, and finally, at the end of telophase, the mitotic centrosomes fold in half to reform the interphase length, three layered centrosome . The large centrosomes in the IC mutant cells appear to result from failure of the centrosome replication cycle. The elongated centrosomes observed during interphase have the morphology of prophase centrosomes, suggesting that the lengthwise separation of prophase centrosomes requires dynein function. The failed replication would prevent the centrosome from effectively serving as spindle poles as they could not nucleate polar or kinetochore MTs. The resulting failure of chromosome separation would result in accumulation of abnormal DNA content, one of the phenotypes we observed. Immediately after the longitudinal separation described above, the two mitotic centrosomes (now spindle poles) must separate in order to segregate chromosomes. The centrosome pairs we observed may be the result of failed spindle pole separation and/or elongation. This failure to separate, followed by the normal postmitotic folding, would produce paired centrosomes. Failure of this step in the centrosome duplication would also lead to accumulation of abnormal DNA content seen in IC mutants. Both centrosome phenotypes appear to result from a disrupted centrosome replication cycle caused by the expression of IC truncations. These results strongly suggest that dynein is required for the early steps in the centrosome replication, mitotic spindle formation, and/or elongation. This is consistent with the report that injection of function-blocking dynein HC antibody blocked spindle formation in cultured mammalian cells . How might dynein be involved in centrosome separation? One possible model is that dynein attached to some cellular anchor point, such as membranous organelles or the cell cortex, produces a minus end–directed force on the astral MTs that draws the two mitotic centrosomes apart. Alternatively, dynein could be required to transport a plus end–directed motor to the centrosome, and this plus end–directed motor actually provides the force for centrosome separation. Several kinesin-like proteins have been implicated in spindle pole separation . Xklp-2, a plus end–directed kinesin-like protein is required for centrosome separation and the maintenance of bipolar spindles in Xenopus oocyte mitotic extracts. This function is dependent on its proper localization to the spindle poles, which requires cytoplasmic dynein and dynactin function . Therefore, defective dynein function may indirectly affect centrosome separation by affecting the localization of kinesin-like motors. Finally, it is possible that dynein exerts its effect indirectly by altering MT organization or dynamics. In sum, our results, together with the work of others, suggests the model shown in Fig. 11 for the role of dynein in MT organization and centrosome separation. Dynein, acting through an association with dynactin, could produce traction forces acting either along the sides or on the plus-ends of cytoplasmic MTs to maintain the radial array or direct the movement of the MT network in interphase. During mitosis, the pulling force on astral MTs could facilitate centrosome replication and spindle pole separation. Cytoplasmic dynein might exert such plus-end–directed forces on MTs and centrosomes by anchoring on membranous organelles, including the nuclear membrane or cell cortex. This anchoring of cytoplasmic dynein is dependent on its proper association with dynactin. The IC serves as a bridge between cytoplasmic dynein and dynactin complex and is crucial for their interaction. Overexpression of IC truncation mutants disrupts the binding of cytoplasmic dynein to dynactin, leading to dissociation of dynein from its cytoplasmic anchoring points. Without anchoring points, dynein could no longer produce tension on MTs, resulting in collapsed MT arrays and abolished centrosome separation. | Study | biomedical | en | 0.999998 |
10601340 | 5′ and 3′ rapid amplification of cDNA ends (RACE)-PCR were performed on adapter-ligated mouse (BALB/c) brain Marathon-Ready™ cDNA (Clontech), and long-range PCR was done on regular mouse (BALB/c) brain QUICK-Clone™ cDNA (Clontech) using Advantage ® cDNA PCR kit (Clontech). All procedures were carried out according to the manufacturer's protocols PT1156-1, PT1150-1, and PT-1580-1 (Clontech). In general, gene-specific 28-nucleotide primers were designed with high guanine cytosine contents (50–70%) and melting temperature >70°C. The following cycling parameters were employed for RACE-PCR: an initial denaturing step of 94°C for 30 s, followed by 5 cycles of 94°C (5 s) and 72°C (4 min), 5 cycles of 94°C (5 s) and 70°C (4 min), 25 cycles of 94°C (5 s) and 68°C (4 min), and a final extension step of 68°C for 5 min. PCR products were cloned into pGEM-T vector (Promega) or pCR2.1-TOPO vector (Invitrogen) for sequencing. Most of the cDNA clones were sequenced using the BigDye™ sequencing kit (Applied Biosystems) and processed by Perkin Elmer/Applied Biosystems Model ABI 377A Sequencers (DNA Facilities, Columbia University, New York). In some cases, cDNA clones were also sequenced manually with Sequenase 2.0 (U.S. Biochemical Corp.). pTOPO-ACF-5A, pTOPO-ACF-5B, pTOPO-ACF-rod, and pTOPO-ACF-3A were generated by cloning the long-range PCR products into pCR2.1-TOPO vector or pCR-XL-TOPO vector (Invitrogen). To engineer pFLAG-ABD, the 0.8-kb PCR fragment of pTOPO-ACF-5A using sense primer 5′-AAGCCAGAATTCTGTGCTGGACCCTGC-3′ and antisense primer 5′-TAAAGTGCCTCGAGTTCAACAGGG-3′ was digested with EcoRI/XhoI and ligated to the EcoRI/XhoI-digested pcDNA-FLAG vector . Similarly, pGEM-mACF7-C was generated by cloning the PCR fragment of pTOPO-ACF-3A using sense primer 5′-CATGGAGAATTCCCGCAGTGGTAG-3′ and antisense primer 5′-TTATCGCTTGGGACCTGGAGTCCTGGGG-3′ into pGEM-T vector (Promega). The 1.3-kb EcoRI-NotI fragment of pGEM-mACF7-C was then ligated to the EcoRI/NotI-digested pcDNA-FLAG vector to make pFLAG-mACF7-C. pGEM-GARt was created by cloning the PCR fragment of pTOPO-ACF-3A using sense primer 5′-CAACAAGAATTCCTATCGGCCAAC-3′ and antisense primer 5′-TTATCGCTTGGGACCTGGAGTCCTGGGG-3′ into pGEM-T vector (Promega). The 0.85-kb EcoRI-NotI fragment of pGEM-GARt was then cloned into pcDNA-FLAG vector to generate pFLAG-GARt. To construct pFLAG-mACF7-mini, three-piece ligation was performed with 2.5-kb Not-NcoI fragment of pTOPO-ACF-5A, 0.85-kb NcoI-XhoI fragment of pFLAG-GARt, and 5.4-kb NotI/XhoI-digested pcDNA3 vector (Invitrogen). To create a FLAG-tagged ACF7-3A clone, the stop codon on ACF-3A was first removed by PCR and the resulting fragment was ligated inframe to a FLAG-epitope tag coding sequence. Clones ACF-5A, ACF-rod, and FLAG-tagged ACF-3A were sequentially ligated together using the unique restriction endonuclease recognition sites KpnI and SalI at cDNA position 3665 and 11728, respectively, to generate a full-length FLAG-tagged ACF7 cDNA, which was subcloned into a multiple cloning site–modified eukaryotic expression vector pCI (Promega) to construct pFLAG-mACF7-fl . All studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Timed pregnant mouse C57BL/6 embryonic day 14.5 embryos were isolated, freshly frozen, and embedded directly in the OCT compound (Sakura Finetek, Inc.). Both sagittal and transverse cryostat sections were prepared. 35 S-labeled cRNA transcripts were synthesized in vitro from pGEM-GARt using Riboprobe Gemini Systems from Promega. Both sense and antisense [ 35 S]UTP-labeled cRNA were prepared. cRNA probes were purified on Sephadex G-50 columns (Boehringer Mannheim). In situ hybridization was performed as described previously . After the nuclear emulsion autoradiography was performed, slides were examined in Leitz microscopes under both bright field and dark field illuminations. Hybridization with control (sense) probes yielded only low background staining in all cases. COS-7 cells were cultured at 37°C with 5% CO 2 in DMEM (Life Technologies, Inc.) supplemented with 10% FBS. Transient transfections for immunofluorescence analysis were performed on 18-mm coverslips using GenePORTER™ transfection reagents (Gene Therapy System) or LipofectAMINE PLUS reagents (Life Technologies, Inc.). 48 h after transfection, coverslips with adherent cells were fixed in cold methanol at −20°C or 4% paraformaldehyde in PBS at room temperature. To optimize stress fiber staining, fixed cells were permeabilized with 0.1% Triton X-100 for 5 min before further processing. After rinsing several times with PBS, cells on coverslips were blocked with 5% normal goat serum and incubated with primary antibodies at room temperature for 1 h. The primary antibody–treated cells were then washed with PBS and incubated with appropriate secondary antibodies or rhodamine-conjugated phalloidin (Sigma Chemical Co.) for 30 min. Subsequently, the coverslips were washed with PBS and mounted onto slides with Aquamount (Lerner Laboratories) for indirect immunofluorescent microscopy. The following primary antibodies were used for immunostaining: mouse monoclonal anti-FLAG M2 antibody (IBI-Kodak); rabbit polyclonal anti–Glu- and anti–Tyr-tubulin antibodies; rat monoclonal anti–Tyr-tubulin YL1/2 antibodies (a gift from Dr. Gregg G. Gundersen, Columbia University, New York, NY). The STP3 system (Novagen) was used to synthesize [ 35 S]methionine-labeled proteins from the pcDNA-FLAG constructs that contain T7 RNA polymerase promoters. In vitro synthesized proteins were prespun before they were used for the binding assays. The actin-binding assays were performed with the Non-muscle Actin Binding Protein Spin-Down Biochem Kit (Cytoskeleton, Inc.). In each testing, 20 μl of crude 35 S-labeled protein was incubated with 0.23 nmol of polymerized F-actin at room temperature for 30 min. After centrifugation for 90 min at 65,000 rpm, supernatants and pellets were separated and subjected to SDS-PAGE analysis. After SDS-PAGE, the gels were dried and exposed to X-ray film to visualize the 35 S-labeled proteins. The MT-binding assays were performed with the Microtubule Associated Protein Spin-Down Assay Kit (Cytoskeleton, Inc.). In each case, 20 μl of crude 35 S-labeled protein was incubated with 0.15 pmol of taxol stabilized MTs at room temperature for 20 min. After centrifugation for 40 min at 55,000 rpm through a 40% glycerol cushion buffer containing 20 μΜ taxol, supernatants and pellets were collected for SDS-PAGE analysis. The gels were dried and exposed to X-ray film to visualize 35 S-labeled proteins. The mRNA transcripts of mACF7 were estimated to be 14–18 kb, of which only 6 kb had been characterized previously . Using a RACE-PCR–based method, we successfully isolated consecutively overlapping clones of the remaining cDNA . The 5′ RACE product contained a putative start codon (ATG) that matched the Kozak consensus . Moreover, an inframe stop codon was located 102 bp upstream of this ATG, making it the most likely translational start site. By sequentially performing 3′ RACE-PCR with primers specific for the end sequence of the previous RACE products, we obtained 10 overlapping cDNA clones that spanned ∼11 kb in length before reaching a polyadenylation signal followed by a polyA stretch. In addition, we also performed long-range PCR to obtain longer cDNA clones for more detailed sequence analysis. The composite cDNA was about 17.3 kb , and the longest open reading frame encoded a 5,327–amino acid polypeptide with a calculated molecular mass of 608 kD . The deduced amino acid sequence of mACF7 was used to search for homologous proteins in the GenBank database. The closest relative of mACF7 is the Drosophila protein Kakapo. mACF7 and Kakapo not only share homology in primary sequence but also the overall protein architecture, indicating that mACF7 is the mouse homologue of Kakapo. As described in the previous study , mACF7 contains a dystonin/BPAG1-n homologous NH 2 -terminal region, which includes a calponin homology ABD and a globular plakin-like domain. Interestingly, following the NH 2 -terminal domains, the sequences of mACF7 and dystonin start to diverge. Sequence comparison of mACF7 with dystrophin revealed that the central region of mACF7 would fold into 23 spectrin repeats . By analogy to β-spectrin, this collection of spectrin repeats would also constitute the rod domain (not a coiled–coil rod) of mACF7. Following the rod domain, two calmodulin-like, EF-hand calcium-binding motifs were identified . Together, these features suggest that mACF7 is a new member of the spectrin superfamily with plakin-like features. A partial human brain cDNA clone, KIAA0465 was found showing >80% sequence identity to mACF7 COOH terminus. According to the UniGene database, the gene coded for KIAA0465 is located on chromosome 1, between markers D1S2843 and D1S417. In mice, the mACF7 gene was mapped to chromosome 4, close to the marker D4mit11 . Based on the chromosome synteny and the extensive sequence homology, the partial KIAA0465 cDNA should encode the human orthologue of mACF7. In addition, a short stretch of the mACF7 COOH terminus displays significant homology to a portion of the recently identified protein, GAR22 (Gas2 related on chromosome 22) . This part of GAR22 is also closely related to the Gas2 protein (growth arrest–specific 2 protein) . Because of this similarity, we designated this region of mACF7 as the GAR region. A schematic structure of mACF7 is depicted in Fig. 2 C. To study the expression pattern of mACF7, mouse embryos at embryonic day 14.5 were hybridized with a mACF7 ribonucleotide probe. As illustrated in Fig. 3 , mACF7 was ubiquitously expressed in all tissues, with higher levels in the nervous system, muscle, lung, heart, and adrenal glands. Full-length Gas2 was shown previously to be a component of the MF network, although the interaction domain had not been fully characterized . Therefore, we examined possible interactions between the COOH-terminal domain of mACF7 (mACF7-C) and MFs by transient transfection assays. In addition, we also analyzed the actin-binding properties of the putative NH 2 -terminal ABD of mACF7. To facilitate the detection of the truncated proteins, the NH 2 termini of mACF7-C and ABD were fused to FLAG epitope tags, which also provided the translational start codons. Transient transfections were performed on COS-7 cells. As shown in Fig. 4A and Fig. B , overexpressed ABD protein colocalized with filamentous actin in stress fibers and membrane ruffles of transfected cells, confirming the interaction of this highly conserved domain with MFs. In contrast, the mACF7-C protein, displayed a filamentous staining pattern that exhibited no significant correlation with the actin network . Therefore, we compared this staining pattern with that of the other two cytoskeletal networks, MTs and IFs, and found that mACF7-C proteins codistributed with MTs , but not with vimentin (data not shown). Within each cell there are dynamic and stable MTs. Stable MTs are a small subset of dynamic MTs and are thought to be selectively generated from dynamic MTs. These stable long-lived MTs accumulate a posttranslationally modified form of tubulins known as detyrosinated or Glu-tubulins. These MTs are distinct from their dynamic counterparts that contain predominantly tyrosinated tubulin (Tyr-tubulin). In transfected cells, the overexpressed mACF7-C proteins colocalized with many but not all Tyr-MTs . Interestingly, mACF7-C proteins decorated all the Glu MTs. The dynamic Tyr MTs at the periphery of the cell did not appear to colocalize with mACF-7, although it is possible that the bound mACF-7 was present in low amounts too scarce to be detected. As expected, the ABD of mACF7 did not associate with MTs . Since long MT-forming whorls were frequently observed in the transfected cells, we considered the possibility that mACF7-C proteins might not only bind to, but also stabilize MTs. To explore this possibility, cells transfected with mACF7-C cDNA were treated with the MT depolymerization agent, nocodazole (10 μM) for 1.5 h before being fixed for immunofluorescence microscopy. These conditions have been shown to be sufficient to cause the complete depolymerization of the endogenous MTs . In contrast to cells without mACF7-C protein, the MT networks of the transfected cells remained intact and were decorated with mACF7-C proteins , implying that the COOH-terminal domain of mACF7 can associate with and stabilize MTs. In similar assays, the ABD of mACF7 still only associated with the actin network . In vitro spin-down binding assays were carried out to ascertain interactions between ABD and actin filaments, and between mACF7-C and MTs. 35 S-labeled proteins were synthesized in vitro and incubated with polymerized actin and tubulin before centrifugation. The bound proteins in the spin-down pellets were resolved on SDS-PAGE and visualized by autoradiography. As demonstrated in Fig. 7 , the taxol stabilized MTs pull down mACF7-C protein but not ABD protein, whereas polymerized actin filament pulls down a significant amount of ABD protein and a small amount of mACF7-C protein. The interaction between mACF7-C and polymerized tubulins is most likely direct, because no MT-associated proteins (MAPs) are present in the taxol stabilized MTs, although proteins in the reticulocyte lysate system could enhance this binding. Since polymerized actin filaments were not able to pull down a partial BPAG1 COOH-terminal protein in similar assays (data not shown), the in vitro interaction between mACF7-C and actin filaments may also be specific. However, no obvious colocalization of mACF7-C and actin structures was observed in transfection studies; therefore, it is not clear that this interaction happens in vivo. To investigate whether mACF7 might be able to cross-link MFs and MTs in vivo, a construct (pFLAG-mACF7-mini) encoding for a chimeric protein, that contained the ABD and the COOH-terminal domain of mACF7 connected by a FLAG epitope tag, was used for transient transfection studies. Triple-labeling with phalloidin, anti-FLAG, and antitubulin was performed and the results are shown in Fig. 8 . Similar to cells expressing mACF7-C protein, long MT-forming whorls and bundles were observed in cells transfected with pFLAG-mACF7-mini. In contrast to the straight stress fibers and membrane ruffles stained by phalloidin in nontransfected cells, many curly actin filaments were detected in cells expressing mACF7-mini. These unusual actin filaments were perfectly colocalized with MTs and decorated with mACF7-mini . These results show that mACF7-mini could enhance the formation of actin filaments and efficiently cross-link them to MTs. To further characterize the function of mACF7, a COOH-terminal FLAG-tagged full-length mACF7 construct was prepared for transient transfections. In some transfected cells, we observed partial coalignment of mACF7, MTs, and actin filaments . However, in most cases, colocalization of mACF7, MTs, and actin was only observed in patches . These differences appear to be due to the differential expression levels of mACF7 in the transfected cells. Cells expressing lower levels of full-length mACF7 had more stress fibers that colocalized with mACF7. A subset of these stress fibers also colocalized with MTs . In contrast, in most transfected cells there were few actin stress fibers . In addition, mACF7 also colocalized with actin at membrane ruffles and decorated all the MTs. These data show that mACF7 can bind to both actin filaments and MTs, and may have the ability to cross-link these cytoskeletal elements. Since vimentin has also been shown to associate with MTs , we studied the distribution of vimentin in mACF7-overexpressing cells, but found no obvious colocalization of IFs with mACF7 (data not shown). The plakin family is a group of sequence-related proteins that associate with IFs and localize to junctional complexes at the plasma membrane. Current interest in plakins has been spurred by the discoveries of their roles in cross-linking cytoskeletal elements, as implied by their other name, cytolinkers . This functional feature is particularly prominent for plectin and dystonin (BPAG1-n) as they contain both actin- and IF-binding domains. Although the precise functions played by each plakin are not fully understood, hereditary diseases and gene targeting experiments vividly demonstrate the detrimental results caused by their loss-of-functions. Defects of the plectin gene in humans cause the skin blistering disease epidermolysis bullosa simplex combined with muscular dystrophy . Mutations in desmoplakin cause striate palmoplantar keratoderma in humans . The loss of mouse dystonin/BPAG1 results in dystonia musculorum , whereas desmoplakin-null embryos cannot survive beyond the egg cylinder stage . Plectin knockout mice die shortly after birth and exhibit severe defects in skin, skeletal muscle, and heart . Therefore, identification of new family members might help us gain further insights into the functions of this family of proteins and unexplained human genetic diseases. In fact, more potential members of the plakin family had been identified, such as ACF7 and a 450-kD epidermal protein . Unfortunately, characterization of these new family members is very time consuming, because all plakins are large proteins encoded by tremendously long mRNAs. Therefore, rather than using the standard hybridization approach, we attempted to isolate the full-length mACF7 cDNA by an efficient PCR-based method. By sequentially performing RACE-PCR, we were able to obtain overlapping clones spanning 11 kb in a relatively short period of time. The success of this approach not only accelerates the characterization of plakins, but can also be applied to other large-sized proteins. Because the partially characterized cDNA of mACF7 displayed 38–76% identity to that of dystonin/BPAG1-n , mACF7 was initially speculated to be a new plakin. However, after careful examination of its completed primary sequence, we found that mACF7 also represents a novel member of the spectrin superfamily. mACF7 can be structurally divided into three domains: NH 2 -terminal head, central rod, and COOH-terminal tail domains. The head domain consists of an ABD followed by a plakin-like globular domain. This portion of mACF7 displays the strongest homology to dystonin/BPAG1-n as described previously . However, unlike plakins, the rod domain of mACF7 does not conform to a coiled–coil; instead it is composed of 23 repetitive motifs that are more closely related to dystrophin's spectrin repeats. The tail domain of mACF7 contains two putative EF-hand calcium-binding motifs and a Gas2-like GAR region. Because mACF7 is structurally related to spectrin, bearing the highly homologous ABD, spectrin repeats, and EF-hand motifs, mACF7 also belongs to the spectrin superfamily. As illustrated by in situ hybridization, mACF7 is ubiquitously expressed in mouse embryos, with the highest expression level in the nervous system. In transfected cells, the transiently expressed mACF7 COOH-terminal protein associated with the endogenous MT networks, and this interaction stabilized the MTs from disassembly by nocodazole. Interaction of the COOH-terminal domain of mACF7 and MTs was also suggested by in vitro binding assays. The putative ABD at the NH 2 terminus of mACF7 was proven to be functional. In vivo, colocalization of overexpressed ABD and actin structures was observed. In vitro, polymerized actin filaments were able to pull down ABD, and to a lesser extent the COOH-terminal domain of mACF7 in spin-down assays. To study the actin-MT cross-linking properties of mACF7, we generated mACF7-mini and COOH-terminal FLAG-tagged full-length mACF7 constructs. Overexpression of mACF7-mini caused coalignment of MTs and actin filaments. In contrast, colocalization of full-length mACF7, MTs, and actin was observed mostly in patches. Most of the stress fibers were disrupted in the transfected cells. Colocalization of actin, MTs, and mACF7 were only occasionally observed in the remaining stress fibers. These differences in the transient transfection results between mACF7-mini and full-length mACF7 are most likely due to the presence of the plakin-like domain and spectrin repeats in full-length mACF7. The ABD and the MT-associated domains are separated by the flexible spectrin repeats in the rod domain, making full-length mACF7 less likely to form a directly observable link between MFs and MTs. In addition, the plakin-like domain and the spectrin repeats may mediate association of mACF7 with membrane proteins and induce dimerization of mACF7. Transient transfections with full-length plectin, another known cytoskeletal linker protein with a homologous ABD as mACF7, also showed that the actin stress fibers appeared considerably reduced in number and complexity. Full-length plectin led to the collapse of the vimentin network in transfected cells into perinuclear aggregates, which were immunoreactive with antivimentin and antiplectin, but colocalization of vimentin, plectin, and actin stress fibers (or MTs) was not readily evident . The transfection results that we obtained with full-length mACF7 are therefore consistent with those described for plectin. The plakin-like domain is highly conserved among the plakin family members. In desmoplakin, this globular domain clusters desmosomal cadherin–plakoglobin complexes and binds directly to plakoglobin . A similar domain in plectin binds integrin β4 both in vitro and in vivo, although the plectin–integrin β4 interaction is probably more complex, involving multiple interfaces of the proteins and even the ABD of plectin . Hence, mACF7 might also interact with membrane proteins. Indeed, some of the Drosophila kakapo alleles were identified from a screen of mutations affecting processes requiring integrin adhesion, suggesting that the mACF7 homologue Kakapo is closely associated with integrin functions . By analogy to that of the other spectrin superfamily members, the spectrin repeats of mACF7 could confer flexibility to the molecule for adjusting to the curvature of the cell membrane and may also mediate dimerization of two antiparallel mACF7 molecules. Accordingly, mACF7 could also cross-link individual MF fibers and/or MT fibers in its dimerized form. Collectively, mACF7 represents a potential cytoskeletal cross-linking protein; the NH 2 -terminal ABD binds to MFs, whereas the COOH-terminal tail domain associates with MTs. Because mACF7 binds to MTs as well as actin, we suggest a modification of its name ACF7 (actin cross-linking family 7) to MACF (microtubule actin cross-linking factor). So far, very few proteins have been reported to be able to bind both MFs and MTs. The yeast protein coronin promotes the rapid assembly and cross-linking of actin filaments and contains sequences homologous to the MT-binding region of MAP1B . However, the actin- and MT-binding domains are contiguous, and the region homologous to MAP1B is unique to yeast coronin. None of the mammalian homologues of coronin was found to possess this putative MT-binding domain . MAP1B is a 320-kD MAP that was originally copurified with MTs from mammalian brain. Microtubule-binding domains were found in both of the polypeptides, heavy chain and light chain, that constitute MAP1B . Recently, transient transfection studies illustrated that a COOH-terminal fragment of the light chain was also able to associate with actin stress fibers . However, the overexpressed native light chain colocalized only with MTs in transfected cells, raising the possibility that the ABD of MAP1B is functional only under certain unknown circumstances. The other protein that has been reported to associate with MFs and MTs is plectin. Plectin contains a functional ABD near its NH 2 terminus and a defined IF-binding domain at the COOH-terminal tail domain . Although plectin has been reported to bind MTs, the association appears to be indirectly through other MAPs in neurons . Nevertheless, plectin can directly associate with MTs in nonneuronal cells as observed by EM , although no specific MT-binding region on plectin has been defined. We have been able to show that the COOH terminus of MACF binds directly to MTs. The MT-binding region of MACF has no obvious sequence similarities to the MT-binding domains of other MAPs, such as the MT-binding repeats of tau, MAP2, and MAP4, or the MT-binding domains of MAP1B. Recently, the Drosophila gene encoding for the MACF homologue, kakapo , was cloned and characterized in three studies by examinations of mutant flies with blistered wings or with a paralytic phenotype, and by library screening with an antibody that stained the epidermal muscle attachment (EMA) cells in a unique pattern . Similar to MACF, Kakapo also contains an ABD, a plakin-like domain, a rod domain composed of spectrin repeats, and a GAR-region containing COOH-terminal tail domain. These studies indicate that Kakapo expression is restricted to ectodermally derived cells and that mutations of the Kakapo gene cause defects in the muscle-dependent tendon cell differentiation and the local development of neuronal processes . In EMA cells, Kakapo is localized to the termini of MT bundles and the absence of Kakapo causes detachment of these MT bundles from the basal membrane . In addition, disorganization of MTs was also observed in the scolopidial sensory neurons of kakapo mutants . Therefore, Kakapo was deduced to mediate the connection between the actin network, MTs, and membrane-associated proteins. Since we have shown that the COOH-terminal region of MACF can associate with MTs, we can therefore infer that Kakapo could also associate with MTs. The ubiquitous expression of MACF implies that it could also function as a linker protein connecting MFs, MTs, and membrane-associated proteins in a variety of tissues. The high level of expression of MACF mRNA that we observe in the nervous system could also indicate that it may have an important function in neurons. The architecture of MACF clearly demonstrates the intricacies of gene evolution. MACF is a union of different kinds of structural domains that have been conserved throughout evolution. Although some of these structural domains have been well-studied in other proteins, together they make MACF bear very unique properties that have not been described previously for other cytoskeletal linker proteins. It has a clearly identified ABD as well as an MT-binding domain that are spatially well-separated. These domains may connect both MFs and MTs. The importance of this kind of connection is indicated by some of the lethal alleles obtained in the kakapo locus of Drosophila . Although the functional significance of MACF in mice as well as in human remains to be studied, it is certain that the ABD of MACF is functional and that its COOH-terminal domain functions like a MAP, i.e., it interacts with and stabilizes MTs. | Study | biomedical | en | 0.999998 |
10601341 | The coding regions of murine CPβ1 and -β2 were amplified using PCR with 5′ GCGCGTCGACGCCACCATGAGCGATCAGC 3′ (primer 1) and 5′ GCGCGTCGACAGAGATGGCGCTGCGTGGTC 3′ (primer 2) and inserted downstream of the α-MyHC promoter in a unique SalI restriction site from plasmid clone 26, provided by Dr. J. Robbins . A 611-bp HindIII–EcoRI fragment containing the human growth hormone poly(A) signal was at the 3′ end of the α-MyHC/CPβ constructs. A third construct, α-MyHC-β1L262R, which contains a T to G transversion at base 839, changing amino acid 262 from leucine to arginine, was amplified using PCR. Primer 1 and 5′ CAGCACTTGAGAACGTTCCCTCTGCAACTGC 3′ were used to make one product. Primer 2 and 5′ GCAGTTGCAGAGGGAACGTTCTCAAGTGCTG 3′ were used to make another product. The products were then used as template in a final PCR reaction with primers 1 and 2 to generate the full-length product. The complete open reading frames of the recombinant plasmids were sequenced using specific primers. No changes were present. To prepare the DNA constructs for injection, plasmid containing α-MyHC promoter/cDNA construct was digested with NotI to release the transgene DNA fragment. The fragment was separated from the vector by agarose gel electrophoresis, purified using QiaexII (Qiagen), phenol/chloroform extracted, alcohol precipitated, resuspended, and dialyzed against endotoxin free 5 mM Tris, pH 8.0, 1 mM EDTA. The DNA was quantitated before injection by optical density and agarose gel electrophoresis using a known standard. The linearized transgene constructs were microinjected into the male pronuclei of one-cell embryos, which were then surgically reimplanted into pseudopregnant female mice. DNA was purified from the tail clips of progeny by digestion at 55°C overnight with 50 mM Tris-HCL, pH 8.0, 100 mM EDTA, 0.5% SDS, and 500 μg/ml proteinase K (Boehringer Mannheim Corp.). DNA was purified using Phase Lock Gel I (Heavy; 5 Prime-3 Prime) and resuspended in 100 μl of 10 mM Tris, pH 8.0, 1 mM EDTA. Progeny were screened for the presence of the transgene by genomic Southern blot analysis using a 32 P-labeled 1.2 kb SalI–EcoRI fragment of the α-MyHC promoter. Three different founder lines were identified for α-MyHC-wtβ1, three for α-MyHC-wtβ2, and four for α-MyHC-β1L262R. Founders were bred against a C57BL/6 background and studied as N1 heterozygotes. Founder lines with the greatest expression and most severe phenotypes are described in this paper. Transgene copy number was quantitated for the progeny of the three founder lines for α-MyHC-wtβ1 by PhosphoImager analysis of genomic Southern blots. The transgene copy numbers for lines 1, 2, and 3 were 2, 4, and 7, respectively. Genomic DNA digested with EcoRI and SalI were probed with a 32 P-labeled 1.2-kb SalI–EcoRI fragment of the α-MyHC promoter. The transgene generated a 1.7-kb band, and the endogenous α-MyHC gene generated a 2.5-kb band. The intensities of the radioactivity associated with each band were measured with the PhosphoImager, and the endogenous gene was used as the standard for a single-copy gene. For the α-MyHC-wtβ2 and α-MyHC-β1L262R transgenic lines, genomic Southern blots were evaluated qualitatively and showed transgene copy numbers of 2 to 10, based on the intensity of the transgene band relative to the endogenous gene band. To evaluate transgene expression by Northern blot analysis, hearts were dissected from 3-wk-old pups, pulverized on dry ice, and homogenized in Trizol reagent (GIBCO BRL). RNA was extracted according to the manufacturer's protocol (GIBCO BRL). Total RNA (10 μg/lane) was size-fractionated in formaldehyde agarose, transferred to Hybond nylon membrane (Amersham Life Science), and hybridized in Rapid-hyb buffer (Amersham Life Science) with a 3′ human growth hormone fragment unique to the transgene. Radiolabeled RNA blots were quantitated with a PhosphoImager (Molecular Dynamics) and each value was normalized to β-actin expression (human β-actin cDNA control probe; CLONTECH). Levels of CPβ1, CPβ2, and CPα protein were assayed by immunoblotting using SDS polyacrylamide gel and two dimensional electrophoresis of mouse hearts with isoform specific antibodies as previously described . The differences in mass and pI of mouse α1 (32.7 kD, 5.3); α2 (32.9 kD, 5.7); β1 (30.6 kD, 5.5); β2 (31.4 kD, 5.8); and β1-L262R (30.6 kD, 5.8) polypeptides were the basis for their identification. Blots were probed with isoform-specific polyclonal antibodies to β1 (R33) and β2 (R25), a pan β antibody (R22), and an mAb (5B12) that recognizes the α1 and α2 isoforms equally well . For the SDS polyacrylamide gels, two whole heart extracts from each transgenic line were prepared in SDS sample buffer. Approximately equal amounts of protein were run on 15% SDS-polyacrylamide gels. One gel was stained with Coomassie blue to confirm equal protein loading for each sample and the other gels were transferred to nitrocellulose and probed. The immunoblots were developed as described and scanned to obtain a digital image. The β1 and β2 protein were quantitated using NIH image and compared with the signal intensities of immunoblots of known concentrations of purified β1 and β2 probed with the same antibody . These measurements were normalized to the amount of protein loaded on the gels. For statistical purposes, Western blot analyses of proteins were performed twice with each sample and mean ± values calculated. Transgenic mice and their nontransgenic littermates, from 9–180-d-old, were anesthetized. Their beating hearts were excised, rinsed, drained of fluid, and weighed. Heart/body weight ratios were calculated and expressed as mg:g. Hearts were excised and fixed overnight in 10% buffered formalin. The hearts were cut in half along a midsagittal plane, paraffin-embedded, and sectioned. Sections were stained with hematoxylin and eosin. For immunofluorescence, sections were deparaffinized with xylene and rehydrated through a series of graded alcohol washes to distilled water. Sections were labeled with antibodies to CPβ1 (R33), CPβ2 (R25), actin (C4, a gift of Dr. J. Lessard, University of Cincinnati), and vinculin (Sigma Immunochemicals). Primary antibodies were detected by fluorescently tagged secondary goat anti–rabbit (Cy3; Jackson ImmunoResearch Laboratories) or goat anti–mouse antibodies (DTAF; Sigma Immunochemicals). Sections were mounted in N -propylgallate/glycerol mounting media. Immunofluorescence microscopy was performed on an epifluorescence microscope (IX70; Olympus) with a 1.35 NA 100× UPlanApo objective and U-MWIBA (DTAF) and U-MNG (Cy3) filter sets. Images were collected with a cooled CCD video camera (RC300, Dage-MIT). For EM, hearts were fixed in 2.5% glutaraldehyde, 100 mM calcium cacodylate overnight. A section of the left ventricular wall parallel to the papillary muscle was removed. This section was postfixed in 1.25% osmium tetraoxide, stained en bloc with 4% uranyl acetate, embedded in Polybed (Polysciences), sectioned, and stained with 4% uranyl acetate/Reynold's lead citrate. Thin sections were examined using a Zeiss 902 electron microscope. To test if CPβ2 can functionally replace CPβ1 in organizing the thin filaments at the Z-lines of the sarcomere, we used the α-MyHC to express the CPβ2 isoform in the mouse myocardium. These transgenic lines are referred to as TG-wtβ2. The well-characterized α-MyHC promoter directs strong and specific expression in the murine ventricles beginning at birth. It is not expressed in smooth or skeletal muscle, or in the ventricular chamber during development . Three TG-wtβ2 founders were identified. None of the founders demonstrated any gross phenotype, reduced viability, or reduced fertility. Backcrossing of the TG-wtβ2 founders produced heterozygous N1 offspring. N1 progeny from two of the founders (2 and 3) showed severe effects. The transgenic mice exhibited stunted growth and an irregular gait beginning at approximately seven days after birth. They expired at 7–26 d after birth . No peripheral edema was noted, but breathing appeared labored for several days before death. The phenotype was completely penetrant, with 100% of heterozygous N1 transgenic mice from these two founders displaying the described phenotypes . Because the founders were heterozygous for the transgene insertion, we anticipated 50% of the N1 progeny would be heterozygous for the transgene insertion. However, heterozygous transgenic N1 progeny with the severe phenotype represented only 20–35% of the litter, suggesting that these founders were chimeric for the transgene insertion. In ∼20–30% of transgenic mice, the foreign DNA is presumed to integrate at a later stage, resulting in transgenic mice that are mosaic for the transgene . This hypothesis can also account for the fact that the founders had far less severe cardiac phenotypes than the transgenic N1 progeny. To test whether the phenotype of the TG-wtβ2 mice was due to defective interaction of the actin-based thin filaments with CP at Z-lines, we generated transgenic mice expressing a dominant negative point mutation of CPβ1. These transgenic lines are referred to as TG-β1L262R. This mutation changes amino acid residue 262 from leucine to arginine and lowers the actin-binding affinity by a factor of 10 4 . Four founders were identified. None of the founders exhibited any discernable gross phenotype. There was no reduction in viability or fertility. The phenotype of the N1 TG-β1L262R heterozygotes (TG-β1L262R lines 2 and 3) was similar to that of the N1 TG-wtβ2 heterozygotes. These mice showed stunted growth, an irregular gait, labored breathing, and death at 7–26 d after birth . This phenotype was almost completely penetrant (16/17 animals). In contrast to the TG-wtβ2 heterozygotes, one eye often failed to open in the TG-β1L262R heterozygotes, presumably due to α-MyHC's weak expression in the ocular musculature . To determine if the TG-wtβ2 and TG-β1L262R phenotype could be caused by increased expression of CPβ protein in general, we used the same approach to generate transgenic mice that overexpress the CPβ1 isoform. These transgenic lines are referred to as TG-wtβ1. Three founders were identified. Neither the founders, nor the N1 heterozygotes, exhibited any of the gross phenotypes as observed in the TG-wtβ2 transgenic mice (see CPβ1 Cannot Functionally Replace CPβ2). To confirm the integrity of the transgenic transcript and to quantify transgene expression, Northern blot analysis using a transgene-specific probe was performed on total RNA from hearts obtained from transgenic and nontransgenic littermates. Nontransgenic animals showed zero expression of the transgene, as expected. All the TG-wtβ2 transgenic lines showed expression. Line 1 showed the lowest level of expression. Line 2 expression was approximately fourfold that of line 1. Line 3 expression was ∼4.5-fold that of line 1 . N1 heterozygotes from lines 2 and 3 had the previously described strong gross phenotype whereas transgenic line 1 did not. Therefore, transgene transcript accumulation correlated with the severity of the gross phenotype. Similarly, in the TG-β1-L262R lines, the levels of transgene expression correlated with the severity of the phenotype. A strong heterozygote (line 2) expressed the transgene approximately fourfold more than a weak line . To determine the level of protein expression of the CPβ1 and CPβ2 isoforms in the hearts of TG-wtβ2, TG-β1L262R, and TG-wtβ1 mice, myocardial protein was quantified by immunoblot with isoform-specific and pan-reactive antibodies. For the TG-wtβ2 lines, an increased level of CPβ2 protein was observed in all of the transgenic lines . The level of CPβ2 protein varied from approximately two- to fourfold that of the wild-type endogenous level. The amount of transgenic protein correlated with the level of transgene transcript, with line 3 containing the highest levels of protein and transcript. Overexpression of β2 led to a decrease in the level of β1 . The total amount of CPβ and CPα did not change . Therefore, the level of active heterodimer was constant, with an increased ratio of α:β2 to α:β1. To determine the CPβ isoform protein expression in TG-β1L262R lines, two-dimensional immunoblots were required to identify the β isoforms . Although the β1 and β1-L262R polypeptides have similar molecular masses, the β1-L262R mutation shifts the pI from 5.5 to 5.8 and then can be discriminated from wild-type . The blots were also probed with an mAb pan-reactive to α1 and α2. An increased level of the transgenic protein was observed in hearts of TG-β1L262R animals that have both a strong (line 2) and a mild (line 1) phenotype . The severity of the phenotype correlated with the amount of protein expressed. The increased expression of the β1-L262R polypeptide was accompanied by a decreased level of the endogenous β1 subunit and a complete loss of the β2 subunit . There was no change in the level of the CPα subunit expression. Therefore, overexpression of the β1-L262R polypeptide changed the ratio of the isoforms in the heterodimer population with an increase of α:β1-L262R and a decrease in α:β1 and α:β2. For the TG-wtβ1 lines, an increased level of the CPβ1 protein was seen in all the transgenic lines . The increased level of CPβ1 protein in the TG-wtβ1 lines was similar to the increased level of CPβ2 protein seen in the TG-wtβ2 lines, when considered as a ratio with respect to wild-type, and was substantially increased when considered in absolute terms. The TG-wtβ1 lines did not show the severe phenotypes of sarcomere disruption and hypertrophic cardiomyopathy seen in the TG-wtβ2 lines (described in detail in CPβ1 Cannot Functionally Replace CPβ2). Therefore, the severe phenotype of the TG-wtβ2 mice was specifically caused by the increased level of CPβ2 protein, and could not be explained by an increased level of β subunit protein in general. To determine if overexpression of CPβ2 and CPβ1-L262R in the murine myocardium leads to cardiac hypertrophy, cardiac weights corrected for body weight were determined for the TG-wtβ1 and TG-β1L262R mice ( Table ). TG-wtβ2 and TG-β1L262R mice had elevated heart to body weight ratios relative to control littermates, ∼30% larger than ratios for nontransgenic mice. Therefore, overexpression of CPβ2 and CPβ1-L262R caused cardiac hypertrophy. Light microscopic analysis of hematoxylin- and eosin-stained heart sections of TG-wtβ2 (lines 2 and 3) and TG-β1L262R (lines 2 and 3) revealed global dilation of the hearts of transgenic, compared with nontransgenic controls . The walls of the ventricles were thickened throughout the chambers. The chambers were not grossly dilated. To determine if cardiac hypertrophy was accompanied by an altered morphology of myofibrils and sarcomere structure, the ventricular walls of control animals (nontransgenic littermates) and transgenic mice were examined by light microscopy and thin section EM. Light microscopic analysis of ventricular walls of nontransgenic mice showed the characteristic pattern of striations. The striations are observed due to the periodicity of the repeating sarcomere units and because the myofibrils are aligned with each other laterally. In contrast, the ventricle wall of the TG-wtβ2 hearts revealed loss of the striations. Also, the size of the myocyte nuclei was increased . In thin section EM, the hearts from wild-type animals had myofibrils aligned laterally from Z-line to Z-line, with distinct A and I bands, and Z- and M-lines . Mitochondria had an elongated shape and were between the parallel arrays of myofibrils. In TG-wtβ2 mice, gross myofibrillar disarray was apparent. The myocytes were arranged in chaotic patterns at oblique and perpendicular angles. In the sarcomere, the I band was difficult to identify. The A band appeared to span the entire length between highly disorganized Z-lines. The Z-lines were truncated, wavy, and appeared slightly thickened. In some areas, the Z-lines were lacking entirely. The myofibrils were filled with numerous swollen mitochondria. The intercalated discs of the TG-wtβ2 myocardium had a relatively normal ultrastructure, but were increased in number and decreased in length. To confirm this result, intercalated discs were examined by light microscopy using antivinculin to show the intercalated discs. In normal myocardium, antivinculin staining was found as a fine line at the intercalated discs and weakly at the border of the myocytes . In TG-wtβ2 myocardium, vinculin labeling appeared as small irregular lines consistent with the multiplicity of intercalated discs in ultrastructure analysis. This could be due to the addition of new intercalated discs or fragmentation of existing discs caused by the underlying myofibril dysgenesis (see Discussion). Abnormal cardiomyocyte structure and organization was also apparent in the left ventricles of TG-β1L262R mice, whose mutation precludes the binding of actin to CP . This suggests that the myofibrillar disarray observed in the hearts of the TG-wtβ2 mice was due to the inability of CPβ2 to attach the actin filaments to the Z-line. The myofibrils of TG-β1L262R had a highly disorganized architecture, including abnormally registered sarcomeres with disoriented or missing Z-lines. The A and I bands were apparent, but lacked their typical striation. The mitochondria had lost their organization and shape. Analysis of the myocardium of eight-day-old TG-β1L262R mice showed milder defects, confirming that the myocyte deterioration was progressive from birth as expected . At eight days after birth, the periodicity of the Z-lines, the registration of the Z-lines, and the alignment of the myofibrils were only slightly altered. Higher magnification showed minor deterioration of the sarcomeres with thinning of the Z-line. The distribution of the CPβ1 and CPβ2 isoforms in murine heart tissue is the same as that described previously for chicken heart tissue and cultured cardiomyocytes . CPβ1 is at the Z-lines of the sarcomere . CPβ2 is at intercalated discs and in a punctate pattern . Two alternative hypotheses can explain the inability of CPβ2 to functionally replace CPβ1 and organize the thin filaments at the Z-line in the TG-wtβ2 mice. The first is that β2 may not localize to the Z-line due to a lack of targeting information. The second is that β2 may localize to the Z-line, but not bind to and correctly orient the thin filaments there. To discriminate between these alternatives, the localization of CPβ2 in TG-wtβ2 hearts was determined. In TG-wtβ2 hearts, CPβ2 localized to the periphery of the myofibrils and in a punctate pattern . Because the Z-lines were truncated and wavy, we used double immunofluorescence labeling with antiactin to identify the Z-line . Double immunofluorescence labeling with anti-β2 revealed that CPβ2 did not localize to the Z-line in the TG-wtβ2 myocardium. This suggests that CPβ2 is unable to functionally replace β1 at the Z-line due to its inability to localize to the Z-line. Western blot analysis showed a decreased level of β1 protein in the TG-wtβ2 hearts. To confirm this result and determine the localization of β1 in the TG-wtβ2 hearts, we examined the localization of the β1 isoform with anti-β1. In TG-wtβ2 hearts, β1 was observed as weakly stained truncated Z-lines and in a punctate pattern. The weak level of staining was consistent with the decreased level of β1 seen in Western blot analysis. The truncation of Z-lines was consistent with the myofibril dysgenesis observed in thin section EM . β1-L262R is a mutant form of the β1 subunit that binds actin poorly in vitro. We hypothesized that in TG-β1L262R hearts, the mutant β1 protein would localize to the Z-line, but be incapable of binding actin filaments, as expected from its biochemical properties. To determine if β1-L262R protein was able to localize to the Z-lines in transgenic hearts, we examined the localization of β1-L262R with anti-β1. β1-L262R protein did localize to the Z-line, appearing as short, wavy lines, reflecting the loss of the parallel alignment of the Z-lines seen by EM . To confirm that the level of β2 protein was reduced in TG-β1L262R hearts, as seen in Western blot analysis, and to examine the localization of β2, we stained heart sections with anti-β2. The intensity of β2 staining was decreased, as expected. β2 localized in a punctate pattern and at cell–cell junctions. We found that CPβ2 could not functionally replace CPβ1 in organizing the actin filaments at the Z-line. We next asked the converse question, whether CPβ1 could functionally replace CPβ2 in organizing the actin cytoskeleton–membrane interactions at intercalated discs. To test this hypothesis, we expressed the CPβ1 isoform in the mouse myocardium using the α-MyHC promoter. Transgenic lines are referred to as TG-wtβ1. Three founders were identified. Neither the founders nor the N1 heterozygotes exhibited any gross phenotype or reduced viability . All of the transgenic TG-wtβ1 lines showed expression . TG-wtβ1 line 1 had the lowest level. Expression in line 2 was twofold that of line 1, and expression in line 3 was tenfold that of line 1. Nontransgenic littermates showed no expression. An increased level of CPβ1 protein was observed in all of the TG-wtβ1 lines, from about two- to fourfold that of endogenous CPβ1 in nontransgenic littermates . The level of transgenic protein correlated with the level of transgene transcript. Line 3 expressed the highest levels of protein and transcript. Overexpression of CPβ1 led to an increase in the total amount of CPβ subunit expressed . The level of the β2 subunit did not change. Importantly, the level of the α1 and α2 subunits did not change. Therefore, the level of active heterodimer was constant, with an increased ratio of α:β1 to α:β2. To determine if overexpression of CPβ1 in the murine myocardium caused cardiac hypertrophy, heart weights were measured and corrected for body weight ( Table ). TG-wtβ1 mice did not have elevated heart to body weight ratios relative to control littermates. Therefore, in contrast to overexpression of CPβ2 and CPβ1-L262R (see above), overexpression of CPβ1 did not cause cardiac hypertrophy. Light microscopic analysis of hematoxylin- and eosin-stained heart sections of the CPβ1 transgenic mice showed a normal structure of the heart with no enlargement of ventricular walls or chambers (data not shown). To determine if there was an altered morphology of intercalated discs or myofibrils in the TG-wtβ1 hearts, ventricular walls were examined by thin section EM. In TG-wtβ1 myocardium, the intercalated discs were fragmented, composed of short, disjointed segments that were misaligned relative to the orientation of the myofibrils . The density of the electron-dense segment parallel to the plasma membrane was diminished. The segment was also discontinuous along its length. The registration and morphology of the sarcomeres of the TG-wtβ1 myocardium were largely unremarkable . The Z-lines of the sarcomere were more electron dense and slightly thickened, which may be caused by the increased level of CPβ1 protein seen in Western blot analysis . Since this phenotype was relatively mild compared with expression of β2, we considered that the β1 isoform might be functioning at the intercalated disc. To address this question, we examined the intercalated discs of transgenic mice expressing a mutant form of the β1 subunit, TG-β1L262R, that should not function because it binds actin poorly in vitro. The TG-β1L262R mice showed intercalated disc remodeling similar to that of the mice expressing the wild-type β1 isoform, with fragmentation of the intercalated disc and a reduction of electron-dense material parallel to the plasma membrane. The intercalated discs were also examined by light microscopy using antivinculin to show the intercalated discs. In TG-wtβ1 and TG-β1L262R myocardium, vinculin labeling at the intercalated discs appeared as slightly thickened, skewed lines, consistent with the misalignment of the intercalated discs seen in ultrastructure analysis . Therefore, the wild-type β1 isoform appears not to function at all in place of the β2 isoform at the intercalated disc. Two alternative hypotheses can explain the inability of CPβ1 to functionally replace CPβ2 and organize the thin filaments at the intercalated discs in TG-wtβ1 mice. The first is that β1 may not localize to the intercalated disc. The second is that β1 may localize to the intercalated disc, but not bind to and correctly orient the thin filaments there. To discriminate between these alternatives, the localization of CPβ1 in TG-wtβ1 hearts was determined. Intercalated discs do contain Z-lines from each of the adjoining myocytes. CPβ1 is present at the intercalated disc for that reason, but its staining intensity is not increased relative to other Z-lines in the myofibril. In contrast, CPβ2 is seen at the intercalated discs, but not at Z-lines . In TG-wtβ1 myocardium, β1 localized to the Z-lines of the sarcomeres, including the Z-line component of the intercalated discs, in a slightly broader pattern than in wild-type . However, β1 staining intensity was not increased at the intercalated discs. This result suggests that CPβ1 is unable to functionally replace β2 at the intercalated disc due to its inability to localize to the intercalated disc. CP, an α/β heterodimer, is a component of the actin cytoskeleton in all eukaryotes . Vertebrates contain three α subunit isoforms (α1, α2, and α3) encoded by three different genes and three β subunit isoforms (β1, β2, and β3), which are produced from one gene by alternative splicing. The sequences of the β1 and β2 isoforms are highly conserved across vertebrates and the β1 and β2 isoforms show different expression and localization patterns . In myocytes, β1 is a component of the Z-lines of sarcomeres and β2 is a component of cell–cell junctions, where myofibrils associate with the plasma membrane. We considered two hypotheses to explain why the β1 and β2 isoforms have conserved sequence differences and unique expression patterns in myocytes. One hypothesis is that the β1 and β2 isoforms are functionally equivalent, but have acquired different expression patterns important for muscle differentiation. This hypothesis predicts that the β1 isoform can replace the function of the β2 isoform and that the β2 isoform can replace the function of the β1 isoform. To test this hypothesis, we shifted the β1:β2 isoform ratio in the mouse heart using expression from a cardiac specific promoter. Our findings exclude this hypothesis because differences in the β1:β2 isoform ratio changed the morphology and physiology of the heart. Another hypothesis is that the β isoforms have acquired novel biochemical functions and fulfill different roles within the heart. This hypothesis predicts that an increase in the β2 isoform will lead to an alteration in the structure and function of the sarcomeres, which contain β1, and that an increase in β1 will lead to an alteration in the structure and function of the intercalated discs, which contain β2. Our findings support this hypothesis. We found that β2 cannot replace β1 at the Z-line. Expression of wild-type β2 subunit or an actin-binding mutant form of β1 caused major structural defects in sarcomere organization, leading to cardiac hypertrophy and juvenile lethality. We also found that β1 cannot replace β2 at the intercalated disc. Expression of wild-type β1 subunit or an actin-binding mutant form of β1 caused structural defects in the intercalated discs. In striated muscle, interactions among actin thin filaments and various contractile proteins contribute to the highly ordered structure and function of muscle. The actin thin filaments of the sarcomere are attached to Z-lines. CP binds the barbed ends of the thin filaments at the Z-line, maintaining the actin filament location, polarity, and length. We found that inhibiting CP's ability to attach the barbed end of an actin filament to the Z-line has severe effects on sarcomere assembly in mice, consistent with the effects seen in cultured cells . Transgenic mice that overexpress the β2 isoform or an actin-binding mutant of the β1 isoform showed juvenile lethality with severe disruption of myofibril architecture and cardiac hypertrophy. This result argues that the isoform-specific function of CPβ1 is to maintain the organization of the sarcomere by attachment of thin filaments to the Z-line. Presumably, the loss of β1 function leads to a loss in the number of thin filaments that can be attached to a Z-line, leading to myofibrillar disarray. The overexpressed wild-type β2 subunit did not localize to the Z-line. This suggests that the unique function of the β1 isoform involves interacting with additional components of the Z-line, not only actin. This conclusion is also supported by previous work on cultured cells, where an anti-CPβ1 antibody that prevents the binding of CP to actin was observed to localize to the Z-line . Together, these results indicate that CPβ1 binds Z-lines independent of actin, by some additional interaction. The components responsible for the isoform-specific interaction remain to be defined. Potential candidates include other sarcomere components present at the Z-line, such as titin , α-actinin , and nebulin . Our studies define CPβ1 as a sarcomeric protein for which mutation can cause hypertrophic cardiomyopathy, a disorder characterized by increased left ventricular mass and myofibrillar disarray. Familial hypertrophic cardiomyopathy in humans is caused by mutations in genes encoding sarcomeric proteins, including α-cardiac actin , β-myosin heavy chain , troponin T, α tropomyosin , myosin-binding protein C , myosin essential light chain, myosin regulatory light chain , and troponin 1 . In addition, dilated cardiomyopathy can be caused by mutations in actin . These actin mutations are hypothesized to affect attachment of thin filaments to the Z-line. The relationships between cardiac hypertrophy and dilatation, and the mechanisms that lead to each of these responses, are being explored in mouse models and humans with cardiomyopathies . Using transgenesis, we have generated a new mouse model displaying hypertrophic cardiomyopathy, based on defective thin filament/Z-line attachment. Mutations in CP genes, therefore, merit consideration as causes of human cardiomyopathies. Ventricular cardiac muscle cells of the myocardium are connected to one another at their ends by the intercalated disc. The intercalated disc links the Z-lines of adjacent myofibrils and the plasma membranes of adjacent myocytes to one another. The intercalated disc ensures that upon contraction, mechanical tension is efficiently transmitted through the myocardium . We found that overexpression of CPβ1 or an actin-binding mutant form of CPβ1 caused changes in the structure of the intercalated disc in the myocardium. The intercalated discs of TG-wtβ1 and TG-β1L262R hearts were disjointed and misaligned relative to the orientation of the myofibrils. Presumably, the altered discs were weakened by the loss of CPβ2 and then fragmented in response to contraction. Overexpression of CPβ2 led to the formation of multiple truncated intercalated discs in the myocardium; the morphology of the intercalated discs was otherwise normal. The increased number of intercalated discs could be a secondary effect of the underlying myofibril dysgenesis or to increased stress that accompanies hypertrophy . However, multiple truncated intercalated discs were not seen in the TG-β1L262R myocardium, in which loss of CPβ1 function caused prominent myofibril dysgenesis. This result does not support the hypothesis that myofibril dysgenesis alone can cause multiple truncated intercalated discs, as seen in TG-wtβ2 myocardium. Alternatively, the multiplicity of intercalated discs may, in TG-wtβ2 mice, be due the increased level of CPβ2, which is a component of the intercalated disc, having dominant effects on other components of the intercalated disc. | Study | biomedical | en | 0.999996 |
10601342 | Livers were recovered from mouse fetuses and single cell suspensions were generated using methods described previously . Between the fourth and sixth day of megakaryocyte culture, cells were placed on a 1.5–3.0% albumin step gradient and sedimented to obtain enriched populations of megakaryocytes. Platelet-sized particles were isolated from culture supernatants by centrifugation at 500 g , and when necessary, were washed using a metrizamide step gradient . Isolated megakaryocytes were diluted into semi-solid medium containing 65% Leibowitz L-15 medium (GIBCO BRL) and 35% MethoCult . Megakaryocytes in suspension were pipetted into chambers formed by mounting a glass coverslip coated with 3% BSA onto a 10-mm petri dish with a 1-cm hole. Preparations were maintained at 37°C using a Bipolar temperature controller (Medical Systems Corp.), and examined on a Zeiss IM-35 inverted microscope equipped with a 40× phase-contrast long working distance condenser. Images were obtained using a Hamamatsu charged coupled device (CCD) camera and frames were captured at 1-, 5-, or 10-min intervals using a Power Macintosh 9500 equipped with a SCION LG3 Frame grabber. Movies were generated using NIH-Image 1.61 software. Samples were prepared for electron microscopy as described . In brief, megakaryocytes in suspension were placed in wells of a 96-well microtiter plate, each containing a poly- l -lysine–coated coverslip, and the plate was centrifuged at 500 g for 5 min at 37°C. Some cells were fixed with 1% glutaraldehyde in PBS to study their surface topology. Cytoskeletons were isolated by permeabilizing cells with PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2 ) containing 0.75% Triton X-100, 2 μM phallacidin, 20 μM taxol, and protease inhibitors for 2 min. Cytoskeletons were washed with PHEM buffer containing 0.1 μM phallacidin and 30 μM taxol, and fixed in PHEM buffer containing 1% glutaraldehyde for 10 min. To examine the membrane skeleton, cells were incubated with permeabilization buffer containing 0.1% glutaraldehyde for 2 min and fixed as described above. Coverslips of fixed cells and cytoskeletons were rapidly frozen, freeze-dried, and coated with tantalum-tungsten and carbon. Replicas were picked up on carbon-formvar–coated copper grids and examined with a JEOL JEM-1200 EX electron microscope at an accelerating voltage of 100 kV. mAbs specific for β-tubulin were obtained from Amersham. Secondary antibodies, FITC, and TRITC conjugates of goat anti–rabbit and goat anti–mouse IgG were obtained from Sigma Immunochemical. Primary antibodies were used at 5 μg/ml in PBS + 1% BSA and secondary antibodies at 1:200 dilution in the same buffer. Megakaryocytes were fixed with 4% formaldehyde in Hanks' balanced salt solution (GIBCO BRL) for 20 min, cytocentrifuged at 500 g for 4 min onto coverslips previously coated with poly- l -lysine, permeabilized with 0.5% Triton X-100 in Hanks' containing 0.1 mM EGTA, and blocked with 0.5% BSA in PBS. The specimens were incubated in primary antibody for 3–6 h, washed, treated with the appropriate secondary antibody for 1 h, and washed extensively. Controls were processed identically except for omission of the primary antibody. Preparations were examined with a BioRad MRC 1024 laser scanning confocal microscope equipped with Lasersharp 3.1 software. Images were obtained using a Zeiss Axiovert S100 equipped with a 100× differential interference contrast (DIC) oil immersion objective (NA 1.4). A fluorescein-phalloidin–based flow cytometric assay was employed to quantitate the actin filament content in blood and culture-derived platelets . Platelets treated with and without 1 U/ml mouse thrombin (Sigma Chemical Co.) were fixed by the addition of an equal volume of 4% paraformaldehyde in platelet buffer (145 mM NaCl, 10 mM Hepes, 10 mM glucose, 0.5 mM Na 2 HPO 4 , 5 mM KCl, 2 mM MgCl 2 , and 0.3% BSA, pH 7.4) for 30 min at 37°C. Fixed platelets were permeabilized with 0.1% Triton X-100, labeled with 10 μM FITC-phalloidin for 1 h at room temperature, and washed twice with PBS. Samples were analyzed in a Becton Dickinson FACScan, gated for platelets, and a total of 10,000 events were analyzed using Lysis II software (Becton Dickinson). Experiments were repeated in triplicate and the results were averaged. Taxol, cytochalasin B and D, and nocodazole were obtained from Sigma Chemical Co. All images were imported into either Adobe Photoshop or Claris Draw and printed on a Kodak 8650 PS color printer. The manuscript includes three movies supplied in Quick Time™ format. Figure 1: Proplatelet formation contains 58 frames taken at 10-min intervals using a 40× phase-contrast objective. This movie shows a megakaryocyte undergoing proplatelet formation and demonstrates the dynamic behavior of proplatelets. Figure 2: (a) Dynamic interconversion contains 30 frames taken at 10-min intervals and shows a higher magnification view of the reversible interconversion between spread lamellar segments and condensed proplatelet forms. (b) Bending/branching contains 10 frames taken at 10-min intervals and shows proplatelet bending and the bifurcation at high magnification. Videos are available at http://www.jcb.org/cgi/content/full/147/6/1299/DC1. To date, proplatelets have been studied and described largely through static images, and most conclusions about mechanisms of proplatelet formation are inferred from studies conducted with only limited benefit of time-lapse cinematography. We used video-enhanced light microscopy of cultured mouse megakaryocytes to reveal a spectrum of highly reproducible morphogenetic changes and cellular movements during formation of proplatelets . A transformation of the entire megakaryocyte cytoplasm results in condensation of cell material into platelet-sized particles, which have the appearance of beads linked by thin cytoplasmic bridges. This process unfolds over 4–10 h, and initiates with erosion of one pole of the megakaryocyte cytoplasm. This generates a unique pseudopodial structure that elongates to yield slender tubules of uniform diameter of 2–4 μm; these in turn develop periodic densities along their length that impart the characteristic beaded appearance of proplatelets. Maturation of proplatelets ends in a rapid retraction that separates a variable portion of the proplatelets from the residual cell body, according to a process that presumably mirrors platelet release in vivo. High resolution time-lapse analysis reveals several striking features that have not been recognized previously, and emphasizes the highly dynamic nature of thrombopoiesis. Remodeling of the megakaryocyte cytoplasm is accompanied by centrifugal spreading that increases the apparent cell surface area and, measured from representative cells , proceeds at a rate of ∼0.2 μm/min. In addition, portions of the proplatelet cytoplasm exhibit striking and dynamic interconversion between spread lamellar segments and condensed structures resembling platelets; this oscillation between proplatelet and spread morphologies may occur multiple times . The distal ends of proplatelet processes periodically flatten to form fan-shaped sheets resembling lamellipodia and crawl away from the cell center, dragging a proplatelet trail . Interestingly, proplatelet fragments and the megakaryocyte cell body move at similar speeds (0.2–0.4 μm/min), and spreading movements are inhibited by 1–10 μM of cytochalasin B, suggesting that they require actin polymerization; upon removal of this drug, megakaryocytes recover the ability to spread on the surface (data not shown). The stable cytoskeletal architecture of mature blood platelets would seem to preclude the degree of dynamic cell motility revealed by these studies. Hence, platelet assembly must at best be incomplete during this phase of proplatelet morphogenesis, and platelets must be assembled de novo in the course of this transition; structural studies detailed below strongly support this conclusion. To better understand the dynamic aspects of proplatelet morphogenesis, we examined selected aspects at high resolution. Branching of proplatelets has been recognized previously but not investigated in detail. Proplatelet bifurcation occurs at a variable distance from the cell body, but only at sites of pronounced bending . The sharp kinks elongate, forming a small loop, and a new proplatelet process formed by this loop extends from the original nexus. Newly generated extensions also develop the periodic constrictions characteristic of proplatelets. Platelet-sized particles and nodules of variable size translocate extensively along the length of megakaryocyte processes , another feature that is incompatible with preassembly of a stable platelet cytoskeleton. Distinct segments move in either direction at an estimated rate of ∼1 μm/min, may move in opposite directions on a single extension, and can even reverse direction. The particles typically do not reveal changes in phase density as they move; however, we have observed several platelet-sized particles fuse together during translocation . Additionally, proplatelets undergo continuous cycles of extension and retraction that are most clearly evident in the video accompanying Fig. 1 . Thus, these observations reveal proplatelet processes to be inherently unstable structures with a capacity for extensive morphogenetic changes. The surprisingly dynamic character of proplatelet formation raises many important questions. (1) What is the structure of the intermediate, platelet-like particles detected within developing proplatelets? (2) Which cellular structures drive megakaryocyte spreading, elongation, and thinning of proplatelets? (3) What is the structural basis for the transition between proplatelets and mature platelets? (4) How does branching of proplatelet processes occur, and what is its significance? In the following experiments, we address some of these questions. Previous studies investigating platelet formation by cultured megakaryocytes have only partially assessed the structure and function of the released particles . Therefore, we first investigated whether the shape changes and movements observed in our studies lead to the release of verifiable platelets. Platelet-sized particles released by cultured mouse megakaryocytes exhibit each of the hallmark features of blood-derived platelets. They are disc-shaped, 2–3 μm in diameter, lack discernible membrane topology or protrusions , and show periodic surface invaginations that demarcate entrances into the open canalicular system . Removal of the plasma membrane exposes an elaborate membrane skeleton composed of 3–5-nm strands identical in appearance to the spectrin-based network of human platelets , and a prominent marginal band derived from the apparent coiling of a single microtubule . A complex three-dimensional network of actin filaments, similar to that observed in mature blood platelets , is also found beneath the membrane skeleton . 200–225 platelet-sized microtubule coils may be seen extending from typical proplatelet-producing cells , a value that is in good agreement with the proposed theoretical range of platelets produced per megakaryocyte and with observations in cultured human cells . Furthermore, the released particles respond to stimulation with thrombin by extending lamellipodia and filopodia , forming microaggregates , and exhibiting a twofold increase in their proportion of filamentous actin (data not shown), which is similar to observations in activated blood platelets . These particles showed increased expression of the activation-dependent antigens P-selectin and the functional fibrinogen receptor (data not shown), as previously reported for human proplatelets . Hence, platelets released in cell culture display all the morphological and functional criteria that distinguish blood platelets. Microtubule assembly has been previously implicated in platelet formation , and our studies confirm its essential role in the progression of initial pseudopodia into proplatelets. The microtubule disrupting agent nocodazole (1–10 μM) completely inhibits formation of all megakaryocyte projections . When nocodazole is added to megakaryocytes after they have formed proplatelet processes, particle transport is completely blocked (data not shown), indicating the requirement for a microtubule-based motile apparatus in this process. To understand the structural basis of the transitions between the megakaryocyte cell body, pseudopodia, proplatelet processes, and nascent platelets, we examined the microtubule cytoskeletons of proplatelet-producing megakaryocytes. Early in the maturation process, before spreading and erosion of the cell body begin, the megakaryocyte cytoplasm is replete with long individual arrays of microtubules that cluster around the nucleus and radiate toward the cell margins . As large and blunt pseudopodia are formed in the area of erosion, the microtubules consolidate into cortical bundles situated just beneath the membrane surface of the protrusion . As the pseudopodia extend and become thinner, they display a prominent band of microtubules along their edges; in some cases, these bundles are seen to curl or spiral inside the pseudopodia . The next recognizable step is the conversion of the blunt pseudopodia, via elongation, into cytoplasmic extensions that continue to harbor thick bundles of microtubules . These processes always have bulbous ends, and electron microscopy reveals a microtubule bundle that loops just beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure . Determining when and where platelet units are finally assembled during megakaryocyte differentiation has been controversial . At the heart of this question is understanding where and how the marginal microtubule band, a stable feature of terminally differentiated platelets , is formed. If platelets preassemble within the megakaryocyte cytoplasm into platelet fields or territories, then microtubule coils would be expected to lie within these areas before cytoplasmic fragmentation. The dynamic process of proplatelet formation and the underlying progression of the microtubule cytoskeleton from pseudopodia to proplatelets argue strongly against this possibility. In addition, in scanning hundreds of antitubulin immunofluorescence profiles of megakaryocytes, we failed to identify microtubule coils within the cell bodies; instead, these are consistently and readily detected only in proplatelet extensions . Most released platelet-sized particles remain connected by cytoplasmic bridges, the most abundant being barbell forms composed of two platelet-like particles joined by one cytoplasmic strand. To ask whether the bulbous ends and thickenings along the shaft of proplatelets show mature cytoskeletal features, we examined representative released particles by antitubulin immunofluorescence confocal microscopy and electron microscopy . A microtubule bundle forms the core of the proplatelet shaft, and the ends have microtubule bundles forming teardrop-shaped loops that are similar in both size (1–3 μm) and appearance to those described in mature blood platelets . Distorted microtubule bundles are also visible in platelet-sized swellings along the shaft of the proplatelet; however, in contrast to the terminal rings, those observed along the proplatelet shafts are uniformly less distinct . Whereas these platelet-sized nodules superficially appear to include microtubule rings, close inspection reveals that they are actually points where microtubule bundles simply diverge for a short distance but fail to form the mature teardrop shape . These nodules consistently reveal membrane and other detergent-insoluble materials, presumably destined for final platelet assembly elsewhere . These results are entirely consistent with the dynamic activity that is observed at the sites of the platelet-sized nodules, including branching , interconversion between condensed and lamellipodial forms , and rapid translocation of particles , and indicate that they are structurally unstable. More importantly, they establish that the mature cytoskeletal feature of a microtubule coil is only detected at the ends of proplatelets and not along their lengths. The separation of proplatelet morphogenesis into stages characterized by specific alterations of the microtubule cytoskeleton and the recognition of distinct dynamic features provide approaches to investigation of the underlying molecular mechanisms. Megakaryocytes cultured in the presence of cytochalasin B, an inhibitor of actin assembly, retain the capacity to extend long, slender proplatelet-like projections but show specific abnormal features. The same results were observed with cytochalasin D. The cell body fails to spread, proplatelets extend from multiple points around the cell margin instead of the typical single erosion site, branching is completely inhibited, and although proplatelet processes retain bulbous ends, they harbor many fewer intermediate swellings. Thus, actin assembly is not required for megakaryocytes to extend proplatelets but is essential for proplatelet branching. Interestingly, one process associated with proplatelet bending is the attachment of a small region to the substratum over a period of 5–10 min and robust ruffling activity of this portion (data not shown), movements classically thought to be mediated by actin. In the electron microscope, the microtubule bundles composing the shaft routinely reveal small filamentous outpouchings . At sites of bending, meshworks of actin filaments extrude processes that connect the microtubule bundles much like tendons attaching muscle to bone . At more pronounced bends, the apparent sites of proplatelet branching, filamentous aggregates form a cusp between the microtubule bundles . Considered together, these observations demonstrate that actin filaments are enriched at the sites of proplatelet bifuraction and probably required to execute this process. The elongation of proplatelet extensions could be explained by forces derived from polymerization of tubulin subunits, from sliding of microtubules, or possibly a combination of both processes. Treatment with cytochalasin B results in substantially reduced complexity of proplatelets and permits isolated examination of the elongation process. In the representative sequence of micrographs shown in Fig. 7 b, we followed the growth of a single proplatelet. As this structure elongates, it rolls up upon itself into a loop. While the distal end subsequently remains stationary , the process continues to elongate and causes the diameter of the loop to increase. This strongly suggests that extension of proplatelet processes can occur without growth at the tip. The microtubule stabilizing agent taxol completely blocks bending and branching along the length of the proplatelet tube and increases both the diameter of individual tubes and the thickness of microtubule bundles within them. Megakaryocytes treated with taxol remain capable of projecting short, thickened extensions with large bulbous tips. Fig. 7 c shows representative video-enhanced light microscopy designed to investigate the mechanics of proplatelet extension after taxol treatment, and illustrates that the processes formed are thickened compared with normal. As processes elongate in the presence of taxol, their distal tip bends and curves back on itself until it makes contact with the shaft and appears to fuse with it to form a teardrop-shaped structure. In the presence of taxol, microtubules still align and compress in the cell cortex from which they enter pseudopodia . In many cases, they do so from two sides and separate within the extension. Immunofluorescence reveals that microtubules either coil at the tips of projections or fold back on themselves to generate teardrop forms. The notion that mammalian platelets assemble through intermediate proplatelet structures was originally hypothesized by Becker and DeBruyn 1976 , developed into a plausible model by Radley and co-workers , is strongly supported by the sum of many previous investigations, and is inconsistent with a competing model of thrombopoiesis that states that platelets are first formed as platelet territories within megakaryocytes. If the platelet territory model is true, as has been suggested by freeze-fracture and thin-section electron microscopy on the internal membranes of megakaryocytes , one would expect to find microtubule rings within such platelet fields . This simple model finds little support here, or historically, as microtubule coils have not been observed in the megakaryocyte body. In contrast, many observers have documented the elaboration of proplatelets in megakaryocyte cultures , and occasional micrographs have captured human megakaryocytes extruding proplatelet-like processes into bone marrow sinusoids . The infrequent sighting of this morphology in situ most likely results from geometric limitations of thin-section transmission electron microscopy. Proplatelets have been recognized in a wide range of mammalian species, including mice, rats, guinea pigs, dogs, cows, and humans; platelet territories are not observed in megakaryocytes from some of these species. Moreover, the early characterization of proplatelets revealed many features that are consistent with known aspects of thrombopoiesis, including the critical requirement for microtubule integrity . Finally, mice lacking two distinct hematopoietic transcription factors have severe thrombocytopenia and fail to produce proplatelets in culture . Taken together, these findings establish the central importance of proplatelets in mammalian thrombopoiesis. Here, we report detailed studies aimed at investigating discrete steps in platelet formation by terminally differentiated mouse megakaryocytes, and our analysis represents the first effort to dissect dynamic aspects of this process. Proplatelets extend by a microtubule-based system, bifurcate repeatedly to increase the number of ends, and deliver packets of platelet material to these ends. Proplatelets and their contents are highly dynamic structures that continuously engage in alternate extension and retraction, reversible spreading, and bidirectional transport of particles. This dynamic behavior is inconsistent with the notion of proplatelets as static, linear arrays of nascent blood platelets, and strongly suggests that platelets mature along and at the ends of proplatelets in the final stages of an elaborate process. Our analysis suggests the model for platelet formation shown in Fig. 9 . Segments of proplatelets clearly demonstrate the capacity to spread reversibly on the substratum and lose their characteristic tubular, beaded appearances. These changes occur both at the ends of tubes and along the shafts and are transient, with rapid reversion to the original, beads-on-a-string appearance . This remarkable plasticity needs to be reconciled with the presence in blood platelets of an elaborate membrane skeleton, where molecules of filamin A (ABP-280) extend through pores in the spectrin network, connecting actin filaments to the cytoplasmic tail of the α chain of GPIb of the von Willebrand factor receptor, and create an arrangement that restricts the mobility of the membrane adherent spectrin lattice . Although not discussed here, the plasma membranes of proplatelet extensions have a spectrin lattice identical in appearance to that of the mature platelet (Italiano, J.E., R.A. Shivdasani, and J.H. Hartwig, unpublished observations). Our observations on the dynamic nature of proplatelet morphogenesis suggest that the linkages holding the membrane skeleton in compression between the plasma membrane and actin cytoskeleton must be formed only during the final stages of platelet maturation. Because lamellar spreading of proplatelets involves the dynamic assembly and disassembly of actin filaments, the filamin A–von Willebrand factor receptor linkage could only be established once the underlying actin cytoskeletal network is stabilized. This reinforces the notion that intermediate swellings do not correspond to assembled platelets, and suggests that platelet assembly completes just before release. An additional dynamic feature of intermediate proplatelet segments is their rapid internal translocation. Viewed in the light microscope, nodular segments move bidirectionally along the proplatelet shaft, collide into each other, and fuse. These observations suggest that they are packets of material destined for assembly into nascent platelets, and the linear arrays of microtubules present throughout proplatelet processes probably serve as tracks for the transport of membrane, organelles, and granules into developing platelets. Therefore, we interpret the observed movement of particles to result from a combination of translocation along the microtubule tracks and proplatelet extension by microtubule growth. Proplatelet processes are generated by the elongation and thinning of blunt pseudopodia, which first appear at one pole of the cell and exhibit a unique organization of microtubule bundles parallel to the cell margins. Pseudopodial growth is associated with extension and apparent compression of these cortical microtubule bundles, and it is likely that the final orientation of microtubule bundles in the shafts of thinner proplatelet processes result from a continuation of this compression reaction. These structural details highlight earlier work demonstrating the central role of microtubules in proplatelet formation; indeed, disassembly of cytoplasmic microtubules with nocodazole blocks formation of pseudopodia, whereas taxol treatment results in the formation of very few abnormal protrusions. Proplatelets terminate in bulbous structures invariably lined by a bundle of microtubules that originate in the shaft, form a teardrop-shaped loop within the bulb, and reenter the shaft. The loop itself reveals at least two discrete bundles, which must have opposing polarity, within the distal portion of the proplatelet. Whether loop formation is part of the mechanism that drives tube extension or a structural consequence of this process is unclear. In the simplest model, assembly of microtubules at their plus ends, either at the tip or tube base, could generate the forces leading to proplatelet extension. The invariable finding of microtubule loops at the termini, however, argues against a model of plus end–driven tip growth, such as that which occurs in axonal extension. Moreover, extension of the proplatelet per se does not require tip elongation because time-lapse microscopy reveals stationary tips during periods of intense shaft growth . Alternatively, tubulin subunits could add selectively to microtubules at the base and thereby elongate the tube; this model makes the testable prediction that the plus ends (the preferred sites of microtubule assembly) of proplatelet microtubules lie near the cell body, an unlikely possibility. Therefore, we favor the third possibility that tubulin subunits add to the plus ends of microtubules dispersed throughout the proplatelet shaft, and that microtubule motor proteins aid in tube elongation by sliding individual microtubules past one another. Such sliding would both lengthen the tube and reduce its diameter, consistent with the present observations, while microtubule-based motor activity might also contribute to the observed bends in proplatelet extensions and deliver platelet components to the ends. Our examination of the sequence of events leading to proplatelet bending and bifurcation reveals critical details and again suggests mechanistic possibilities. For a proplatelet extension to bifurcate into two processes, three unique events must occur. First, the proplatelet shaft must bend sharply until it folds over on itself to form a loop that in turn elongates until it is structurally indistinguishable from its parent form. Second, microtubules within the tube must fragment, branch, or bend into loops, as depicted in Fig. 5 , which elongate to form new processes. Third, either two sections of the shaft must fuse or looping must occur within the confines of the proplatelet shaft. The transient cell spreading and actin-based movements observed at sites of bifurcation may be necessary to accomplish this feat. The mechanical forces leading to tube branching require the activity of the actin cytoskeleton because treatment with cytochalasins prevents all branch formation. Regions with focal actin filament assembly may, thus, transmit contractile forces to the microtubule bundle to help compress it during loop formation. In the last step, proplatelets separate from the residual cell mass and are released into the culture medium by a retractile mechanism. The tension generated by this retraction can be observed as a snapping of the strands between individual proplatelet extensions (data not shown). Although individual platelet-sized particles frequently appear in the culture, most of the released material consists of chains containing two or more platelet-sized particles. Release of individual platelet-sized particles in vivo may be accelerated by a fragmentation mechanism that is not expressed in cell culture or by shear forces normally encountered in the circulation. The process of proplatelet formation may also be modified by factors present in vivo. The bone marrow environment is composed of a complex adherent cell population containing endothelial cells, macrophages, and preadipocytes, which could play a role in platelet formation by direct cell contact or secretion of cytokines. Our observations support the view that proplatelets are essential intermediates in platelet assembly, but refute the widely assumed concept that proplatelets are simply chains of fully and equally mature blood platelets. While platelet-sized units found along the shaft of proplatelets may represent intermediate stages in platelet maturation, these segments are surprisingly motile and unstable. Therefore, we suggest a conservative reinterpretation of previous models that imply that each platelet-sized swelling observed along proplatelet shafts is a nascent blood platelet. Only the particles at the ends of proplatelets consistently display the mature feature of a single microtubule rolled into a coil. This finding posits the ends of proplatelets as the best sites of platelet assembly. One implication of these results is that proplatelet branching represents an elegant mechanism designed to increase the number of termini for productive thrombopoiesis. The process of proplatelet branching, therefore, appears to play an essential role in platelet biogenesis and provides a mechanism for increasing the final number of released platelets. By looping microtubules, the mechanism of proplatelet bifurcation may also initiate the process of rolling individual microtubules into the marginal ring destined to be incorporated into each mature blood platelet. Reconstitution of platelet formation from murine megakaryocytes in vitro provides a powerful system for identifying the cytoskeletal components involved in the remarkable mechanism of platelet formation and release. More detailed structural analysis, coupled with the ability to study knockout mice lacking specific proteins will allow us to probe the contribution of individual components in thrombopoiesis. The principles learned from studying cytoskeletal dynamics in megakaryocytes are likely to provide insights into mechanisms of shape changes and movements of other cells. | Study | biomedical | en | 0.999998 |
10601343 | Goldfish were anesthetized by a brief exposure to hypothermic water and killed by decapitation, followed by pithing. Scales were removed and placed in fish Ringer's supplemented with 1% Fungizone. Individual scales were teased loose from clumps and placed on 22 × 22-mm coverslips that had been cleaned with Chromerge and wetted with fish media (70% fish Ringer's, 30% Phenol red-free DME supplemented with 20% FCS, 2 mM l -glutamine, 10,000 U penicillin/streptomycin, and 20 mM Hepes). A second clean coverslip was wetted and placed over the first coverslip, creating a scale sandwich. Once the keratocytes began to crawl off the scale, usually 1–2 h, the petri dish containing the coverslips was gently flooded with fish media. Cultures were kept in a humidified chamber at room temperature until use (12–24 h). Keratocytes were fixed in 2% paraformaldehyde in PHEM for 10 min. Cells were permeabilized for 0.5 min in 0.5% Triton X-100 in PBS and then blocked with 1% serum, and 0.5% BSA for 20 min. Cells were then incubated for 1 h in primary antibody, followed by a 1-h incubation in secondary antibody. Finally, coverslips were mounted in Slow Fade Light (Molecular Probes, Inc.). Three washes in PBS were performed between each step. Oregon green phalloidin was obtained from Molecular Probes, Inc., secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc., and the antibody to the conserved sequence of the β1 integrin was the generous gift of Richard Hynes (MIT, Cambridge, MA). Acid washed coverslips were silanized to block any surface charge. Cloning cylinders were attached to the coverslips with Sylgard 184 Elastomer (Dow Corning). The center of the coverslip was then coated with either fibronectin (FN) 120 kD (GIBCO BRL) or 2 mg/ml BSA in PBS overnight at 4°C. The adhesion assay is based on the protocol described by Ruoslahti et al. 1982 . In brief, the coverslips were rinsed in PBS and blocked with 2 mg/ml BSA in PBS for at least 1 h. Cells were rinsed in Ca 2 +/Mg 2 + free HBSS and then trypsinized with 0.05% trypsin 0.53 mM EDTA 4 Na in HBSS. Cells were rinsed in a threefold excess volume of soybean trypsin inhibitor at 1 mg/ml in PBS and gently centrifuged. Cells were resuspended in stage media (70% fish Ringer's, 30% Phenol red-free DME supplemented with 1% FCS, 2 mM l -glutamine, 10,000 U penicillin/streptomycin, and 20 mM Hepes). An equal volume of cells was added to each cloning cylinder and allowed to incubate for 60 min at room temperature. The cloning cylinder was washed with PBS and then fixed for 15 min with 4% paraformaldehyde. The cells were then fixed for an additional 1 h with 0.5% filtered Toluidine blue in 4% paraformaldehyde. The coverslips were washed with copious amounts of water, the cloning cylinders removed, and mounted with 50% glycerol in PBS. Cells in 20 nonoverlapping fields on each coverslip were scored as either adhered and spread (spread) or adhered and not spread (round). The number of spread and round cells for each of the coverslips within a given repetition of the experiment was normalized by the maximum number of cells that adhered and spread on the FN-coated substratum. A micromachined substrata was developed to measure ventral traction forces of subcellular regions . In brief, this device is based upon a system of several thousand cantilever beams buried beneath its surface. On the free end of each beam is a pad that is planar with the surface of the device. The pads range in size from 4–25 μm 2 . A small square hole surrounds each pad, allowing the cell to displace the pad. The force that the region of the cell in contact with the pad exerts is calculated from the displacement of the pad and the stiffness of the beam. The beams used in this study are 0.18-mm long and have a stiffness of ∼76 nN/μm as determined by calibrated microneedles . Keratocytes were grown on micromachined substratum in fish media as either explant cultures that migrated off the scale or as individual cells that had been dissociated from an explant grown on glass coverslips. In neither case was the substratum coated with matrix in addition to serum or deposited by the cell; these are the same substratum conditions that were used in the laser trap experiments. We did not coat the substratum with a more defined matrix because the micromachined devices are reused several times due to cost constraints. The micromachined devices are subjected to 10× trypsin after each use, but this does not remove all of the matrix deposited by the cells. Harsher acid treatments would remove the matrix, but they would also destroy the micromachined device. In repeated use, we have found no variation in the force produced after the device had been used once; the first use of the device typically results in poor cell adhesion, and this plating is only used to build up matrix. Furthermore, unpublished data on traction forces generated by fibroblasts where the substrate coating of laminin was varied by twofold did not show any changes in traction force, suggesting that force generation is insensitive to matrix coatings in excess of saturation level. The substratum and cells were visualized with polarized reflection microscopy. The experiment was recorded on S-VHS tape, and individual frames were captured with a Scion LG-3 frame grabber in a Macintosh Power PC 7100. A threshold between two grey levels was applied to the image, and the centroids of the pad and the surrounding hole were calculated with NIH IMAGE 1.60 (developed at the National Institutes of Health and available by anonymous FTP from zippy.nimh.nih.gov). The centroid of the hole was subtracted from the centroid of the pad. Carboxylate beads were coated with a fragment of FNIII 7–10 (bacteria expressing FNIII 7–10 were the generous gift of Harold Erickson, Duke University, Durham, NC). Beads were constrained against the surface of keratocytes by the laser trap, and the displacement of the bead within the trap, as well as the stiffness of the trap were used to calculate the force that the cell exerted against constrained beads. Cells were prepared as described above, and the experiment was performed in stage media. An optical gradient laser trap was calibrated by moving the stage in a sinusoidal pattern and measuring the displacement of the bead from the center of the optical trap due to movement of the fluid. Using Stokes' Law, the force that the moving fluid generated against the bead was calculated. The stiffness of the trap was then determined from the slope of the fluid force vs. bead displacement curve . The displacement of the bead within the trap was measured with a nanometer-tracking routine within the program Isee (Inovision, Inc.). The force per unit area on a 1-μm diam bead was calculated by equating the amount that the bead displaced into the cell to the centroid displacement due to focus change when the bead was initially pressed to the cell surface. The contact area was then calculated as the surface area of a zone of a sphere with this height. The total traction force exerted on the ventral surface of a migrating fish keratocyte is ∼45 nN , an order of magnitude smaller than the migration force exerted by fibroblasts . The predominant ventral traction forces are perpendicular to the direction of migration. These forces are ∼20 nN in size, and they produce wrinkles in deformable substratum that are parallel to the direction of migration . To identify additional traction forces, ventral traction forces generated by subcellular regions were measured with a micromachined substratum. This device has several advantages over other techniques for measuring ventral traction forces. The device can determine the force generated by only the region of the cell contacting the measurement unit, and the measurement unit is elastic, so there is no relaxation time of the substratum to limit temporal resolution. However, since the device can only measure displacements in one direction, orthogonal to the long axis of the measurement unit, the predominant axis of force generation was derived from either deformable substrata or laser tweezer measurements. We measured the predominant traction force generated on either side of the nucleus to compare the magnitude of the force determined by our device with the force determined by other substrata. As the force vs. time trace in Fig. 2 illustrates, the maximum force that is generated by the pincer region on either side of the nucleus is ∼13 nN. Measurements made of the same region with other substratum report values as high as 11 nN . Thus, both substrata measure forces of very similar magnitude in the pincer region of the cell. With the micromachined substratum, we were able to measure traction forces in the front region of the lamella. An example of a typical experiment is shown in Fig. 3 . Initially, the lamella generated a small force directed opposite to the direction of motion; this is consistent with the forward movement of lamella fragments . This force was not detectable by our measurement device until the majority of the lamella was over the pad , and then the force was only two- to threefold greater than our measurement noise. The force increased in magnitude from the front of the cell toward the perinuclear region. As the perinuclear region of the cell crossed the pad the force changed direction, and it was now oriented in the direction of motion. The maximal rearward forces were ∼4.5 nN, or 0.2 nN/μm 2 . This was similar to the traction forces generated by fibroblast lamella , and was ∼75% less than the smallest force measured with deformable substrata . Thus, keratocytes, like fibroblasts, pull rearward under their lamella, change traction force direction near the nucleus , and pull forward under the nucleus (data not shown). Since most previous work on cytoskeletally mediated movement of integrin and ECM has been performed on the dorsal surface, we asked whether the keratocyte generates similar forces against the ECM on both the dorsal and the ventral surfaces. To measure the traction forces generated along the dorsal surface of the fish keratocyte, we probed the surface of the cell with ligand-coated beads that were held by an optical gradient trap. The generation of cellular traction forces against ECM substratum depends critically on the ligand activation of specific cell surface integrins . Therefore, to characterize keratocyte forces, it was necessary to identify the binding specificity of receptors mediating substratum recognition. Specifically, we investigated whether keratocytes had an integrin that would couple FN to the cytoskeleton. FN was a likely candidate, since it had been shown that keratocyte motility is inhibited by a peptide that inhibits both FN- and vitronectin- (VN) binding to integrins . To examine the ability of keratocytes to bind FN, we performed adhesion assays. Keratocyte explant cultures were trypsinized and plated on surfaces coated with either the 120-kD fragment of FN or BSA. The percentages of cells that bound and spread on the FN-coated substrata were significantly greater than those that bound and spread on BSA-coated substrata . The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP, a peptide suggested to specifically inhibit cell binding to FN. Moreover, the addition of 1 mM GRGDNP, a peptide that more strongly inhibits cell binding to FN, but has some cross-inhibition with VN , decreased the percentage of cells bound on FN to the same level as the BSA control . Although 1 mM GRGdSP has been shown to produce half-maximal inhibition of adhesion by CHO cells to FN , it was less effective in our assay, possibly due to species differences or differences in the receptor that binds FN in keratocytes. These results indicate that integrin-dependent binding and spreading does occur. To examine ligand binding to the dorsal surface, polystyrene beads coated with either a small fragment of FN (FNIII 7–10) or with BSA were held against the cell surface by the laser trap for three seconds and then released . Most of the FN-coated beads, ∼63%, bound and started to move rearward along the cell surface. In contrast, 78% of the BSA-coated beads did not bind to the cell surface, and almost none of the bound BSA-coated beads exhibited retrograde movement. Binding of FN-coated beads was not uniform across the surface, showing preferential attachment at the leading edge of the cell. The binding and retrograde movement of FN-coated beads decreased to ∼30% when the beads were placed 0.5–1.0 μm behind the leading edge of the cell , consistent with earlier observations on fibroblasts . To determine whether preferential binding reflects integrin distribution, keratocytes were fixed and labeled with an antibody to β1 integrin, a component of the α5β1 heterodimer, a common FN receptor. The antibody is directed against the conserved cytoplasmic domain of β1 . The staining was localized to the leading edge of the cell , with an approximately twofold increase in fluorescence intensity in this region. To quantify dorsal traction forces, a 1-μm diam FN-coated bead was placed on the cell surface and constrained within an optical gradient laser trap. An example of a typical experiment is shown in Fig. 7 . As the cell pulled on the bead that was constrained by the laser trap, the bead displaced from the center of the trap , but it did not escape from the trap. From the displacement of the bead within the trap and the stiffness of the trap, we were able to calculate that the cell exerted a force on the bead of 158 pN, or 0.4 ± 0.3 nN/μm 2 ( n = 7, ± SD). Eventually , the cell exerted enough force on the bead to pull the bead from the trap. Once the bead escaped the trap, it traveled rearward with approximately the same velocity as the forward traveling cell, until it reached the nuclear region when the bead stopped moving. Moreover, this velocity is equivalent to the rate of retrograde flow of the actin cytoskeleton (data not shown). We were unable to measure the large forces orthogonal to the direction of motion on the dorsal surface using the laser trap. FN-coated beads placed on this region of the cell were pulled by the cell with a force that exceeded the force of the trap. This result is in agreement with our ventral force measurement in the same region . We asked whether the regional differences in binding of FN-coated beads correlated with differences in the traction forces exerted by the leading edge, compared with other regions of the lamella. To measure the strength of cytoskeletal attachment and force generation against the bead, we tested for reinforcement of the fibronectin beads . A laser trap was used to place and hold FN-coated beads near the leading edge of the cell. When placed on the cell at this location, the beads frequently escaped the restraining force of the trap . We then tried to recapture the beads into the center of the trap to determine if the force exerted on the bead by the cytoskeleton was less than or greater than that exerted by the trap. If the strength of the force exerted on the bead by the cytoskeleton was greater than the force exerted by the trap, then we could not recapture the bead, and the linkage between the bead and the cytoskeleton was considered to be reinforced. This response frequently (∼80%) occurred when FN-coated beads were placed on 3T3 fibroblasts ; however, as shown by the rapid displacement of the bead when the trap was turned on , beads could easily be recaptured near the edge of the keratocyte lamella (100%, n = 10). Further into the lamella, at the region where it thickens, beads frequently (67%, n = 12) could not be recaptured , and beads could also not be recaptured in the perinuclear region . Control experiments in which beads were continuously recaptured in the front of the lamella indicate that this phenomenon is a function of location, not a function of the number of times that a recapture is attempted. Thus, there appears to be a positional reinforcement of the linkage between the bead and the cytoskeleton as the bead travels into the lamella. To determine if this positional reinforcement could be due to an increase in cytoskeletal density rather than an enhancement of the linkage, keratocytes were labeled with Oregon green phalloidin, and the intensity of fluorescence from the leading edge to the perinuclear region was determined . Similar to the results shown by others , we observed a decrease in fluorescence between the front and the perinuclear region of the lamella, with the fluorescence intensity in the perinuclear region being ∼75% of the intensity at the leading edge. Since we were able to recapture beads located above the region where the actin cytoskeleton is the densest, the positional reinforcement is probably not completely due to entanglement with the underlying cytoskeleton, but rather an enhanced integrin–cytoskeletal linkage. The definition of traction forces at a subcellular level has uncovered several new aspects of the motile machinery in keratocytes that were unexpected. Propulsive forces are generated by the keratocyte lamella. The myosin-dense perinuclear region of the lamella is the major propulsive force-generating region. The thin leading edge of the lamella does not generate substantial forces, but it does act as a region of preferential binding between the rearward moving cytoskeleton and the ECM. Furthermore, the similarity in propulsive force per unit area generated against a submicron bead contact on both the dorsal surface and a portion of the ventral surface suggests that traction forces are generated by a similar mechanism on subcellular regions of both cell surfaces in the keratocyte. These findings indicate a possible mechanism of migration where traction forces are generated by a myosin-based, actin network condensation. The largest ventral traction forces generated by the fish keratocyte lamella were ∼4.5 nN. This force yields a traction force per unit area of 0.2 nN/μm 2 . Although early measurements with deformable substrata were unable to detect this force on the ventral surface , in later studies, the lamella of some cells appear to have rearward and forward directed traction forces . The laser trap and the micromachined substratum measure forces generated in subcellular regions, without the influence of other forces exerted on the substratum. Because both of these methods measure rearward forces of roughly the same magnitude in the front lamella, we conclude that rearward directed forces are generated in the lamella of keratocytes, and these forces are similar to those observed in fibroblasts. Our measurement of dorsal traction forces per unit area yields a traction force per unit area of 0.4 ± 0.3 nN/μm 2 ( n = 7, ± SD). The largest possible source of variability in determining this number comes from the measurement of the contact area between the bead and the cell. Our estimate is based on the contact area during initial placement of the bead with the laser trap, and this height could vary during the course of the experiment. However, other studies that have evaluated out method for determining the z-position of a bead by noting the change in the area of a DIC image of a bead suggest that it is accurate within ±20 nm (Suzuki, K.W., R.W. Sterba, and M.P. Sheetz, manuscript submitted for publication). This accuracy in height is within 15% of our area calculation and significantly less than our SD. The agreement between dorsal and ventral traction forces in the keratocyte lamella suggests that a similar mechanism of force generation operates on both cell surfaces, and it is in contrast to the disagreement between ventral and dorsal traction forces measured in locomoting fibroblasts. Although the normalized ventral traction force generated by the lamella is in good agreement for both keratocytes and fibroblasts, the normalized dorsal traction forces measured in fibroblasts are two orders of magnitude smaller. There are several explanations for this discrepancy. The dorsal measurements on fibroblasts were made with a microneedle contacting the cell surface, and it is unclear what the area of contact is between the needle and the cell. Alternatively, the variations in the cytoskeletal structure between the ventral and dorsal surfaces of fibroblasts could account for the different forces measured on these two surfaces. The former explanation is more likely since studies examining the forces that a fibroblast can exert against an extension of the cell created by attaching a needle to the cell and pulling, are of a similar magnitude, but different time course than those observed in this study. Our results indicate that keratocytes use an FN receptor for adhesion to their substrata and for linking ECM to the rearward moving cytoskeleton. Other investigators have used immunofluorescence to demonstrate that keratocytes have a β1 integrin , part of the α5β1 heterodimer of the fibronectin receptor. It has also been shown, by peptide and antibody competitive inhibition, that moving keratocytes have RGD and β1 dependent adhesion and locomotion . We have demonstrated that keratocytes use an FN receptor to generate preferential adhesions at the leading edge, and this receptor-ligand interaction allows the cytoskeleton to exert traction forces against the substratum for migration. The preferential leading edge binding that we report for FN receptor–ligand interaction has also been observed in other cell types. In fibroblasts, there was a preferential attachment to the cytoskeleton of FN-coated beads at the leading edge , but there was no preferential localization of FN-receptor at the leading edge. Similarly, in keratocytes, Concanavalin A–coated beads attached to the cytoskeleton when brought to the leading edge, but there was no preferential localization of Concanavalin A at the leading edge, as judged by immunofluorescence . These results suggest that preferential binding at the leading edge could be due to something other than receptor localization, perhaps due to an avidity change caused by receptor cross-linking to the cytoskeleton. Alternatively, the curvature of the membrane at the leading edge could cause greater binding, but our inability to see a preferential binding at the leading edge with BSA-coated beads indicates that this is probably not the mechanism responsible for our results. The enhanced localization of β1 integrin, which we observe at the leading edge of keratocytes, suggests that receptor localization is at least partly responsible for the enhanced binding of FN-coated beads to the rearward moving cytoskeleton at the leading edge of keratocytes. Although the leading edge is a site of enhanced attachment of ECM to the force generating cytoskeleton, it is not a site of enhanced force generation. Our measurements of the ventral traction force demonstrated that force increased in magnitude with distance from the leading edge. Additionally, measurements of the dorsal traction variation using the positional reinforcement assay demonstrated that it was more difficult to recapture a bead as it traveled rearward, away from the leading edge of the lamella, suggesting that a stronger link was formed, enabling the cell to exert greater force against the bead. The ability to recapture a bead was not related to the number of times that we attempted to retrap the bead (data not shown). Nor was it related to the distance traveled across the lamella as beads that escaped the trap could be brought back to the front of the cell, allowed to escape again, and retrapped again. These results indicate that the cell generates larger traction forces in the thickened lamella and the perinuclear region. Furthermore, the location-specific traction forces did not correlate with the density of the underlying actin cytoskeleton. Low traction forces and easily retrapped beads were characteristic of the region of the lamella near the leading edge, an area of high F-actin concentration . However, the myosin II density increase from the leading edge to the perinuclear region in the keratocyte lamella correlates with the increase in traction force from the leading edge to the perinuclear region that we report. Thus, the magnitude of the traction force appears to be dependent upon the density of myosin II, suggesting that the cytoskeleton is a dynamic structure in which the linkages are dynamic force-generating components. Perhaps, myosin filaments cross-link actin filaments to create a cytoskeletal network that would generate a contractile force in proportion to the density of active myosin. Several models have been proposed for explaining the migration of keratocytes: adhesion–propulsion , network condensation , and dynamic network contraction . None of the models are able to describe both of the key features of our traction force data: a rearward force in the lamella that increases with distance from the leading edge and a change in force direction in the perinuclear region. Therefore, we propose a new model that reconciles our observations and the observations that led to the previous models. The adhesion–propulsion model suggests that the adhesive and the propulsive forces driving migration are equal and opposite, and there are no traction forces along the direction of migration. The only forces powering movement in that model are the large forces on either side of the nucleus that are oriented perpendicular to the direction of migration. These forces, which are approximately threefold larger than the lamella forces, are important in keratocyte migration. However, the adhesion–propulsion model, which only predicts net forces perpendicular to the direction of migration, cannot account for the rearward lamella traction forces that we measure on both the dorsal and the ventral surfaces. Furthermore, the rearward forces that we measure are not completely balanced by forces resisting motion since we are able to measure them over the small spatial region of the pad in the micromachined device. The network condensation model proposes that the balance between polymerization of actin at the front of the cell and depolymerization at the rear drives the cell forward. Therefore, the network appears to move rearward in front of the cell, forward in the rear of the cell, and is stationary at a point in the lamella where the network's velocity equals the cell's velocity. The dynamic network contraction model proposes that the force for forward movement is generated by contraction of an actin–myosin II network in the perinuclear region. As the actin and myosin contract, they form bundles that are oriented perpendicular to the direction of migration; the actin and myosin in this region move forward relative to the lamella and pull the cell body forward. Both models predict a change in traction force direction in the transition zone, which we observe experimentally. However, neither model can explain the increase in traction force that occurs between the leading edge and the perinuclear region of the lamella, nor can they account for the strong inward traction forces located on either side of the nucleus. A model of keratocyte migration must account for the pattern of traction forces. The model needs to explain the rearward traction force in the lamella that increases in magnitude from the leading edge to the perinuclear region where it changes direction, and the model must provide a mechanism for generating the strong inward traction forces located on either side of the nucleus. We propose a model in which the actin–myosin condensation in the perinuclear region generates the rearward traction forces under the lamella and forward movement of the cell body . This mechanism is different from the dynamic network contraction model because it postulates that the condensing myosin pulls on the orthogonal array of lamella actin that is stationary with respect to the substratum. Support for this model comes from the polarity of the peripheral lamella actin indicating that it could generate a rearward force, the continuity of the lamella actin with the condensed perinuclear fibers , and the magnitude of the traction force correlating well with the density of the myosin clusters. The proposed model also suggests that the forces generated in the condensation region are responsible for the change in traction force direction. The lamella generates rearward traction forces, and the periodic contractions of the condensation zone (Waterman-Storer, C., unpublished results) may exert sufficient force to pull the rear of the cell forward. Finally, the condensed perinuclear bundles probably generate the large forces on either side of the nucleus that are oriented perpendicular to the direction of migration. The perinuclear bundles are organized with their barbed ends oriented toward the cell periphery , which is the correct organization for myosin to generate the strong traction forces that are orthogonal to the direction of migration. These traction forces need to be stronger than the lamella traction forces to overcome the tighter adhesions to the substratum in these regions . Moreover, the mixed polarity of these filaments in the center of the cell indicates that the mechanism of actin–myosin interaction is not sarcomeric, but is a contraction similar to that used to move fibroblasts on their ventral actin filaments . Thus, the condensation of actin and myosin in the perinuclear region may provide the force necessary for rearward lamellar traction force while organizing the longitudinal fibers that generate the traction forces orthogonal to the direction of migration. Our measurements of the traction forces generated by subcellular regions of slow moving fibroblasts and fast moving keratocytes have some similar features, suggesting that there is some commonality to portions of the mechanism of motility for both cell types. In the lamella of both cell types, there is a small retrograde force, the actin remains fixed with respect to the substratum , and myosin II contracts and organizes the actin network . Myosin moves rearward relative to the substratum in fibroblasts, but it does not move rearward in keratocytes unless the rear of the cell is tethered to the substratum or another cell, suggesting that a similar mechanism may generate the rearward lamella force in both cells, especially when the adhesions to the surface are comparable. The ventral actin fibers in fibroblasts and the perinuclear actin bundles in keratocytes are similarly organized. Both sets of fibers have their barbed ends oriented outward toward the cell periphery and have mixed polarity in the center. However, the ventral fibers in a fibroblast are oriented along the axis of migration, and the perinuclear bundles are oriented perpendicular to the direction of motion. This orientation of the actin bundles reflects the orientation of the maximum traction forces in both cell types. Additionally, when keratocytes become tethered to the substratum in their rear, they adopt the trigonal morphology observed in migrating fibroblasts, and their nucleus rotates 90° so that its long axis is parallel to the direction of migration . The keratocytes then exert strong forward directed traction forces underneath the tethered rear that are comparable to those exerted by fibroblasts . If the keratocyte perinuclear fibers rotate with the nucleus, then the ventral actin polarity, barbed ends facing the cell periphery and mixed polarity in the center of the cell under the nucleus, would be the same for both cell types, allowing the keratocyte to move forward with a contraction mechanism that is similar to that used in fibroblasts . The similarity in the amount of force generated by keratocyte and fibroblast lamella, and in the adhered tails of both cells suggests that this scheme may be possible. Therefore, we suggest that myosin contraction on the actin cytoskeleton–integrin–fibronectin links provide a dynamic tension that is responsive to both the sites of greatest adhesion and the formation of new links. | Study | biomedical | en | 0.999998 |
10601344 | The mouse anti-α3A and anti-α3B antibodies and the rat anti-α6 antibody have been described previously. The rabbit polyclonal antisera to synthetic peptides derived from the cytoplasmic domain of the α1, α2, and αv integrin subunits were kindly provided by Dr. G. Tarone (Universita di Torino, Torino, Italy). The rat anti-α5 antibody was the generous gift of Dr. B.M. Chan (J.P. Robarts Research Institute, London, Ontario, Canada). The rabbit polyclonal antibody recognizing β5 was kindly supplied by Dr. E. Roos (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The rabbit polyclonal antibody against the cytoplasmic domain of β1A (U19) was a kind gift of Dr. U. Mayer (Max Planck Institute for Biochemistry, Martinsried, Germany). The mouse anti–human β1 (TS2/16) and the mouse anti–human β1 (K20) antibodies were obtained from the ATCC and Biomeda, respectively. The hamster anti-β3 and the rat anti-α4 antibodies were purchased from PharMingen. The mouse mAb against vinculin , rabbit anti-vinculin , anti-keratin 8 , and the anti-desmoplakin , and the anti–NCAM-1 antibodies were kindly provided by Dr. M. Glukhova (Institut Curie, Paris, France), Dr. B. Geiger (The Weissmann Institute of Science, Rehovot, Israel), Dr. R. Kemler (Max Planck Institute for Immunobiology, Freiburg, Germany), Dr. D. Garrod (University of Manchester, Manchester, U.K.), and Dr. R. Michalides (The Netherlands Cancer Institute, Amsterdam, The Netherlands), respectively. The mouse anti–interleukin-2 receptor (IL2Rα) (TB30) was a gift of Dr. R. van Lier (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The rabbit anti–pan-cadherin, the rabbit anti–α-catenin, and the mouse antitalin (8D4) antibodies and rhodamine-labeled phalloidin were obtained from Sigma Chemical Co. The mouse anti–β-catenin and mouse anti–γ-catenin antibodies were purchased from Transduction Laboratories, and the rabbit anti–ZO-1 and the rabbit anti-occludin antibodies were from Zymed. The monoclonal 9E10 antibody against the myc-epitope tag was purchased from Oncogene Research Products. Mouse anti-RhoA (26C4) and rabbit anti-Cdc42 antibodies were obtained from Santa Cruz Biotechnology, and mouse anti-Rac1 antibody from Transduction Laboratories. Secondary antibodies used were: FITC-conjugated goat anti–mouse antibody (Jackson ImmunoResearch Laboratories), Texas red–conjugated goat anti–mouse and anti–rabbit antibodies (Molecular Probes), FITC-conjugated rabbit anti–hamster antibody (Nordic Immunological Laboratories), and FITC-conjugated goat anti–rabbit antibody (Zymed). Fibronectin was purchased from Sigma Chemical Co., and laminin-1 from Collaborative Biomedical Products. The previously described fibroblast-like GD25 cell line was obtained by in vitro differentiation and immortalization of β1-knockout embryonic stem (ES) cells. For the establishment of epithelial-like GE11 cells, β1-null ES cells were grown, injected into blastocysts, and transferred into foster mice as reported earlier . Pregnant females were killed at around embryonic day 10.5 and β1-null chimeric embryos removed and subsequently freed of membranes. After incubating embryos in trypsin/EDTA, they were broken up by pipetting embryos repeatedly using pasteur pipettes. Cells were plated on gelatin-coated tissue culture dishes overnight, infected with SV-40 large T transducing retrovirus for 1 h at 37°C, and immediately selected at high concentration of G418 (1 mg/ml). After 2 more days in mass culture, cells were trypsinized, counted, and distributed over 96-well plates and cultured in selection medium. 10 96-well plates were prepared with 3 cells/well, 10 plates with 6 cells/well, and 10 plates with 12 cells/well. Cells in these 96 wells were cultured in the presence of G418. After 3 wk in culture, 14 wells contained polarized cells which had formed small colonies (GE1–GE14). They were trypsinized and expanded. Whereas 13 clones stopped growing during the expansion period, one clone (GE11) continued to grow and was clearly polarized. Although all cells of clone GE11 showed a uniform morphology, cells were recloned by limiting dilution using 96-well plates (15 cells/plate). 12 clones were isolated and analyzed immunohistochemically for β1 integrins and histochemically for lacZ expression. All clones lacked β1 integrins on the surface and stained strongly for lacZ in their cytoplasm, suggesting that GE11 cells were clonal even before the limiting dilution experiment. A cDNA encoding full-length human β1A was obtained by screening an human λ-phage keratinocyte library with two β1 oligonucleotide probes and subsequently cloned into pUC18. A Kozak consensus sequence was introduced by PCR, and the Kozak-containing human β1A cDNA was then ligated into the retroviral LZRS-IRES-zeo expression vector, a modified LZRS retroviral vector conferring resistance to zeocin . Full-length β1D was obtained by exchanging the sequence encoding the cytoplasmic domain of β1A by a β1D reverse trancription PCR product in the LZRS-IRES-zeo retroviral vector. The IL2Rα encoding sequence of the pCMV-IL2R vector , which was kindly provided by Dr. K. Yamada (National Institutes of Health, Bethesda, MD), was cloned into the retroviral LZRS-IRES-zeo vector. The IL2R-β1A chimeric construct was obtained by cloning the β1A cytoplasmic cDNA into the LZRS-IL2Rα-IRES-zeo vector. Myc epitope-tagged dominant negative N17Cdc42, N17Rac1, N19RhoA, and dominant active V12Cdc42, V14RhoA, and V12Rac1 were cloned in the LZRS-IRES-zeo vector . The mouse α(E)-catenin cDNA was kindly provided by Dr. R. Kemler (Max Planck Institute for Immunobiology, Freiburg, Germany) and was cloned in the same vector. Phoenix packaging cells were transfected with retroviral constructs as described previously to produce culture supernatants containing virus. Then, 3 × 10 4 GE11 or GD25 cells were infected with virus by culturing the cells for 8 h in 1 ml of cell-free Phoenix supernatant in the presence of 10 μg/ml DOTAP (Boehringer Mannheim). Cells were then cultured in fresh DME medium supplemented with 10% FCS and penicillin/streptomycin (GIBCO-BRL Life Technologies). Zeocin (0.2 mg/ml; Invitrogen) was added to the culture medium 48 h after transduction. Expression of the β1 subunit or the IL2R-β1 chimera was determined by FACS ® analysis. Expression of the mutant forms of Rho-like GTPases was checked by immunoblotting as described previously , using a HRP-conjugated goat anti–mouse antibody (Amersham Pharmacia Biotech). Immunoreactive proteins were visualized using enhanced chemiluminescence as described by the manufacturer (Amersham Pharmacia Biotech). Cells were grown on coverslips in DME, 10% FCS, fixed in 2% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.2% Triton X-100 for 5 min. Cells were blocked in PBS, 2% BSA for 1 h, and incubated with primary antibodies for 1 h at room temperature. After washing in PBS, cells were incubated in the presence of FITC- or Texas red–conjugated secondary antibodies or in the presence of rhodamine-labeled phalloidin for 1 h. Preparations were then washed in PBS, mounted in Vectashield (Vector Laboratories Inc.), and analyzed with a confocal Leica TCS NT microscope. For flow cytometry and cell sorting, cultured cells were trypsinized, washed twice in PBS, 2% FCS, and incubated with primary antibodies for 45 min at 4°C. Cells were then washed in PBS, incubated with FITC-conjugated secondary antibodies for 45 min at 4°C, washed again, and analyzed in a FACScan ® using Lysys II software (Becton Dickinson) for determination of integrin expression levels. Cells were sorted on a FACStar Plus ® (Becton Dickinson). For the Transwell migration assay, 3 × 10 4 or 10 5 cells in DME, 0.5% BSA were seeded in the upper compartment of 8-μm Transwells (Costar) previously coated with 10 μg/ml fibronectin on the lower side on the filter, and allowed to migrate for 2 h at 37°C. Cells in the upper chamber were removed with a cotton swab and cells on the lower side of the filter were fixed in methanol and stained with crystal violet. The number of cells that had migrated was counted on photographs taken from the filters. For each filter, a total of three different 5-mm 2 fields were photographed to obtain an average cell count. For in vitro wound healing assay, cells were seeded for 2 h in DME, 10% FCS. After cell spreading, a cross was scratched in the cell monolayer to analyze wound closure and facilitate the localization of the same spot in time. Cells were photographed at the indicated time points (magnification 500×). Subconfluent cell cultures were lysed for 10 min on ice in 1% Triton X-100, 50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA in the presence of protease inhibitors and the lysates were centrifuged at 14,000 g for 15 min to obtain the soluble protein fraction. The pellet (the cytoskeletal, insoluble fraction) was resuspended in Laemmli sample buffer. For detection of total protein samples, cells were extracted with radio immunoprecipitation assay (RIPA) lysis buffer. Samples were adjusted to 50 μg of total proteins, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). The membranes were incubated for 1 h with anti–pan-cadherin, anti–α-catenin, or anti–β-catenin antibodies and then further incubated for 1 h with HRP-conjugated secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence. The biochemical activity assays were performed essentially as described previously . For the RhoA activity assay, a glutathione S -tranferase (GST) fusion protein of the Rho effector protein rhotekin was employed. For the Rac1 and Cdc42 assays, we used a GST fusion protein of the binding domain PAK1b, which binds Cdc42 and Rac in the GTP-bound form only. The GST-rhotekin or GST-PAK precoupled to Sepharose-glutathione beads (Amersham Pharmacia Biotech) were used to precipitate GTP-bound RhoA, Rac1, or Cdc42 from cleared lysates of cells. For each measurement, two T75 flasks of subconfluent GE11 or GD25 cells were lysed for 5 min at 4°C in 1% NP-40, 50 mM Tris, pH 7.4, 10% glycerol, 100 mM NaCl, 2 mM MgCl 2 , in the presence of protease inhibitors. Lysates were clarified by centrifugation and the appropriate GST fusion protein was added for 30 min at 4°C, followed by three washes in lysis buffer. The beads were boiled in Laemmli sample buffer and protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The blots were probed with anti-RhoA, anti-Rac1, or anti-Cdc42 antibodies and developed by enhanced chemiluminescence. GE11 cells were isolated from a β1 integrin subunit knockout chimeric embryo aged 10.5 d postcoitum and their ontogeny is unclear. They grow in epithelial colonies , contain keratin 8, no desmoplakin, and express the neural cell adhesion molecule NCAM-1. Electron microscopic studies showed that GE11 cells are polarized, have microvilli at their apical surface, and organized tight junctions, but do not assemble desmosomes (data not shown). Together, these observations suggest that GE11 cells are epithelial cells of neural origin, possibly the neuroepithelium. The previously described GD25 cells were obtained by in vitro differentiation and immortalization of β1-knockout ES cells . GD25 cells have a more fibroblastic phenotype than GE11 cells, although they maintain intercellular adhesions . We have expressed two cytoplamic splice variants of the β1 integrin subunit, β1A and β1D, in GE11 and GD25 cells, by retroviral transduction. FACS ® analysis revealed that 90–95% of the cells express β1 at their plasma membrane 24 h after the start of retroviral transduction (data not shown). Stable transfectants were selected with zeocin. Immunoprecipitation experiments showed that GE11 cells expressed the β3 and β5 subunits in association with αv (data not shown). GE11-β1A cells express several integrins of the β1 family, including α3β1, α5β1, α6β1, and α4β1 at low levels, but not α1β1 or α2β1 (data not shown). Infection of cells with the empty retroviral vector (GE11- and GD25-control) did not alter the epithelial phenotype of GE11 cells or the intercellular adhesions in GD25 colonies . In both cell types, the expression of the β1A integrin subunit resulted in disruption of cell–cell contacts and dissociation of cell colonies. Cells originally present in epithelial cell colonies separated from them and assumed a morphology resembling that of motile fibroblasts. These morphological changes were detected as early as 24 h after the start of retroviral infection . Similar effects, although somewhat less pronounced, were observed after the expression into GE11 and GD25 cells of the muscle-specific β1D splice variant . The lower levels at which β1D was expressed (50% of those of β1A) probably account for this less pronounced phenotype. Because the effect of β1 expression was most dramatic on intercellular adhesions in GE11 cells, we concentrated our studies on stable GE11 cells expressing β1A (GE11-β1A). The distribution of various proteins associated with the actin cytoskeleton and with intercellular adhesions was analyzed in GE11 and GE11-β1A cells . In GE11-control cells, actin filaments were organized in heavy peripheral bundles, which ran parallel to the outer membrane of cells at the periphery of the epithelial cell colonies. Actin filaments were also present in cortical bundles under the plasma membrane, along cell–cell boundaries, and in stress fibers at the cell basis, where they were attached to the plasma membrane at sites of focal contacts . In GE11-β1A cells, peripheral bundles of actin filaments were absent and stress fibers crossed the entire cell. Intercellular staining of cadherins, typical of epithelial cells, was observed in GE11-control cells, and no staining was found at the free cell border at the periphery of colonies . α-, β-, and γ-catenins had a similar localization (data not shown). In contrast, cadherins and catenins were more diffusely distributed over the membrane of GE11-β1A cells, and although there were some residual adherens junctions at high confluency, these proteins were also found in regions of the plasma membrane that were not in contact with other cells . In GE11-control cells, vinculin was found in regions of cell–cell contacts, where it was colocalized with cadherins . In contrast, although GE11-β1A cells developed cell–cell contacts at high confluency, vinculin and cadherins were not colocalized in these cells . Using interference reflection microscopy, we found that the number and size of focal contacts were different in GE11-control and GE11-β1A cells (data not shown), and this was confirmed by the distribution of vinculin and talin (data not shown) at the basal surface of the cells. Typically, focal contacts were small and numerous in GE11-control cells, and distributed over the entire basal cell surface. However, at the periphery of the colonies they were more concentrated at the outer region of the cell, thus forming a characteristic interrupted ring-like structure. In GE11-β1A cells, focal contacts were thick, and appeared to be arranged in long streaks frequently found at the end of actin stress fibers . In confluent GE11-β1A cells, focal contacts were also found between two cells and sometimes on their apical surface, as seen by staining for talin and vinculin. Electron microscopic analysis revealed that this was likely to be due to the presence and assembly of secreted ECM proteins between cells and on their apical surface (data not shown). Another specialized membrane domain involved in intercellular adhesion of epithelial cells is the tight junction. A marker of tight junctions is ZO-1, but in cell types lacking these structures, such as fibroblasts or cardiac muscle cells, ZO-1 is colocalized with cadherins at adherens junctions . Upon expression of the β1A integrin subunit, ZO-1 became redistributed from tight junctions to the adherens junctions formed by GE11-β1A cells at high confluency . This relocalization was correlated with the transition from polarized epithelial cells to fibroblast-like cells. Another marker of tight junctions, occludin, was also found at the apical lateral border of GE11 cells, and became diffusely distributed in GE11-β1A cells (data not shown). Furthermore, EM showed that tight junctions present in GE11-control cells were no longer present in GE11-β1A cells (data not shown). Finally, we have found that β1 and the endogenous β3 integrin subunits were both present in focal contacts formed by GE11-β1A cells in the presence of FCS (data not shown). In conclusion, these results show that the expression of the β1 integrin subunit in GE11 cells induces a reorganization of the actin cytoskeleton and of focal contacts, accompanied by the disruption of both cadherin-based intercellular adhesions and tight junctions in epithelial cell colonies. However, adherens junctions of the type formed by fibroblasts can assemble in the presence of β1 integrins at high cell confluency. When GE11 cells were cultured on plastic in the presence of FCS, the mere expression of the β1 subunit was sufficient for inducing the disruption of intercellular adhesions and the dissociation of cell colonies. Because GE11-β1A cells express several integrins that can bind to fibronectin and vitronectin present in FCS, we investigated whether the change in morphology was due to the expression of β1 or whether it was triggered by the interaction of β1 integrins with their ligands. Although GE11-β1A cells expressed the laminin receptor α6β1, we have generated GE11 cells expressing α6β1 at higher levels by coexpression of the human α6 integrin subunit in GE11-β1A cells and by further selection by FACS ® . The overexpression of α6 in GE11-α6β1A cells resulted in a strong decrease of the percentage of cells expressing the fibronectin receptor α5β1 as well as in a decrease of its average expression levels , probably because α6 associated with most of the available β1 subunit. Although these cells could spread, they poorly scattered and developed strong cell–cell adhesions when cultured on fibronectin , suggesting that the expression of all fibronectin-binding β1 integrins (α5β1 as well as αvβ1) was reduced. However, scattering was induced when they were cultured on laminin-coated dishes . Together, these results indicate that the interaction of β1 integrins with their ligand is required for the disruption of cadherin-based cell–cell adhesion and cell scattering. They also show that several integrins of the β1 family, which bind to various ECM proteins (α5β1 or αvβ1 to fibronectin, and α6β1 to laminin), can trigger the described morphological transition. The localization of a particular integrin in focal contacts is regulated by its α subunit and requires the binding of the integrin to ligand . In contrast, when chimeric molecules containing the extracellular and transmembrane domains of the human IL2R and the cytoplasmic domain of the β1A integrin subunit were expressed at relatively low levels, they were colocalized with endogenous integrins in focal contacts and were able to transduce signals leading to the phosphorylation of the focal adhesion kinase . These properties indicate that IL2R-β1 chimerae mimic endogenous ligand-occupied integrins. We have analyzed the effects of an IL2R-β1A chimera on the morphology of GE11 cells. Fig. 4 shows that expression of the IL2R alone did not either alter the morphology of GE11 cell colonies or cause any changes in the subcellular distribution of actin, α-catenin, or ZO-1 . Expression of the IL2R-β1A chimera, on the contrary, induced the disruption of most intercellular adhesions. A few small epithelial-like colonies remained and cell–cell adhesions had a tendency to reform, although they did not appear to be as stable as those between GE11-control cells. IL2R-β1A expression induced an alteration of the peripheral bundles of actin filaments and changes in the localization of cadherins, catenins, and ZO-1 , similar to the full-length β1A subunit. In addition, the IL2R-β1A chimera promoted cell spreading and induced a redistribution of vinculin: the ring of focal adhesions at the periphery of the colonies was no longer assembled in GE11-IL2R-β1A cells and was replaced by thick and long streaks of vinculin at the base of cells. The arrows in Fig. 4 indicate vinculin-positive rings in cells in which IL2R-β1A was not expressed. In cells expressing IL2R-β1A, the chimera was colocalized with vinculin and the endogenous β3 subunit (data not shown) in focal contacts. Together these results indicate that IL2R-β1A induces both the disruption of intercellular adhesions and the reorganization of the cytoskeleton, thus mimicking the effects of full-length β1A. Whether IL2R-β1A is primarily incorporated into β3-containing, preexisting focal adhesions and induces their remodeling, or whether it participates in the formation of new adhesion structures into which β3 is eventually recruited will be discussed. To quantify potential changes in the motility of GE11-β1A cells as compared with that of GE11-control cells, we have performed migration experiments using fibronectin-coated Transwells. As shown in Fig. 5 A, although GE11-control cells are able to migrate to some extent on this substrate, the expression of β1A strongly increased cell motility. The expression of IL2R-β1A failed to enhance cell migration in any of the conditions tested. In addition, we tested random cell migration using an in vitro wound healing system. Fig. 5 B shows that GE11-β1A cells spread fast when grown under standard conditions on plastic, and that they migrate into the introduced wound. GE11-β1D cells showed similar migration kinetics (data not shown). In contrast, GE11-control cells maintained stronger cell–cell adhesions and spread and migrated more slowly. Together, these results show that the motility of GE11-β1 cells is increased. The fact that IL2R-β1A is sufficient to trigger the disruption of intercellular adhesions, although it does not increase cell motility, indicates that cell migration is not simply the cause of the disruption of cell–cell adhesions. Because the expression of β1 integrins altered the integrity of intercellular adherens junctions, we compared the cadherin and α- and β-catenin protein levels as well as their detergent solubility in GE11-control, and GE11-β1A and -β1D cells. Immunoblotting of total cellular proteins revealed that GE11-β1A cells contained smaller amounts of cadherin and α-catenin than their control counterparts . The amount of β-catenin also appeared to be reduced in GE11-β1A and GE11-β1D cells, but to a lesser extent than the amounts of cadherin and α-catenin. Results of a detergent solubility assay, using 1% Triton X-100, indicated that the ratios between the insoluble and soluble fractions of cadherin and α-catenin were reduced in GE11-β1A cells as compared with GE11-control cells. Thus, smaller amounts of these two proteins are associated with the detergent-insoluble cytoskeletal fraction in GE11-β1A cells. Although the amount of β-catenin is reduced in GE11-β1A cells, its distribution in the cytoskeletal and soluble fractions is apparently not affected by β1 expression. This might be due to the existence of a detergent-insoluble pool of β-catenin in the nucleus of GE11-β1A cells, although we could not confirm this hypothesis by immunofluorescence. These results indicate that β1 integrins might cause the disruption of intercellular adhesions by inducing a downregulation of cadherin and/or catenin function. Moreover, immunofluorescence studies performed on cells that were first permeabilized in Triton X-100 and subsequently fixed in paraformaldehyde showed stainings for cadherins and catenins similar to those obtained with paraformaldehyde-fixed cells without prior solubilization (data not shown). This suggests that the redistribution of cadherins and α-catenin from the Triton-insoluble to the -soluble fraction upon β1 expression is due to a reduced number of cell–cell adhesions rather than a decrease in their rigidity. By providing a link between cadherin complexes and the cytoskeleton, α-catenin plays a major role in the establishment and maintenance of cadherin-based adhesions . Moreover, α-catenin has been implicated in the formation of tight junctions . Because we have found that both the protein level and the detergent solubility of α-catenin were affected by β1 integrins in GE11 cells, we have investigated the role of this protein in the β1-induced phenotypic changes. First, α-catenin was retrovirally overexpressed in GE11 cells (GE11–α-catenin cells). The effect of subsequent β1 expression in GE11–α-catenin cells was less pronounced than in GE11 cells or in cells previously transduced with the empty LZRS vector. Several clones were isolated in which the expression of β1 and α-catenin was analyzed by FACS ® and immunoblotting, respectively. Fig. 7 shows the results obtained with three of them. The expression levels of α-catenin were higher in GE11-control cells and in cells from clones 3, 4, and 9, than in GE11-β1A cells . In addition, β-catenin and cadherin protein levels were greater in GE11-control cells and in the three GE11–α-catenin–β1A clones than in GE11-β1A cells. Thus, when α-catenin is overexpressed in GE11 cells, protein levels of β-catenin and cadherin are not decreased by the expression of β1 integrins. Although the surface expression levels of β1 integrins in clones 3 and 9 were similar to those in GE11-β1A cells , cells from these clones remained clustered in epithelial cell colonies , indicating that overexpression of α-catenin prevented the β1-induced morphological change. In spite of β1 expression, cells from clone 9 exhibited the peripheral bundles of actin filaments and the vinculin-positive ring found in GE11-control cells but not in GE11-β1A cells. In addition, these cells presented well-developed intercellular adhesions containing both vinculin and cadherins . However, results obtained with clone 4 showed that a further increase of β1 levels could overcome the inhibitory effect of α-catenin and allow cell scattering. These results suggest that there is a tight balance between intercellular adhesion and β1-mediated attachment to ECM proteins, which can be regulated at the level of α-catenin. Small G proteins of the Rho family are involved in the regulation of the actin cytoskeleton, the turnover of focal adhesions, in cell migration, and in the assembly of intercellular adhesions . Therefore, we have studied their role in the cell scattering induced by β1 integrins. GST-rhotekin and GST-PAK fusion proteins were used to precipitate the active form (GTP-bound) of RhoA, and of Rac1 and Cdc42, respectively, from cell lysates of stably transfected GE11 cells expressing either β1A, β1D, IL2R, or IL2R-β1A. The total amounts of GTPases (guanosine diphosphate (GDP)- and GTP-bound) were measured in parallel in the same lysates. Increased amounts of GTP-RhoA were precipitated from the lysates of cells expressing either β1A or β1D, when compared with vector controls . Experiments performed with transduced GD25 cells confirmed the activation of RhoA by β1A, and to a lesser extent, by the β1D integrin splice variant. Similarly, the expression of either splice variant of the β1 integrin subunit induced the activation of Rac1 in both GE11 and GD25 cells. Fig. 8 also shows that the activation of RhoA and Rac1 mediated by IL2R-β1A was less efficient than that observed with full-length β1A. Finally, in contrast to RhoA and Rac1, the activity of Cdc42 was not significantly increased upon expression of β1 integrins. These data demonstrate that expression of either the β1A or the β1D integrin splice variant activates RhoA and Rac1 in both GE11 and GD25 cells. Next, we investigated whether the activation of RhoA and Rac1 is required for the phenotypic conversion induced by β1 integrins. We first transduced dominant negative mutants of RhoA, Rac1, and Cdc42 in GE11 cells, and 2 d later, we introduced β1A. Because the same retroviral vector was used for both proteins, the clones expressing β1A could not be selected with antibiotics. Therefore, we isolated the cells expressing β1 at high levels by FACS ® analysis and sorting. In the second step, several individual clones were isolated from this β1-positive cell population. The expression of both N17Rac1 and β1 (data not shown) was finally measured in these clones. We show that even when β1 was strongly expressed at the cell surface, substoichiometric amounts of N17Rac1 inhibited the disruption of cell–cell adhesion and cell scattering . It has been reported previously that amounts of RhoA or Rac1 mutants well below those of endogenous RhoA and Rac1 levels caused changes in the cellular organization , presumably because their ability to rapidly bind and dissociate from guanine-exchange factors is impaired by the mutation. The β1-positive clones that displayed a more fibroblast-like phenotype did not express N17Rac1 at detectable levels (data not shown). Fig. 9 C shows that N17Rac1 was associated with the entire plasma membrane of GE11-N17Rac1-β1A cells, but that it was especially concentrated in regions of cell–cell contacts. Cadherins and catenins remained present in intercellular junctions . Pictures taken at both the basal and medial plane of the cell show that the β1A subunit was found in both focal contacts and in regions of cell–cell contacts . This indicates that N17Rac1 prevented β1-mediated disruption of cadherin-based adhesion and further scattering. We have not been successful in isolating GE11 clones expressing detectable levels of dominant negative forms of RhoA and Cdc42, probably because these constructs have a toxic effect. This was most obvious for N19RhoA, of which expression induced cell rounding and detachment 2 d after the retroviral transduction. Therefore, we followed another approach in which the dominant negative mutants were expressed subsequently to the β1 integrin subunit. All three RhoA, Rac1, and Cdc42 mutants inhibited cell scattering, but the morphology of the cells in the colonies was different from that of untransfected GE11 cells . In particular, the morphology of the cells in which N17Rac1 had been expressed after β1 was clearly different from that of the cells that were first retrovirally transduced with N17Rac1 . The expression of all three mutants N19RhoA, N17Rac1, and N17Cdc42 in GE11 cells subsequent to β1 correlated with the localization of cadherins (data not shown), α-catenin, and vinculin at cell–cell junctions . The inhibition of cell scattering was directly dependent on the amount of dominant negative GTPases: cells expressing N19RhoA, N17Rac1, or N17Cdc42 at high levels formed islands, whereas cells with low expression levels maintained a fibroblastic morphology and remained scattered . As a control, immunofluorescence staining for β1 and FACS ® analysis (data not shown) demonstrated that the inhibition of cell scattering by dominant negative mutants of the Rho-like GTPases was not due to a decrease in the levels of surface expression of β1 integrins. Finally, we have found that both N17Rac1 and N17Cdc42, but not N19RhoA, were enriched at cell–cell contacts . This localization of N19RhoA and N17Rac1 in epithelial cells has been described previously. It is now well-established that RhoA plays a role in actin stress fiber and focal contact formation . As expected, strong expression of N19RhoA inhibited the formation of actin stress fibers in GE11 cells . Moreover, in those cells that developed stable intercellular adhesions, vinculin remained diffusely distributed, indicating that focal contact formation is inhibited by N19RhoA . These results suggest that under conditions in which the formation of stress fibers and focal contacts is inhibited by N19RhoA, GE11-β1A cells can still form intercellular adhesions. To determine whether the activation of Rac1 or RhoA by β1 integrins was sufficient for inducing the morphological change, dominant active mutants of RhoA (V14RhoA) or Rac1 (V12Rac1) were retrovirally expressed in GE11 cells. Although V14RhoA cells appeared to be more contracted and V12Rac1 cells displayed larger lamellipodia, neither of the constitutively active mutants separately nor when they were combined induced cell scattering (data not shown). Taken together, these results suggest that Rac, RhoA, and Cdc42 are required but not sufficient for the morphological changes induced by the expression of the β1A integrin subunit. In this study we have shown that the expression of either β1A or β1D integrin splice variants in two different β1-deficient cell lines, GE11 and GD25 cells, induces the disruption of intercellular adhesions followed by cell scattering. This phenotypic conversion, which depends on the interaction of β1 integrins with their respective ligands, is accompanied by the reorganization of the actin cytoskeleton and of focal contacts, and an increased ability of cells to migrate. However, loss of cell–cell adhesions and the reorganization of the cytoskeleton does not require cell migration, since the expression of an IL2R-β1A chimera had both these effects on cell morphology without stimulating cell motility. Disruption of cell–cell adhesions by β1 integrins was correlated with a decrease in cadherin and α-catenin protein levels and with their redistribution from the cytoskeleton-associated fraction to the soluble fraction. When the levels of α-catenin were increased in GE11 cells by retroviral transduction, the β1-induced phenotypic changes did not occur, suggesting an important role for this catenin in the regulation of cadherin-based adhesions by β1 integrins. We have also found that the activity of RhoA and Rac1, but not that of Cdc42, was enhanced upon expression of β1A, β1D, or IL2R-β1A in both GE11 and GD25 cells. These findings suggest that activation of these two Rho-like GTPases by β1 integrins contributes to the loss of cell–cell adhesions. Indeed, expression of either N17Rac1 or N19RhoA prevented the morphological transition induced by β1 integrins. However, additional β1-induced intracellular signals are required for the phenotypic change, since constitutively active mutants of either RhoA or Rac1 or both could not induce the disruption of cell–cell contacts in GE11 cells. The β1-deficient GD25 cells, which have been described previously as fibroblast-like cells , develop intercellular adhesions but scatter upon expression of the β1 integrin subunit. GE11 cells, on the other hand, displayed several features of a simple epithelium, including their morphology, polarization, the presence of apical microvilli and tight junctions, and the expression of keratin-8. Both adherens and tight junctions were disrupted upon the expression of full-length β1A. The interaction of β1 integrins with their ECM ligand appeared to be required for the disruption of cell–cell adhesions, since GE11 cells expressing α6β1 as the major β1 integrin, scattered only when laminin-1 was provided as a substrate. The IL2R-β1A chimera is distributed to focal contacts, triggers phosphorylation signals independently of binding to the ligand, and functions as a constitutively active integrin when expressed at relatively low levels . The expression of IL2R-β1A in GE11 cells caused the disruption of intercellular adhesions and a dramatic reorganization of the ECM cell adhesion structures, similar to those induced by the intact β1 subunit. The characteristic ring of focal contacts at the periphery of the colonies of GE11 cells was replaced by more prominent, streak-like focal adhesions in which IL2R-β1A was colocalized with β3 integrins. The incorporation of IL2R-β1A into preexisting, β3-containing focal contacts, might lead to the recruitment of larger amounts and/or other cytoskeletal proteins, ultimately causing the remodeling of these adhesion structures. However, it is also possible that IL2R-β1A participates in the formation of complexes of cytoskeletal proteins at the basal membrane in a ligand-independent manner. β3 integrins could eventually be incorporated in these complexes and stabilize them by interactions with ECM proteins. Such ligand-independent formation of cytoskeletal complexes by the cytoplasmic domain of integrins have been reported previously . Although the expression of IL2R-β1A led to the disruption of intercellular adhesions, it did not promote cell migration, probably because the ligand-binding domain of the β1 integrin subunit is not present in the chimera. This indicates that the disruption of cell–cell adhesion is not merely the consequence of the stimulated cell migration. In many epithelial cells, and in particular those forming desmosomes, the mere expression of β1 integrins is not sufficient to induce cell scattering. However, it has been shown previously that specific ECM–integrin interactions may lead to such changes in MDCK cells . In addition, EMT, which occurs during normal development and in epithelial tumorigenesis, is often correlated with an increase in the expression of β1 integrins and functional studies using inhibitory antibodies or chimeric proteins containing the cytoplasmic domain of β1 have indicated a role for these receptors in EMT . We show here that expression of the β1 integrin subunit induces an EMT-like transition in GE11 cells. However, it is likely that β1 integrins are involved in some but not all stages of EMT, because keratin 8, an epithelial marker, remained expressed in GE11-β1 cells, whereas vimentin, a mesenchymal marker, was expressed in both GE11-control and GE11-β1 cells (data not shown). Monier-Gavelle and Duband 1997 have suggested previously that β1 integrins are not involved in the regulation of cadherin activity during EMT of neural crest cells. Rather, they might potentiate αvβ3-mediated cell migration by an unknown mechanism. In contrast, our results suggest a requirement for β1-mediated effects for the regulation of cadherin activity, at least in some cell types. αvβ3 has been shown previously to be involved in cell migration, and it is not clear why it does not efficiently support the motility of GE11 cells, given the strong homology between the cytoplasmic domains of β1 and β3 subunits. One possibility could be that the surface levels of αvβ3 are too low to induce migration. Our results suggest that a decrease in cadherin and α-catenin protein levels, together with their redistribution from the cytoskeleton-associated, Triton X-100–insoluble to the -soluble fraction play a major role in the β1-induced phenotypic changes. This was further supported by our finding that high levels of α-catenin expressed by retroviral transduction could inhibit cell scattering. Cadherin protein levels were increased in GE11–α-catenin–β1A cells as compared with those in GE11-β1A cells, probably because overexpression of α-catenin stabilizes cadherin-based adhesions and prevents protein degradation. In addition, because α-catenin is also involved in the formation of tight junctions between epithelial cells , its downregulation by β1 integrins might be responsible for the disassembly of these structures between GE11 cells. Although our results clearly show that intercellular adhesions can be regulated by β1 integrins via α-catenin, the molecular mechanisms of this regulation remain to be elucidated. Integrin ligation triggers multiple intracellular events, among them the activation of various protein kinases . Although the physiological relevance of catenin phosphorylation in the regulation of adherens junction has been questioned previously , other studies have shown that changes in catenin phosphorylation correlated with the regulation of cadherin-based adhesions . We have not observed any changes in the tyrosine phosphorylation of catenins in GE11-β1A stable transfectants versus GE11-control cells (our unpublished data). Phosphorylation generally occurs on β- and γ-catenins and not on α-catenin, leading to their dissociation from the cytoskeleton. However, if the disruption of intercellular adhesions by β1 integrins would be the result of β- or γ-catenin phosphorylation, we would not expect that α-catenin could compensate for such an effect, which suggests another type of regulation by β1 integrins. It is also possible that β1-induced phosphorylation of α-catenin-binding proteins other than β- or γ-catenin contributes to the disruption of intercellular adhesions. Alternatively, overexpression of α-catenin might stabilize adherens junctions by compensating for the redistribution of structural proteins. While many components of intercellular adherens junctions and focal contacts are specific to one or the other structure, other proteins such as vinculin and α-actinin, are found in both complexes. Previous studies have suggested that α-actinin and vinculin play an important role in the establishment and maintenance of intercellular adhesions. Notably, vinculin, which shares homology with α-catenin , mediates anchorage of the cadherin complexes to the actin cytoskeleton in certain cell types by direct binding to β-catenin . In addition, the binding of vinculin to α-catenin might provide alternative and possibly stronger links between cadherins and the actin cytoskeleton . The redistribution of vinculin from adherens junctions to well-developed focal contacts that we observed in GE11 cells upon β1 expression might thus contribute to the disassembly of cell–cell adhesions and overexpression of α-catenin might compensate for this redistribution. However, this hypothesis implies that the amount of vinculin in the cell is a limiting factor, which does not seem to be the case, since it was found to reassociate with intercellular junctions in GE11–α-catenin–β1A cells. Our data show that in addition to α-catenin, Rho-like small G proteins also play a major role in the regulation of cell scattering by β1 integrins. Recently, cell adhesion was found to regulate the activity of RhoA and that of the Rac1 and Cdc42 downstream effector, PAK . We show in this report that the stable expression of β1A or β1D in either β1-deficient GE11 or GD25 cells induces an increase in RhoA and Rac1 activity that is correlated with enhanced cell motility. That the stimulation of RhoA and Rac1 activity by IL2R-β1A does not correlate with increased cell migration is due to the absence of the ligand-binding domain of the β1 integrin subunit in the IL2R-β1A chimera. Results of experiments performed with dominant negative mutants of RhoA, Rac1, and Cdc42 indicated that all three GTPases are involved in GE11 cell scattering induced by β1 expression. In addition, our data confirm previous findings that Rho-like GTPases regulate the formation of focal contacts and focal complexes . Together, these results indicate that RhoA and Rac1 act simultaneously upstream and downstream of integrins, and that positive feedback mechanisms probably regulate the relationship between integrins and Rho-like GTPases. The finding that β1 integrin–mediated adhesion activates both RhoA and Rac1 is consistent with the changes in the organization of the cytoskeleton and in cell behavior that we have observed in GE11 cells. Upon β1 expression, GE11 cells developed extensive lamellipodia and started to migrate. Whereas Rac1 stimulates the formation of lamellipodia and small focal complexes at the leading edge of migrating cells, RhoA stimulates the formation of new focal contacts and regulates the interaction of myosin-based motors with actin filaments to generate contractile forces required for cell motility. Although previous work suggested that cell adhesion induces the activation of the Cdc42 and Rac downstream effector PAK , β1 integrins did not significantly activate Cdc42 in our cells. Nevertheless, basal levels of Cdc42 activity seem to be required for scattering of GE11 cells, since expression of N17Cdc42 in GE11-β1 cells inhibited their scattering and partially restored intercellular adhesions. A role for Cdc42 in cell migration has been documented for correct cell polarization during migration, but not for random migration . Alternatively, the effects of N17Cdc42 might be mediated by unspecific inhibition of the Rac1 signaling pathway, by binding to guanine-nucleotide exchange factors common to both Rac1 and Cdc42. Immunofluorescence microscopy revealed that cadherins and catenins are redistributed to intercellular adherens junctions in GE11-β1A cells upon expression of dominant negative mutants of RhoA, Rac1, and Cdc42. This indicates that in these cells, Rho-like GTPases are not strictly required for the assembly of cadherin-based adhesions. These observations are in contrast to previous studies that reported decreased staining intensity of cadherins and catenins induced by N17Rac1 or the RhoA inhibitor C3 in keratinocytes , MDCK cells , and a mammary epithelial cell line . However, other investigators showed that dominant negative mutants of Rho-like GTPases affect cadherin distribution in MDCK at low, but not at high cell density . Finally, inhibition of these GTPases had no effect on the organization of adherens junctions in intestinal epithelial cells . These discrepancies might reflect specific regulation mechanisms in different cell types, but also different methods used for the expression of dominant negative mutants, i.e., microinjection of recombinant proteins versus inducible expression systems. An explanation we favor is that the amounts of dominant negative mutants of Rho-like GTPases expressed by retroviral transduction are sufficient to prevent the migration of cells derived from the originally transduced single cell, which results in the formation of small epithelial-like colonies but still allows cell–cell contacts to be formed. When expressed before β1, N17Rac1 was able to prevent GE11 cell scattering. Expression of N17Rac1 did not appear to affect the distribution of cadherins and catenins, and cells remained tightly attached to each other even in the presence of high levels of β1. This suggests that Rac1 activation is required not only for cell migration but also for the disruption of cadherin-based adhesions in these cells. However, expression of either constitutively active RhoA, Rac1, or their simultaneous expression was not sufficient for dissociating the cells in GE11 colonies, suggesting that β1 integrins trigger additional types of signals involved in the disruption of cell–cell adhesions. Moreover, the phenotypic reversion induced by the dominant negative mutants of small GTPases was only partial, and the morphology of both cells and colonies was different from that of control GE11 cells. This suggests either that the prior expression of β1 integrins induces irreversible morphogenetic events or that β1 integrins continue to transduce GTPase-independent signals that modify the cell phenotype. In conclusion, we have shown that the expression of β1 integrins in two different β1-deficient cell lines downregulates intercellular adhesions and stimulates cell scattering by inducing intracellular events involving both α-catenin and Rho-like GTPases. Further studies are now in progress to elucidate the molecular mechanisms underlying this regulation. | Other | biomedical | en | 0.999994 |
10601345 | LM-1 from the mouse EHS tumor, type IV collagen, and fibronectin were purchased from Becton-Dickinson, and LM-2 was obtained from GIBCO BRL. A polyclonal rat antibody to EHS LM which recognizes LM α1, α2, and β1/γ1 chains (LM-1/LM-2) , was a gift from Dr. Amy Skubitz (University of Minnesota, Minneapolis, MN). A rat monoclonal antibody to the NH 2 terminus of mouse LM α2 chain was obtained from Alexis Inc. A mouse monoclonal antibody to SM α-actin was obtained from Boehringer Mannheim, rabbit polyclonal antibodies to SM myosin were purchased from Biomedical Technologies, and a mouse monoclonal antibody to desmin was purchased from Dako (Carpinteria, CA). HRP-labeled secondary antibodies were purchased from Bio-Rad and normal rat IgG was purchased from Cappel. To obtain embryonic mesenchymal cells, Crl: CD-1 ® (ICR) BR mice (Charles River) were mated and the day of finding a vaginal plug was designated as day zero of embryonic development. Lungs were removed at days 12–15 of gestation, minced, and then trypzinised into a single cell suspension, and the mesenchymal cells were isolated by differential plating as previously described . The cells were directly cultured under conditions that promoted either cell rounding or cell spreading/elongation. The cell's shape was controlled by means of three different cell culture systems: (a) mesenchymal cells were plated on 10- or 20-μm-diam culture microsurfaces, the first to force cell rounding and the second to allow cell spreading/elongation . (b) Mesenchymal cells were plated on culture dishes pretreated with 0.05% poly- l -lysine or 0.05% poly- l -lysine was added to the cultures after cell spreading/elongation was completed, the first to force cell rounding and the second to allow cell spreading/elongation . (c) Mesenchymal cells were plated in untreated culture dishes at high density or at subconfluent densities without further treatment, the first to force cell rounding and the second to allow cell spreading . In some experiments, after 24 h in culture, the cells were switched from conditions that promoted cell rounding to conditions that promoted cell spreading/elongation and vice versa. In these cases all the cells were trypsinized after an additional 24 h in culture. Surface-anchored epithelial-mesenchymal cocultures (organotypic) were generated by plating at high density (2–4 × 10 6 /ml) a mixture of epithelial and mesenchymal cells isolated from embryonic lungs . Lung embryonic epithelial cells and intestine and kidney embryonic mesenchymal cells were used in some studies. These were also isolated by differential plating and cultured under conditions that promote cell rounding or cell elongation. To obtain adult mesenchymal cells, the lungs of adult CD-1 mice, C57BL/6J dy/dy mice, or C57BL/6J +/+ normal mice (both from Jackson ImmunoResearch Laboratories) were minced in 0.1% collagenase and incubated at 37°C for 60 min. Single cell suspensions were obtained and cultures of round or spread/elongated mesenchymal cells were generated as described above. All the cultures were incubated in MEM with nonessential amino acids, 0.29 μg/ml l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and 10% fetal bovine serum. In some studies, tissues were isolated from the embryo, immediately lysed, and used for immunoblot analysis. Whole lungs were microdissected from day 10–10.5 mouse embryos. Fragments of undifferentiated, round mesenchymal cells were microdissected from the periphery of day 12 mouse embryonic lungs, and fragments of elongated mesenchymal cells were microdissected, together with epithelial cells, from the proximal/central part of day 12 lungs. Cells and tissues from CD-1, C57BL/6J dy/dy mice and C57BL/6J +/+ normal mice were lysed and resolved under reducing conditions by PAGE. Immunoblotting for LMs and SM proteins was then done as described . For immunoprecipitation studies the cells were lysed and 50 μg of sample were cleared with protein A–conjugated sepharose (Sigma) and immunoprecipitated with 1 μg of monoclonal antibody against LM α2 chain for 16–24 h at 4°C in a rotary shaker. The immune complex was precipitated with protein A–conjugated Sepharose and resolved by 3.5% PAGE. The samples were transferred to nitrocellulose membranes and blotted with polyclonal antibody to LM-1/LM-2 (1:100 dilution) and bands were detected by chemiluminescence as previously described . Mesenchymal cell cultures were briefly washed with PBS, lysed with Trizol (GIBCO BRL) and total RNA was extracted and used for RT-PCR and ribonuclease protection assay (RPA). RT-PCR was performed with the GeneAmp ® RNA PCR kit (Perkin Elmer) following the manufacturer's instructions. 25 cycles were run for amplification of β1 and γ1 chains and 30 for amplification of α1, α2 chains, α4 and α5 chains. The following primers were used for PCR: LM α1 chain 5′ forward primer: 5′-tgggtgtgggatttcttagc-3′ and 3′ reverse primer: 5′-cctgaccgtctacccagtgt-3′, LM α2 chain 5′ forward primer: 5′-gcctgccaactctgagaaac-3′ and 3′ reverse primer: 5′-tcccaagagaatgatcccag-3′, LM α4 chain 5′ forward primer: 5′-gccatcgaagaagtagctgg-3′ and 3′-reverse primer: 5′-cccctgaggacactgtgttt-3′, LM α5 chain 5′ forward primer: 5′-tgaaggcagagggtagcagt-3′ and 3′ reverse primer 5′-gttgtctgcctggcttcttc-3′, LM β1 chain 5′ forward primer: 5′-gttcgagggaactgcttctg-3′ and 3′ reverse primer: 5′-tgccagtagccaggaagact-3′. LM γ1 chain 5′ forward primer: 5′-gagctttgaggacgaccttg-3′ and 3′ reverse primer 5′-ctggggtgtgtatgctgatg-3′. For RPA a 356-bp LM α2 chain fragment which was cloned in the pCR II vector was linearized and transcribed in vitro using the MAXIscript kit (Ambion Inc.) and UTP α-[ 32 P] (400–900 Ci/mmol and 10 mCi/ml; NEN). The hybridization was carried out using an RPA kit (Ambion Inc.) and following the manufacturer's instructions. A 256-bp GAPDH linearized fragment obtained from Ambion Inc. was used as a control. The protected RNA fragments were resolved on a 5% denaturing polyacrylamide gel and autoradiographed. Freshly isolated lung mesenchymal cells were cultured in the presence of anti-LM α2 chain antibody, anti-LM α2 chain antibody preincubated with 10-fold excess of LM-2 (molar:molar) or IgG control. The antibody and IgG were added at concentrations of 1, 2, 5, and 10 μg/ml to the cultures either at the time of cell plating to determine their effect on cell attachment or 40 min after plating, when attachment was completed, to determine their effect on cell spreading. The cultures were maintained for 2 and 4 h for cell attachment studies and for 4 and 24 h for cell spreading studies. At the end of the culture period the cells were either fixed or lysed. Fixed cells were counted and their diameters were determined on images projected on a computer screen with the aid of a digitized pad and the image analysis program Optimas 5.1. . Statistical significance was determined by the Student's t test. Lysed cells were used for immunoblotting with anti-SM α actin, anti-desmin and anti-SM myosin antibodies as described above. In additional experiments mesenchymal cells were isolated from adult C57BL/6J dy/dy and C57BL/6J +/+ normal mice and plated on culture dishes uncoated or coated with 10 μg/ml of LM-2 as indicated in the manufacturer's instructions for cell attachment assays or with the same concentration of LM-1 or fibronectin. Cell attachment and spreading was then determined as just described. Lungs with trachea, stomach, and intestine from adult C57BL/6J dy/dy , C57BL/6J +/+ normal mice and CD-1 mice were formalin-fixed and paraffin-embedded. 5-μm sections were mounted on glass slides and stained with hematoxilin-eosin for light microscopic examination. Since the nuclei of SM cells are proportional to the cell's length and the cell boundaries are indistinct in vivo, nuclear length was determined as an indicator of cell elongation. Up to 460 nuclei (lung, 216; stomach, 120; intestine, 124 nuclei) were measured as described above in sections from lung, stomach, and intestine dissected from four dy/dy , four C57BL/6J +/+ and two CD-1 mice. Mesenchymal cell cultures and organotypic epithelial-mesenchymal cocultures were immunostained with anti-LM α2 antibodies and frozen sections from dy/dy and normal lungs were immunostained with anti-SM α-actin following previous protocols . Both antibodies were used at a concentration of 1 μg/ml. The corresponding secondary antibodies were used at a 1:50 dilution and the immunostaining was completed using a commercial peroxidase-anti-peroxidase kit (Dako), following the manufacturer's instructions. Fast red (red color) was used for detecting LM α2 and diaminobenzidine tetrahydrochloride (DAB) (brown color) was used for detecting SM α actin. LM α2 chain expression is prevented by cell rounding and is induced by cell spreading/elongation. Regardless of the culture system used to facilitate cell rounding or cell spreading, we consistently observed that only spread cells expressed LM α2 chain. Immunoblots of round and elongated lung mesenchymal cells plated on culture microsurfaces showed LM α2 chain as a protein with a M r of ∼350 kD present only in lysates from elongated cells . The band was not detected during the first 4 h in culture, but progressively appeared within the next 20 h. Unlike the LM α2 chain, no modulations were found in expression of the LM β1 and γ1 chains, which was similar for both round and elongated cells. We used a monospecific antibody to confirm the identity of LM α2 chain. Since this antibody is not suited for immunoblotting, we used it for immunoprecipitation, followed by immunoblotting with the anti–LM-1/LM-2 antibody mentioned above. The lack of LM-2 in the round cell immunoprecipitates confirmed that only elongated cells synthesize LM α2 . RT-PCR and RPAs demonstrated that the increment in LM α2 chain is, at least in part, the result of an increment in its steady-state mRNA levels . RT-PCR demonstrated no changes in mRNA levels for LM α1, β1, and γ1 chains between round and elongated cells as well as no changes in LM α4 and α5 message levels (not shown). As expected, LM α1 polypeptide chain was not detected under any of these culture conditions. Our previous studies showed that LM α1 chain synthesis requires epithelial-mesenchymal cell–cell contact . No differences were seen in cell spreading and LM α2 chain synthesis between cells plated on uncoated dishes or dishes coated with LM-1, LM-2, collagen IV, and fibronectin (not shown). Immunohistochemical studies confirmed the absence of LM α2 chain in round mesenchymal cells and its presence in elongated cells . In organotypic cultures epithelial cells rearrange into spheres and mesenchymal cells surround them forming also a surface-anchored monolayer . A basement membrane is the novo assembled at the epithelial-mesenchymal interface . The mesenchymal cells apposed to this new basement membrane elongate and become SM cells, while the rest remain round and undifferentiated . The elongated cells synthesized LM α2, whereas the rest remained negative . Over time the mesenchymal cells formed cysts with central lumens and additional mesenchymal cells spread/elongated around them and synthesized LM α2 chain . In vivo undifferentiated (round) mesenchymal cells do not express LM α2 chain, but elongated cells do. Here we determined whether the observations made in tissue cultures were relevant to the lung in vivo. At day 10.5 of gestation all the cells in the lung are round in shape. At this stage, the embryonic lung synthesizes LM α1 but not LM α2 . At day 12 of gestation, most of the proximal peribronchial mesenchymal cells become spread/elongated and the distal epithelial cells become spread/flat. The undifferentiated mesenchymal cells, however, are still round. Western blots of microdissected airways and surrounding mesenchyme showed that the elongated cells express both LM α1 and α2 chains , whereas Western blots of microdissected undifferentiated mesenchyme showed absence of LM α2 chains in round cells . LM α2 chain expression is turned on and off by changes in cell shape. Switching cells from culture conditions that promote cell rounding to culture conditions that promote cell elongation and vice versa demonstrated that synthesis of LM α2 chain can be switched on and off by changes in cell shape . Replating efficiency was similar for round and elongated cells (48 ± 12 and 50 ± 10% of replated cells attached, respectively). The control of LM α2 chain expression by the cell shape is not restricted to lung embryonic mesenchymal cells. During lung development, both epithelial and mesenchymal cells undergo changes in shape. The most dramatic changes take place on distal epithelial cells that become totally flat and spread by day 14–15 of gestation. Here we determined the role of cell shape on lung embryonic epithelial cells as well as on embryonic mesenchymal cells from kidney and intestine. Immunoblots showed that cell shape also regulates LM α2 chain expression in these cells in a similar manner as observed in mesenchymal cells. Like in embryonic mesenchymal cells, rounding prevented and spreading induced LM α2 chain synthesis . Cell spreading is blocked by monoclonal antibodies to LM α2 chain. Effect on SM differentiation. In functional studies, 1 and 2 μg /ml of monoclonal antibody against LM α2 chain were sufficient to block mesenchymal cell spreading in a statistically significant manner . Higher concentrations of antibody did not further reduce spreading. Therefore, these studies indicated a reciprocal interaction between cell shape and LM α2 by showing that not only is the LM α2 chain induced by cell spreading, but also that cell spreading is induced by the LM α2 chain. Mesenchymal cells exposed to the anti-LM α2 chain antibodies showed a decrease in synthesis of SM-specific proteins, including SM α-actin, desmin and myosin when compared with cells exposed to control immunoglobulin . Attachment assays in the presence of anti-LM α2 chain antibody or IgG control showed that the antibody also inhibited cell adhesion (64% less cells attached in the presence of 20 μg/ml of anti-LM α2 chain antibodies than in the presence of same concentration of control IgG; not shown). SM cells from adult dy/dy mouse lung spread poorly in culture and contain less SM-specific protein. Over 40% of the attached mesenchymal cells, whether isolated from normal or mutant mice, reacted with anti-desmin antibodies (not shown). All mesenchymal cell cultures isolated from adult dy/dy mouse lungs showed poor cell spreading and lower levels of SM-specific protein than controls. The deficiencies in cell spreading and SM protein synthesis disappeared by plating the cells on LM-2 . LM-1 or fibronectin coating did not improve the abnormalities in cell spreading and SM protein synthesis seen in dy/dy mouse cells . Dy/dy mice have SM abnormalities in vivo, including shorter cells and deficiencies in SM-specific protein production. Cell morphometry studies showed that bronchial SM cells in dy/dy mice were shorter than in control animals in a statistically significant manner . No differences in size were found, however, in lung vascular SM cells or bronchial columnar epithelial cells (not shown). Immunoblots demonstrated that adult dy/dy mouse lungs contained less SM-specific proteins, including SM α-actin, desmin, and myosin . On histological examination, the most abnormal SM was that of the trachea and main bronchi. In those sites, the visceral SM cells were shorter and underdeveloped . For comparison, A and C show tracheal and bronchial SM in normal animals of the same strain. Unlike the extraparenchymal airway, the changes in the intraparenchymal bronchial SM were subtle and detected mainly by cell morphometry (shorter cells) and immunoblotting as already shown in Fig. 8 . On immunohistochemistry using an anti-SM α-actin antibody, the large and medium sized bronchial muscle exhibited slightly, but clear thinning and more discontinuity than the normal counterparts . The SMs of stomach and intestine were also studied. Histological examination and morphometric studies of the stomach revealed no alterations in dy/dy mice when compared with controls. The intestine, however, exhibited shorter SM cells, similar to what was seen in the lung . Unlike the trachea and bronchi, the SM cells in the intestinal wall were arranged in more layers (hyperplasia) resulting in a thicker muscularis wall. In the thinnest areas, the muscular circular layer was 2 ± 1-SM cell–thick in dy/dy mouse intestine and 1 ± 1-SM cell–thick in control animals. In the thickest sites, the circular layer was 15 ± 3-cell-thick in dydy intestine and 12 ± 2-cell-thick in controls. Reflecting this hyperplasia, immunoblotting showed a slight increase in SM-specific proteins in the intestine of dy/dy mice compared with controls . During development, embryonic cells undergo significant changes in shape. In the early stages of embryogenesis, essentially all cells are round, but along with differentiation part of them become spindly whereas others adopt a columnar, cuboidal, or flat configuration. Our studies and those of others indicate that these changes in shape can be part of a mechanistic cascade directing precursor cells to specific differentiation lineages. Among the multiple factors controlling cell shape, we originally identified LM-1 as relevant for bronchial myogenesis . We observed that during lung development, LM α1 chain synthesis is induced by epithelial-mesenchymal contact. LM α1 is deposited at the airway epithelial-mesenchymal interface, where it stimulates peribronchial mesenchymal cells to spread and to synthesize SM-specific proteins . Here we show that this change in cell shape from round to elongated results in the concomitant induction of LM α2 chain expression. In these studies we used different culture approaches to modulate cell shape and to determine its impact on LM α1, α2, β1, and γ1 chain expression. To confirm our in vitro findings, we analyzed tissue fragments microdissected from developing lungs containing either round cells or mostly elongated cells. Our studies demonstrated that, while LM β1 and γ1 chains are equally synthesized by round and elongated cells, LM α2 expression is under the control of cell shape in such a manner that round cells do not synthesize LM α2 chain, either in vivo or in vitro, but its expression is turned on by cell spreading/elongation. Furthermore, the induction of LM α2 chain is a reversible process that can be switched on and off in culture by cyclic changes in cell shape. It has been shown that limiting the degree of cell spreading in culture may decrease protein secretion . In our study, however, LM α2 chain expression was not suppressed due to nonspecific inhibition in protein synthesis, as indicated by the fact that round and elongated cells synthesized the same amounts of LM β1/γ1 chains. Furthermore, we recently showed that round and elongated cells cultured on microsurfaces have similar metabolic rates, and round cells even synthesize higher levels of α-fetoprotein than their spread/elongated counterparts . Finally, LM α2 chain was not found in undifferentiated mesenchyme freshly isolated from the lungs, confirming its absence in round cells in vivo. As previously stressed, among the several LM chains analyzed, the effect of cell shape on message and protein levels was specific to LM α2. Message RNA for LM α1, α4, α5, β1, and γ1 chains was not significantly altered by cyclic changes in cell shape, neither were LM β1 and γ1 polypeptide chain levels. In regard to LM α1 chain, its synthesis requires epithelial-mesenchymal cell contact and as expected it was not detected in fragments of mesenchyme alone or mesenchymal cell cultures. However, its presence in the early embryonic lung, where essentially all cells are round, and the lack of significant oscillations at the mRNA level , indicated that LM α1 chain synthesis does not cycle with changes in cell shape. Functional studies in which embryonic mesenchymal cell spreading/elongation was blocked by a monoclonal antibody against LM α2 chain demonstrated that, once secreted, LM α2 chain becomes a powerful stimulus for cell spreading. The antibody used in our functional studies reacts with the NH 2 -terminal of LM α2 chain, a region that has been shown to be involved in heparin binding, LM-2 polymerization and cell binding through integrins α1β1 and α2β1 . The functional role of the NH 2 terminus is also underscored by an animal model of congenital muscular dystrophy, the dy 2J /dy 2J mouse. This mutant animal has a truncated LM α2 chain which lacks only 57 amino acids at its NH 2 -terminal domain , and nevertheless this deletion is enough to cause muscular dystrophy. Integrins α1β1 and α2β1 are expressed by developing bronchial SM and therefore may participate in LM α2 chain–mediated mesenchymal cell spreading. Supporting such a possibility, deletion of integrin α1 by homologous recombination permits normal murine development but gives rise to specific deficit in mesenchymal cell adhesion and spreading . In addition, knockout of integrin β1 chain, which cause early embryonic death , results in retardation of myogenic differentiation of embryonic stem cells in culture . We recently observed that most embryonic mesenchymal cells are potential SM precursors and if allowed to spread/elongate, they will differentiate into SM cells . Our functional studies demonstrated that LM α2 chain deposition in the extracellular matrix is per se a stimulus for cell spreading. This led us to determine the effect of inhibiting LM α2 chain–mediated cell spreading on SM differentiation. Mesenchymal cells exposed to the anti-LM α2 chain antibodies showed a decrease in synthesis of SM-specific proteins when compared with controls. Therefore, a feedback interaction seems to exist between the cells' shape, the levels of LM α2 chain and SM myogenesis. The molecular pathways linking LM α2 chain, cell shape, and SM myogenesis are currently unknown. One possibility is that by promoting cell spreading/elongation, LM α2 chain stimulates specific cell receptors known to function as mechanotransductors, such as integrins . These then could activate gene expression by a signaling mechanism similar to that exerted by the breast extracellular matrix upon the mammary gland epithelium . An important player in cell shape signaling is the Rho family of G proteins . Interestingly, searching a subtracted cDNA library of undifferentiated, round mesenchymal cells, we found RhoA to be highly expressed in round mesenchymal cells with minimal expression in elongated cells (Liu, J., and L. Schuger, unpublished studies). It should be stressed, however, that cell shape controls gene expression through multiple mechanisms including signaling pathways not necessarily involving Rho , changing the cytoskeleton and nuclear matrix architecture , inducing synthesis of auto/paracrine factors , etc. Therefore, multiple mechanisms may be involved in the process of LM α2 chain–induced SM myogenesis. Dy/dy mice express lower levels of LM α2 chain, due to a spontaneous genetic mutation . Therefore, we studied SM cells isolated from dy/dy lungs to determine how these cells behave in culture. These mutant mice have been used for many years as a model of skeletal muscular dystrophy, and although there are no previous reports describing SM defects in dy/dy mice, we predicted, based on our findings, that these animals should have abnormal SM cells. Indeed, we found that lung SM cells isolated from dy/dy mice spread defectively in culture and synthesize less SM proteins than controls. These deficiencies were overcome by the addition of exogenous LM-2 but not LM-1 or fibronectin. On histological examination of lung sections, bronchial SM cells in dy/dy mice were shorter than in control animals and their lungs contained less SM-specific proteins. The morphological abnormalities diminished in severity from trachea, where the muscle was also underdeveloped, to the distal bronchi. Although the SM changes in the intraparenchymal bronchi were subtle, our current studies seem to indicate that these are sufficient to affect the electrical and contractile properties of dy/dy SM cells in culture (Yang, Y., and L. Schuger, ongoing studies). The SM cells of stomach and intestine were also examined. While no abnormalities were detected in the former, the intestine exhibited shorter SM cells, as seen in the bronchi. However, unlike the airways, the intestinal wall showed more SM cell layers, indicative of compensatory hyperplasia. The latter resulted in a net increase of SM-specific proteins in immunoblots. It is unclear why some SMs were hyperplastic and others not. One reason could be related to the mechanical challenge sustained by the muscle. Muscles with peristalsis, such as the intestine, are rhythmically stimulated to contract. Since SM cells are able to proliferate, the dy/dy intestinal muscle may overcome the functional deficit caused by low LM α2 chain with an increment in cell number, thus, becoming hyperplastic. In contrast, muscles with minimal mechanical activity, such as the trachea and main bronchi, experience no mechanical challenge and therefore have no need to compensate. Between these two extremes, SMs with moderate contractile activity, such as the intraparenchymal bronchi, may require only intermediate grades of compensation. The gastric and vascular musculature may also fall under this category. The effect of cell shape on LM α2 chain expression was not restricted to lung mesenchymal cells, but it was seen in other cells as well. Therefore, our findings suggested that cell shape–controlled LM α2 chain expression may play a broader role in development. In this regard, Virtanen et al. 1996 found that during the preglandular stage of human lung development, LM α2 chain is deposited in the distal airway basement membranes, where epithelial cells are undergoing a change in shape from round/cuboidal to flat/spread . Similarly, Lefebvre et al. 1999 showed that during intestinal development, LM α2 chain is synthesized by elongating mesenchymal cells, but not by epithelial cells that are round/cuboidal in shape. Although neither of these papers intended to draw correlations between the cell shape and LM α2 chain expression, both support the possibility that epithelial cell shape and LM α2 chain may be functionally connected in vivo. Fig. 11 depicts the proposed roles of LM α1 and α2 chains in bronchial myogenesis. Our studies suggest that the development of new epithelial-mesenchymal contacts during lung organogenesis results in the induction of LM α1 chain expression and deposition of LM-1 at the epithelial/mesenchymal interface . LM-1 molecules polymerize, contributing to the formation of a basement membrane . The mesenchymal cells apposed to the newly formed basement membrane utilize this meshwork to spread/elongate through binding to LM α1 . This change in cell shape induces them to synthesize LM α2 chain and concomitantly to differentiate into SM. Once secreted as part of LM-2, LM α2 chain stimulates spreading and SM differentiation of nearby mesenchymal cells, eventually leading to the establishment of a multilayered visceral muscle. The process of myogenesis could stop when LM α2 chain expression drops dramatically at the end of the pseudo glandular period whereby preventing excessive SM formation. | Other | biomedical | en | 0.999996 |
10601346 | Mouse anti–ZO-1 mAbs (T8-754 and T7-713), rabbit anti–ZO-2 pAb (pAb62), guinea pig anti–claudin-1 pAb, rat anti–claudin-1 mAb, and rat anti–claudin-2 mAb were described previously . The mAbs, T8-754 and T7-713, recognize the NH 2 - and COOH-terminal halves of ZO-1, respectively. Anti–ZO-3 pAb was raised in rabbits using GST fusion protein with COOH-terminal region (amino acids [aa] 758–904) of mouse ZO-3 as an antigen. Mouse anti–c-myc tag and anti-His tag mAbs were purchased from Sigma Chemical Co. Mouse L cells, dog MDCK cells, and their transfectants were cultured in DME supplemented with 10% FCS. C1L and C2L cells were described previously . Using the BLAST search program, we found that the sequence of mouse EST clone AA016964 was highly homologous but not identical to the PDZ1 domains of ZO-1 and ZO-2, leading to the isolation of full-length cDNA encoding this sequence. During the course of this isolation, a cDNA encoding dog ZO-3 was reported and its sequence was almost the same as that of AA016964, suggesting that AA016964 encodes part of mouse ZO-3. Then, specific primers were synthesized from the sequence of AA016964, and a partial cDNA of mouse ZO-3 was obtained by RT-PCR, which was used as a probe to screen a mouse lung λ ZAP cDNA library. Four positive clones were obtained and sequenced to clarify the entire open reading frame of mouse ZO-3 cDNA. The full-length mouse ZO-3 cDNA was subcloned into pSK- to produce pSK-ZO-3. The mouse ZO-3 sequence has been submitted to EMBL/GenBank/DDBJ under accession number AF157006. To fuse a c-myc epitope tag onto the COOH terminus of each construct, the eukaryotic expression vector pME18S-7myc was used. For production of mammalian expression vectors for c-myc–tagged full-length ZO-2 and ZO-3 (pME18S-ZO-2-7myc and pME18S-ZO-3-7myc, respectively), cDNA fragments encoding the entire open reading frame of mouse ZO-2 and ZO-3 were produced by PCR using pSK-ZO-2 and pSK-ZO-3 as template, respectively, and subcloned into pME18S-7myc. Various deletion mutant constructs with a c-myc tag at their 3′-end were also produced by PCR using the following primers; sense primer from position 344 containing a StuI site and antisense primer from position 3549 containing a SalI site for ZO-2 lacking PDZ1 (pME18S-ΔPDZ1-ZO-2-7myc), sense primer from position 1208 containing a StuI site and antisense primer from position 3549 containing a SalI site for ZO-2 lacking both PDZ1 and -2 (pME18S-ΔPDZ1,2-ZO-2-7myc), sense primer from position 492 containing an EcoRV site and antisense primer from position 2918 containing an XhoI site for ZO-3 lacking PDZ1 (pME18S-ΔPDZ1-ZO-3-7myc), sense primer from position 993 containing EcoRV site and antisense primer from position 2918 containing an XhoI site for ZO-3 lacking both PDZ1 and -2 (pME18S-ΔPDZ1,2-ZO-3-7myc). For the production of ZO-2 lacking PDZ2 (pME18S-ΔPDZ2-ZO-2-7myc), two fragments were amplified by PCR using the sense primer from position 44 containing an EcoRI site and antisense primer from position 886 containing an ApaI site, and sense primer from position 1208 containing an ApaI site and antisense primer from position 3549 containing a SalI site. Two fragments were simultaneously ligated into pME18S-7myc after digestion with the appropriate restriction enzymes. Similarly, for production of ZO-3 lacking PDZ2 (pME18S-ΔPDZ2-ZO-3-7myc), two fragments were amplified by PCR using the sense primer from position 198 containing an EcoRV site and antisense primer from position 761 containing a SacI site, using the sense primer from position 993 containing a SacI site and antisense primer from position 2918 containing an XhoI site, then simultaneously ligated into pME18S-7myc. For production of claudin-1 lacking its COOH-terminal YV (pCAGCL-1ΔYV), the cDNA fragment was amplified by PCR using the sense primer from position 1 and antisense primer from 626 containing a stop codon. The obtained fragment was blunted with T4 polymerase and ligated into pCAGGSneodelEcoRI , which was provided by Dr. J. Miysazaki (Osaka University). Cells (10 7 ) were cotransfected with 2 μg of each expression vector and 0.1 μg of pPGKpuro using Lipofectamine reagent for 5 h in Opti-MEM (GIBCO BRL). After 43 h, cells were replated onto four 9-cm dishes in the presence of 8 μg/ml of puromycin to select stable transfectants. Colonies of puromycin-resistant cells were isolated and screened by either immunoblotting or immunofluorescence with anti–c-myc tag mAb or guinea pig anti–claudin-1 pAb. To construct GFP-tagged expression vectors, GFP cDNA was excised from pQBI25 and introduced into pCAGGSneodelEcoRI generating pCAG-GFP. The following cDNAs were amplified by PCR and subcloned into SwaI-digested pCAG-GFP to express each PDZ domain tagged with GFP at its COOH-terminal end: PDZ1 (aa 19–113), PDZ2 (aa 181–292), or PDZ3 (aa 423–503) of ZO-1, PDZ1 of ZO-2 (aa 4–100), and PDZ1 of ZO-3 (aa 5–96). These GFP-tagged proteins were introduced into cultured MDCK cells as described previously . Cells plated on glass coverslips were rinsed in PBS and fixed with 1% formaldehyde in PBS for 15 min at room temperature. Cells were then treated with 0.3% Triton X-100 in PBS for 15 min and washed three times with PBS. After soaking in PBS containing 1% BSA, the samples were treated with primary antibodies for 1 h in a moist chamber. They were then washed three times with PBS, followed by incubation for 30 min with appropriate secondary antibodies. The samples were washed with PBS three times, embedded in 95% glycerol-PBS containing 0.1% para-phenylendiamine and 1% n -propylagate, and examined with a photomicroscope (model Axiophoto; Carl Zeiss). Frozen sections of the intestine of occludin-deficient or control mice were immunofluorescently stained as described previously . The cDNA fragments encoding the cytoplasmic domain of claudin-1 to -8 and the cytoplasmic domain of claudin-1 lacking its COOH-terminal YV were amplified using specific primers and subcloned into pGEX vector . Recombinant NH 2 -terminal half of ZO-1 (N-ZO-1; aa 1–862), COOH-terminal half of ZO-1 , 6xHis-tagged NH 2 -terminal portion of ZO-2 (N-ZO-2; aa 1–938), and 6xHis-tagged COOH-terminal portion of ZO-2 were produced in Sf9 cells by baculovirus infection . For various mutants of ZO-1/ZO-2/ZO-3 carrying 6xHis tag, the following cDNAs were amplified by PCR and subcloned into pET32 (Novagen): PDZ1 of ZO-1 (P1-ZO-1; aa 19–113), PDZ2 of ZO-1 (P2-ZO-1; aa 181–292), PDZ3 of ZO-1 (P3-ZO-1; aa 423–503), PDZ1 of ZO-2 (P1-ZO-2; aa 4–100), PDZ2 of ZO-2 (P2-ZO-2; aa 282–388), and PDZ3 of ZO-2 (P3-ZO-2; aa 491–571), full-length of ZO-3 (aa 1–904), PDZ1 of ZO-3 (P1-ZO-3; aa 5–96), PDZ2 of ZO-3 (P2-ZO-3; aa 189–263), and PDZ3 of ZO-3 (P3-ZO-3; aa 371–449). These recombinant proteins were expressed in E . coli (BL21). In vitro binding assay was performed basically as previously described . In brief, GST fusion proteins with the cytoplasmic domains of claudins expressed in E . coli were purified using glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech Ltd.). After washing with PBS, the beads were incubated with lysate of Sf9 cells or E . coli expressing each recombinant protein of ZO-1, ZO-2, and ZO-3 described above followed by washing with PBS containing 1% Triton X-100. Then, bound proteins were eluted with 300 μl of 50 mM Tris-HCl buffer (pH 8.0) containing 20 mM glutathione. Each eluate was analyzed by SDS-PAGE. To estimate the dissociation constant between the cytoplasmic domain of claudin-1 and the PDZ1 domains of ZO-1/ZO-2/ZO-3, 200 μl of the glutathione–Sepharose bead slurry containing 40 μg of GST-claudin-1 was incubated with 2 ml of the E . coli lysate containing 0.01–0.5 μg of the PDZ1 domain of ZO-1, ZO-2, or ZO-3. The beads were washed and bound proteins were eluted with 1 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM glutathione. The amounts of recombinant PDZ1 domains in the cell lysate or in each eluate were estimated by comparing the intensity of their Coomassie brilliant blue–stained bands with those of various amounts of BSA using Adobe Photoshop™ 3.0J histogram, and Scatchard plot of the data was generated. Experiments were repeated three times for each estimation of K d . For metabolic labeling of transfectants, cells cultured on 9-cm dishes were washed twice with methionine-free medium supplemented with 2% FCS, and then incubated with 3 ml of the same medium containing 0.2 mCi [ 35 S]methionine for 5 h. After washing with ice-cold PBS three times, cells were lysed in 2 ml of extraction buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 10 mM Hepes [pH 7.4], 2 mM phenylmethylsulfonyl, 2 mg/ml leupeptin). Cell lysates were clarified by centrifugation at 100,000 g for 30 min and incubated with 50 μl of protein G–Sepharose bead slurry (Zymed Laboratories Inc.) coupled with anti–c-myc tag mAb for 3 h. After washing five times with the extraction buffer, immunoprecipitates were eluted from beads with the SDS-PAGE sample buffer. One-dimensional SDS-PAGE (10–12.5%) was performed based on the method of Laemmli 1970 . Gels were stained with Coomassie brilliant blue R-250 and exposed to imaging plates and analyzed using a BAS2000 (Fuji Film). For immunoblotting, proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose sheets, which were then incubated with primary antibodies. The antibodies were detected with a blotting detection kit (Amersham Pharmacia Biotech Ltd.). We previously produced occludin-deficient embryonic stem cells, and differentiated them into epithelium-like cells (visceral endoderms) . Unexpectedly, these cells bore well-developed TJs, where ZO-1 was still concentrated. In this study, we first examined whether ZO-2 and ZO-3 as well as ZO-1 are recruited to TJs of highly polarized epithelial cells such as intestinal epithelial cells in occludin-deficient mice, which were born normally (Saitou, M., unpublished data). Frozen sections of occludin-deficient intestinal epithelial cells were double stained with anti-occludin mAb/anti–claudin-3 pAb, anti–claudin-3 pAb/anti–ZO-1 mAb, anti–ZO-1 mAb/anti–ZO-2 pAb, or anti–ZO-1 mAb/anti–ZO-3 pAb. As shown in Fig. 1 , not only ZO-1 but also ZO-2 and ZO-3 were targeted to and concentrated at TJs of these well-polarized occludin-deficient epithelial cells. These findings suggested the possible interactions between claudins and ZO-1/ZO-2/ZO-3, prompting us to in vitro binding analyses. As shown in Fig. 2 , various constructs for ZO-1, ZO-2, and ZO-3 were used in this study for in vitro biding assays and in vivo colocalization analyses. First, we produced recombinant N-ZO-1, C-ZO-1, N-ZO-2 (6xHis tag), and C-ZO-2 (6xHis tag) in Sf9 cells by baculovirus infection , and full-length ZO-3 in E . coli , and their cell lysates containing the same amount of recombinant proteins were mixed with beads conjugated with the GST-fusion protein with the cytoplasmic domain of claudin-1. Bound proteins were then eluted from beads and each eluate was subjected to SDS-PAGE followed by immunoblotting with corresponding antibodies. As shown in Fig. 3N -ZO-1, N-ZO-2, and full-length ZO-3, but not C-ZO-1 or C-ZO-2, appeared to specifically bind to the GST-fusion protein with the cytoplasmic domain of claudin-1. Next, using similar in vitro binding assays, we narrowed down the domains responsible for the ZO-1/ZO-2/ZO-3–claudin-1 interaction. Because preliminary experiments showed that PDZ domains of ZO-1/ZO-2/ZO-3, but not SH3 or GUK domains, are involved in this interaction, we produced recombinant 6xHis-tagged PDZ1, -2, and -3 domains of ZO-1, ZO-2, and ZO-3 in E . coli , and performed the in vitro binding analyses with the GST fusion protein with the cytoplasmic domain of claudin-1. In these analyses, the direct binding was evaluated by Coomassie brilliant blue staining of each eluate. As a result, among nine recombinant PDZ domains, only PDZ1 domains of ZO-1, ZO-2, and ZO-3 were specifically associated with the GST–claudin-1 fusion protein . Interestingly, these PDZ1 domains lost their binding affinity to the cytoplasmic domain of claudin-1, when the YV sequence was deleted from the COOH terminus of claudin-1 . We then estimated the dissociation constant between the cytoplasmic domain of claudin-1 and PDZ1 domains of ZO-1/ZO-2/ZO-3 . For this purpose, various concentrations of these PDZ1 domains were incubated with beads bearing the GST fusion protein with the cytoplasmic domain of claudin-1 or GST (for control). The binding constant was estimated by subtracting the binding to the GST-conjugated Sepharose beads . For each PDZ1 domain the binding was saturable, and Scatchard analysis revealed a single class of affinity binding sites with K d 's of ∼13, 11, and 18 nM for the PDZ1 domain of ZO-1, ZO-2, and ZO-3, respectively. Finally, with the similar binding assays, we examined the binding ability of the PDZ1 domain of ZO-1 to the cytoplasmic domains of claudin-1 to -8 . Since some GST fusion proteins with the cytoplasmic domains of claudins were easily degraded, it was difficult to estimate the amount of GST fusion proteins precisely, but it is clear that the PDZ1 domain of ZO-1 directly bound to the cytoplasmic domain of claudin-1 to -8. PDZ1 domains of ZO-2 and ZO-3 also showed binding affinity to claudin-1 to -8 (data not shown). In vitro binding analyses indicated that the PDZ1 domains of ZO-1, ZO-2, and ZO-3 are directly associated with the COOH termini of claudins. To examine these bindings in vivo, the immunoprecipitation experiments with epithelial cells would be one of the most appropriate approaches, but this method does not work in the case of claudins; the claudin molecules are polymerized into strands which are highly insoluble in detergents. The transfection experiments with epithelial cells would also be another appropriate approach, but the data obtained are not easily interpreted, since multiple species of claudins as well as ZO-1/ZO-2/ZO-3 are endogenously expressed in epithelial cells. To avoid these technical difficulties, we used L transfectants expressing various types of claudin. As shown previously, parental L cells lack either TJs or the expression of claudins and occludin . When claudin-1 or -2 was singly transfected into L cells (C1L and C2L cells, respectively), introduced claudins were concentrated at cell–cell borders as planes in an elaborate network pattern at the immunofluorescence microscopic level, where they reconstituted well-developed networks of TJ strands at the electron microscopic level . Using these C1L and C2L cells, we evaluated the interactions of various constructs of ZO-1/ZO-2/ZO-3 with claudins by examining whether they were recruited to the claudin-based networks. As shown in Fig. 6 , ZO-1, but not ZO-2 or ZO-3, was expressed endogenously in parental L cells (and also in C1L and C2L cells). This endogenous ZO-1 did not show characteristic concentration in L cells . In contrast, in both C1L and C2L cells in which claudin-1 and claudin-2 were concentrated at cell–cell borders in an elaborate network, respectively, endogenous ZO-1 was colocalized with claudins . Close inspection revealed that ZO-1 was also concentrated as elaborate networks, which almost overlapped with those based on claudin . When claudin-1 mutant lacking its COOH-terminal YV was transfected into L cells (C1ΔYVL cells), introduced claudin-1 mutant was again concentrated at cell–cell borders as elaborate networks . Interestingly, however, ZO-1 was not recruited to these claudin-based networks in C1ΔYVL cells . These findings, together with the in vitro binding data suggested that ZO-1 also directly binds to the COOH-terminal YV of claudins through its PDZ1 domain inside cells. Since ZO-2 was not expressed endogenously in L cells , the full-length cDNA encoding c-myc–tagged ZO-2 was introduced into C1L cells. As shown in Fig. 8 , a and b, this introduced ZO-2 was recruited to the claudin-based networks. However, since ZO-2 can directly bind to ZO-1 through its PDZ2 domain , it was not clear whether this recruitment of full-length ZO-2 was based on the direct interaction of ZO-2 with claudin-1 or endogenous ZO-1 that was recruited to the claudin-based networks as shown in Fig. 7 . Therefore, we next transfected cDNAs encoding c-myc–tagged ZO-2 mutant lacking both PDZ1 and -2 (ΔPDZ1,2-ZO-2), PDZ1 alone (ΔPDZ1-ZO-2) or PDZ2 alone (ΔPDZ2-ZO-2) into L transfectants , confirmed their expression by immunoblotting, and their subcellular localization was followed by anti–c-myc mAb staining. ΔPDZ1,2-ZO-2 was not recruited to the claudin-based networks , whereas ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 were recruited . These findings can be interpreted as indicating that ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 are recruited by the direct association with endogenous ZO-1 (through PDZ2) and claudin-1 (through PDZ1), respectively. Since ΔPDZ1,2-ZO-2 cannot bind to either endogenous ZO-1 or claudin-1, it would not be recruited. In good agreement with this, in C1ΔYVL cells, exogenous full-length ZO-2 was not concentrated at cell–cell borders, probably because in these cells endogenous ZO-1 as well as exogenous ZO-2 cannot bind to mutant claudin-1 . The same results were obtained when C2L cells were used (data not shown). ZO-3 was reported to directly bind to ZO-1 , but the domain responsible for this binding has not yet been determined. We then transfected cDNAs encoding c-myc–tagged full-length ZO-3 and mutants lacking both PDZ1 and -2 (ΔPDZ1,2-ZO-3), PDZ1 alone (ΔPDZ1-ZO-3) or PDZ2 alone (ΔPDZ2-ZO-3) into C1L cells. When these introduced ZO-3 and ZO-3 mutants were immunoprecipitated with anti–c-myc mAb from the cell lysate of stable transfectants, endogenous ZO-1 was coimmunoprecipitated with full-length ZO-3 and ΔPDZ1-ZO-3, but not with ΔPDZ1,2-ZO-3 or ΔPDZ2-ZO-3 . Therefore, we concluded that ZO-3 binds to ZO-1 through its PDZ2 domain inside cells. Then, to examine the direct interaction of ZO-3 with claudins in vivo, we transfected cDNAs encoding c-myc–tagged full-length ZO-3, ΔPDZ1,2-ZO-3, ΔPDZ1-ZO-3, or ΔPDZ2-ZO-3 into C1L cells, confirmed their expression by immunoblotting, and their distribution was examined by immunofluorescence microscopy with anti–c-myc mAb . Similarly to the transfection experiments with ZO-2 , full-length ZO-3, ΔPDZ1-ZO-3, and ΔPDZ2-ZO-3, but not ΔPDZ1,2-ZO-3, were recruited to claudin-based networks. Furthermore, full-length ZO-3 was not recruited in C1ΔYVL cells (data not shown), and similar results were obtained in C2L cells (data not shown). Based on these findings, we concluded that ZO-3 also directly binds to the COOH-terminal YV of claudins in vivo, and that ZO-3 can be recruited to the claudin-based TJ strands through interactions either with endogenous ZO-1 or claudins themselves. A question naturally arose as to whether PDZ1 domains of ZO-1/ZO-2/ZO-3 interact with the cytoplasmic domain of claudins in epithelial cells. To evaluate this point, we constructed expression vectors for green fluorescent protein (GFP)-fusion proteins with PDZ1, PDZ2, or PDZ3 domains of ZO-1 and introduced them into cultured MDCK cells . For an unknown reason, all these fusion proteins were concentrated in the nucleus. In addition to the nuclear staining, PDZ1-GFP and PDZ2-GFP were clearly recruited to and concentrated at claudin-1–positive TJs . Similarly to L transfectants, these findings can be interpreted as indicating that PDZ1-GFP and PDZ2-GFP are recruited by the direct association with endogenous claudins and ZO-2/ZO-3, respectively. In epithelial cells, PDZ3-GFP also appeared to be concentrated at TJs, although very faintly as compared with PDZ1-GFP and PDZ2-GFP , probably due to unidentified binding partners for PDZ3 domain of ZO-1 localized at TJs in epithelial cells. GFP-fusion proteins with PDZ1 or PDZ2 (data not shown) domains of ZO-2/ZO-3 also showed significant concentration at claudin-1–positive TJs in MDCK cells. TJs are essential intercellular junctions, because their barrier and fence functions are indispensable for the establishment of compositionally distinct compartments in multicellular organisms . Furthermore, evidence has accumulated that TJs recruit various important intracellular signaling molecules to their cytoplasmic surface, which may be involved in the polarization of epithelial cells . Thus, elucidation of the molecular architecture of the TJ plaque is very important to understand the molecular mechanism of the regulation of barrier and fence functions of TJs as well as the relationship between TJs and epithelial polarization. To date, three MAGUKs, ZO-1, ZO-2, and ZO-3, have been identified as components of the TJ plaque , and occludin has been thought to be a binding partner for these MAGUKs to recruit them to TJs . However, even in occludin-deficient visceral endoderms and human Sertoli cells , ZO-1 was shown to be concentrated at TJs. Furthermore, in this study, we found that not only ZO-1 but also ZO-2 and ZO-3 were localized at TJs of highly polarized intestinal epithelial cells in occludin-deficient mice. These findings prompted us to examine the interaction between these MAGUKs and claudins, major constituents of TJ strands in vitro as well as in vivo. We then found that the PDZ1 domains of ZO-1, ZO-2, and ZO-3 directly bind to the COOH-terminal YV sequence of claudins. Interestingly, we detected no differences in affinity for claudin-1 among ZO-1, ZO-2, and ZO-3 ( K d = ∼13, 11 and 18 nM, respectively). ZO-1, ZO-2, and ZO-3 shared the same portion of the cytoplasmic domain of claudins (i.e., their COOH-terminal YV sequence) as their binding sites. Furthermore, ZO-1/ZO-2/ZO-3 bound to claudin-1 to -8, although it is possible that the binding affinity varies depending on the claudin species. These findings led to the molecular architecture model for the TJ plaque shown schematically in Fig. 11 . In this scheme, TJ strands are represented as linear co-polymers of occludin and various species of claudin . It is likely that these kinds of co-polymers present large linear clusters of YV sequences toward the cytoplasm, which may strongly attract cytoplasmic proteins containing PDZ domains with high affinity to the COOH-terminal sequence of claudins. Since the PDZ1 domains, but not PDZ2 or PDZ3 domains, of ZO-1, ZO-2, and ZO-3 has an affinity to the COOH-terminal sequence of claudins, they are recruited to TJ strands . In good agreement, GFP-fusion proteins with PDZ1 domains of ZO-1/ZO-2/ZO-3 were recruited to TJs in MDCK cells . These three MAGUKs were also reported to bind to the COOH-terminal 150–amino acid sequence of the cytoplasmic domain of occludin . Since occludin does not end in valine, the molecular basis for the occludin-MAGUKs interaction may be different from that of the claudin-MAGUKs interaction. In ZO-1, the GUK domain was reported to be responsible for occludin-ZO-1 binding . If this is the case also for ZO-2 and ZO-3, each MAGUK molecule can bind to occludin and claudins simultaneously through GUK and PDZ1 domains, respectively. Furthermore, to complicate the molecular architecture model for the TJ plaque, ZO-1/ZO-2 and ZO-1/ZO-3 interactions were also reported, suggesting the existence of heterodimers of MAGUKs . The results of this and previous studies revealed that these interactions were dependent on the PDZ2/PDZ2 interaction . GFP-fusion proteins with PDZ2 domains of ZO-1/ZO-2/ZO-3 were recruited to TJs in MDCK cells , probably through this PDZ2/PDZ2 interaction. ZO-1 does not form homodimers reportedly , but no information is available regarding the homodimers of ZO-2 and ZO-3. Furthermore, it remains unclear whether ZO-1, ZO-2, and ZO-3 exist in the TJ plaque as monomers. On the other hand, the COOH-terminal region of ZO-1 and ZO-2 directly binds to actin filaments . These findings indicated that ZO-1, ZO-2 and ZO-3 function as scaffolds of the TJ plaque to cross-link TJ strands to the actin-based cytoskeleton. The molecular linkage described in Fig. 11 would be dynamically regulated within cells. For example, phosphorylation is expected to play an important role in the protein-protein interactions in the TJ plaque. The ZO-1/ZO-2 interaction has been detected by immunoprecipitation from cell lysates, but not by in vitro binding assay with recombinant proteins, probably because some modifications such as phosphorylation on ZO-1 and/or ZO-2 are required for the interaction . Furthermore, occludin was shown to be heavily serine/threonine phosphorylated when it was incorporated into TJ strands . Therefore, it should be examined whether these phosphorylated occludin still showed binding affinity for ZO-1/ZO-2/ZO-3. Elucidation of the molecular architecture of the TJ plaque will provide insight into the functions of ZO-1, ZO-2, and ZO-3. As discussed above, the model shown in Fig. 11 favors the notion that these TJ-specific MAGUKs function as cross-linkers between TJ strands and actin filaments, although the actin binding ability has not been examined for ZO-3. Accumulating evidence has suggested that the barrier function of TJs is regulated through the association of actin filaments with TJs . Therefore, it appears reasonable to speculate that ZO-1, ZO-2, and ZO-3 are directly involved in the mechanism of regulation of the barrier function of TJs by modulating the actin filament/TJ strand association. In general, MAGUKs are believed to cluster integral membrane proteins to establish and maintain specialized plasma membrane domains such as pre- and post-synaptic regions . Therefore, it is interesting to speculate that ZO-1, ZO-2, and ZO-3 are required for the clustering of claudins and occludin within the plasma membranes, i.e., the formation of TJ strands. In our previous study , we obtained L transfectants expressing claudin-1 and -2 tagged with a FLAG sequence at their COOH termini (C1FL and C2FL cells, respectively), and found that the tagged claudin-1 and -2 were concentrated at cell–cell borders to reconstitute TJ strands. Interestingly, however, in contrast to C1L and C2L cells used in this study, in these C1FL and C2FL cells endogenous ZO-1 was not recruited to the claudin-based TJ strands because the COOH-terminal end of claudin-1 and -2 was masked by the FLAG sequence causing loss of its binding activity to the PDZ1 domain of ZO-1 (data not shown). Furthermore, recently we found that claudin-1 deletion mutant lacking almost all portion of its COOH-terminal cytoplasmic domain still reconstituted a well-developed network of TJ strands in L transfectants . Therefore, we concluded that claudins can be polymerized to form TJ strands without interacting with ZO-1 (and probably ZO-2 and ZO-3). This study improved our understanding of the molecular architecture of the major scaffolds of the TJ plaque. Further studies are required to identify various proteins that are recruited to these ZO-1/ZO-2/ZO-3–based scaffolds directly or indirectly. To date, three other TJ-specific peripheral membrane proteins, cingulin, 7H6 antigen and symplekin have been identified . An integral membrane protein called JAM, which belongs to the immunoglobulin superfamily, has also been shown to be concentrated at TJs . Furthermore, TJs were reported to recruit various types of molecules involved in intracellular signaling (heterotrimeric G proteins, atypical protein kinase C λ/ζ and their specific binding protein called ASIP) and vesicle targeting/fusion (Rab3B/Rab13 and sec6/8) . On the other hand, as shown in Fig. 11 , the binding partners for the PDZ3 and SH3 domains of ZO-1, ZO-2, and ZO-3 have not yet been identified, although a putative kinase, ZAK (ZO-1–associated kinase), was reported to be associated with the SH3 domain of ZO-1 . Therefore, it is possible that these PDZ3 and SH3 domains have some important roles in recruiting the molecules mentioned above to TJs. Further studies to clarify the molecular linkage within the TJ plaque will lead to a better understanding of the functions of TJs as well as the molecular mechanism of their regulation. | Study | biomedical | en | 0.999996 |
10601349 | Human CRP 26 , human serum amyloid P component (SAP; reference 27 ), rat CRP 28 , rat C3 28 , and cobra ( Naja naja ) venom factor 29 were isolated and purified and/or assayed precisely as previously described. The preparations of human CRP and SAP and of cobra venom factor used in vivo were all >99% pure. Female Wistar rats bred at Imperial College School of Medicine, Hammersmith campus were used at age 9–10 wk and weighed 230–280 g. Within each experiment, the whole body and heart weights were the same in control and experimental groups. All animals were carefully examined and were healthy before fasting overnight before surgery. General anesthesia was induced with inhaled isoflurane, and supplemental oxygen was provided at 1.0 liter/min. The chest was opened through the sixth intercostal space, anesthesia was discontinued but supplemental oxygen continued at 0.5 liter/min, and the pericardium was opened to allow the heart to be lifted out of the thorax. The left anterior descending coronary artery was immediately ligated at a constant distance just below the atrium with a 5/0 silk suture, the heart was replaced, and the chest was closed while active ventilation with 100% oxygen was continued. The duration of pneumothorax was ∼30 s. Resuscitation was completed by gentle chest massage with the rat lying supine, and there was usually rapid recovery of consciousness and the righting reflex. Buprenorphine (0.05 mg/kg) was given immediately for postoperative analgesia and repeated 12-hourly. Development of myocardial infarction was confirmed by early postoperative four lead electrocardiogram showing marked ST segment elevation in leads I and aVL as well as various arrhythmias. Intraoperative and immediate postoperative mortality was up to 20%, but thereafter there were few deaths before day 5, when the rats were killed for measurement of infarct size. Deep anesthesia was induced with isoflurane, and the hearts were excised while still beating in sinus rhythm and immediately arrested in diastole by immersion in 10 ml of 30 mM KCl. They were then cleaned of any extraneous adherent tissue and weighed before being briefly chilled at −20°C to produce sufficient rigidity to facilitate cutting into defined sections. Each heart was cut into four slices of equal thickness perpendicular to the course of the left coronary artery, starting from the apex of the heart and ending at the position of the ligature around that artery. These slices were designated A, B, C, and D starting from the apex, and each was washed with pure water and then immersed in 0.02 M phosphate buffer, pH 7.4, containing 0.5 mg/ml of nitroblue tetrazolium (NBT; Sigma Chemical Co.) at 37°C for 30 min. NBT stains viable but not infarcted myocardium. After staining with NBT, the slices were washed briefly in cold water before fixation in 10% buffered formalin for 48 h, and the proximal cut surface of each slice was then imaged under standard conditions with a high resolution digital camera. The captured images were coded and analyzed ‘blind,’ without knowledge by the operators of the treatment received by the rat in question. Lines were drawn around the total area of the slice, the NBT-negative infarct zone, and any parts of the image to be excluded from the estimation, including valves, chordae tendinae, shadows, and edges, and the area of the infarct was determined as a percentage of the total area. The infarcts were confined to slices B, C, and D, and the mean percentage in these three was taken as the infarct size in each heart. Statistical significance of differences between infarct sizes in different treatment groups within each experiment was sought by one-way analysis of variance and by Bonferroni t tests. Complement depletion was induced in vivo by a single intraperitoneal injection of cobra venom factor at 250 U/kg. Human CRP or SAP was injected intraperitoneally at 40 mg/kg 1 h after coronary artery ligation and then at 24-h intervals until the rats were killed on day 5. The proteins were dissolved in 10 mM Tris and 140 mM NaCl, pH 8.0, and also 2 mM CaCl 2 in the case of human CRP, and the control rats received injections of the buffer alone at the same intervals. In one experiment, rats were killed 24, 48, and 72 h after coronary artery ligation. For analysis of the effect of cobra venom factor on plasma C3, the usual dose was given and normal, unoperated rats were bled from the tail at intervals up to 6 d. For measurement of plasma clearance of human CRP and SAP, normal unoperated rats received a single 40 mg/kg dose of each protein and were bled at intervals up to 72 h later. Plasma clearance of human CRP was also studied in the same protocol in normal unoperated rats that had received cobra venom factor 24 h previously. These experiments could not be performed in rats that had undergone coronary artery ligation because they do not survive the anesthesia required for bleeding, but there is no reason to suppose that the results in those rats would be significantly different. The formalin-fixed heart slices were embedded in wax and sectioned at 2-μm thickness for routine hematoxylin and eosin staining and for immunohistochemical staining. Optimal dilutions of the IgG fractions of monospecific goat anti–human CRP, rabbit anti–rat CRP, and sheep anti–rat C3 antisera, raised by immunization with the respective isolated pure antigens, were used as primary antibodies after blocking the sections by incubation in 20% vol/vol normal serum of the species of origin of the secondary antibody. In all cases, endogenous peroxidase activity was blocked by incubation with H 2 O 2 before testing. Secondary antibodies were optimal dilutions of goat anti–rabbit IgG or rabbit anti–goat IgG (both from DAKO Corp.), which cross-reacts with sheep IgG. Finally, rabbit or goat peroxidase–antiperoxidase complexes (DAKO Corp.) were used, followed by visualization with diaminobenzidine. All positive staining was completely abolished when the primary antibodies had been absorbed before use with an excess of the respective specific antigen. In the case of rat C3, which was not available in pure form, parallel absorptions of the sheep anti–rat C3 antibody were performed with normal rat serum and with C3-depleted serum from a cobra venom–treated rat. The latter did not affect anti-C3 staining, whereas C3-sufficient rat serum abolished it completely. To confirm that the antigens under investigation were not affected by formalin fixation and tissue processing, sample hearts were snap frozen without fixation, and 5-μm cryostat sections were cut for immunoperoxidase staining. For all antigens, appearances were identical to those of the fixed sections, except for poorer preservation of histological detail as expected. Frozen sections of a heart from a human CRP–treated rat killed on day 3 were also stained for human CRP by indirect immunofluorescence and the nuclei counterstained with ethidium bromide to visualize binding of CRP to cell surfaces. We have previously shown that rat CRP does not activate rat complement, whereas human CRP does so very efficiently 28 . The rat model therefore permits specific analysis of the complement-dependent effect of human CRP on infarct size in vivo. Rats that received 40 mg/kg of isolated pure human CRP by intraperitoneal injection 1 h after coronary artery ligation rapidly became clinically less well than buffer-treated controls, and some died during the next 3 d while receiving further daily injections of the same dose of human CRP. In contrast, rats that had not been operated on but received the same doses of human CRP showed absolutely no ill effects. Injections of human SAP, the pentraxin protein very closely related to CRP 30 , had no adverse clinical effects in either normal or coronary artery–ligated rats. When all surviving animals were killed on day 5 after coronary artery ligation, the infarcts in those receiving human CRP were ∼40% larger than in control rats treated with either buffer or human SAP ( Table ). The plasma clearance of human CRP and SAP after single intraperitoneal injections in control, nonoperated rats and the clearance of human CRP in decomplemented rats is shown in Fig. 1 . The clearance of human CRP was not affected by complement depletion. The peak values for human CRP were comparable to massive acute phase responses in humans 31 , and those for human SAP were much higher than ever seen in humans 32 . Rats that have undergone coronary artery ligation do not survive the anesthesia required for bleeding and were therefore not bled before they were killed on day 5. However, when these animals were killed 24 h after the last of five daily injections, the serum concentrations of human CRP were still in the range of 13–53 mg/liter, typical of a moderate clinical acute phase response in humans. In animals that had received human SAP, the serum concentrations of this protein were between 45 and 175 mg/liter. Values for rat CRP in serum at the time of exsanguination 5 d after coronary artery ligation were 215–525 mg/liter, which is within the normal range for this species 28 . Administration of cobra venom factor in vivo rapidly produces profound and sustained depletion of C3 29 , with no active C3 remaining in the circulation at 6 h. Traces of C3 antigen detectable thereafter are inactive cleavage fragments 33 . With the dose of cobra venom factor used here, 250 U/kg 29 , active C3 starts to reappear in the circulation after ∼4 d and is within the normal range by day 5 or 6. When rats had been decomplemented by in vivo administration of cobra venom factor 24 h before coronary artery ligation, their infarcts at 5 d were ∼60% smaller than those in control untreated animals ( Table ). Complement-sufficient rats injected daily with human CRP developed, as before, infarcts ∼40% larger than those in control untreated animals. However, injection of human CRP had no effect at all on the reduced infarct size in decomplemented rats ( Table ). The damaging effect of human CRP in this model is thus absolutely complement dependent. Human CRP and rat C3 and rat CRP (not shown) were all deposited in the infarcted myocardium. On day 5, human and rat CRP were present homogeneously throughout the infarcted muscle and also in a more intense, speckled pattern in multiple foci that occasionally coincided with hematoxyphil nuclear remnants. These foci may be nuclear ghosts from which chromatin has been cleared but that retain the small nuclear ribonucleoprotein particles to which human CRP binds avidly 34 35 and possibly other CRP ligands. Rat C3 was present predominantly in the same speckled foci, with no diffuse immunoreactivity on the infarcted muscle cells, in contrast to the distribution of CRP. Immunofluorescence staining of sections of unfixed, snap-frozen myocardial tissue taken 72 h after coronary ligation clearly demonstrated the presence of CRP on the surfaces of damaged myocardial cells in and around the infarct, as well as the same distribution seen in fixed sections stained by the immunoperoxidase method. Staining for human CRP was always more intense than for rat CRP, although it is not clear whether this represents greater abundance of the human protein in the tissue sections or just greater sensitivity of the respective immunostaining procedure. Numerous mononuclear cells in the dense periinfarct infiltrate also stained strongly for human CRP but not for rat CRP (not shown) or rat C3 . Marked reductions in infarct size and in ischemia/reperfusion injury have previously been demonstrated in animals in which complement activation had been blocked by treatment, either with cobra venom factor or with recombinant soluble complement receptor type 1, before or at the time of induction of ischemia 4 5 6 7 8 . The therapeutic implications of this observation are obvious, but for clinical implementation, it is critical to know whether complement depletion initiated after the onset of coronary occlusion can still have a protective effect. We therefore administered cobra venom factor at various times between 24 h before and 24 h after coronary artery ligation. There was, as shown before, a marked, ∼60% reduction in infarct size in rats that had received treatment 24 h beforehand and that were maximally complement depleted at the time of operation ( Table ). Although injection of cobra venom factor 6 or 24 h after ligation had no effect, the infarcts in animals that received cobra venom factor either 0.5 or 2 h after ligation were almost 50% smaller ( Table ). Maximal complement depletion is achieved only from 6 h after intraperitoneal injection of cobra venom factor, and in this rat model there is therefore a window of therapeutic opportunity of perhaps up to 8 h after acute coronary artery occlusion during which immediate inhibition of complement activation could potentially reduce infarct size. CRP has been very stably conserved in evolution 30 36 37 , and no structural polymorphism or deficiency of CRP has yet been reported in humans, suggesting that this protein has important normal functions that contribute to survival. In experimental models, CRP is protective against pneumococcal infection 38 39 and may contribute to innate immunity to other microorganisms to which it binds 40 . By analogy with this role of SAP in relation to chromatin that we have recently demonstrated 41 , CRP also probably plays an important role in scavenging autologous ligands and preventing development of autoimmunity. This does not mean, however, that CRP may not also contribute to pathogenesis of disease, especially conditions developing in postreproductive later life. Natural selection is ‘blind’ to phenomena occurring after reproduction, provided they do not affect viability of the species as a whole 42 . The results presented here unequivocally demonstrate, in a robust experimental model, that human CRP markedly enhances the extent of myocardial damage produced by ischemic injury. Although the time–concentration profile of human CRP produced by daily CRP injections was not the same as the monophasic acute phase response that follows uncomplicated naturally occurring myocardial infarction in humans, it was comparable to the persistent, high, and fluctuating CRP pattern typically found in patients with postinfarct complications 11 and was therefore not ‘unphysiological.’ Endogenous rat CRP, as well as the injected human CRP, was deposited in the infarct in vivo, but rat CRP does not activate rat complement 28 . In contrast, human CRP is a potent activator of rat complement 28 , and the enhancement of infarct size caused by administration of human CRP is completely abrogated by in vivo complement depletion. Human CRP production is always greatly increased after acute myocardial infarction, CRP is always deposited in human myocardial infarcts, and early and late clinical outcomes are significantly associated with peak postinfarction plasma levels of CRP. It is therefore very likely that CRP contributes significantly to the extent of damage in human acute myocardial infarction, and, based on our results here, it probably does so via complement activation. Although complement activation by CRP is not efficient in generating the terminal lytic complement complex, it very effectively cleaves C3, the critical step for opsonization by C3b and liberation of the C3a anaphylatoxin. These findings have important therapeutic implications, suggesting that a drug capable of inhibiting the binding of human CRP to its target ligands in vivo and thereby preventing it from activating complement should reduce infarct size, with corresponding clinical benefit. Furthermore, increased CRP production is a feature of the nonspecific acute phase response to a very wide range of traumatic, infectious, inflammatory, and neoplastic tissue-damaging conditions 31 . In all of these, including disorders as diverse as burns, surgical trauma, rheumatoid arthritis, sepsis, and invasive neoplasia, there are nonirremediably damaged cells that, by analogy with myocardial infarction, are likely to be targeted by the opsonic and proinflammatory actions of CRP and complement. Specific inhibition of CRP binding in vivo might thus be expected to be of wide clinical benefit, and suitable compounds are currently being sought, supported by our recent description of the high resolution 3-dimensional structure of the physiological CRP–ligand complex 43 . The cardioprotective effect of total complement depletion at the time of experimental coronary artery occlusion is well established 4 5 6 7 8 . However, in the clinical context of patients presenting with acute myocardial infarction, complement depletion could only be initiated after the onset of symptoms. We show here that injection of cobra venom factor up to 2 h after ligation of the coronary artery significantly reduces infarct size, despite the fact that maximal depletion of plasma C3 is only achieved 6 h after cobra factor administration. This encouragingly suggests that, after acute ischemia, the major pathogenic action of complement occurs after some hours, and there is therefore a window of therapeutic opportunity during which inhibition of complement activation could still reduce infarct size, with corresponding reduction in morbidity and mortality. Although no anticomplement drugs are yet in clinical use, our results support their development and testing. | Study | biomedical | en | 0.999996 |
10601350 | 8–10-wk-old female C57BL/6 mice were purchased from Japan SLC. Bone marrow (BM) cells were freshly prepared from femur and tibia and used as a source of hematopoietic precursors. 1,25-dihydroxyvitamin D 3 (1,25-[OH] 2 D 3 ) was provided by Dr. Ishizuka (Teijin Institute for Biomedical Research, Tokyo, Japan). Recombinant M-CSF and anti–mouse M-CSF neutralizing antibody were purchased from R & D Systems, Inc. Recombinant IL-7 and recombinant IL-3 were provided by Toray Industries Inc. Recombinant stem cell factor (SCF) was a gift from Chemo-Sero-Therapeutic Co., Ltd. Erythropoietin (Epo) was a gift from Snow-Brand Milk Product Co. Mice were killed by cervical dislocation, and the tibiae and femora were removed and dissected free from adhering soft tissues. The bone ends were cut off with a scalpel, and the marrow was flushed with α-modified (α-)MEM (GIBCO BRL) containing 10% FCS (JRH Biosciences). Mononuclear cells were isolated by centrifugation of total BM cells on Lymphep™ (Nycomed Pharmaceuticals) according to the manufacturer's instructions. A DNA fragment encoding the extracellular domain (Asp 76 –Asp 316 ) of RANKL was prepared using reverse transcriptase (RT)-PCR from total RNA of ST2 stromal cells that were cultured in the presence of 1,25-(OH) 2 D 3 (10 −8 M) for 4 d. PCR primers were as follows: 5′-CCGCTCGAGCGTCTATGTCCTGAACTTTGA-3′ (sense) and 5′-CCCAAGCTTGATCCTAACAGAATATCAGAAGACA-3′ (antisense). The PCR product was digested with HindIII and XhoI and ligated into the HindIII and XhoI sites of the pSecTag2 vector (Invitrogen Corp.) to yield pSecTag2–soluble (s)RANKL containing His 6 and myc tags. pSecTag2–sRANKL was transfected into COS7 cells cultured in DMEM (Life Technologies, Inc.) containing 10% FCS, and the supernatant was collected every 4 d for 12 d. sRANKL was purified from the supernatant using TALON and TALON Superflow Metal Affinity Resin (Clontech). The supernatant was applied to a chromatography column (Bio-Rad Labs.) filled with affinity resin, and the trapped proteins were eluted in 125 nM imidazol and fractionated. The high concentration fraction was collected and applied to a PD-10 column (Amersham Pharmacia Biotech, Inc.), eluted in PBS to eliminate the imidazol, and fractionated. Fractions containing a large amount of sRANKL were concentrated by a centricon concentrator (Amicon, Inc.). Mouse BM–derived stromal cell line ST2 (a gift from Dr. Hayashi, Tottori University, Yonago, Japan) was maintained in RPMI 1640 (GIBCO BRL) supplemented with 10% FCS with 10 −5 M of 2-ME (GIBCO BRL) at 37°C in humidified 5% CO 2 air. Cells were harvested every 3 d using 0.05% trypsin–EDTA (GIBCO BRL), and 10 5 cells were passaged on 100-mm culture dishes . The newborn calvaria-derived mouse stromal cell line OP9 (a gift from Dr. Kodama, Bayer Yakuhin Ltd., Kyoto, Japan) was maintained in α-MEM supplemented with 20% FCS at 37°C in humidified 5% CO 2 air. Cells were harvested every 3 d using 0.05% trypsin–EDTA, and 1.5 × 10 5 cells were resuspended on 100-mm cell culture dishes. The cell staining procedure for flow cytometry was performed as previously described 33 . The mAbs used in immunofluorescence staining were anti–c-Kit antibody (ACK2; a gift from Dr. S.-I. Nishikawa, Kyoto University, Kyoto, Japan), anti–Mac-1 (CD-11b) antibody (M1/70; PharMingen), anti–c-Fms antibody (AFS98; a gift from Toray Industries Inc., anti-RANK antibody (muRANK-M395; a gift from Immunex Corp.), anti-CD51 antibody (H9.2B8; PharMingen), and anti-B220 antibody (RA3-6B2; PharMingen). The mAbs used were either biotinylated or fluoresceinated. Biotinylated mAbs were detected with streptavidin-conjugated allophycocyanin (Caltag Labs.) or streptavidin-conjugated Red613 (GIBCO BRL). The following rat Igs were used as isotype controls: biotinylated IgG2a and IgG2b and fluoresceinated IgG2a and IgG2b (PharMingen). Cells were incubated for 15 min on ice with CD16/32 (FcγIII/II receptor; 1:100; FcBlock™; PharMingen) before staining with the first antibody. 10 6 cells/100 μl were suspended in 5% FCS/PBS (washing buffer). Cells were stained with the first antibody, incubated for 30 min on ice, and washed twice with washing buffer. The secondary antibody was added, and the cells were incubated for 30 min on ice. After incubation, cells were washed twice with washing buffer and suspended in washing buffer for FACS ® analysis. The stained cells were analyzed and sorted by FACSVantage™ (Becton Dickinson). Sorted cells were centrifuged onto microscope slides using a cytospin centrifuge (Shandon Southern Products) and stained with May-Grünwald-Giemsa staining solution (Merck KGaA). 5 × 10 3 ST2 cells were plated in 96-well plates (PRIMARIA; Becton Dickinson Labware) 1 d before coculture with sorted BM cells. Cultures were maintained in α-MEM/10% FCS with 10 −8 M 1,25-(OH) 2 D 3 and 10 −7 M dexamethasone (Dex). Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 4, and the TRAP solution assay (TRAP activity) was performed on day 7 or 10. Cocultures were scaled down to 100, 50, 40, 30, 20, 10, 8, 6, 5, 4, 2, and 1 cell in 100 μl for a limiting dilution assay. To assay osteoclast formation without a stromal layer, sorted BM cells were cultured in α-MEM/10% FCS in the presence of recombinant M-CSF (100 ng/ml) and sRANKL (25 ng/ml). After aspiration of medium, cells were fixed with 1% glutaraldehyde (Wako Pure Chemical Industries, Ltd.) in PBS for 15 min at room temperature. Cells were stained for TRAP using a histochemical kit (no. 387; Sigma Chemical Co.) according to the manufacturer's instructions. TRAP + cells were scored as osteoclasts microscopically. In the TRAP solution assay, enzyme activity was examined by the conversion of α-naphthyl phosphate (4 mmol/liter; Sigma Chemical Co.) to α-naphthol in the presence of 2 mol/liter l -tartrate solution (Sigma Chemical Co.) in each well. Absorbance was measured at 405 nm using a microplate reader (model 550; Bio-Rad Labs.). c-Kit + Mac-1 dull c-Fms − cells were cultured for 2 d in α-MEM/10% FCS in the presence of SCF (100 U/ml), and c-Fms expression was examined. Sorted c-Kit + Mac-1 dull c-Fms + cells derived from BM were cultured for 3 d in α-MEM/10% FCS plus either M-CSF (30 ng/ml) or IL-3 (100 U/ml), and the expression of RANK was analyzed. To investigate whether c-Kit + Mac-1 dull c-Fms − cells and c-Kit + Mac-1 dull c-Fms + cells differentiate into B cells, these cells were cocultured with OP9 stromal cells for 10 d in RPMI 1640/10% FCS in the presence of IL-7 (20 U/ml). After 10 d in culture, the expression of B220 was analyzed using FACSVantage™. An RNeasy mini Kit (QIAGEN GmbH) was used for isolation of total RNA from total BM cells or fractionated BM cells. Total RNA was reverse transcribed using an RT for PCR kit (Clontech). The cDNAs were amplified using an Advantage polymerase mix (Clontech) in a GeneAmp PCR system for 25–30 cycles. Sequences of gene-specific primers for RT-PCR were as follows: 5′-mRANK, CCAGGGGACAACGGAATCAG; 3′-mRANK, GGCCGGTCCGTGTACTCATC; 5′-mβ-actin, TCGTGCGTGACATCAAAGAG; and 3′-mβ-actin, TGGACAGTGAGGCCAGGATG. Each cycle consisted of 30 s denaturation at 94°C and 4 min annealing/extension at 70°C. To compare CFU-C (culture) activity between c-Kit + Mac-1 dull c-Fms ± cells, 10 3 cells were cultured in 1 ml of culture medium containing α-MEM, 1.2% methylcellulose (1,500 centipoise; Aldrich Chemical Co.), 30% FCS, 1% deionized BSA, 50 mM 2-ME, 100 U/ml IL-3, 2 U/ml Epo, and 100 ng/ml SCF. After 7 d in culture, aggregates of 50 or more cells were counted as a single colony. For in vitro osteoclast colony formation assay, 10 3 R3 cells were plated in methylcellulose medium. In brief, cells were embedded in 1 ml of 1.2% methylcellulose, 30% FCS, 1% deionized BSA, 50 mmol/liter 2-ME, and 100 ng/ml M-CSF in the presence or absence of sRANKL (25 ng/ml) in α-MEM. The culture dishes were incubated in humidified atmosphere at 37°C with 5% CO 2 . After 7 d, the colonies were counted, picked up, and stained for nonspecific esterase, May-Grünwald-Giemsa, and TRAP. To characterize and isolate osteoclast precursor cells, expression of cell surface markers was analyzed . Of the c-Kit + BM mononuclear cells, 37.5% were c-Fms + and 56.1% were Mac-1 + . Using contour blot analyses, four populations of c-Kit + cells were detected: Mac-1 dull c-Fms + (R3), Mac-1 high c-Fms + (R4), Mac-1 dull c-Fms − (R5), and Mac-1 high c-Fms − (R6). These cells were fractionated, and further analyses were performed. The percentages of these populations in both BM mononuclear cells and c-Kit + cells is shown in Table . The morphology of cells of each fraction was examined by May-Grünwald Giemsa staining . R3 and R5 cells were immature cells showing a large N/C ratio, whereas most of the R4 and R6 cells were mature cells, including macrophages or neutrophils. TRAP is an enzyme highly expressed in both immature and mature osteoclasts. To determine which fractionated cells differentiate into TRAP + cells, 10 3 cells of each fraction were cocultured with an ST2 stromal cell line in the presence of 1,25-(OH) 2 D 3 and Dex . To detect TRAP + mononuclear or multinuclear osteoclasts, TRAP staining was performed on day 4 and TRAP activity (TRAP solution assay) was measured on days 7 and 10 . The number of TRAP + cells in the R3 fraction was 10-fold higher than observed in unfractionated cells . Relative TRAP activity (TRAP activity in each cell fraction/TRAP activity in unfractionated BM mononuclear cells) was highest in fraction R3 on day 7. In contrast, on day 10, the highest TRAP activity was detected in fraction R5 (1.55 ± 0.20) compared with R3 (1.17 ± 0.01). The R4 and R6 fractions showed low TRAP activities on both days 7 and 10. These data suggest that the R3 fraction contains a higher proportion of osteoclast precursor cells than do other populations (R4, R5, and R6) and that R5 cells also contain osteoclast precursor cells that are less mature than R3 cells. To investigate whether R5 cells (c-Kit + Mac-1 dull c-Fms − ) differentiate to R3 cells (c-Kit + Mac-1 dull c-Fms + ), the expression of c-Fms was analyzed after cultivation . c-Fms − cells in R5 or R6 were sorted and cultured in SCF (100 ng/ml), as they expressed c-Kit receptors. After 2 d in culture, 42.2% of cultured R5 cells (R5′) expressed c-Fms and 7.1% of c-Fms + cells were also c-Kit + . In contrast, of cultured R6 cells (R6′), 9.3% were c-Fms + cells and 0.4% were c-Kit + c-Fms + cells. Moreover, of R5′ cells, c-Kit + c-Fms + cells were mainly Mac-1 dull . To determine if c-Fms + cells in R5′ or R6′ could undergo osteoclastic differentiation, both cell fractions were cocultured with ST2 stromal cells for 4 d in the presence of both 1,25-(OH) 2 D 3 and Dex and assayed for TRAP activity . Although c-Fms + cells of R5′ differentiated to osteoclasts, c-Fms + cells of R6′ did not differentiate into TRAP + cells. R6′ cells were mature granulocytes and macrophages. To analyze the proportion of osteoclast precursor cells in unfractionated BM cells, R3, R5, or c-Kit + c-Fms + R5′ cells, each fraction was cultured for 4 d on ST2 stromal cells and scored for TRAP + cells by limiting dilution analysis . Limiting dilution of BM cells, R3, R5, and c-Kit + c-Fms + R5′ cells revealed 2.0% (1/49.3; unfractionated BM), 20.4% (1/4.9; R3), 3.4% (1/29.8; R5), or 16.4% (1/6.1; c-Kit + c-Fms + R5′) TRAP + cells. R3 or c-Kit + c-Fms + R5′ cells contained 10-fold more osteoclast precursor cells than did unfractionated BM cells. These data suggest that c-Fms − cells differentiate to osteoclasts through the induction of c-Fms. c-Fms expression increased during cultivation. As c-Kit + c-Fms + R5′ cells contained the same proportion of osteoclast precursor cells as did R3 cells, we speculated that R5 cells are less mature than R3 cells. To examine the capacity of those osteoclast precursor cells to differentiate to other lineages, a colony assay using R3 or R5 cells was performed ( Table ). 10 3 cells from each fraction were cultured with methylcellulose semisolid medium in the presence of SCF (100 ng/ml), IL-3 (100 U/ml), and Epo (2 U/ml) for 7 d. The numbers of colonies (CFU-GEMM [granulocyte, erythrocyte, macrophage, megakaryocyte] burst forming unit-erythrocyte, CFU-GM [granulocyte, macrophage], CFU-G, and CFU-M [macrophage]) were counted under an inverted microscope. Most colonies derived from R3 cells were macrophage colonies. In contrast, R5 cells contained more CFU-GM than CFU-M. Moreover, CFU-GEMM or burst forming unit-erythrocyte–derived colonies were observed in R5 cells. To analyze the ability of R3 or R5 to differentiate into a B cell lineage, 10 4 cells of each fraction were cocultured with the OP9 stromal cell line and IL-7 (20 U/ml; Table ). After 10 d in culture, the number of expanded nonadherent cells was 73-fold more in R5 (7.9 ± 0.6 × 10 6 cells) than in R3 (1.0 ± 0.2 × 10 5 cells), and 47.0% of R3 or 93.9% of R5 cells were B220 + cells. These data suggest that R5 cells are more immature than R3 cells and that R5 cells can differentiate not only into osteoclast but also into myeloid, erythroid, or B cell lineages. Expression of RANK in fractionated cells was examined by RT-PCR and FACS ® . To examine whether M-CSF induces RANK mRNA expression, unfractionated BM mononuclear cells were cultured for 72 h in the presence of IL-3 (100 U/ml) or M-CSF (30 ng/ml). Both IL-3 and M-CSF are able to support the differentiation of macrophagic differentiation. The expression of RANK mRNA was detected in a 24-h incubation with M-CSF and a 72-h incubation with IL-3 . Subsequently, fractionated cells were cultured with M-CSF for 48 h. Before induction with M-CSF, very low levels of RANK mRNA were detected in all fractionated cells except R6 cells . After incubation for 48 h, the expression of RANK mRNA was obvious in the R3 fraction. FACS ® analyses demonstrated that 5.4% of unfractionated BM cells were RANK + (data not shown). Of R3 cells, 15.7% were RANK + , and 1.5% of R5 cells were RANK + . The expression of RANK protein in R3 or R5 cells was analyzed by FACS ® after incubation with M-CSF for 24 or 72 h . The percentage of RANK + cells in R3 increased with longer incubations with M-CSF (41.3% for a 24-h incubation and 58.4% for a 72-h period). Although a similar increase was also observed in R5 cells, the overall percentage of RANK + R5 cells was lower than that of R3 cells (2.6% after a 24-h incubation and 11.5% after 72 h). To characterize RANK + or RANK − cells in the R3 fraction cultured with M-CSF, cells were sorted with a RANK mAb, and the expression of c-Kit, Mac-1, and c-Fms was analyzed. After 24 h in culture with M-CSF, sorted RANK + cells were c-Kit − Mac-1 high c-Fms + , and sorted RANK − cells were c-Kit − Mac-1 dull c-Fms + (data not shown). RANK expression in R3 cells was induced by stimulation of M-CSF; however, R3 cells originally contained RANK + cells. To examine whether M-CSF induces the expression of RANK on RANK − R3 cells, R3 cells were subdivided into RANK + and RANK − fractions. RANK − R3 cells were cultured in the presence of M-CSF with or without anti–mouse M-CSF neutralizing antibody, and the expression of RANK was analyzed by RT-PCR. The expression of RANK was detected in freshly isolated RANK + R3 cells and cultured RANK − R3 cells. However, RANK expression was significantly reduced by the addition of mouse M-CSF neutralizing antibody to the culture . These results indicate that M-CSF stimulates the expression of RANK on osteoclast precursor cells. Next we analyzed how osteoclast precursor cells differentiate into TRAP + cells in the presence of M-CSF and sRANKL rather than ST2 stromal cells. Fractionated R3 cells were cultured for 72 h in IL-3 (100 U/ml) or M-CSF (30 ng/ml). As shown in Fig. 4 A, M-CSF induced RANK expression more efficiently than did IL-3. RANK + or RANK − cells precultured with IL-3 or M-CSF for 72 h were sorted and cultured in the presence of sRANKL and either IL-3 or M-CSF. The percentage of TRAP + cells is shown in Fig. 4 B. Both IL-3– and M-CSF–precultured cells differentiated into TRAP + cells in the presence of sRANKL and M-CSF. Moreover, RANK − cells in each preculture condition showed a higher percentage of TRAP + cells than RANK + cells in the presence of sRANKL and M-CSF. M-CSF–precultured cells showed a higher percentage of TRAP + cells than did IL-3–precultured cells. To examine whether RANK + cells differentiate to TRAP + cells in the presence of sRANKL alone, primary R3 cells or R3 cells precultured with M-CSF were cultured for 2, 4, or 6 d and analyzed for TRAP staining. RANK + cells precultured for 24 h differentiated into TRAP + cells, and the percentage of TRAP + cells decreased in the presence of sRANKL alone . However, primary R3 cells (RANK + or RANK − ) or RANK − cells precultured for 24 h did not differentiate into TRAP + cells with sRANKL alone. No TRAP + cells were observed when cells were cultured for 6 d with M-CSF alone (data not shown). These data suggest that M-CSF affects not only the survival factor but also the competence factor for osteoclast precursor cells during their differentiation. To clarify the synergistic effect of M-CSF and sRANKL, the relationship between the onset of RANK expression and osteoclast differentiation was examined. Fig. 5 shows the flow chart of cell sorting and conditions of further cultivation. Sorted RANK + or RANK − cells from the primary R3 fraction were cultured for 2, 4, and 6 d with both sRANKL and M-CSF . On days 2 and 4, the percentage of TRAP + cells was higher in RANK − cells (11.8 ± 1.9% on day 2 and 97.5 ± 1.4% on day 4) than in RANK + cells (7.0 ± 1.7% on day 2 and 42.5 ± 6.1% on day 4). On day 6, however, the percentage of TRAP + cells was similar in RANK + and RANK − cells. Interestingly, the percentage of TRAP + cells in R3 cells precultured for 24 h with M-CSF was similar between RANK + and RANK − cells , whereas in the case of R3 cells that were not precultured, RANK + cells did not efficiently differentiate into TRAP + cells . RANK − cells showed a higher percentage of MNCs, which are fully matured osteoclasts, than did RANK + cells. These MNCs from RANK − cells were extremely large and contained a large number of nuclei. Growth of RANK + and RANK − cells after 72 h of preculture with M-CSF was less than that of primary or 24-h–precultured R3 cells . Although the percentage of TRAP + cells was higher in RANK − cells than RANK + cells on day 4 (9.16 ± 4.3% in RANK + and 95.6 ± 1.8% in RANK − cells), the percentage was similar on day 6 . A delay in differentiation of TRAP + cells from RANK + cells was observed. Also RANK − cells showed a higher percentage of TRAP + MNCs than did RANK + cells, but this number was lower than that observed in RANK − cells precultured for 24 h. Moreover, the size of MNCs was reduced and the number of nuclei contained in MNCs was smaller in RANK − cells precultured for 72 h than in cells precultured for 24 h. In all culture conditions, especially 24-h preculture with M-CSF, the potential of cell growth of RANK − cells was greater than that of RANK + cells. Also, RANK − cells formed a large number of MNCs, which contained high numbers of nuclei because of the increment of cell density induced by cell proliferation. In addition, when RANK + and RANK − cells were precultured with M-CSF for 24 h, a higher number of TRAP + cells was observed in the latter, suggesting that RANK expression is necessary but not sufficient for osteoclast differentiation and that the timing of RANKL binding to RANK + cells may be critical for commitment of osteoclast cells. Fractionated cells (R3 or R5) can differentiate to lineages other than osteoclast ( Table and Table ); however, it is not clear whether a single osteoclast precursor cell is committed to differentiate into a TRAP + cell or whether it can differentiate into other lineages. To understand the mechanism of osteoclast differentiation, R3 cells were cultured in methylcellulose instead of liquid culture . 10 3 R3 cells were cultured with methylcellulose medium containing M-CSF (100 ng/ml) in the presence or absence of sRANKL (25 ng/ml) for 7 d. In the absence of both cytokines, colony formation was not detected, whereas 139 ± 14.5 and 131 ± 1.7 colonies were observed in M-CSF alone and in M-CSF and sRANKL, respectively ( Table ). Whereas colonies formed in the presence of M-CSF alone were tightly compacted , colonies observed in M-CSF and RANKL were of mixed type . Individual colonies in the presence of M-CSF and sRANKL contained not only nonspecific esterase–positive macrophages but also TRAP + cells . The percentage of TRAP + cells in 20 colonies was 0 ± 0% (M-CSF alone) and 43.8 ± 30.4% (13.3–94.2%; M-CSF and sRANKL; Table ). Any homogeneous colonies consisting of all TRAP + cells were not observed. These data strongly suggest that single precursor cells can differentiate into TRAP + cells and macrophages at a late stage of osteoclast differentiation. Here we identify early and late stages of osteoclast precursor cells and describe the differentiation pathway of osteoclasts from hematopoietic precursor cells by coculture of those cells with the ST2 stromal cell line and 1,25-(OD) 2 D 3 . We also analyze the osteoclast commitment process by substituting M-CSF and sRANKL for the stromal cells. Hematopoietic precursor cells exist in the c-Kit + fraction 34 35 . This fraction was clearly subdivided by the expression of c-Fms and Mac-1. Mac-1 + cells contain mainly mature granulocytes and macrophages, whereas Mac-1 dull cells are multipotential progenitor cells 36 37 . We demonstrate that c-Kit + Mac-1 dull c-Fms + cells in murine BM are early stage precursors of osteoclasts using a limiting dilution method. These cells are shown to be derived from c-Kit + Mac-1 dull c-Fms − cells. We show directly that c-Fms − cells differentiate into c-Fms + cells after 2 d in culture with SCF. It has been previously reported that c-Fms is a key determinant in the differentiation of monocyte–macrophage lineage cells 38 . c-Fms expression is regulated by a tissue-specific promoter. Although the precise mechanism of c-Fms expression is not known, transcription factors c-ets-1, c-ets-2, and PU.1 mediate induction of c-Fms 39 40 41 . Thus, it is reasonable that c-Kit + Mac-1 dull c-Fms + cells differentiate into TRAP + osteoclasts after 4 d, whereas c-Kit + Mac-1 dull c-Fms − cells require >7 d. In addition, c-Kit + Mac-1 dull c-Fms + cells express the RANK mRNA and protein in 24 h in the presence of M-CSF. Lacey et al. 21 demonstrated that osteoclast precursor cells were identified by sRANKL–FITC. They sorted sRANKL–FITC + cells after cultivation of mouse BM cells in the presence of M-CSF and sRANKL for 1 d, and these precursor cells were therefore regarded as RANK + cells. These sRANKL–FITC + cells formed a larger number of multinucleated TRAP + cells than sRANKL–FITC − cells. In contrast to this finding, we found that a large number of multinucleated TRAP + cells were derived from RANK − rather than RANK + cells, regardless of precultivation with M-CSF . By cultivation with M-CSF and sRANKL for 1 d before cell sorting, it is speculated that sRANKL–FITC + cells have already been committed to osteoclast lineage. We show a sequential change of phenotype in the differentiation pathway of osteoclasts . Although an mAb against c-Fms suppresses osteoclastogenesis 42 , most osteoclast precursors in BM are c-Kit + c-Fms − , and their numbers decrease along with expression of c-Fms 43 . This observation is based on an assay of TRAP + cells on day 6 of culture, which is consistent with our observation. We show that a later stage of osteoclast precursor cells (c-Fms + RANK + ) differentiates into TRAP + osteoclasts in 2 d. It was reported that osteoclast precursor cells were significantly higher in the c-Kit low fraction, whereas myeloid cells of other lineages were higher in the c-Kit high fraction 44 . However, the c-Kit high fraction contained a similar number of osteoclast precursor cells 45 . We showed that c-Kit + Mac-1 dull c-Fms − cells can differentiate not only into the myeloid lineage but also into B220 + B cells in the presence of IL-7. On the other hand, c-Kit + Mac-1 dull c-Fms + cells differentiate into osteoclasts at a frequency of 1:5 on stromal cells. Stromal cells such as ST2 express RANKL, and its expression can be upregulated by the bone-resorbing factors 1,25-(OH) 2 D 3 , IL-11, prostaglandin E2, and parathyroid hormone 46 . However, the ST2 coculture system with 1,25-(OH) 2 D 3 does not maximally stimulate osteoclastogenesis, as ST2 also produces the inhibitory molecule OPG/OCIF, which belongs to the TNFR family 47 48 . We investigated the commitment process of c-Kit + Mac-1 dull c-Fms + cells to osteoclasts using M-CSF and sRANKL instead of stromal cells. Our data suggest that M-CSF stimulates c-Kit + Mac-1 dull c-Fms + cells to induce RANK mRNA and protein in 24 h more efficiently than does IL-3. These cells can differentiate into osteoclasts in the presence of both M-CSF and sRANKL. With M-CSF only, they differentiate into macrophages but not into osteoclasts. RANKL is a differentiation factor for osteoclasts but not an exclusive osteoclast commitment factor, as RANK is expressed not only in osteoclasts but in T cells and dendritic cells 24 . Mice with a disrupted RANKL ( opgl ) gene show severe osteopetrosis and lack all lymph nodes 32 , suggesting that RANKL–RANK signaling plays several roles in organogenesis. Preculture of c-Kit + Mac-1 dull c-Fms + cells with M-CSF for 24 or 72 h results in different fates for osteoclast precursors. RANK + cells after 24-h preculture differentiate into osteoclasts more efficiently than after 72-h preculture. RANK + cells may autonomously differentiate into the macrophage lineage in the absence of sRANKL for 72 h. By contrast, in the presence of both sRANKL and M-CSF after 24-h preculture with M-CSF, RANK + cells efficiently differentiate into osteoclasts. RANK − cells, after preculture with M-CSF, may differentiate into RANK + cells if they are continuously exposed to M-CSF. Once the cells express RANK, existing RANKL binds to the receptors. This is the most productive system for osteoclast differentiation, which may take place in vivo on bone surfaces. Thus, RANK − cells differentiate into osteoclasts more efficiently than RANK + cells. Moreover, as their proliferative activity is higher, multinuclear osteoclast formation from RANK − cells is greater than that of RANK + cells. This is dependent upon the cell density (data not shown). c-Kit + Mac-1 dull c-Fms + cells differentiate exclusively into osteoclasts (∼100%) in the presence of both M-CSF and sRANKL. As even RANK + cells can differentiate into macrophages, we conclude that the stage of commitment of osteoclasts is late in the differentiation process. A clonal assay using colony formation in semisolid methylcellulose medium provided us with precise identification of constituent cells and revealed no pure osteoclast colonies in the presence of both M-CSF and sRANKL. A single colony contained 13.3–94.2% TRAP + osteoclasts. This observation suggests the very low incidence of osteoclast colony forming cells, which are exclusively committed to osteoclasts. The growth of osteoclasts in methylcellulose is poorer than that observed in a liquid culture system, which enables osteoclasts to attach to the culture dish. Cell anchoring or polarity is critical for osteoclast differentiation. A study of cell adhesion molecules expressed by osteoclasts is underway. Our findings suggest that M-CSF plays three roles in osteoclastogenesis: (a) it induces RANK, (b) it is a competence factor for differentiation, and (c) it stimulates cell survival and proliferation . Although upregulation of RANK by M-CSF is higher than that by IL-3 and GM-CSF (data not shown), RANK induction is not M-CSF specific. Even though expression of RANK on RANK − cells was upregulated in the presence of M-CSF and its expression was significantly inhibited by addition of neutralizing M-CSF antibody , further analysis in association with the signaling pathway would be necessary to elucidate the mechanism of c-Fms–RANK interaction. As shown in Fig. 4 C, TRAP + cells differentiated from M-CSF–precultured RANK + cells but not from primary RANK + cells in the presence of sRANKL alone, suggesting that unknown events induced by M-CSF are competent for differentiation to osteoclasts. Without M-CSF, RANKL–RANK could not induce osteoclasts even in the presence of IL-3. It will be interesting to define the molecular event induced by M-CSF in precursors. As RANK belongs to the TNFR family and does not stimulate cell proliferation, M-CSF–precultured RANK + cells could not survive and proliferate without the continuous presence of M-CSF. It has been shown that RANKL stimulates mature osteoclasts to activate bone resorption 49 . In the absence of RANKL, precursor cells autonomously differentiate into macrophages in the presence of M-CSF, a situation regarded as the default pathway of macrophagic differentiation. The determination of osteoclastic differentiation is achieved to avoid the default pathway of macrophagic differentiation from the signal of the RANK–RANKL system . Spontaneous recovery of adult op/op mice suggests the presence of unknown molecules 50 51 . The osteoclast defect in M-CSF mutant osteopetrotic mice can be rescued by overexpression of the antiapoptotic protein Bcl-2 in the monocyte lineage 14 . Although M-CSF is an indispensable factor for osteoclastogenesis, other molecules may induce RANK less efficiently than M-CSF. It is noteworthy that the intracellular domain of RANK directly binds TRAF2, TRAF5, and TRAF6 26 28 , and TRAF6 deficiency results in osteopetrosis 29 . In conclusion, we have identified osteoclast precursor cells and clarified the function of M-CSF and RANKL. The cooperation of these two factors is critical for osteoclast differentiation. Osteoclastogenesis is thus a unique model system in the lineage determination of blood cell differentiation. | Study | biomedical | en | 0.999997 |
10601351 | FITC-conjugated mAbs used were anti-CD1a, anti-CCR5, and anti–B7-2 (PharMingen); anti-CD2 and anti-CD11b (Becton Dickinson); and anti-CD3 (Immunotech). PE-conjugated mAbs included anti-CD14 (Becton Dickinson), anti-CD1a and anti-CXCR4 (PharMingen), and anti-CCR2 and anti-CCR6 (R&D Systems). The peridinin chlorophyll protein (PerCP)-conjugated anti-CD34 and anti-CD3 mAbs were from Becton Dickinson, and anti-CD1a CyChrome was from PharMingen. Biotin-conjugated anti-CCR1 was from R&D Systems. The anti–E-cad mAb (Immunotech) was biotinylated according to standard protocols. The binding of biotinylated mAbs was revealed either by PE- (Becton Dickinson) or Cy5 R-phycoerythrin (RPE)–conjugated streptavidin (SA-Cy5; Dako). Isotype control mAbs included biotinylated, FITC-, PE-, or PerCP-labeled mouse IgG1 or IgG2a (Becton Dickinson). Recombinant human (rh)GM-CSF and IL-4 were from Novartis Forschungsinstitut. rhTNF-α and rhFlt3-ligand (Flt3-L) were obtained from Genzyme and Serotec, respectively. rhMIP-1α, MIP-3α, MIP-3β, SDF-1α, RANTES, and MCP-1 were obtained from R&D Systems. Cord blood (CB) was obtained according to institutional guidelines. CD34 + cells were separated from CB-MNCs by a positive immunoselection procedure (CD34 MultiSort Kit; Miltenyi Biotec). In brief, CB mononuclear cells (MNCs [1–2 × 10 8 ]) were incubated with anti-CD34 mAb–coated paramagnetic microbeads for 30 min at 4°C. After several washings, bead-bound CD34 + HPCs were isolated on MiniMACS separation columns using a magnet (MiniMACS; Miltenyi Biotec). CD34 + cells (0.5–1.5 × 10 6 ) were recovered at a purity of >95%, as determined by immunostaining with a PerCP-labeled anti-CD34 mAb (clone HPCA-2) recognizing a CD34 epitope distinct from that bound by the mAb used for immunoselection. CD34 + HPCs were cultured in RPMI 1640 medium containing 10% FCS (both from GIBCO BRL) supplemented with 200 U/ml GM-CSF, 50 U/ml TNF-α, and 50 ng/ml Flt3-L. CD34 + HPCs were cultured in 75-cm 2 tissue culture flasks (Costar) at a density of 1–2 × 10 4 /ml. At day 3 or 4, cell suspensions were split and diluted in fresh RPMI/10% FCS supplemented with GM-CSF and TNF-α. At day 10, cells were collected and resuspended in fresh cytokine-conditioned medium at a density of 1–2 × 10 5 /ml, and further cultured until days 12 and/or 14. Where indicated, cells were harvested at day 6 and labeled with anti-CD1a–FITC and anti-CD14–PE. CD1a + CD14 − cells (24.6 ± 2.0% of the total population, mean ± SEM, n = 20) and CD1a − CD14 + cells (36.1 ± 2.2%) were isolated using a FACStar PLUS™ flow cytometer (Becton Dickinson). The purity of the sorted cell populations was always >98%. Sorted cells were either used in chemotaxis assays, subjected to lysis and mRNA extraction, or further propagated in the presence of GM-CSF and TNF-α until days 12 and/or 14. Normal human skin was obtained from patients undergoing plastic surgery upon informed consent. Keratomed split-thickness skin was incubated in dispase (50 U/ml; Collaborative Biomedical Products) for 1 h at 37°C, or overnight at 4°C. Thereafter, epidermal sheets were peeled off and exposed to trypsin/0.05% EDTA (GIBCO BRL) containing 0.025% DNase 1 (Sigma Chemical Co.) for 15 min at 37°C, and cell suspensions were prepared by mechanical agitation. Residual aggregates and cellular debris were removed by filtering and density gradient centrifugation on Lymphoprep (Nycomed Amersham plc). For cell sorting experiments, epidermal cell suspensions were exposed to anti-CD11b/CD2–FITC, anti-CD1a–PE, and anti-CD3–PerCP. After several washings, CD1a + CD2 − CD3 − CD11b − LCs and keratinocytes (CD1a − CD2 − CD3 − CD11b − cells) were isolated using a FACStar PLUS™ flow cytometer. Resulting cell populations were resorted to yield purities of >99%. In some experiments, sorted epidermal LCs or LC-enriched epidermal cell suspensions were cultured for the indicated time periods in GM-CSF (200 U/ml)– and TNF-α (50 U/ml)–conditioned RPMI 1640/10% FCS. PB-DCs were prepared as described previously 23 24 . In brief, PB-MNCs from healthy donors were prepared by density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech) and subjected to counter current elutriation centrifugation (JE-6B elutriation system; Beckman Instruments ). From the resulting cell population, T, B, and NK cells, HPCs, monocytes, and basophils were removed by anti-CD3/CD11b/CD16/CD19/CD34/CD56 immunolabeling and anti–mouse IgG immunomagnetic depletion (MACS; Miltenyi Biotec). PB-DC–enriched cell populations were further reacted with anti-CD11c–FITC, anti-CD13–PE, and anti-CD4–Cy5 mAbs, and CD11c + CD13 + CD4 + cells were isolated on a FACStar PLUS™ flow cytometer. Purified CD11c + PB-DCs were seeded at a density of 0.5–1 × 10 6 /ml in 96-well flat-bottomed microtiter plates (Costar) in RPMI 1640/10% AB serum supplemented with GM-CSF (1,000 U/ml) and IL-4 (800 U/ml). Dermal microvascular ECs (DMECs) were isolated from human foreskins according to a protocol approved by the institutional ethics committee. Foreskins were cut into 1-cm 2 pieces and exposed to dispase (25 U/ml) for 20 min at 37°C. Cells recovered after gentle teasing of the tissue were resuspended in low-serum endothelial cell medium (PromoCell) and plated on dishes coated with fibronectin (1 μg/ml; Endogen, Inc.). For passaging, cells were recovered from the dishes by trypsin/0.05% EDTA treatment. Cells were used for further experiments at passage four. Human umbilical vein ECs (HUVECs) were isolated as described previously 25 and passaged in IMDM/20% FCS, streptomycin (100 μg/ml), penicillin (100 U/ml), l -glutamine (2 mM; all from GIBCO BRL), EC growth factor (50 μg/ml; Collaborative Biomedical Products), and heparin (5 U/ml; Sigma Chemical Co.). Activated DMECs and HUVECs were prepared by exposing the cells for 6 h to 50 ng/ml IFN-γ (Endogen, Inc.). Primary human cells isolated and cultured as described above were lysed at a cell density of 4 × 10 6 /100 μl in Tris/lithium-dodecylsulfate (LiDS)-containing buffer (100 mM Tris-HCl, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol, pH 8.0) for 5 min at 4°C. Poly(A) RNA was isolated using oligo (dT) 25 -conjugated Dynabeads (Dynal) according to the manufacturer's recommendations. In brief, 30 μl of the Dynabead oligo (dT) 25 suspension was added to 100 μl cell lysate, and beads were allowed to hybridize with mRNA poly(A) tails for 5 min at room temperature. Bead-bound poly(A) RNA was retrieved using a magnetic particle concentrator (Dynal), and mRNA was eluted for 3 min at 65°C in H 2 O. Reverse transcription (RT) of purified mRNA was performed using the avian myeloblastosis virus (AMV) first strand cDNA synthesis kit (Boehringer Mannheim). In brief, eluted mRNA was incubated with 10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl 2 , 1 mM dNTP, 0.04 A 260 U Oligo-p(dT) 15 primer, 50 U RNase inhibitor, and 20 U AMV reverse transcriptase for 1 h at 42°C. Thereafter, the reverse transcriptase was inactivated by heating the reaction mix to 99°C for 5 min. PCR amplifications were performed in 50 μl reaction buffer (20 mM [NH 4 ] 2 SO 4 , 75 mM Tris-HCl, pH 8.8, 0.01% Tween 20, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, and 2.5 U thermostable DNA polymerase; all from Advanced Biotechnologies Ltd.), and 20 pM of each primer. Sequences of primer pairs used are given in Table . cDNA derived from mRNA of 10 4 cells was used as template for individual PCR reactions. Amplified products were subjected to 2% agarose gel electrophoresis and visualized by ethidium bromide (Sigma Chemical Co.) staining. Chemotaxis assays were performed as described previously 26 . Cells, either directly recovered from cultures at the indicated days or FACS ® sorted and recultured overnight in GM-CSF/TNF-α–conditioned medium, were washed and resuspended in migration buffer (HBSS [GIBCO BRL], 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% BSA [Sigma Chemical Co.)] at a density of 2–3 × 10 6 cells/ml. 600 μl of chemokine solution or buffer alone was added to individual wells of 24-well plates (Costar) on ice. Immediately thereafter, Costar transwell devices with 5-μm pore size, polyvinylpyrrolidone-free polycarbonate membranes were inserted into the wells, and 100 μl cell suspension was layered on top of the membrane. In experiments addressing directional versus random migration, different concentrations of chemokines were placed above and/or below the membrane. Cells were allowed to attach to and transmigrate through the membrane for 2 h at 37°C. The fluid phase above the membrane was then removed, transwell inserts were taken out of the wells, and membrane-bound cells were stained with Hemacolor (Merck) for enumeration, or were fixed with 4% paraformaldehyde (PFA; Fluka AG) for 30 min at 4°C, washed, and processed for confocal laser scanning analysis as described below. Non–membrane-bound, transmigrated cells were recovered by complete aspiration of the remaining solution in the well. These samples (i.e., 600 μl) were pooled with the eluates obtained after two washes of the same well with 200 μl HBSS. Complete removal of cells from the wells was checked by light microscopy. 10% of the volume (i.e., 100 μl) of individual samples was subjected to cell enumeration on a FACScan™ (Becton Dickinson). Using appropriate forward scatter threshold settings, all cells contained in individual samples were acquired. The number of cells fulfilling the same forward/side scatter criteria as the cells before migration was multiplied by 10 to reveal the absolute number of migrated cells. Cell numbers calculated by this flow cytometry measurement correlated well with those obtained by conventional trypan blue exclusion counting using a hemocytometer (data not shown). Cells recovered from the remaining 9/10 of the samples were double/triple-stained with anti-CD1a– FITC and anti-CD14–PE (cells recovered from 6-d cultures) or with anti-CD1a–FITC, anti-CD11b–PE, and biotinylated anti–E-cad/SA-Cy5 (cells recovered from 12- or 14-d cultures). After PFA fixation, cells bound to the transwell membrane were stained with 1 μg/ml propidium iodide in PBS/0.001% saponin (both from Sigma Chemical Co.) for 1 h at room temperature. Membranes were then cut out of the transwell inserts, mounted onto slides, and embedded in VectaShield medium (Vector Laboratories). Slides were examined using a confocal laser scanning microscope system (LSM 410; Carl Zeiss, Inc.) equipped with a laser emitting light at 488 nm. Membranes were scanned horizontally by acquiring 32 sections within a depth of 64 μm. Vertical images were obtained by three-dimensional transformation of data obtained by horizontal sectioning. For comparison, cell migration was also evaluated using the microchamber chemotaxis assay (NeuroProbe) as described previously 27 . Chemokines were diluted as described above, and 30 μl of chemokine solution or buffer alone was pipetted into individual wells of the 48-well bottom chamber. Wells were covered with a 5-μm pore size, polyvinylpyrrolidone-free polycarbonate membrane and a plate forming the upper compartments. 50 μl of cell suspension (10 6 cells/ml) was placed into the upper compartment of each well. Chambers were incubated for 2 h at 37°C, filters were harvested, nonmigrated cells were removed, and the migrated filter-bound cells were stained with Hemacolor. Chemokine-induced cell migration was analyzed by computer-assisted counting (Optomax V; Bestobell Mobrey) of the cells adherent to the bottom side of the membrane. For two- or three-color immunolabeling, cells were washed twice in ice-cold PBS and incubated in 50 μl PBS containing the appropriate biotin- and/or fluorochrome-labeled mAbs (2.5 μg/ml each) for 30 min on ice. For the detection of cell-bound biotinylated mAbs, cells were further exposed to 1 μg/ml SA-PE or SA-Cy5. At least 10,000 cells were analyzed on a FACScan™ (Becton Dickinson). For the detection of intracellular antigens, cells were subjected to fixation and permeabilization after surface immunostaining using a kit according to the manufacturer's recommendations (Fix & Perm; An der Grub Bioresearch). HPC-derived cells were harvested on day 6, washed, and resuspended in migration buffer (1 × 10 6 cells/ml). After allowing the cells to equilibrate for 15 min at 37°C, buffer only or MIP-1α at the indicated final concentration was added. After the indicated incubation periods, the reaction was stopped by adding PFA (final concentration 0.1%), and individual samples were incubated for 30 min at 4°C. Thereafter, cells were washed and sequentially exposed to anti-CD1a–CyChrome/anti-CD14–PE and saponin (final concentration 0.1%)/phalloidin–FITC (final dilution 1:5,000; both from Sigma Chemical Co.), and subjected to FACS ® analysis. Normal human skin was removed during elective plastic surgery upon informed consent. The skin samples were snap-frozen in liquid nitrogen–chilled isopentane. Frozen sections were mounted onto glass slides and fixed for 10 min in acetone at room temperature. Thereafter, the slides were kept at −20°C for up to 8 wk, until the staining procedure. For immunostaining, the slides were rehydrated and incubated with the following primary Abs: rabbit anti–MIP-3α, anti–MIP-3β (Peprotech), and anti-CCR6 (gift of Joshua Farber, National Institutes of Health, Bethesda, MD ); monoclonal mouse anti–HLA-DR (Biotest), mouse anti-CCR6, anti–MIP-1α, anti–MIP-1β, and anti-RANTES; and goat anti–SDF-1α and anti–MCP-1 (all from R&D Systems). The bound primary Abs were detected by sequential incubations with alkaline phosphatase–conjugated goat anti–rabbit, rabbit anti–mouse, or rabbit anti–goat secondary Abs and an alkaline phosphatase–anti-alkaline phosphatase staining kit (Dako), according to the manufacturer's instructions. To control the specificity of Ab binding, rabbit, goat, and mouse Ig and IgG control Abs (Dako) were used at equimolar concentrations. Each immunostaining protocol was performed on skin from at least three different donors. In a first series of experiments, we investigated whether chemokines can induce the migration of well-defined DC types and their precursors in a selective manner. As a model system, we used the cellular progeny of GM-CSF/TNF-α–stimulated CD34 + HPCs. Under these culture conditions, virtually nonoverlapping CD1a + and CD14 + cell subsets appear around day 6. Until day 12, they develop into E-cad + CD11b − CD1a + LCs and E-cad − CD11b + CD1a + non-LC DCs, respectively ( 29 ; data not shown). Chemokines that have been found to induce the migration of bulk DC populations of various maturational stages and lineages (i.e., MIP-1α, RANTES, MIP-3α, MIP-3β, MCP-1, and SDF-1α) were tested. Fig. 1 shows that, on the basis of their chemokine response pattern, CD1a + LCs and CD14 + non-LC DC progenitors are functionally diverse cell populations. Most importantly, MIP-3α, a cytokine with chemotactic activity for CD34 + HPC-derived DCs, but not mdDCs 18 , attracted almost exclusively CD1a + LC precursors . The maximal MIP-3α–induced response measured for CD14 + cells and for the residual, presumably poorly differentiated, CD14 − CD1a − cell population was at least threefold lower and only occurred at an MIP-3α concentration 100-fold higher than that needed to achieve a similar migratory response with CD1a + LC precursors . We also confirmed, by a checkerboard assay, that the CD1a + LC precursors, as well the few CD14 + cells that were attracted by MIP-3α, migrated in a directional rather than a random fashion (data not shown). In addition to being a selective chemoattractant for LC precursors, MIP-3α was the only chemokine in the panel tested that induced potent and efficacious migratory responses of these cells. LC precursors responded only weakly to SDF-1α and MIP-3β , and did not migrate at all in response to MCP-1, which, in contrast, attracted some CD14 + non-LC DC precursors . Another chemoattraction profile was observed for the CD14 − CD1a − cell population. As seen previously with HSCs, these cells were attracted by SDF-1α and responded to MCP-1 , but not to MIP-3α or MIP-3β . As shown in Fig. 2 A, MIP-1α failed to induce migration of day 6 CD34 + HPC-derived DC precursors. The apparent lack of migratory response to this chemokine was surprising, since day 6 DC precursors express CCR1 and CCR5, both of which are receptors for MIP-1α (see below). The transwell migration assay used allows only the analysis of cells that have passed the membrane and detached into the lower chamber, whereas the cells that remain membrane bound escape detection. To control for the cells adherent to the lower side of the membrane, we also tested MIP-1α in the Boyden-type chamber assay, which detects the migrated, membrane-bound cells only. The direct comparison of the results obtained in these two migration assays, plus the analysis of the transwell membranes by confocal microscopy, showed that MIP-1α was eliciting migratory and/or adhesive responses in CD34 + HPC-derived DC precursors that were different from those induced by MIP-3α . In both the transwell and the Boyden-type assay, the cells that had been attracted by MIP-1α remained adherent to the membrane . In contrast, the cells that had responded to MIP-3α detached from the lower side of the membrane in the transwell system and remained membrane bound only in the Boyden-type assay . The proadhesive effect of MIP-1α was pronounced also when an additional migration-inducing stimulus (e.g., MIP-3α) was provided at the same time . To better characterize the cell populations that respond to chemokines by adhesion versus transmigration, CD1a + and CD14 + DC precursor subsets were flow sorted on day 6, recultured overnight, and then tested in the transwell chemotaxis assay. In concordance with our results obtained with nonsorted DC precursor subsets, MIP-3a induced vigorous transmigration of sorted CD1a + LC, but not of CD14 + non-LC DC precursors . The enumeration of cells that migrated but did not detach from the transwell membranes revealed that only a minor part of the migrated CD1a + LC precursors remained membrane bound, and that only very few CD14 + cells adhered to the filters in response to MIP-3α . The argument that LC, rather than non-LC DC precursors, are the main MIP-3α–responsive cell population was further corroborated in the Boyden-type microchamber chemotaxis assay using flow-sorted CD1a + and CD14 + DC precursors (data not shown). In another series of experiments, we used the same experimental set-up to study MIP-1α–induced migration of isolated DC precursors. However, unlike their nonsorted founder population, neither of the two sorted DC precursor subsets significantly migrated to MIP-1α either in the transwell or in the Boyden-type microchamber chemotaxis assay (data not shown). The reason for this discrepancy is not currently understood, but may be due to desensitization of MIP-1α receptors by chemokines 3 4 21 released after the sorting procedure. To still be able to identify the principal MIP-1α–responsive DC precursor, we measured MIP-1α–induced actin polymerization, a cellular event occurring early after chemokine receptor triggering 8 . As revealed by filamentous (F)-actin and mAb co-staining experiments performed on nonsorted day 6 DC progenitor subsets, CD14 + non-LC DC precursors and, to a lesser extent, CD1a − CD14 − cells, but no CD1a + LC precursors, responded to MIP-1α . In summary, our cumulative data indicate that MIP-1α and RANTES (not shown), which act on the same set of receptors, induce non-LC DCs rather than LC precursors to migrate and to firmly adhere to the substratum. It is possible that the differential responses of defined DC precursors to MIP-3α and MIP-1α may be important for the fine-tuning of migration- versus adhesion-dependent immobilization and, as a result, for the correct localization of the various types of DCs in the tissues. When we compared the effect of chemokines on the migration of E-cad + CD11b − CD1a + LCs and of E-cad − CD11b + CD1a + non-LC DCs between days 12 and 14, we observed that neither of the DC types responded to MIP-3α . In fact, it appears that LCs have completely lost the MIP-3α reactivity characteristic of their precursor stage, and that the non-LC DCs did not acquire responsiveness to MIP-3α. The finding of a selective but transient involvement of MIP-3α in the migration of defined LC precursors, but not of monocyte-related DC/DC precursors , together with the observation of a temporary appearance of MIP-3α–responsive cells in bulk progenies of cytokine-stimulated CD34 + HPCs 18 , suggests that MIP-3α could have a specific role in guiding LC precursors to their correct anatomical location in vivo. The refractory state for chemokine-induced migration seen at day 12 of the cultures lasts for only a short time period, since at day 14 the LC/DC progeny displayed pronounced migratory responses to MIP-3β . Data presented in Fig. 4 C also show that day 14 LCs and non-LC DCs display quantitatively comparable chemotactic responses to MIP-3β. Thus, DCs show subtype-restricted chemokine responses at the committed precursor stage only. To see (a) whether a correlation exists between the chemokine expression pattern and the migratory properties of different types of DCs, and (b) whether our findings are relevant to the in vivo situation, we analyzed the chemokine receptor profile of in vitro–generated LCs and freshly isolated epidermal LCs. As shown in Fig. 5 B, epidermal LCs express mRNAs encoding CCR6, CCR7, and CXCR4, which are the receptors for MIP-3α, MIP-3β/SLC, and SDF-1, respectively. In contrast, these cells are virtually devoid of CCR1, CCR3, and CCR5 transcripts, and express only limited amounts of CCR2 mRNA. Although a similarly restricted set of chemokine receptor mRNAs was expressed by in vitro–generated LCs , other types of immature DCs and DC precursors, i.e., the progeny of cytokine-stimulated CD34 + HPCs at day 6 , blood DCs , and monocyte and/or mdDCs 10 13 31 , express mRNA for a wide array of different chemokine receptors, including CCR1, CCR2, CCR3, CCR5, and CXCR4. These two different patterns were mirrored by the FACS ® data of chemokine receptor expression on the surface of in vitro–generated LC versus non-LC DC precursors . CCR6 was the only chemokine receptor among those studied that was present on the surface of CD1a + LC precursors . Curiously, CXCR4 was not detected on the surface of these cells, but was found in their cytoplasm . In contrast, CD14 + DC precursors expressed several chemokine receptors, including CCR1 and CCR5, thus suggesting that the migratory responses to MIP-1α seen in Fig. 2 are indeed confined to this subset. Immunohistochemistry using both mouse and rabbit Abs demonstrated strong in situ CCR6 expression by dendritic epidermal cells of normal human skin . In serial sections, these cells were weakly HLA-DR + , and thus represent LCs in their nonactivated state (data not shown). In most experiments we also observed a weak, albeit distinct, granular anti-CCR6 reactivity in keratinocytes of the Malpighian layer . Our additional finding of CCR6 mRNA in purified keratinocytes (data not shown) is indicative of receptor synthesis by these cells. CCR6 surface expression by a major subset of freshly isolated HLA-DR + CD1a + LCs was also demonstrated by FACS ® . In vitro culture of these LCs under maturation-promoting conditions resulted in a rapid loss of anti-CCR6 surface immunoreactivity that even preceded the maturation-related upregulation of the costimulatory molecule B7-2 . These data suggest that epidermal LCs, although CCR6 + in their nascent state, downregulate this receptor during the process of in situ maturation. In correlation with these results and the maturation-dependent responses of LCs to either MIP-3α or MIP-3β , we observed a complete loss of CCR6 mRNA production with continued synthesis of CCR7 mRNA during cytokine-mediated maturation of epidermal LCs . Similarly to CCR6 mRNA, CCR2 transcripts disappeared during the in vitro maturation of epidermal LCs, whereas the expression of CXCR4 mRNA remained unchanged . In conclusion, it appears that immature LCs ex vivo and those generated in vitro display a similar and restricted set of chemokine receptors, and that mature cells no longer produce chemokine receptor mRNAs, with the notable exception of CCR7 and CXCR4. In view of data published previously by Zaitseva et al. 32 , the apparent lack of CCR5 mRNA expression by epidermal LCs appears surprising. Although unlikely, it is conceivable that LCs express CCR5 mRNA at levels too low to be detected by our RT-PCR assay. It is important to note that the LC-purification strategy employed here strictly excluded CD2 + and/or CD3 + skin T cells that express CCR5 (data not shown). Moreover, using two different mAb clones and RT-PCR, we also failed to detect CCR5 protein and mRNA expression by in vitro–generated LCs , but could amplify CCR5-specific transcripts from day 6 cytokine-stimulated HPCs and from freshly isolated and cytokine-stimulated PB-DCs . To get information about the expression of chemokines in normal human skin and to correlate it with the chemokine receptor pattern of LC precursors, we performed immunohistochemistry and RT-PCR on frozen skin sections and FACS ® -purified skin cells, respectively. Keratinocytes were found to express MIP-3α mRNA ; accordingly, MIP-3α immunoreactivity was seen in the basal and suprabasal layers of the epidermis . RT-PCR revealed no MIP-3β mRNA in isolated keratinocytes . Together with our finding of only sparse and focal epidermal MIP-3β immunoreactivity (not shown), this suggests that MIP-3β, unlike MIP-3α, is not constitutively produced in the epidermis. Conversely, we observed strongly MIP-3β–immunoreactive cells throughout the dermis . SDF-1α immunoreactivity was seen in the epidermis but, in contrast to MIP-3α, its expression was strictly confined to the basal keratinocyte layer . As shown in Fig. 7 E, normal human epidermis is devoid of MCP-1 immunoreactivity, an observation that finds support in a previous publication 33 . Also, no MIP-1α, MIP-1β, or RANTES immunoreactivity was seen in normal epidermal keratinocytes (data not shown). Thus, it appears that two chemokines, MIP-3α and SDF-1α, are produced constitutively by human keratinocytes, and therefore could both be involved in LC homing to the epidermis. Our findings that SDF-1α does not attract LC precursors and that these cells also fail to express CXCR4 on their surface speak against a role of SDF-1α in LC homing. Since ex vivo–purified LCs express CCR2 transcripts , one could argue that this receptor is involved in the attraction of LCs into the epidermis. This is unlikely because (a) resting keratinocytes do not express the CCR2 ligand MCP-1 at the protein level , and (b) LC precursors do not express CCR2 on their surface , and do not migrate in response to MCP-1 . In this context, it is noteworthy that mice genetically manipulated to express MCP-1 in the epidermis have close to normal LC numbers while accumulating dermal DCs and macrophage-like cells 34 . In conclusion, among all the chemokine–chemokine receptor pairs studied, only MIP-3α–CCR6 fulfills the spatial and temporal expression, as well as function requirements, for a ligand–receptor pair responsible for LC homing to the epidermis. This is evidenced by (a) the constitutive expression of MIP-3α in normal human epidermis ; (b) the selective in vitro chemotactic responses of CD1a + LC precursors to MIP-3α ; (c) the expression of CCR6 by in vitro–generated LC/LC precursors , by skin-derived LCs , and by LCs in situ ; (d) the absence of CCR6 from non-LC DCs, e.g., PB-DCs , mdDCs 17 , and dermal DCs (data not shown); and (e) the rapid loss of CCR6 surface expression during LC maturation, as illustrated by a reverse regulation of CCR6 and B7-2 expression during LC culture , and the lack of CCR6 expression by cytokine-matured epidermal LCs . MIP-3α, although absent from spleen and bone marrow, is also expressed in appendix, thymus, and tonsils as well as in fetal liver and lung 35 36 37 38 39 . Curiously, in tonsils 18 , skin (this study), and perhaps also in the other organs containing epithelial cells, MIP-3α is expressed constitutively by ectodermal rather than bone marrow–derived cells. Because keratinocytes produce MIP-3α , and at the same time apparently also express CCR6 , it is possible that, in addition to driving LC homing, this chemokine plays a previously unrecognized autocrine role in keratinocyte homeostasis. If MIP-3α is expressed in several different organs, why do immature LCs appear only in stratified epithelia? It is conceivable that additional factors, and not MIP-3α expression alone, determine whether LC precursors can populate a given organ. A prerequisite for homing into the epidermis is the emigration of LC precursors from the circulation, which, by analogy with other leukocytes, is likely to be mediated by a cascade of discrete multistep interactions with ECs 40 . Organ-specific differential expression of endothelial adhesion molecules may provide an important additional source for the selectivity of LC homing into skin. The multistep adhesion paradigm may also explain how the lack of expression of leukocyte adhesion molecules, e.g., cutaneous lymphocyte-associated antigen (CLA), may be responsible for the fact that not all CCR6-bearing blood leukocytes 28 can enter the skin in response to MIP-3α. We argued above that MIP-3α is ideally positioned to guide the migration of LC precursors into the epidermis, but can it also drive their emigration from blood? To do this, MIP-3α would have to be associated with dermal ECs and appear on their luminal surface 41 42 . This could be achieved either by EC binding and transcytosis of keratinocyte-derived MIP-3α, as has been demonstrated previously for IL-8 and RANTES 42 , or by ECs themselves producing MIP-3α, as was shown for other chemokines 43 . Postcapillary and small venular ECs in normal human skin display MIP-3α immunoreactivity , and isolated DMECS produce MIP-3α mRNA , showing that, in addition to keratinocytes, dermal ECs also produce this chemokine under normal conditions. The lack of MIP-3α mRNA in HUVECs underlines the site-specific differences among ECs. Cumulatively, these findings suggest that, in addition to navigating LCs towards the epidermis, MIP-3α may be the chemokine responsible for the transendothelial migration of LC precursors. Additionally, the immunohistochemical studies revealed MIP-3α and MIP-3β immunoreactivity to be associated with the ECs of afferent lymphatics . This observation is in line with our previous in situ binding studies, which demonstrated the presence of saturable, broad specificity binding sites for CC (MCP-1, MCP-3, RANTES) but not CXC chemokines on the lymphatic ECs in human dermis 44 . Additionally, SLC immunoreactivity was shown to be associated with lymphatic vessels 45 . The functional significance of chemokine binding to this microanatomical location is not yet clear. It is possible that chemokines immobilized on lymphatic ECs facilitate the entry of LCs into the lymphatics or, alternatively, promote their movement within the lymphatic channels toward the draining lymph node. In both cases, the primary role is likely to be played by CCR7 ligands, since LCs at this stage of their maturation downregulate CCR6 expression. In this study, we show that LCs display a chemokine response pattern that is much narrower than perhaps anticipated. Thus, the restricted set of chemokine receptors expressed by constitutively trafficking LCs stands in striking contrast to the very broad chemokine receptor repertoire of DCs that appear under inflammatory conditions ( 5 10 13 31 ; this study). These findings suggest a scenario where the differential behavior of two ontogenetically different DC lineages is encoded by their differential expression of chemokine receptors. One cell type, the LC, follows only one tissue-derived constitutive signal, MIP-3α, and is remarkably ignorant to inflammation-related chemokine stimuli. As a result, it resides in the skin and plays a role in the homeostatic host defense. Another cell type, exemplified by the PB-DC, lacks CCR6 but displays a broad repertoire of chemokine receptors and, consequently, can home to any inflammatory site where it perpetuates or modulates the inflammatory tissue response. It also appears that the ultimate step in the DC odyssey, i.e., their migration into draining lymph nodes, follows one pattern common to all DC subtypes: LCs, non-LC DCs (this study; 18, 45), and monocyte-derived DCs 10 13 19 20 start to express CCR7 and respond to MIP-3β upon receipt of maturation-promoting stimuli. This chemokine, and probably also the alternative CCR7 ligand SLC 22 45 , may guide DCs into T cell areas of draining lymph nodes. | Study | biomedical | en | 0.999995 |
10601352 | The embryonic stem (ES) cell line WW6 65 was generated by F. Poirier and E.J. Robertson (Harvard University) from a mouse (50% 129Sv, 25% C57BL/6, 5% SJL, and 20% unknown) and provided by Dr. P. Stanley (Albert Einstein College of Medicine, New York, NY). Cells were cultured on γ-irradiated STO feeder cells (SNL2; developed by E.J. Robertson) in DMEM (GIBCO BRL) containing 20% FCS (Hyclone). Genomic clones of mouse PSGL-1 were isolated from an adult 129/SvJ mouse liver genomic DNA library as described previously 55 . Two contiguous EcoRI fragments together containing a portion of the 5′ flanking region, the complete coding region, and the complete 3′ untranslated region of the PSGL-1 gene were cloned independently into the pBluescript vector to create A1.6 and A1.1 . An SmaI-EcoRI fragment from A1.6 and an EcoRI-BamHI fragment from A1.1 were recombined in pBluescript at the EcoRV and BamHI sites to generate A SMB . A HindIII (blunt ended)-BamHI fragment released from A SMB was ligated 3′ to the Neo cassette in pNT at Xbal (blunt ended) and BamHI sites to create KOIII. The vector pNT was provided by Dr. B. Spiegleman (Dana-Farber Cancer Institute, Boston, MA). Primer pairs P1/P2 and P3/P4 were used to amplify the DNA sequence by PCR to be inserted 5′ to the Neo cassette. The PCR product from NotI-tagged P1 (5′-CCCCA ACGGG TTGTT TTGGC CCACC-3′) and EcoRI-tagged P2 (5′-GAAGC CGGAA GGGTC TGGGC ATGG-3′) was subcloned and recombined at the EcoRI site with the PCR product from EcoRI-tagged P3 (5′-AGAATC TCATT GAGTT ACACA GCC-3′) and XhoI-tagged P4 (5′-GGGGT CCTGC AGCTG AAGGC TG-3′). The resulting 1.3-kb fragment was inserted at the NotI and XhoI sites of KOIII to create KO.pNT. WW6 cells (2 × 10 7 ) were electroporated (GenePulser, 500 μF, 240 V; Bio-Rad) with the targeting vector DNA (∼30 μg/ml) that was linearized by NotI digestion. Cells were plated onto monolayers of γ-irradiated feeder cells. After 24 h, medium was replaced with medium containing 200 μg/ml of G418 (GIBCO BRL) and 2 μM ganciclovir (Syntex). Individual colonies were picked 7 d after application of selective media. Genomic DNA from individual colonies was isolated, digested with HindIII, and analyzed by Southern blotting using probes B or C and Neo. One positive clone, W19, was expanded. ES cells from clone W19 were injected into C57BL/6 blastocysts. The blastocysts were transferred to pseudopregnant foster mothers. Four chimeric male mice were obtained and bred with C57BL/6 mice. Germline transmission was identified by Southern analysis of EcoRI-digested DNA using probe A. Heterozygous animals were bred to generate mutant mice. All studies and procedures were approved by the Animal Care and Use Committee of Beth Israel Deaconess Medical Center. Total RNA samples (20–60 μg) were prepared from spleen and thymus obtained from PSGL-1 −/− and PSGL-1 +/+ mice using the SV Total RNA Isolation System (Promega). RNA samples were separated by electrophoresis in 0.8% agarose-formaldehyde gels using 1× 3-( N -morpholino)propanesulfonic acid (Mops) buffer (40 mM Mops, 10 mM sodium acetate, and 1 mM EDTA, pH 7.0). The RNA was transferred to a Duralon-UV™ membrane (Stratagene) and affixed to the membrane by incubation at 80°C for 1 h. Prehybridization or hybridization was at 65°C overnight in 50 mM piperazine- N , N ′-bis(2-ethanesulfonic acid) (Pipes), pH 6.5, 100 mM sodium chloride, 50 mM sodium phosphate, pH 7.0, 1 mM EDTA, 5% SDS, and 60 μg/ml salmon sperm DNA. The probe was a 1.0-kb PCR fragment amplified from the coding region of mouse PSGL-1 (probe D). Mice were bled from the retroorbital sinus into EDTA-anticoagulated tubes while under anesthesia with Metofane (Mallinckrodt Veterinary). Leukocytes were prepared by red cell lysis followed by washing and centrifugation. Cells were incubated for 10 min with Fc Block (anti-CD32/16; PharMingen) before all staining procedures. Cells were incubated on ice for 30 min with 10 μg/ml of affinity-purified anti–PSGL-1 antibody or rabbit nonimmune IgG in PBS containing 1% FCS and 0.04% azide, washed, and stained with FITC-labeled goat anti–rabbit IgG (Sigma Chemical Co.) and PE-conjugated anti-Ly6G (PharMingen). Analysis was performed on a FACSCalibur™ flow cytometer with CELLQuest™ software (both from Becton Dickinson). Light-scatter gating procedures were used to enrich for granulocytes. Fresh frozen sections were fixed with 80% acetone, and samples were treated with normal goat serum for 1 h at 25°C. The sections were then incubated with rabbit anti–mouse PSGL-1 antibody at 0.5 μg/ml in PBS for 16 h at 4°C. The tissue was washed three times for 5 min with PBS at 23°C. The washed sections were incubated with biotinylated goat anti–rabbit antibody (VectaStain™ Elite ABC staining kit, 1:200 dilution; Vector Labs) for 1 h at 23°C. Sections were washed three times and then incubated with peroxidase-conjugated streptavidin for 1 h at 25°C. After three washes, the tissue sections were stained using 3,3′-diaminobenzidine in 40 mM Tris buffer, pH 7.4, and then counterstained with methylene blue. A polyclonal antibody directed against PSGL-1 was prepared using a synthetic peptide (QVVGDDDFEDPDYTYC) based on residues 42–56 of mouse PSGL-1 as immunogen 55 . A cysteine residue was added at the COOH terminus to facilitate coupling to KLH. The peptide was synthesized using FMOC/ N -methylpyrolidone chemistry on a peptide synthesizer (model 430A; Applied Biosystems). After cleavage and deprotection, the peptide was purified to homogeneity by reverse phase HPLC using a C18 column. The peptide was covalently coupled to KLH through the free cysteine, and the conjugate was injected intradermally (550 μg/ml) in complete Freund's adjuvant into a New Zealand White rabbit. Subsequent injections of immunogen (250 μg) were performed weekly for 2 wk, then monthly thereafter. Antipeptide antibodies were purified from rabbit immune serum by immunoaffinity chromatography. The serum was applied to a KLH-Sepharose column to remove antibodies against KLH. The serum proteins that failed to bind to KLH-Sepharose were then applied to a PSGL-1 (residues 42–56)–Sepharose column in which the peptide was covalently attached to Sulfo-Link Resin (Pierce Chemical Co.). Bound antibodies were eluted with 4 M guanidine and dialyzed into PBS. Blood was collected from 8–10-wk-old male wild-type and PSGL-1 −/− mice as well as P-selectin −/− mice and their matched controls. Complete blood counts were performed using a Coulter Counter. Differential counts were performed on blood smears stained with Wright-Giemsa stain (Sigma Chemical Co.). Differentials were performed in duplicate on 100 leukocytes per slide. PSGL-1 −/− mice and wild-type mice of similar mixed genetic background were used at 8–12 wk of age. P-selectin −/− mice (C57BL/6 background) and wild-type C57BL/6 mice (The Jackson Laboratory) were of the same age. Mice were injected intraperitoneally with 1 ml of 4% Brewer thioglycollate medium (Difco). At each time point after stimulation, the mice were killed and 10 ml of PBS containing 1% bovine serum albumin, 0.5 mM EDTA, and 10 U/ml heparin was injected into the peritoneal cavity. After gently massaging the peritoneal wall, the injected wash was withdrawn. Total cell numbers in the peritoneal lavage were determined on a Coulter Counter. Cytospin preparations of the cells were stained with Wright-Giemsa stain, and a differential leukocyte count was determined. From the total cell count in the peritoneal lavage and the percentage of neutrophils on cytospin preparations, the absolute number of neutrophils was calculated. Mice were preanesthetized with an intraperitoneal injection of 125 mg/kg of ketamine HCl (Parke Davis), 12.5 mg/kg xylazine (Phoenix Pharmaceuticals), and 0.25 mg/kg atropine sulfate (American Reagent Laboratories). A trachea tube was inserted to facilitate spontaneous respiration, and the mice were kept on a 37°C thermocontrolled rodent blanket (Thermal Care) during the experiment. To maintain anesthesia and neutral fluid balance, Nembutal (5 mg/ml in saline) was administered in 30–50 μl boluses through a cannula placed in the jugular vein. Mean arterial blood pressure ranged between 60 and 100 mmHg as measured on a physiological pressure transducer (model 60-051; Harvard Apparatus) connected to a carotid artery cannula. When TNF-α was used, mice were treated with an intrascrotal injection of mouse TNF (0.5 μg in 200 μl saline; R&D Systems) 2 h before exteriorization of the cremaster muscle. In experiments where the neutralizing anti–E-selectin mAb 9A9 was used, mice were injected with 80–90 μg of antibody through the jugular cannula just after exteriorization of the cremaster muscle. The cremaster muscle was prepared for intravital microscopy as described by Ley et al. 7 . Through an incision made in the scrotum, the testicle and surrounding cremaster muscle were exteriorized onto an intravital microscopy tray. After removal of the connective tissue, a longitudinal incision was made in the cremaster, and the muscle was carefully stretched and pinned across the intravital microscopy stage while the testis and epididymis were tacked to one side. The cremaster preparation was superfused with thermocontrolled (36°C) and aerated (5% CO 2 , 95% N 2 ) bicarbonate buffered saline throughout the experiment. The cremaster exteriorization surgery was typically accomplished in 4–7 min. Microvessel data were obtained using an intravital microscope (Axioskop; Carl Zeiss, Inc.) fitted with an Achroplan (40×, 0.80 numerical aperture) water immersion objective, long distance condenser, and stabilized D.C. power supply. Leukocyte rolling in the venules was recorded for 75–90 s using a Sony CCD camera (model SSC-S20) connected through an IBM-compatible computer to a Sony SVHS video recorder . The centerline red blood cell velocity ( V cl ) in each venule was measured in real time with a dual photodiode velocimeter running a digital cross-correlation program on an IBM-compatible computer (Microvessel Velocity OD-RT; CircuSoft Instrumentation). A Coulter Counter (model T890) was used to determine the systemic leukocyte counts from 30–50 μl blood samples taken from the carotid cannula at 30–40-min intervals or after injection of antibodies. After the start of the cremaster surgery, data were acquired for 30 or 40 min in the untreated and TNF-treated mice, respectively. Video recordings from the intravital microscopy experiments were analyzed on a computer-based image acquisition system, and vessel diameter was determined using the IBM-adapted version of NIH Image (Scion Corp.). The centerline red cell velocity recorded from each vessel was used to determine the volumetric blood flow ( Q ) via the equation Q = ( V cl )(0.625)( A cs ), where A cs is the cross-sectional area of the cylindrical vessel, and 0.625 is an empirical correction factor 66 . For each vessel, wall shear rate ( W sr ) was determined: W sr = 2.13 × [(8 × 0.625 × V cl )/ D v )], where D v is the vessel diameter. Critical velocity ( V crit ) was also calculated: V crit = ( V cl × 0.625)( D cell / D v )[(2 − ( D cell / D v )]. Recordings of each vessel were analyzed for 30–60 s, and rolling leukocytes were identified as the visible cells passing through a plane perpendicular to the vessel axis. In certain cases, the rolling velocity of visible high velocity cells ( V cell ) was estimated in order to determine whether these cells qualified as rolling leukocytes, defined V cell < V crit . Total leukocyte flux was determined as the product of the measured systemic leukocyte concentration and microvessel volumetric blood flow. To compensate for differences in systemic leukocyte count, the rolling behavior of leukocytes in the cremaster muscle is presented as the rolling leukocyte flux fraction, which is the number of rolling leukocytes in the vessel as a percentage of the total leukocyte flux. We have included six videoclips from representative experiments depicted in Fig. 6 and Fig. 7 . These experiments correspond directly to those used to prepare the still photos in Fig. 6 and Fig. 7 . Leukocyte rolling in the venules was recorded for 75–90 s using a CCD camera connected through an IBM-compatible computer to an SVHS video recorder. Fig. 7 C was videotaped at an accelerated rate to compress the images into a shorter time span. Several ubiquitous transposon-like repetitive sequences B1 and B2 67 were found in the 5′ and 3′ flanking regions of the PSGL-1 coding region, and two segments containing CT repeats similar to those in ribosomal DNA 68 were located 5′ to the PSGL-1 coding region . These repetitive sequences were excluded from the knockout vector, KO.pNT, in which a small region of the PSGL-1 coding sequence from 69 to 94 nucleotides (nt) was replaced by a neomycin-resistance gene cassette . A deletion of ∼300 bp in the 5′ flanking region of the PSGL-1 coding region was generated in the vector in order to exclude a segment containing CT repeats. A thymidine kinase gene cassette was inserted 3′ of the construct as a negative selection marker. ES cells were transfected with linearized KO.pNT vector, selected with G418 plus ganciclovir, and screened by Southern blot analysis. Of 200 ES cell colonies analyzed, two exhibited a targeted band at 6.1 kb by HindIII digestion using probe B or probe C in addition to the expected wild-type band at 4.3 kb. Negative clones including W34, shown in Fig. 1 B, showed only the wild-type band. One of the positive clones, W19, displayed a single band corresponding to the targeted band when the Neo probe was used, indicating a single integration event in the cells . As a result of random integration, the Neo probe hybridized to a band of irrelevant size in W34 . A homologous recombination event in W19 cells was also confirmed by EcoRI digestion followed by Southern blot analyses using probe A or the Neo probe (data not shown). In all cases, a targeted band of the predicted size was detected. Results from the EcoRI digest using probe A and PCR analyses using primers P1 and P4 suggested that homologous recombination occurred 3′ of the EcoRI site of the targeting vector. Thus, there is no deletion in the 5′ flanking region of the PSGL-1 coding region in W19 cells. W19 cells were microinjected into blastocysts from C57BL/6 mice and transferred to recipient female mice, and chimeric mice were obtained. Breeding of the chimera with C57BL/6 mice yielded heterozygous animals. Homozygous mutant mice were obtained from subsequent breeding between heterozygotes. The animals were genotyped by Southern blot analysis as shown in Fig. 1 C. Using probe A, animals with the mutated allele showed an EcoRI band at 7.1 kb, whereas those carrying the wild-type allele yielded a band at 5.3 kb. Northern blot analysis of thymus and spleen RNA showed high expression of PSGL-1 messenger RNA in wild-type mice but did not show any transcript in PSGL-1 −/− mice , indicating that the targeted mutation created a null allele. To confirm that PSGL-1 −/− mice were deficient in cell surface expression of PSGL-1, leukocytes from the blood of wild-type, heterozygous, and homozygous mutant animals were stained with a rabbit anti–PSGL-1 antibody that was raised to an NH 2 -terminal peptide of PSGL-1, and analyzed by flow cytometry. Granulocytes were identified by light-scatter properties and positive staining with anti–Ly-6G antibody. All granulocytes from wild-type animals expressed high levels of PSGL-1. In contrast, granulocytes from the PSGL-1 −/− mice did not show specific staining , confirming a null mutation. The mean fluorescence intensity of PSGL-1 on granulocytes from heterozygous (+/−) mice was ∼50% of that observed on wild-type granulocytes, consistent with the presence of only one PSGL-1 gene allele in the heterozygotes. Lymphocytes from the blood of PSGL-1 −/− mice did not express PSGL-1, as assessed by flow cytometry (data not shown). The absence of PSGL-1 expression in lymphoid organs was also confirmed by immunohistochemistry. Thymus, spleen, and lymph node from wild-type mice showed positive staining on lymphocytes, whereas no specific staining was observed in PSGL-1 −/− mice . Bone marrow was similarly negative for PSGL-1 antigen, in contrast to that from wild-type mice. PSGL-1 −/− mice developed normally, and their postnatal growth rate was comparable with that of wild-type mice. Both sexes were fertile. P-selectin and PSGL-1 were previously shown to be cell surface components of the sperm and ovum, respectively, leading to the suggestion that cell adhesion involving P-selectin and PSGL-1 might mediate sperm–ova interaction 69 . Our results indicate that the presence of PSGL-1 on the ova, if confirmed, is not required for fertilization. Similar results were obtained for the P-selectin–deficient mouse 6 . Histological analysis of thymus, spleen, and peripheral and mesenteric lymph nodes, where PSGL-1 expression is high, showed no abnormalities, indicating that PSGL-1 is not required for normal lymphoid organ structure. This is in contrast to the abnormal lymph node architecture that characterizes L-selectin–deficient mice 11 . Examination of the bone marrow revealed normal erythroid and myeloid maturation; no abnormalities in cell morphology were observed. PSGL-1 −/− mice showed no signs of infection up to 12 mo of age, and specifically lacked the ulcerative cutaneous infections exhibited by P- and E-selectin double-deficient mice 12 13 . Leukocytosis is well known to be associated with an inadequate leukocyte adhesion and emigration phenotype. Modest elevation of neutrophil counts is observed in P-selectin–deficient mice 6 and in C2GlnNAc-deficient mice that exhibit partial deficiency of the selectin ligands 70 , whereas a remarkable granulocytosis is observed in P- and E-selectin double-deficient mice 12 and fucosyltransferase (Fuc-T) VII–deficient mice that lack ligand activity for all three selectins 71 . Therefore, we examined the peripheral blood counts of wild-type and PSGL-1 −/− mice. The total leukocyte counts were not significantly different between wild-type and PSGL-1 −/− mice, whereas the neutrophil counts in PSGL-1 −/− mice were 2.9-fold higher than those in the wild-type mice ( Table ). The eosinophil counts tended to be higher in PSGL-1 −/− mice (3.5-fold elevation, P = 0.07). The hemoglobin levels and platelet counts in PSGL-1 −/− mice are essentially identical to those of the matched wild-type controls. The mild neutrophilia observed in PSGL-1 −/− mice is very similar to the 2.4-fold elevation reported with P-selectin–deficient mice 6 . The elevation of neutrophil counts in the PSGL-1 −/− mice suggests a defect in neutrophil adhesion and migration. Neutrophil migration in the PSGL-1 −/− mice was examined in a thioglycollate-induced peritonitis model. Chemical peritonitis was induced by intraperitoneal thioglycollate injection in both PSGL-1–deficient mice and matched wild-type controls. At 2 h after thioglycollate injection, the number of neutrophils migrating into the peritoneal cavity in PSGL-1 −/− mice was 4.3-fold lower than wild-type mice . At the 4-h time point, the difference in neutrophil recruitment between wild-type and PSGL-1 −/− mice was reduced to 1.9-fold . At 8 h after thioglycollate injection, the absolute number of neutrophils in the peritoneal cavity of PSGL-1–deficient mice was only slightly lower (1.5-fold) than in the matched wild-type controls. These results demonstrate that the kinetics of neutrophil influx into the peritoneal cavity in PSGL-1 −/− mice are characterized by an early defect after the initiation of the inflammatory stimuli. Neutrophil migration approaches normal levels 8 h after exposure to the inflammatory stimuli. To directly compare the results obtained with the PSGL-1–deficient mice to those with P-selectin–deficient mice, we reexamined neutrophil migration in the P-selectin–deficient mice. We observed a 15.7-fold decrease in neutrophil migration at 2 h , a 3.9-fold decrease at 4 h, and a 1.7-fold decrease at 8 h compared with wild-type matched controls , thus confirming earlier results 6 . Our observation in PSGL-1–deficient mice of an early defect followed by a significant recovery at later time points is very similar to that of P-selectin–deficient mice, but distinct from that observed in P- and E-selectin double-deficient mice in which neutrophil influx remains low at all time points 12 . Although PSGL-1 −/− mice and P-selectin −/− mice showed similar kinetics of neutrophil migration in the peritonitis model, PSGL-1 −/− mice recruited more neutrophils at each time point than P-selectin–deficient mice when each strain was compared with its wild-type control. It is possible that the difference in the genetic background of the PSGL-1 −/− mice (mixed 129Sv/C57BL/6J/SJL) and the P-selectin −/− mice (100% C57BL/6J) may affect the neutrophil extravasation defect in these strains. Alternatively, these results are consistent with the presence of an additional P-selectin ligand. Leukocyte rolling in the postcapillary venules of the cremaster muscle was examined by intravital microscopy in wild-type mice, PSGL-1–deficient mice, and P-selectin–deficient mice to determine the relative role of P-selectin and PSGL-1 in leukocyte trafficking in vivo. This model enables direct examination of granulocytes, which comprise >90% of the adherent leukocytes, with the blood vessel wall 72 . In the first series of experiments, leukocyte rolling was assessed at time points <30 min after introduction of the inflammatory stimuli in a model of mild trauma-induced inflammation. A severe early defect in leukocyte rolling was observed in the PSGL-1–deficient animals . The rolling flux fraction was 1.2% compared with 20.9% in matched wild-type controls The microvessel and hemodynamic parameters in the observed vessels were closely matched across all of the genotype groups ( Table , top). In agreement with the results of other investigators 6 , P-selectin–deficient animals had a pronounced defect in leukocyte rolling, with an average leukocyte rolling flux fraction of 0.4% compared with 18.4% in matched wild-type controls . These results emphasize that in mild trauma-induced inflammation, leukocyte rolling is markedly decreased but not absent in both PSGL-1–deficient mice and P-selectin–deficient mice within 30 min of introduction of inflammatory stimuli. To determine the role of PSGL-1 in cytokine-induced inflammation that is thought to be both P-selectin and E-selectin mediated, we compared the rolling behavior of leukocytes in TNF-α–treated cremaster muscles of PSGL-1–deficient mice and matched wild-type controls. Mice were treated with TNF-α, then rolling in the venules of the cremaster muscle was evaluated 2 h later. In contrast to trauma-induced inflammation, PSGL-1–deficient mice had a significant number of rolling leukocytes. A leukocyte rolling flux fraction of 5.2% was observed. The rolling flux fraction of the TNF-α–treated wild-type mice was 13.9% . TNF-α treatment led to reduced blood flow in the microcirculation of the cremaster muscle and resulted in lower wall shear rate values for TNF-α–treated animals than was observed under conditions of trauma alone ( Table , bottom). Within the TNF-α–treated group, the microvessel and hemodynamic parameters were similar in both the wild-type control and PSGL-1 null animals ( Table , bottom). Comparable results were obtained with the P-selectin–deficient mice . To prove that the residual leukocyte rolling in the TNF-α–treated PSGL-1–deficient mice is mediated by E-selectin, the blocking anti–E-selectin antibody 9A9 was infused into the PSGL-1–deficient mouse 8 73 . In TNF-α–treated PSGL-1 −/− mice, the leukocyte rolling flux decreased to 0.6% in the presence of 9A9 . Leukocytes visible in the still photograph of the cremaster venules from a PSGL-1–deficient mouse treated with both TNF-α and antibody 9A9 are stationary on the vessel wall and likely adhered via integrins before the infusion of 9A9. In contrast, antibody 9A9 infused into TNF-α–treated wild-type mice had a leukocyte rolling flux of 24.9% . This is likely due to the increased average leukocyte rolling velocity when slow E-selectin–mediated rolling is substituted by faster P-selectin–mediated rolling. In sum, these results indicate that PSGL-1 is not required for E-selectin–mediated leukocyte rolling. The basis for residual rolling after blocking of E-selectin–mediated leukocyte–vessel wall interaction is uncertain, but is consistent with interaction of P-selectin with a ligand on leukocytes other than PSGL-1. The counterreceptors on cell surfaces that bind specifically to the selectin family of adhesion molecules have been difficult to identify, although many candidate selectin ligands have been put forth. As lectins, the selectins bind predominantly oligosaccharide sequences of sialylated fucosylated lactosamine. These sequences are recognized by the selectins in vitro, albeit with low affinity (for a review, see Varki 74 ). Many carbohydrates, glycoproteins, or glycolipids bearing these oligosaccharide sequences will bind to the selectins in vitro when the receptor or ligand is presented at high density or under static conditions. The issue in understanding the biology of selectin ligands has been to prove specific interaction of candidate ligands with selectins under physiological conditions 47 75 . Although in vitro studies performed with isolated selectins and potential ligands or by cell adhesions assays have been invaluable in defining candidates in the inflammatory pathway, the determination of physiological import of each of these adhesion molecules requires study in animal models. The three selectins contribute to leukocyte rolling on the endothelial cell lining of venules in a cooperative and sequential manner in the early stages of the inflammatory process. Genetic disruption of the P-selectin gene in mice demonstrated that P-selectin is required for neutrophil rolling in postcapillary venules and for neutrophil influx into inflamed peritoneum at early time points 6 7 . These studies defined a critical role for P-selectin in early inflammatory events and led to the understanding that P-selectin initiates leukocyte rolling at sites of inflammation and tissue injury. Disruption of the L-selectin gene in mice established that this molecule is also essential for normal leukocyte rolling and emigration into the inflamed peritoneum 7 11 . L-selectin mediates leukocyte rolling in inflamed venules sequentially after P-selectin at time points between 40 and 120 min 7 . In addition to defects in neutrophil rolling and migration, the L-selectin–deficient mice had significant defects in lymphocyte homing to peripheral lymphoid tissue 11 76 . Mice lacking E-selectin initially demonstrated no defects in the inflammatory response 77 . However, detailed analysis later showed that slow, stable granulocyte rolling was absent in E-selectin null mice 10 and that E-selectin was required for firm leukocyte attachment 78 . Further studies using blocking E-selectin antibodies demonstrated that E-selectin adhesive functions overlap with P-selectin in the later phases (>2 h) of inflammation 79 . The cooperative and overlapping function of the selectins is demonstrated in mice that are doubly deficient in both P-selectin and E-selectin. Unlike mice deficient in a single selectin, mice deficient for both P-selectin and E-selectin are characterized by spontaneous skin infections, leukocytosis, and drastic reduction in leukocyte rolling and neutrophil migration into the inflamed peritoneum at both early and late time points 12 13 . Thus, animal studies have demonstrated that P-selectin is required to initiate early contact of leukocytes with the vessel wall, and that after this initial step there is significant cooperativity of function of all three selectins. Using a PSGL-1–deficient mouse, we have explored the role of this glycoprotein in selectin-mediated leukocyte adhesion with the specific goal of determining whether PSGL-1 is the sole ligand for P-selectin and whether PSGL-1 interaction with E-selectin is important for the propagation of the normal inflammatory response. We have now demonstrated that early neutrophil migration after chemically induced peritonitis is impaired in PSGL-1–deficient mice. Previous reports demonstrated that neutrophil migration into inflamed peritoneum is impaired in mice that lack P-selectin or L-selectin but not E-selectin 6 11 77 . P-selectin–deficient mice and L-selectin–deficient mice demonstrate depressed neutrophil migration at the early time points (2–4 h) after chemical insult. In contrast, mice that lack Fuc-T VII or are deficient in both P-selectin and E-selectin show a marked reduction in neutrophil migration at time points up to 8 h 12 71 . Thus, in this assay deficiency of multiple selectin functions results in a more severe phenotype. In our chemical peritonitis studies, the PSGL-1–deficient animals demonstrated a defect in neutrophil migration that was marked at 2 h (77% reduction) but was only minimally abnormal at the later time points. This phenotype more closely resembled that of P-selectin–deficient mice than the severe migration defect seen in mice deficient in both P-selectin and E-selectin 12 13 . When compared with the P-selectin–deficient mice, the PSGL-1–deficient mice had significantly more neutrophils migrating into the inflamed peritoneum at parallel time points. These findings suggest the presence of additional P-selectin ligands other than PSGL-1, although PSGL-1 nonetheless appears to be an important and dominant P-selectin ligand. Our leukocyte rolling experiments have shown that early trauma-induced leukocyte–endothelial cell interaction is greatly reduced, whereas cytokine-induced leukocyte rolling is only modestly impacted in the PSGL-1–deficient mice. Trauma-induced neutrophil rolling in the postcapillary venules of P-selectin–deficient and L-selectin–deficient mice is reduced initially but approaches normal levels as inflammation progresses 6 7 11 . In contrast, neutrophil rolling is virtually absent in trauma- and TNF-α–induced inflammation in mice deficient in both P-selectin and E-selectin 12 13 . In our current work, we have established that PSGL-1 is required for the early, P-selectin–mediated phases of leukocyte rolling at times <30 min after the inflammatory stimulus. Similar to the P-selectin–deficient mice, neutrophil rolling in PSGL-1–deficient mice is reduced by ∼95% compared with wild-type mice. Based on this observation, PSGL-1 appears to be the dominant P-selectin ligand early in trauma-induced inflammatory events. TNF-α is a cytokine that induces E-selectin biosynthesis in endothelial cells and promotes leukocyte rolling in part mediated by E-selectin. Mice deficient in both P-selectin and E-selectin have profound leukocyte rolling defects in TNF-α–treated venules 12 13 . In our studies, the PSGL-1–deficient mice demonstrated a significant amount of leukocyte rolling (5.2% flux) after 2 h of pretreatment with TNF-α. The leukocyte rolling flux for the TNF-α–treated PSGL-1–deficient mice (5.2%) was identical to that of TNF-α–treated P-selectin–deficient mice (5.1%) and is significantly higher than the leukocyte rolling flux reported in TNF-α–primed mice deficient in both P-selectin and E-selectin 12 13 . This finding suggests that PSGL-1 functions mainly as a P-selectin ligand in this system; the preserved function of E-selectin and L-selectin with their complementary counterreceptors supports a TNF-α–induced rolling flux in the absence of P-selectin or PSGL-1. Furthermore, the presence of a measurable leukocyte rolling flux in the PSGL-1–deficient mouse raises questions of the importance of leukocyte–leukocyte rolling interactions mediated by L-selectin and PSGL-1. Leukocytosis, the hallmark of a major defect in host defense, is often associated with recurrent infection or chronic ulceration as a manifestation of blockade of leukocyte activity. Mice deficient in both P-selectin and E-selectin 12 13 and mice deficient in the fucosyltransferase required for synthesis of the selectin ligands 71 exhibit leukocytosis. However, our PSGL-1–deficient mice exhibited only a mild elevation of neutrophils. Since genetic deficiency of P-selectin, E-selectin, L-selectin, or, from these studies, PSGL-1, is not associated with significant leukocytosis, it would appear that the presence of 5% of normal leukocyte rolling and delayed neutrophil migration comprises an adequate host defense. Thus, no single deficiency of a selectin or selectin ligand causes a phenotype of leukocytosis, chronic infection, and early demise. This observation speaks to the importance of redundancy in these host defense systems. It is also consistent with our observation that PSGL-1 is the dominant ligand for P-selectin, but not required for the function of E-selectin within the context of leukocyte rolling. These experiments using PSGL-1–deficient mice have established that PSGL-1 is a critical P-selectin ligand early in inflammation. Comparison of the phenotype of the PSGL-1–deficient mouse and the mouse deficient in both P-selectin and E-selectin 12 13 provides evidence that PSGL-1 is not a required neutrophil ligand for E-selectin–mediated neutrophil rolling. In contrast to the mice deficient in both P-selectin and E-selectin, the PSGL-1–deficient mice have no leukocytosis, no susceptibility to spontaneous infection, and have detectable leukocyte rolling and adequate neutrophil migration into the inflamed peritoneum after inflammatory stimuli. If PSGL-1 were the sole ligand for both P-selectin and E-selectin, the PSGL-1–deficient mice and the mice deficient in both P-selectin and E-selectin would have the same phenotype. These data provide strong evidence for the existence of additional physiological ligands for E-selectin, perhaps ESL-1 31 , and do not support a requirement for PSGL-1 in E-selectin–mediated neutrophil rolling. The relative importance of PSGL-1–E-selectin interaction during T cell homing 40 80 is unresolved, but further studies using PSGL-1–deficient mice will address this important question. | Study | biomedical | en | 0.999996 |
10601353 | Chemicals were obtained from Sigma Chemical Co., and tissue culture reagents from GIBCO BRL. Antibodies to host proteins were obtained from the following sources: American Type Culture Collection, mAb IM7.8.1 (CD44) and mAb E13 161-7 (Sca-1); Michael Kashgarian (Yale University, New Haven, CT), mAb c464.6 (alpha subunit Na + /K + ATPase); Francis Brodsky, University of California at San Francisco, San Francisco, CA), mAb AIIB2 (β1-integrin); Chemicon International Inc., mAb RR1/1 (CD54); PharMingen, mAb IA10 (CD55); and Transduction Laboratories, mAb C34120 (caveolin-1). Biotinylated cholera toxin B (CTB) was obtained from List Biological Laboratories, Inc. Rabbit anti-SAG1 was provided by Lloyd Kasper (Dartmouth Medical School, Hanover, NH), and rabbit anti- Toxoplasma ACT1 was produced commercially (Cocalico Biologicals, Inc.). Secondary antibodies were obtained from Jackson ImmunoResearch Labs or Molecular Probes, Inc. Toxoplasma tachyzoites of the RH strain were propagated by serial passage in monolayers of human fibroblasts (HFs) as described previously 4 . For invasion assays, host cells on LabTek chamber slides (Fisher Scientific) were challenged with either parasites or collagen-coated zymosan in DMEM/3% FCS for 5 min at 37°C 5 . The cytochalasin-resistant clone Cyd R-1 of Toxoplasma was compared with its wild-type parental line, PLK, using similar invasion protocols supplemented with 0.5 μM cytochalasin D as described previously 4 . Monolayers were either fixed immediately after challenge or washed in PBS, and returned to culture at 37°C in DMEM/10% FCS for defined intervals. All cell cultures were free of mycoplasma contamination as verified by testing with the Gen-Probe rapid detection kit. Monolayers of HF cells were surface-labeled with 1.1′-dihexadecyl-3-3′-3-3′-tetramethylindocarbocyanine (DiIC 16 ; Molecular Probes, Inc.) before challenge with parasites or zymosan as described previously 5 . In experiments that required permeabilization of cells, monolayers were labeled with CM-DiI (Molecular Probes, Inc.), an analogue of DiIC 16 that is retained in cells throughout permeabilization. To isolate PVs, infected monolayers were disrupted by gentle scraping and passage through a 23-g needle in PBS containing 3% FCS. This procedure released a mixture of intact PVs and free parasites that were distinguished by phase–contrast and examined by fluorescence microscopy for the presence of DiIC 16 . The percentage of vacuoles or parasites that were stained with DiIC 16 was determined by examining 50 or more vacuoles in each of 2 or more experiments, and results are presented as the mean ± SE. Monolayers of 3T3 fibroblasts were incubated with 10 μg/ml of biotinylated CTB in DMEM/10% FCS for 5 min at 10°C, rinsed, and challenged with parasites or zymosan as described previously 5 . Biotinylated CTB was detected with Oregon green–conjugated streptavidin (Molecular Probes, Inc.) and examined by epifluorescence or confocal microscopy. Monolayers of HF cells were rinsed with PBS (pH 7.8), then incubated with 1 mg/ml sulfo-NHS-LC-LC biotin (Pierce Chemical Co.) in PBS (pH 7.8) for 20 min at 10°C. After permeabilization, biotinylated proteins were detected with Oregon green–conjugated streptavidin (Molecular Probes, Inc.) and viewed by epifluorescence or confocal microscopy. For immunofluorescence (IF) and confocal microscopy, monolayers were fixed and processed for IF as described previously 6 . Slides were rinsed in PBS and mounted in ProLong ® Antifade (Molecular Probes, Inc.), and examined using Zeiss Axioplan or Bio-Rad Confocal 1024 microscopes. To confirm that cell-associated parasites were internalized, monolayers were incubated with rabbit anti-SAG1 before permeabilization, then with secondary antibodies conjugated to Texas red. For quantitative analysis, cells were examined microscopically and vacuoles were scored as positive or negative based on a prominent, continuous rim of fluorescence around the vacuole. Percentages represent the mean and SD from 3 separate counts of 25 PVs each, unless otherwise stated. We have shown previously that collagen-coated zymosan is taken up into compartments that resemble phagosomes in HFs 5 . Here, we used this system to study the early kinetics of phagosome formation. After a 5-min pulse and extensive washing, fibroblast monolayers were examined by phase–contrast microscopy to distinguish internalized zymosan by their phase dark outline. The percentage of zymosan-containing phagosomes that were positive for labeled host proteins or lipids, as determined by a rim of fluorescence staining, was determined by counting 25 vacuoles. Each experiment was performed at least twice. Monolayers of host cells were grown on tissue culture plates and challenged with freshly isolated parasites at a multiplicity of 20:1. After incubation for 2 min, cells were washed in cold PBS, then removed by trypsinization and centrifuged at 200 g for 10 min. Cells were fixed and processed as described previously 5 . For quantification, the density of gold label was determined from 25 separate negatives (magnification ×20,000) from 2 or more experiments, and expressed as density of gold particles per vacuole or per micron of membrane length. Baby hamster kidney (BHK) cells were transfected with human intercellular adhesion molecule 1 (ICAM-1) cDNA subcloned into the CDM8 expression vector (wild-type ICAM-1; Invitrogen), or with a construct that replaced the transmembrane and cytoplasmic domain with the glycosylphosphatidylinositol (GPI) signal sequence from CD58 (ICAM-1–GPI), or with a construct that had the cytoplasmic domain deleted (ICAM-1–Cyt − ). Transfections were performed using Lipofectamine (GIBCO BRL) as instructed by the manufacturer. Cells were plated on chamber slides 24 h after transfection and cultured for an additional 24 h before being challenged with parasites for 5 min. Cells were fixed, permeabilized, and then stained with an antibody against human ICAM-1 and an anti- Toxoplasma SAG1 antibody, and examined by confocal microscopy. For quantitative analysis, cells were examined by confocal microscopy, and PVs were scored as positive or negative based on a prominent, continuous rim of fluorescence around the PV within a 0.5-μm section. Percentages represent the mean ± SD from two separate experiments in which >20 PVs were counted per experiment. For densitometry analysis, images were converted to 256 gray levels, and plots were obtained using the linear transect feature of NIH Image v1.61 (available at http://rsb.info.nih.gov/nih-image/). Previous studies have demonstrated that Toxoplasma invasion is completed within 20–30 s of initial contact with the host cell 3 . To examine the formation of the PV from the host cell plasma membrane, we relied on recently developed protocols for pulse invasion of Toxoplasma 5 15 . In brief, host cells were challenged with a high multiplicity of infection (ratio 50:1) for 2.5–5 min, washed extensively, and either directly fixed (T 0 ) or returned to culture for chase intervals of 5, 10, 15, 30, or 60 min before fixation and examination by confocal, IF microscopy, or immunoelectron microscopy (immuno EM). Staining with rabbit anti-SAG1 antibodies before permeabilization revealed that >95% of cell-associated parasites were internalized during this protocol (data not shown). Internalization of host cell plasma membrane proteins was examined by surface biotinylation, followed by challenge with Toxoplasma or zymosan particles. Vacuoles were classified as positive for the host surface proteins based on a prominent rim of fluorescence staining surrounding the PV or zymosan particle as detected with fluorescent streptavidin. Biotinylated surface proteins were abundantly detected on the cell surface, but were absent or very faintly present in PVs containing Toxoplasma . The short pulse used for infection (5 min) implies that these compartments were negative because of exclusion of the majority of surface proteins at the time of formation of the vacuole. In contrast, surface biotinylated proteins were readily internalized during phagocytosis of zymosan particles . Host cell plasma membrane lipids were simultaneously followed using the fluorescent label DiIC 16 . The long-chain carbocyanine dye DiIC 16 was loaded into the outer leaflet of the cell, where it is stably maintained 16 17 . DiIC 16 -lipids were detected in 85% of Toxoplasma -containing PVs and zymosan-containing vacuoles. The presence of DiIC 16 -lipids in the PV was not due to direct transfer of the DiIC 16 to the parasite membrane, since liberated Toxoplasma parasites were invariably negative for DiIC 16 (0.8 ± 0.6%), whereas isolated PVs remained positive . Over time, the percentage of Toxoplasma PVs that was positive for DiIC 16 decreased, whereas it remained relatively constant for zymosan . To examine the internalization of endogenous host plasma membrane lipids, the distribution of the surface glycolipid G M1 was monitored by selective staining with biotinylated CTB 18 before parasite invasion. CTB bound to G M1 was detected in the PV membrane, and was also incorporated into zymosan-containing phagosomes . Over time, the percentage of vacuoles containing CTB-G M1 decreased in Toxoplasma PVs and remained relatively constant in zymosan vacuoles. We also examined the internalization of specific host cell surface proteins into Toxoplasma -containing PVs, since surface biotinylation may underestimate the distribution of individual proteins. Simultaneously, the internalization of host cell surface G M1 was monitored by prelabeling with biotinylated CTB. Although the surface membrane lipid G M1 was incorporated into the PV, the cell surface protein CD44 was efficiently excluded from the vacuole . When fibroblasts were infected with Toxoplasma in the absence of CTB, CD44 and two additional plasma membrane proteins, Na + /K + ATPase (100-kD subunit) and β1-integrin (130 kD), were also excluded from the PV (data not shown). In marked contrast, CD44 and β1-integrin (data not shown) were readily internalized into zymosan-containing phagosomes. Consistent with the normal remodeling that accompanies phagosome maturation, CD44 was gradually removed from zymosan-containing phagosomes after initial internalization . IF staining may misrepresent the distribution of a protein because of insensitivity of detection or low spatial resolution, making it difficult to resolve subcellular localizations precisely. Therefore, the distribution of CD44 in newly infected monolayers of 3T3 cells (2-min pulse) was also examined by cryoimmunoEM. Immunogold staining revealed that CD44 was present along the outer surface of the plasma membrane, but not within the PV membrane . Quantitative analysis of the distribution of CD44 demonstrated that the majority (>75%) of PVs were negative, with a few vacuoles containing limited staining. In contrast, the density of plasma membrane labeling ranged from two to seven particles per micron of membrane . Collectively, these findings indicate that transmembrane proteins are efficiently excluded from the PV during its formation. We reasoned that the observed exclusion of host transmembrane proteins from the PV could be a property of the size of the extracellular domain or a consequence of their membrane anchoring. GPI-anchored proteins exhibit increased lateral mobility in the membrane 19 ; therefore, we examined internalization of cell surface GPI-anchored proteins into the PV. When 3T3 cells were challenged with Toxoplasma , fixed, and stained for Sca-1, this 18-kD GPI-anchored protein was readily internalized from the host cell plasma membrane into 95–100% of PVs . To determine if larger GPI-anchored proteins were also capable of entering the PV, we examined CD55, a 75-kD GPI-anchored protein on the surface of HeLa cells, and found it was also internalized into 85% of PVs during parasite invasion . Consequently, independent of the size of the extracellular domain, GPI-anchored proteins are internalized from the plasma membrane into the PV. Taken together with our finding that surface biotinylated proteins were not detected within the PV, these data suggest that GPI-anchored proteins represent a minor population on the surface of fibroblast cells. The finding that the PV selectively incorporates plasma membrane lipids and GPI-anchored proteins suggests that invasion could occur selectively through specialized lipid-rich microdomains, possibly including such specialized structures as caveoli 20 . To determine if parasite invasion resulted in the incorporation of caveolar proteins into the PV, we monitored caveolin-1 distribution during parasite invasion. Unlike G M1 , caveolin-1 was not detected in the PV , indicating that the parasite does not interact substantially with caveoli or vesicles derived from them. The observation that GPI-anchored proteins are incorporated into the PV suggests that membrane anchoring determines internalization. However, to eliminate potential differences due to the size and configuration of the extracellular domain, parasite invasion was examined in BHK cells transiently transfected with recombinant forms of ICAM-1 (CD54) that were anchored into the host cell plasma membrane by different means, as diagrammed in Fig. 5 C. Wild-type ICAM-1 (CD54) is a 90-kD molecule that possesses a single transmembrane segment and short cytoplasmic domain 21 . We compared the wild-type ICAM-1 to a lipid-anchored form (ICAM-1–GPI) and a cytoplasmic deletion form (ICAM-1–Cyt − ) of the protein 22 23 . Like other transmembrane proteins, wild-type ICAM-1 was excluded from the PV, whereas ICAM-1–GPI and ICAM-1–Cyt − were both incorporated into PVs. All three forms of ICAM were abundantly expressed and visualized with the same antibody to the extracellular domain, ruling out possible differences due to detection. Exclusion was also not due to size constraints, since the molecular masses of wild-type ICAM-1 and ICAM-1–Cyt − only differ by 3 kD (90 and 87 kD, respectively ). To quantify the distribution of the three forms of ICAM-1 in the PV membrane, confocal images were scaled to 256 gray levels and analyzed using the line transect feature of NIH Image. The relative intensity of a line extending through the plasma membrane and across the PV was determined for each of the three forms . These analyses revealed that ICAM-1–GPI and ICAM-1–Cyt − were enriched by two- to threefold in the PV membrane relative to the plasma membrane, whereas wild-type ICAM-1 was found at background levels . The internalization of the ICAM-1–Cyt − form suggested that restricted access to the PV is mediated by interactions with the host cell cytoskeleton. To test this hypothesis, we examined the invasion of a cytochalasin-resistant mutant of Toxoplasma into HF cells in the presence of sufficient concentrations of the drug to disrupt actin microfilaments. During invasion of the Cyd R-1 mutant, the host cell surface protein CD44 was excluded from 94% of vacuoles formed in the absence of cytochalasin D and from 87% of the vacuoles formed in the presence of the drug. Moreover, the prominent constriction seen at the junction during invasion 3 was still observed in the presence of cytochalasin D (data not shown). We show here that the PV is formed by invagination of the host cell plasma membrane, while simultaneously excluding transmembrane proteins. The formation of a moving junction at the interface of the host cell plasma membrane and the parasite appears to mediate this differential sorting process. In contrast, during uptake of zymosan, both plasma membrane lipids and transmembrane proteins are internalized into phagocytic vacuoles. Despite the efficient exclusion of transmembrane proteins at the moving junction, the presence of a GPI anchor or the absence of a cytoplasmic tail is sufficient to allow internalization of membrane proteins into the PV. Thus, sorting occurs by a mechanism that acts within the membrane spanning region or on the cytoplasmic region of the protein, and which is largely independent of their extracellular domains. Several observations indicate that, during parasite invasion, the bulk of the PV is derived from internalization of the host cell plasma membrane. First, electrophysiological studies have shown that during Toxoplasma invasion there is no net change in capacitance across the host cell plasma membrane, implying that the host cell surface area remains constant until the PV pinches off from the host cell plasma membrane 9 . Second, our results demonstrate that the PV membrane is formed by invagination of lipids from the host cell plasma membrane. For example, when the fluorescent lipophilic dye DiIC 16 16 24 was inserted into the host cell plasma membrane before parasite invasion, it was readily internalized into the PV membrane. Endogenous plasma membrane lipids are also incorporated into the PV, as shown by the internalization of surface G M1 sphingolipid. The presence of these labels in the PV was necessarily due to internalization at the time of entry, as neither one is prone to rapid diffusion, and we have shown previously that there is no vesicular lipid traffic to preformed PVs 5 . However, in both cases the percentage of PVs stained with these labels decreased over time, which may be a result of diffusion, transfer to the parasite, or vesicular traffic from an unidentified source that results in dilution of the label. Although the vacuole surrounding Toxoplasma is derived from the host cell plasma membrane, the transmembrane proteins Na + /K + ATPase, β1-integrin, and CD44 were excluded from the PV, even though they were readily incorporated during formation of zymosan-containing phagosomes. CryoimmunoEM examination of newly formed PVs revealed that the vacuolar membrane contained at least 20-fold lower levels of host cell proteins than the plasma membrane. Similar to our observations on Toxoplasma , when Plasmodium invades red blood cells, host cell plasma membrane lipids are incorporated into the parasite-containing vacuole, whereas transmembrane proteins in the red cell membrane, including glycophorins and band 3, are excluded 25 . Sorting of plasma membrane proteins during formation of phagosomes has been ascribed previously to their involvement as receptors in particle uptake 26 . In contrast, our results indicate that protein exclusion from Toxoplasma -containing PVs is a property of the membrane-anchoring and cytoplasmic extension of proteins, and is independent of the extracellular domain. Initially, this conclusion was supported by examining a variety of different surface proteins that were anchored by either a transmembrane domain (CD44, β1-integrin, Na + /K + ATPase) or a GPI moiety (Sca-1, CD55). Although there was a perfect correlation between mechanism of anchoring and exclusion versus inclusion, these findings may also be influenced by variability in the size and configuration of the extracellular domains of the proteins examined. To eliminate potential differences due to the size of the extracellular domain, we compared the internalization of recombinant ICAM-1 that was anchored in the membrane by different means. ICAM-1 is a heavily glycosylated 90-kD protein containing a single transmembrane segment and five tandem Ig-like extracellular domains 21 22 27 28 . Removal of the cytoplasmic tail from ICAM-1 eliminates the molecule's association with the actin cytoskeleton 14 , and replacement of ICAM-1's transmembrane region with a GPI anchor is likely to increase the molecule's lateral mobility 19 20 . Either the presence of a GPI anchor or the deletion of the cytoplasmic region was sufficient to allow ICAM-1 internalization into Toxoplasma -containing PVs. These results conclusively demonstrate that internalization is largely independent of the extracellular domain, and instead support the conclusion that sorting is based on the lateral mobility of the protein within the membrane. Exclusion of transmembrane proteins from the PV is likely due to establishment of a barrier to protein diffusion that operates at the moving junction, and which may be due to interactions between the cytoplasmic domain and proteins in the cytosol. Lateral diffusion of transmembrane proteins is limited by their direct attachment to the cytoskeleton (tethering) and indirect constraints imposed by the cytoskeleton 29 30 31 . Despite the observation that treatment with cytochalasin D did not allow access of CD44, a transmembrane-anchored protein, past the moving junction, we cannot rule out the possibility that short actin filaments remaining after this treatment were still sufficient to retard its mobility. Alternatively, the colloidal nature of the cytosol may be sufficient to retard the mobility of proteins with cytoplasmic extensions such that they have restricted access to the vacuole. In the case of ICAM-1, the lateral mobility of wild-type ICAM-1 is restricted by its direct association with the cytoskeleton and by general constraints due to its cytoplasmic tail. Based on analogous mutations that have been analyzed for other transmembrane proteins 19 29 30 32 , ICAM-1–Cyt − and ICAM-1–GPI are expected to display increased lateral mobility that evidently allows them to pass through the moving junction and gain access to the Toxoplasma -containing PV. Free diffusion of plasma membrane lipids and restricted passage of transmembrane proteins past the moving junction indicate that this unique structure acts as a molecular sieve. Classical tight junctions act as a diffusion barrier in the outer leaflet of the plasma membrane, but allow diffusion in the inner bilayer 33 34 . In contrast, our results indicate that GPI-linked proteins anchored in the outer leaflet of the plasma membrane freely enter the PV, yet proteins that extend beyond the membrane bilayer are excluded. Freeze–fracture EM analysis during Plasmodium invasion of erythrocytes has shown a band of rhomboidally arrayed particles at this junction that may represent aggregates of proteins that are prevented from entering the vacuole 13 . The molecular basis of this junction is not understood, and identification of host or parasite proteins that are exclusively localized to this structure is an important area for future research. The selective enrichment of GPI-anchored proteins within the Toxoplasma PV indicates that they are specifically recruited or retained within the vacuole. Enrichment could occur if the parasite binds to one or more GPI-anchored proteins, thereby assuring their inclusion in the vacuole. Such a process occurs during internalization of Escherichia coli into macrophages through FimH-mediated attachment to CD48 35 . However, our data do not support this model for Toxoplasma invasion for two reasons: (a) the wide range of different GPI-anchored proteins that are internalized is inconsistent with use of a specific receptor; and (b) the extracellular domain of ICAM-1 was specifically excluded when attached to a transmembrane domain, yet it readily gained access to the vacuole when GPI anchored or when the cytoplasmic tail was truncated. There are two alternative explanations for the enrichment of GPI-anchored proteins in the PV: (a) exclusion of transmembrane proteins from this membrane allows GPI-anchored proteins to be more densely packed than in the plasma membrane; and (b) GPI-anchored proteins are enriched in the PV because of a selective alteration in membrane lipids. Although the first alternative does not necessitate a reorganization of membrane lipids, the second model suggests that lipid microdomains are also selectively recruited into the PV during invasion. Coalescence of lipid microdomains, or rafts, to form the PV would result in the enrichment of GPI proteins and lipids commonly associated with them (i.e., cholesterol and sphingolipids ). Recent evidence indicates that lipid microdomains exist as small patches in the plasma membrane where GPI-anchored proteins are clustered in association with cholesterol and sphingolipids, and that these structures become enlarged by cross-linking 36 37 . How such domains, which are estimated to be ∼50 nm in size, might be reorganized during formation of the PV is uncertain, but a similar process of selective lipid recruitment has been described during influenza viral budding from mammalian cells 38 . Our findings are best explained by a model based on parasite-induced invagination of the plasma membrane in combination with tethering of transmembrane proteins at the moving junction. Plasma membrane lipids, GPI-anchored proteins, and proteins without cytoplasmic domains readily diffuse past the moving junction and gain entry into the nascent PV, whereas transmembrane proteins are efficiently excluded. A similar process of protein and lipid sorting has been proposed for erythrocyte membrane vesiculation, caused by mechanical disruption, that results in the restriction of cytoskeleton and cytoskeletal-associated proteins to the cell body while lipids and GPI-anchored proteins are enriched in the vesicle 39 . Toxoplasma appears to generate such a process in reverse by distending the host cell plasma membrane inward to form the PV, a process that is driven by the active penetration by the parasite 4 . The restricted access of host cell proteins to the PV is likely to underlie its subsequent segregation from the exocytic and endocytic networks, which allows for the parasite's unique and highly successful intracellular lifestyle. | Other | biomedical | en | 0.999996 |
10601354 | Cell lines LE9211-RCC, LE9211-EBV, and LB23-SAR have been described previously 2 . Lines LB2043-PTEC and LB2046-PTEC were derived from surgical samples of normal kidney cortex and grown in Iscove's medium supplemented with ACL4 7 and epithelial growth factor (10 ng/ml) 8 . They were confirmed to derive from the proximal tubule by testing the expression of aquaporin-1 by immunohistochemistry 9 . Stable transfectant LB23-SAR-4.1 was obtained by transfection of cDNA 4.1 cloned into plasmid pEF-BOSpuro-PL3 10 using the calcium phosphate precipitation method, selection with puromycin, and cloning by limiting dilution. CTL clones were obtained exactly as described 2 . Chromium-release and TNF stimulation assays were performed as described previously 2 . Before being used as stimulators for a TNF assay, cells from LB2043- and LB2046-PTEC were transiently transfected with the HLA-B7 cDNA using DMRIE-C (GIBCO BRL) according to the instructions of the manufacturer. IFN-γ production was measured by ELISA using antibodies from Biosource. We used the same cDNA library as that described previously 2 , using the procedure detailed elsewhere 11 . In brief, mRNA was converted to cDNA with the Superscript Choice System (GIBCO BRL) using an oligo-dT primer containing a NotI site at its 5′ end. cDNAs were then ligated to BstXI adaptors and digested with NotI. After size fractionation, the cDNAs were unidirectionally cloned into the BstXI and NotI sites of plasmid pcDNAI/Amp (Invitrogen). Plasmid DNA was purified from pools of ∼100 independent cDNA clones and transfected together with DNA from an HLA-B7 cDNA construct into COS cells by the DEAE-dextran-chloroquine method. Transfected COS cells were then screened for their ability to stimulate the release of TNF by CTL 361A/21. The 5-terminal sequence was obtained by rapid amplification of cDNA ends (RACE) PCR using the 5′ RACE System (GIBCO BRL). The genomic library was prepared in phage vector lambdaGEM-11 (Promega Corp.) using standard techniques. Sequencing reactions were performed by PCR with the dideoxy-termination method and analyzed either manually or on an ABI310 Genetic Analyzer (Perkin-Elmer Applied Biosystems). Expression of RU2S and RU2AS was measured by reverse transcription (RT)-PCR on cDNA produced from total RNA as described previously 2 . Primers used were VDE87 and VDE93 for RU2S, and VDE119/VDE120 for RU2AS. PCR conditions were: 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 54°C for primers VDE87/VDE93 or at 62°C for primers VDE119/VDE120, and 1 min at 72°C, and a final elongation step of 15 min at 72°C. Primer sequences were: 5′-CCGTCAGGAACATCTACA-3′ (VDE87), 5′-CCAACAGCCACATAAAAC-5′ (VDE93), 5′-TAAATGGGTGGGCGGTGGGGGAGAC-3′ (VDE119), and 5′-TAGGCTGTTTGGAAAGGGT-AGCACA-3′ (VDE120). Because primers VDE119 and VDE120 also amplify genomic DNA, positive samples were retested after DNase treatment, to exclude any false positives due to DNA contamination of the RNA samples. Control RT-PCRs were also performed in which the addition of reverse transcriptase was omitted during cDNA synthesis. For the quantitative PCR shown in Fig. 8 , the PCR conditions were first optimized to ensure linearity of the reaction according to De Plaen et al. 12 . Optimized conditions for primers VDE119/VDE120 were: 33 cycles with 1 min at 94°C, 2 min at 68°C, and 3 min at 72°C. Serial twofold dilutions of each sample were then tested together with dilutions of positive control sample LE9211-RCC, which was chosen as a reference. PCR products were then visualized on agarose gels stained with ethidium bromide, and the level of expression of each sample was inferred from the comparison with the reference control. The results were then normalized for RNA integrity by quantifying the actin message in the same way 12 . Results were expressed as percentages of the expression level measured in reference sample LE9211-RCC. CTL clone 361/A21 was isolated by stimulating blood lymphocytes of a kidney cancer patient in vitro with autologous tumor cells LE9211-RCC, and by cloning the responding cells by limiting dilution 13 . This clone was able to lyse both autologous and allogeneic tumor cells sharing the HLA-B7 specificity, but it did not lyse autologous EBV-transformed B cells or NK target K562 . The HLA-B7 restriction of this CTL clone was confirmed by the observation that an anti–HLA-B7 antibody blocked the recognition of the tumor cells . To identify the target antigen of CTL 361A/21, we prepared an oligo-dT–based unidirectional cDNA library with RNA from LE9211-RCC cells, and we transfected DNA from this library into COS cells together with DNA from an HLA-B7 cDNA plasmid construct. The transfected cells were screened by adding the CTLs to the microcultures and measuring the production of TNF. We isolated cDNA 4.1, which was able to stimulate the CTLs when transfected into COS cells together with the HLA-B7 cDNA . This cDNA clone was 924 bp long. Its sequence was new and contained an open reading frame coding for a protein of 84 amino acids . To exclude that recognition of the transfected COS cells was an artifact of high expression levels after transient transfection, we transfected an HLA-B7–positive sarcoma line with cDNA 4.1. A stable transfectant was obtained, and it was recognized by the CTLs . To identify the antigenic epitope recognized by CTL 361A/21, we tested several synthetic peptides encoded by cDNA 4.1 and bearing the binding motif for HLA-B7, which is P in position 2 and L/F in position 9 14 . We found that peptide LPRWPPPQL was able to sensitize autologous EBV-B cells to lysis by the CTLs . To obtain the full-length sequence of the transcript corresponding to cDNA 4.1, we amplified its 5′ end by PCR and sequenced the product. This extended the 4.1 sequence by 458 nucleotides . The 1,382-bp sequence appeared to represent the full-length message, since it corresponded to a unique band observed by PCR amplification of the 5′ end. However, when cDNA 4.1 was used as a double-stranded probe on a Northern blot prepared with RNA from LE9211-RCC cells, it hybridized with a band of 2.2 kb, substantially larger than our putative full-length sequence. To solve this paradox, we screened our cDNA library by hybridization using cDNA 4.1 as a double-stranded probe. We obtained three identical clones of 2.2 kb, represented by clone I.1. Remarkably, the coding strand of clone I.1 did not contain the sequence of the coding strand of cDNA 4.1, but contained a 595-bp sequence that was complementary to the coding strand of cDNA 4.1. Since both cDNAs contained a poly(A) tail at their 3′ end and derived from an unidirectional library, their coding strands appeared to correspond to two genuinely distinct messages, one read in the sense and the other in the antisense direction of the same gene. We cloned the genomic sequence by screening a phage library constructed with genomic DNA from LE9211-RCC cells, using cDNA 4.1 as a probe. We isolated a positive phage containing a 14-kb insert, from which we sequenced the relevant part. The comparison of the three sequences revealed that cDNA I.1 corresponds to the fully spliced transcript of the gene, whereas cDNA 4.1 corresponds to an aberrant message that starts on the antisense strand of the first intron, is transcribed backwards on the antiparallel strand of exon 1, and ends with a polyadenylation site that is located on the reverse strand of the promoter . The gene, which we called RU2 , is therefore transcribed in both directions, producing a message of 2,167 bp on one strand and another message of 1,382 bp on the other strand. The longer message, which corresponds to cDNA I.1, appears to be the “normal” RU2 message, since it encodes a longer protein with potentially functional domains and is expressed at higher levels and in a wider range of tissues (see below). We compared the sequence of the RU2 gene present in tumor cells LE9211-RCC to that of the RU2 gene present in autologous EBV-B cells and found no mutation. By fluorescent in situ hybridization (FISH) analysis, we mapped gene RU2 to chromosome 6p22.1 (data not shown). We tested the expression of the two opposite RU2 transcripts by RT-PCR. For the sense message, which we named RU2S and which is represented by cDNA I.1, we used primers located in different exons, as indicated in Fig. 6 . We found that RU2S was expressed in all of the tissues tested ( Table ). This suggests a housekeeping function for this gene, which is consistent with the presence of a CpG island in its promoter and in most of exon 1 . We then tested the expression of the antisense transcript, named RU2AS and represented by cDNA 4.1, using a primer located in the promoter region and a primer located in the first intron . As opposed to the sense transcript, the antisense was only found in normal kidney, bladder, liver, and testis ( Table ). However, a very high proportion of tumors of various histological origins also express RU2AS, including tumors derived from tissues that are negative for RU2AS, such as melanomas, sarcomas, and colorectal carcinomas ( Table ). To determine the level of expression of RU2AS in the normal tissues that were found to be positive, we repeated the RT-PCR using a limiting number of cycles and an optimized annealing temperature, to allow a quantitative amplification of the messenger RNA 15 . The results shown in Fig. 8 are expressed as percentages of the level of expression found in cell line LE9211-RCC, which is arbitrarily considered as expressing 100% of RU2AS. We found that testis expresses 16–31%, kidney 7–19%, and bladder and liver 6%. We previously observed, with a CTL clone directed against a MAGE-1 peptide, that cell lines expressing the MAGE-1 mRNA below a threshold of ∼10% were not recognized by the CTLs 15 . Therefore, to determine whether the level of RU2AS in normal kidney is above the threshold for CTL recognition, we derived short-term cell lines from two samples of normal kidney. These two lines appeared to derive from the proximal tubule epithelium, since they expressed aquaporin-1, a specific marker of those tubules ( 9 ; data not shown). We measured the level of RU2AS in those lines by RT-PCR, and found that line LB2043-PTEC expressed 50% of the control level, whereas LB2046-PTEC expressed 25%. We then tested those cell lines for recognition by CTL 361A/21, after transient transfection of the HLA-B7 cDNA. As shown in Fig. 9 , both lines were able to stimulate the production of IFN-γ by the CTLs. Line LB2043-PTEC was also recognized without transfection of HLA-B7, suggesting that it already expressed the HLA-B7 molecule. We conclude that this antigen is not tumor specific and is expressed in at least a subtype of normal kidney cells. Expression of antisense transcripts is well known in prokaryotes, where it constitutes an important mechanism of gene regulation 16 . Recently, several natural antisense RNA were also described in eukaryotes. They are believed to control gene expression, either by targeting the sense transcript for rapid degradation or by preventing its translation (for a review, see reference 17 ). To find out whether the two opposite RU2 transcripts exert a similar regulatory role, we transfected COS cells with the HLA-B7 cDNA and with both the sense and the antisense cDNAs at various ratios. We found that a 200-fold excess of RU2S over RU2AS did not prevent recognition of the transfected cells by our CTLs directed against the RU2AS-encoded peptide, indicating that an excess of sense transcript does not significantly affect stability and translation of the antisense transcript (data not shown). Our results contribute to the dissection of the immune response against renal cancer cells and provide the first molecular definition of the antigenic target of CTLs recognizing both normal and tumoral kidney cells. Such CTLs have been isolated from several patients by different groups, and they represent an important part of the T cell response against kidney cancer, at least in vitro 5 6 . In one case, such CTLs were isolated from the lymphocytes of a patient who had been successfully treated with IL-2/LAK therapy, suggesting a possible in vivo relevance of this type of immune response 6 . Interestingly, this patient did not show clinically detectable signs of autoimmunity despite the complete tumor response and the presence in his blood of CTLs against normal kidney cells. However, whether kidney differentiation antigens can be successfully and safely used for cancer immunotherapy is as yet uncertain. The mechanism of expression of the RU2AS antigen is unprecedented and further supports the notion that many antigens recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene, but rather result from nonclassical mechanisms acting at the level of transcription, translation, or processing 18 . One of the first such examples was a melanoma antigenic peptide, which was found to be encoded by the intronic part of an aberrant transcript starting on a cryptic promoter in an intron of the gene coding GnTV, a ubiquitous enzyme involved in protein glycosylation 19 . This mechanism resembles that of RU2AS, except that the cryptic promoter is located on the sense and not the antisense strand of the intron. Other antigens have been found to be encoded by retained introns resulting from splicing defects in the MUM-1 , gp100, and TRP2 genes 20 21 22 . Translation of alternative open reading frames of genes TRP1 and NY-ESO-1 was also found to provide antigenic peptides recognized by CTLs on melanoma 23 24 25 . Finally, a tyrosinase peptide was found to be modified after translation, with the asparagine residue of a glycosylation site changed into an aspartic acid, apparently after removal of the glucid moiety before processing of the peptide by the proteasome 26 . Altogether, these findings indicate that the variety of peptides presented to T lymphocytes is larger than expected, and that some potentially useful antigens cannot be predicted from the sequence of the cellular protein content. | Study | biomedical | en | 0.999997 |
10601355 | Table contains a list of PCR primers used in this study. For PCR, an annealing temperature of 55°C was used throughout. Oligonucleotide probes were hybridized to Southern blots at 50°C and washed twice briefly in 2× SSC/1% SDS at room temperature and then for ∼30 min at 50°C in 1× SSC/1% SDS before exposure to film. Sequencing reactions were performed using the Big Dye Terminator Ready Mix (PE Biosystems) and were resolved and analyzed using an ABI Prism 310 sequencer (PE Biosystems). To generate anti-CD94/NKG2 mAbs, male Lewis rats were immunized three times, at ∼2-wk intervals, with stable Chinese hamster ovary (CHO) cell transfectants expressing the B6 alleles of CD94 and NKG2A. The rats were then boosted with the same transfectants, and 3 d later spleen cells were fused to P3X63-Ag8.653 (subclone of ATCC TIB-9) using PEG1500 (Boehringer Mannheim) according to the manufacturer's instructions. HAT-resistant hybridoma supernatants were screened for staining of NK1.1 + spleen cells and for staining of CHO–CD94/NKG2A but not untransfected CHO cells. Hybridoma 20d5, specific for NKG2, and hybridoma 18d3, specific for CD94, were cloned by serial dilution three times. Both mAbs are rat IgG2aκ, as assessed with a rat mAb isotyping kit (PharMingen) and were purified from hybridoma supernatants over protein G agarose (Boehringer Mannheim). Purified 20d5 was used to block NKG2 receptors at a concentration of 100 μg/ml. Anti-NK1.1 mAb was purified from PK136 supernatants and conjugated to FITC. PE-conjugated PK136 was purchased from PharMingen, and streptavidin–PE was from Molecular Probes, Inc. In the course of amplifying NKG2 cDNA ends (as described previously; reference 9), one clone similar to human NKG2C was obtained but was lacking its 5′ end and start codon. Based on this clone, a sequencing primer (NKG2C3′ex2) was designed and used to obtain the sequence of the 5′ coding and untranslated regions (UTRs) directly from bacterial artificial chromosome (BAC) B6-3 (clone 91i19). 3′ coding and untranslated sequence was obtained by sequencing the end of a genomic fragment, H3.5. Together, these 5′ and 3′ sequences were used to design two sets of primers to amplify full length NKG2C and -E open reading frames (ORFs). The NKG2C 5′ and 3′ UTR primers recognized sites just outside of the predicted ORF, whereas the NKG2C5′ATG and NKG2C3′#3 primers bound just within the ORF. Both sets of primers were used to amplify NKG2 sequences from oligo dT–primed cDNA generated by standard methods from IL-2–cultured NK cell RNA. The products, generated with Taq polymerase (Promega Corp.), were cloned directly into the T-tailed T easy vector (Promega Corp.) and sequenced on both strands. Clones generated with the UTR primer pair were designated ‘C-UTR’ or ‘E-UTR,’ whereas clones generated with the ATG primer pair were designated ‘C-ORF’ or ‘E-ORF.’ Two clones, E-UTR4 and C-ORF4, were found to encode full length NKG2E and NKG2C, respectively, and the sequences were judged free of PCR-induced coding errors based on comparisons of multiple independent clones. Clone C-ORF4 is identical to sequences previously reported to GenBank 26 27 . Clone E-UTR4 contains a silent T→C mutation at nucleotide 414. BACs were identified from the Genome Systems C57BL/6 BAC library as previously described 9 . Two HindIII fragments of 3.5 and 9 kb that hybridized to an NKG2A exon 5/6 probe were cloned into HindIII-digested pBluescript SKII(+) (Stratagene Inc.) to generate clones H3.5 and H9. These clones were sequenced using T3 and T7 primers, as well as the following primers, to determine the exon/intron boundaries for exons 4–7: NKG25′ex5, NKG23′ex5, NKG25′ex6, NKG23′ex6, NKG25′ex7, and NKG23′#3. Sorted purified (>98% pure) NK1.1 + CD3 − IL-2–cultured NK cells or nylon wool–passed, IL-2–cultured NK cells were used as starting material to make total RNA using the Ultraspec II reagent (Biotecx Labs.). Approximately 2 μg of RNA was reverse transcribed using an oligo-dT primer (GIBCO BRL), murine leukemia virus reverse transcriptase (GIBCO BRL), dNTPs (Promega Corp.), and RNasin RNase inhibitor (Promega Corp.) in a 20 μl volume. 1 μl of the resulting cDNA was used as a template for 27 cycles of PCR using the NKG2C 5′ 437 and NKG2C3′#3 primers, which bind to identical sites in all three NKG2 genes and generate a product of 298 bp. 0.1 μl of α-[ 32 P]dCTP was added to each 100-μl PCR reaction. 1–5 μl of each PCR reaction was digested with gene-specific restriction enzymes: MboI digests only NKG2A, at nucleotide 541; StuI digests only NKG2C, at nucleotide 562; and PvuII digests only NKG2E, at nucleotide 617. The digests were resolved on 6.5% PAGE, and band intensities were quantified using PhosphorImager analysis (Molecular Dynamics). As controls, plasmids containing each of the cDNAs were used as templates for the identical PCR and digestion protocol. The expression vector for NKG2A has been described 9 . To generate expression constructs, NKG2C and -E were amplified from clones C-ORF4 and E-UTR4 by PCR using Pfu polymerase (Stratagene Inc.), primer NKG2C5′ATG, and one of either primer NKG2C3′#3 (used for NKG2C) or NKG2AHANot3′ (used for NKG2E). The products were digested with XhoI and NotI, and cloned into the pME18S expression vector , and confirmed by sequencing. The latter primer added a nine–amino acid HA epitope tag (amino acid sequence YPYDVPDYA) to the COOH terminus (extracellular) of the NKG2E molecule, allowing its detection with an anti-HA mAb. Previous results with NKG2A–HA 9 showed the tag is unlikely to affect ligand binding. For our studies, the tag proved unnecessary, as we were subsequently successful in developing anti-NKG2 mAbs. The expression construct for NKG2C did not encode an HA-tagged molecule. The pME18S expression vectors were stably transfected into CHO cells along with a pIRES vector (Clontech) encoding mouse CD94 and the neo cDNA. NKG2E (but not NKG2C) was also cotransfected with pME18S-DAP12, but resulting clones were not assessed for DAP12 expression. Transfection was with the Lipofectamine reagent (GIBCO BRL) as previously described 38 . 48 h after transfection, CHO cells were passaged into RPMI supplemented with 1 mg/ml G418 (GIBCO BRL). Once drug-resistant cells started to grow out, bulk transfectants were sorted for bright surface expression of NKG2A, -C, or -E using the 20d5 anti-NKG2 mAb or Qa-1 tetramer as staining reagent. After sorting, cells were cloned by limiting dilution, and positive clones were expanded without G418. Qa-1 tetramers, complexed with the Qdm peptide (AMAPRTLLL), were generated and used as described previously 9 39 , except that a Superdex200 gel filtration column (Pharmacia) was used in all purification steps. Nucleotide sequences were aligned with CLUSTAL W 40 , using the portion of each sequence corresponding to the carbohydrate recognition domain (CRD) of NKG2A (taken here as the last 363 nucleotides of the mNKG2A ORF). For each pair of aligned sequences, the program DnaSP 3.0 41 was used to calculate the number of synonymous substitutions per synonymous site (Ks) and the number of nonsynonymous substitutions per nonsynonymous site (Ka). This program uses the unweighted algorithm of Nei and Gojobori 42 , which can be explained in brief. First, the total number of nonsynonymous sites ( N , at which nucleotide changes give rise to amino acid changes) and synonymous sites ( S , at which nucleotide changes are silent) are determined for each sequence, and the average values of S and N for each pair of sequences are used. Then, for each pair of sequences, the number of synonymous ( S d ) and nonsynonymous ( N d ) nucleotide differences observed is determined. For a pair of codons that differ by more than one nucleotide, the different possible evolutionary paths that could give rise to the observed change need to be taken into account (different evolutionary paths can involve different numbers of synonymous and nonsynonymous substitutions). In the unweighted algorithm, all of the different possible pathways are considered equally likely. S / S d is the proportion of synonymous differences, which is then used to estimate Ks. See Nei and Gojobori 42 for more details. In the course of amplifying mouse NKG2A cDNA ends (see Materials and Methods), we obtained the 3′ end of a cDNA that exhibited similarity to human NKG2C. Based on genomic sequence obtained from a BAC clone (clone 91i19), we were able to design PCR primers to amplify the entire coding region of an NKG2C cDNA from C57BL/6 mice. The consensus sequence obtained was identical to the sequence also reported recently by others 26 27 . In addition, we also obtained a novel NKG2C-like cDNA that we termed mouse NKG2E. It must be emphasized that mouse NKG2C is no more related to human NKG2C than to human NKG2E, nor does mouse NKG2E exhibit more similarity to human NKG2E than to human NKG2C . Thus, the mouse NKG2C and NKG2E genes are named in the order of their discovery, rather than on the basis of homology to their human ‘counterparts.’ Mouse NKG2C and NKG2E are 91% identical to each other at the amino acid level and are new members of the rapidly expanding family of lectin-like receptors expressed by NK cells. Mouse NKG2C and -E exhibit greatest homology with the extracellular CRD of mouse NKG2A (∼95% similar and ∼93% identical at the amino acid level), but this similarity disappears almost entirely outside of the CRD . The previously identified mouse NKG2D molecule 38 43 , on the other hand, differs markedly from the other NKG2 molecules and is in fact no more related to these molecules than it is to CD94 . These comparisons confirm that NKG2D is likely not a bona fide NKG2 family member. Unlike mouse NKG2A, mouse NKG2C and -E do not contain immunoreceptor tyrosine-based inhibitory motif–like sequences in their cytoplasmic tails. Instead, like human NKG2C and -E, mouse NKG2C and -E appear to contain a positively charged residue (arginine) in their transmembrane domains. Notably, the position of the charged residue within the transmembrane domain is slightly different between the mouse and human molecules . Similar charges have been shown to play an important role in other receptors by mediating associations with activating signaling subunits such as DAP12 44 or FcRγ 45 , but there are no data on the relevant signaling partner for mouse NKG2C or -E. The cytoplasmic tails of mouse NKG2C and -E do not appear to contain any obvious signaling motifs themselves, although their length (64 amino acids) allows for the possibility that at least some component of the signal transduction by NKG2C/E might be through their own receptor tails. The tails do not share any regions of substantial homology with the human NKG2C/E molecules, with the exception of an invariant four–amino acid motif (PPEK) that is presumed to directly abut the cytoplasmic face of the lipid bilayer. The role of this motif is not known. To study CD94/NKG2 expression by NK cells, we have generated mAbs against the mouse NKG2 and CD94 molecules. The specificity of these antibodies was confirmed by staining COS cells that had been transiently transfected with CD94, NKG2, or control (Ly49A) cDNAs . The 18d3 mAb recognized COS cells transfected with CD94 alone as well as CD94/NKG2 cotransfectants, but it did not recognize COS cells transfected with NKG2A alone. In contrast, the 20d5 mAb recognized COS cells transfected with NKG2A alone as well as CD94/NKG2A cotransfectants. The 20d5 mAb also appears to recognize NKG2C and NKG2E, as 20d5 did not stain COS cells transfected with CD94 alone but did stain CD94/NKG2C/DAP12 and CD94/NKG2E/DAP12 transfectants . NKG2C and NKG2E reach the cell surface inefficiently when transfected alone into COS cells (data not shown). Cotransfection with CD94 improved surface expression, and the further addition of DAP12 had a small additional effect (data not shown). As was previously documented 9 , CD94 and NKG2A did not require each other to reach the cell surfaces of COS transfectants. As shown previously 9 37 and in Fig. 2 B, tetrameric complexes of Qa-1/β 2 microglobulin/Qdm stain roughly half of adult mouse NK cells. Although the Qa-1 tetramers presumably bind CD94/NKG2 receptors on NK cells, this has not been formally demonstrated, and the possibility remains that NK cells express additional Qa-1 receptors. We observed staining of NK1.1 + splenocytes with both the 18d3 (anti-CD94; data not shown) and 20d5 (anti-NKG2) mAbs , confirming that these molecules are indeed expressed on the surfaces of NK cells. As expected, the frequency of tetramer-reactive NK1.1 + cells was similar to the frequency of NKG2 + cells . The frequency of tetramer-reactive NK cells was somewhat variable and sometimes lower than (but never higher than) the frequency of 20d5 + cells, particularly as the tetramer preparation aged. Similar variability has been previously observed with class I tetramer recognition of Ly49 receptors and its possible explanations discussed 46 . To confirm that CD94/NKG2 receptors are the predominant Qa-1 receptors on NK cells, we attempted to block Qa-1 binding with the 20d5 (anti-NKG2) mAb . We found that 20d5 completely blocked all binding of Qa-1 tetramers to NK1.1 + splenocytes, strongly arguing that CD94/NKG2 receptors are indeed the predominant, if not the only, Qa-1 receptors on mouse NK cells. Similarly, it has been observed that anti–human CD94 blocks all binding of HLA-E tetramers to the human NKL cell line 10 . Based on the high degree of similarity between the extracellular ligand binding domains of NKG2A, -C, and -E, it is reasonable to hypothesize that CD94/NKGC and CD94/NKG2E heterodimers, like CD94/NKG2A heterodimers, would recognize Qa-1 b . We generated stable transfectants of CHO cells expressing high levels of CD94/NKG2A, CD94/NKG2C, and CD94/NKG2E . All three stable cell lines, but not untransfected CHO cells, stained brightly with soluble, tetramerized Qa-1/β 2 microglobulin/Qdm complexes. We previously showed that CD94 alone is incapable of binding Qa-1 b 9 . The results therefore provide direct evidence that both CD94/NKG2C and CD94/NKG2E recognize Qa-1 b –Qdm complexes. The degree of staining appeared to correlate roughly with the levels of NKG2 and CD94, as independently assessed by staining with anti-NKG2 and anti-CD94 mAbs. In particular, the CD94/NKG2A transfectant stained most brightly with tetramer and with anti-CD94 and anti-NKG2 antibodies, whereas the CD94/NKG2C transfectant stained least brightly with all three staining reagents. Thus, our data do not reveal a gross difference in the avidity of the various CD94/NKG2 subunits for Qa-1 b . Evidence in humans using quantitative surface plasmon resonance techniques suggests that CD94/NKG2A binds HLA-E with higher affinity than does CD94/NKG2C 21 . Such differences may also exist for the mouse CD94/NKG2 receptors, as our data is not quantitative. Because the Qa-1 protein was produced in Escherichia coli and is therefore unglycosylated, our results also demonstrate that CD94/NKG2C and CD94/NKG2E, like CD94/NKG2A 9 , can recognize carbohydrate-independent epitopes on their ligands. Previous Southern blot data were consistent with a small cluster of NKG2 genes located within the NK gene complex near mouse CD94. To determine if the NKG2C and NKG2E cDNAs we obtained might map to the CD94/NKG2 locus, we designed an oligonucleotide probe specific for NKG2E, as well as a probe that recognized NKG2C and NKG2A but not NKG2E. These probes were tested for specificity on their corresponding cDNAs (not shown) and were then used on Southern blots shown in Fig. 4 . We found that a 3.5-kb HindIII fragment, present in all the BACs, hybridized specifically to the NKG2E probe. In contrast, the NKG2A/C probe hybridized specifically to a 9-kb HindIII fragment, present only in BACs B6-1 and B6-4. The NKG2A/C probe also recognized a ∼20-kb fragment present only in BAC B6-4, the only BAC known to contain NKG2A 9 . The 3.5- and 9-kb HindIII fragments were subcloned and partially sequenced and were found to contain sequence that perfectly matched exons 4–7 of NKG2E and NKG2C, respectively (exon numbering based on homology to the human NKG2A gene; reference 47). The boundaries for the mouse exons are presented in Table . As described below, the boundaries of exons 4 and 5 appear to be somewhat flexible and can give rise to alternatively spliced transcripts. Together with previously presented data 9 48 , these results allow us to infer the order of mouse CD94 and NKG2 genes within the NK complex on mouse chromosome 6 . Interestingly, the overall structure of the locus is well conserved with that of the human CD94/NKG2 locus. In particular, the gene order CD94–NKG2D–NKG2E/C–NKG2A appears to be conserved 49 . The human locus differs by the presence of NKG2F, a truncated NKG2 gene that lies between NKG2D and NKG2E 49 50 . We have not seen evidence of a mouse NKG2F gene, although its existence cannot be ruled out. In addition to the cDNA clones encoding full length NKG2C and NKG2E, we obtained multiple clones encoding NKG2C- or NKG2E-like sequences that differed by various insertions and deletions . The positions of the insertions and/or deletions corresponded precisely to canonical splice acceptors and donors determined from the genomic sequencing ( Table ), and so it seems unlikely that these variants were an artifact of PCR. Rather, they probably arose by alternative splicing of primary NKG2C and NKG2E transcripts. The splice variants all involve alternative splice donors or acceptors present in exons 4 and 5, encoding the extracellular stalk and part of the CRD. Alternative splicing of other exons was not observed. Some of the variants produce reading frame shifts that result in premature stop codons and truncated receptors that would not be predicted to bind ligand . A previously identified NKG2C cDNA called NKG2C2 26 likely arose from alternative use of the splice acceptors and donors identified here. One particularly interesting clone was E-UTR1, which was found to contain a chimeric NKG2C/NKG2E cDNA . It is possible that this transcript was generated as an artifact of PCR. Alternatively, if NKG2C and -E share the same transcriptional orientation in the genome as they do in humans, it is possible that a single primary message encoding both genes was transcribed and spliced to form the chimeric receptor. Alternative splicing, particularly involving exons encoding the transmembrane and stalk domains, has also been observed for other C-type lectin-like receptors, including mouse Ly49 C, D, G, H, and J, human CD94, and human and mouse NKG2B, a splice variant of NKG2A 27 47 51 52 53 54 . Despite their apparent prevalence, the function of these alternative splices is not clear. To estimate the relative abundance of the splice variants in NK cells, we amplified the alternatively spliced region from various NK cell cDNA samples using flanking primers that recognize NKG2C and NKG2E but not NKG2A. The heterogeneous PCR product was resolved by gel electrophoresis and then analyzed by Southern blot and probed with NKG2C- and NKG2E-specific oligonucleotide probes . The results confirm that NKG2C and -E are indeed extensively alternatively spliced, and a total of eight different cDNA variants could be detected. The pattern of splicing detected was remarkably consistent in five different RNA samples , including RNA from highly purified (>98% pure) NK1.1 + CD3 − NK cells. Control PCR reactions using an equimolar mix of templates suggested that shorter products were not preferentially amplified (data not shown). Interestingly, although NKG2C and NKG2E are closely related genes, they exhibit quite different patterns of mRNA splicing. The majority of NKG2C cDNAs are identical in size to the NKG2C1 cDNA and would therefore be capable of producing a full length protein. On the other hand, the majority of NKG2E cDNAs are considerably shorter and identical in size to the E-UTR2 cDNA. This cDNA contains a frame shift as compared with the full length ORF. Consequently, most NKG2E cDNAs are predicted to encode a truncated polypeptide that is unable to bind ligand. We conclude that a substantial fraction of the NKG2C and -E message in NK cells is of an alternatively spliced form. Previously, we observed that Qa-1 tetramer–reactive NK cells (presumably expressing CD94/NKG2A, -C, and/or -E) were inhibited, not activated, by the presence of Qa-1–Qdm complexes on target cells. These data raised the possibility that the activating NKG2C and NKG2E receptors might be expressed less frequently than the inhibitory NKG2A receptor. Alternatively, mouse NKG2C and -E might recognize Qa-1 relatively poorly or transmit weaker signals. To distinguish these nonexclusive possibilities, we developed a PCR-based assay to quantitate the relative levels of NKG2A, -C, and -E message in purified NK cells. We took advantage of the high degree of nucleotide identity among the three cDNAs to design a primer pair that would match perfectly to all three cDNAs. As the primer binding sites and the size of the amplified product is identical for all three sequences, PCR should faithfully amplify each cDNA in proportion to its representation in the original sample. The various amplified cDNA products could be distinguished after PCR by differential digestion with gene-specific restriction enzymes. Radioactive nucleotides were included in the amplification reaction to permit quantification of the various digested PCR products by PhosphorImager analysis. The results show that the majority of NKG2 PCR product could be digested with an NKG2A-specific restriction enzyme, MboI, whereas only a small fraction of the product could be digested with NKG2C- and NKG2E-specific restriction enzymes (PvuII and StuI). Using this method, we estimated that NKG2A transcripts are at least 20-fold more abundant than NKG2C and -E transcripts combined. It is unclear whether this difference is best accounted for by the presence of relatively few NKG2C/E + NK cells or by relatively low NKG2C/E transcript levels per cell. Nevertheless, our molecular analysis suggests that the net inhibition of NK cells by Qa-1 expressed on target cells may be explained, at least in part, by the low abundance of activating NKG2C/E receptors on NK cells. It should be emphasized that activating NKG2 genes have been evolutionarily conserved, and it thus seems likely that NKG2C/E genes play an important role that does not depend on abundant mRNA transcripts. The above findings document the striking conservation of the function and genomic organization of CD94/NKG2 receptors between mice and humans. In contrast, other class I–specific receptors, such as Ly49 receptors in mice and KIRs in humans, do not appear to be functionally conserved. Given the availability of NKG2 sequences from three species, it is interesting to analyze the evolutionary history of these genes. We therefore aligned the extracellular CRDs for each possible pair of NKG2 genes from mice, rats, and humans. Using the DnaSP algorithm 41 , each aligned nucleotide position was designated as a synonymous site (at which nucleotide substitutions do not generate amino acid substitutions) or as a nonsynonymous site (at which nucleotide substitutions generate amino acid substitutions; see Materials and Methods). For each pair of aligned sequences we could then estimate Ks, a commonly used parameter that represents the number of synonymous substitutions that have occurred per synonymous site ( Table ). The number of nonsynonymous substitutions per nonsynonymous site, Ka, could be similarly calculated. Synonymous (or ‘silent’) substitutions are usually considered selectively neutral, or nearly so. Thus, a high Ks value reflects the substantial selection-independent accumulation of nucleotide differences 55 . The analysis demonstrates that within a species, NKG2 sequences are closely related, whereas between species they are not. The intraspecies similarity is most dramatically exemplified by the finding that there are, for example, no synonymous substitutions within the CRDs of mouse NKG2A and mouse NKG2C. Other intraspecies pairs of NKG2 genes give similarly low frequencies of synonymous substitution, i.e., 0.03–0.13 ( Table ). In contrast, the frequency of synonymous substitutions for interspecies pairs of NKG2 genes is substantially higher, i.e., 0.60–0.68 ( Table ). These data are consistent with two broad evolutionary histories: (a) humans and rodents share a single ancestral NKG2 gene that underwent duplications separately in each lineage to generate ‘independent’ and relatively young multigene families; or (b) the common ancestor of humans and rodents contained all three genes, but the genes have since been undergoing ‘concerted evolution,’ a process by which homologous loci within a species maintain similarity to each other. Concerted evolution has been inferred in several multigene families 55 . The mechanisms by which it occurs remain unclear but are thought to involve homogenizing events, such as unequal crossing over and/or gene conversion–like events 55 . Although it is difficult to adjudicate between the two above evolutionary histories, the second model may be more likely for the following reasons. First, all three species examined contain multiple NKG2 genes, including at least one inhibitory and one activating type, consistent with a primitive duplication event that was transmitted to all lineages. Second, the 5′ (cytoplasmic) portions of the inhibitory and activating NKG2 genes tend to vary substantially from each other, even within species, suggesting that the genes have been diverging for some time. Unless gene conversion–like events—which can affect discrete parts of genes—are invoked, it is difficult to reconcile the evolutionary divergence apparent in the 5′ portions of mouse NKG2A and -C with the finding that they do not exhibit any synonymous substitutions in their CRDs. Lastly, the remarkable overall similarity seen in the genomic structures of the mouse and human NKG2 loci suggests that the gene families did not result from independent duplication events. It is interesting that gene conversion events have also been proposed to play a role in the evolution of MHC class I genes 56 . Our previous work on mouse CD94/NKG2A provided evidence that recognition of class I was a primitive function of NK cells that predated the divergence of mouse and human ancestors. Assuming that NKG2 genes underwent a primitive duplication, as argued above, our work seems to suggest that both activating and inhibitory class I recognition may have been primitive functions of NK cells. This is interesting, as it suggests some fundamental role for activating receptors in the recognition of class I. This notion is further supported by the observation that families of class I–specific receptors, including Ly49, KIR, and leukocyte Ig-like receptors/Ig-like transcript families, invariably consist of both activating and inhibitory paralogs. Indeed, the pairing of inhibitory and activating isoforms extends beyond receptors specific for class I, and includes the paired Ig-like receptors on B cells and myeloid cells 57 and the CD28/CTLA-4 coreceptors on T cells. Although well understood in the case of T cells, the molecular logic behind the existence of paralogous activating and inhibitory receptors on NK cells is still a matter of conjecture. We anticipate that the identification of mouse CD94/NKG2C and CD94/NKG2E as activating receptors will permit the appropriate in vivo experimental manipulations required to dissect their function. | Study | biomedical | en | 0.999997 |
10601356 | MRL/MpJ ++ (MRL ++ ), MRL/MpJ- Fas lpr /Fas lpr (MRL- Fas lpr ), C3H/FeJ, and C57BL/6 mice were purchased from the The Jackson Laboratory. MCP-1–intact and –deficient B6/129 mice (129SV/J × C57BL/6)F 1 were constructed as described 20 . Control MCP-1–intact B6/129 mice were derived from matings of mice heterozygous for the disrupted allele. All mice were maintained in a pathogen-free animal facility. MCP-1–deficient (MCP-1 −/− ) MRL -Fas lpr mice were created by a series of genetic backcrosses using the cross-backcross-intercross scheme. MRL- Fas lpr mice were mated with MCP-1 −/− (129/Sv × C57BL/6) mice to yield heterozygous F1 offspring. We intercrossed F1 mice and screened the progeny by PCR amplification of tail genomic DNA for the Fas lpr mutation and MCP-1 using specific primers 21 24 . Double homozygotes ( Fas lpr /Fas lpr MCP-1 −/− ) N1F1 progeny were backcrossed to MRL- Fas lpr mice. B1 progeny, homozygous for the Fas lpr mutation and heterozygous for MCP-1 (MCP-1 +/− ), were intercrossed, and mice homozygous for the disrupted MCP-1 gene were selected by PCR typing for continued backcrossing. After three generations of backcross–intercross matings, this breeding scheme generated a colony of MRL- Fas lpr mice (94% MRL- Fas lpr background) homozygous and heterozygous for the disrupted MCP-1 gene. We analyzed the third generation, as we have previously established that there are sufficient MRL -Fas lpr background genes to result in phenotypic changes characteristic of the wild-type MRL- Fas lpr strain 25 . In addition, we compared sex-matched littermates to minimize variability. The MCP-1 −/− MRL- Fas lpr mice are termed MCP-1 deficient, whereas the MCP-1 +/+ MCP-1 +/− MRL- Fas lpr mice are termed MCP-1–intact MRL- Fas lpr mice. Urine protein levels in MCP-1–intact and –deficient MRL- Fas lpr mice were assessed semiquantitatively by dipstick analysis (Albustix; Bayer Diagnostic Division) on a monthly basis beginning at 2 mo of age. On the day of analysis, dipstick proteinuria measurements were taken from individual mice in the morning and evening. If the measurements were inconsistent, the animal was reassessed the following day. Protruding lymph nodes (cervical, brachial, and inguinal) and skin lesions were assessed monthly beginning at 3 mo of age. Lymph node score based on palpable nodes: 0 = none; 1 = small, at one site; 2 = moderate, at two different sites; and 3 = large, at three or more different sites. Skin lesion score by gross pathology: 0 = none; 1 = small (face or ears); 2 = moderate, <2 cm (face, ears, and back); and 3 = severe, >2 cm (face, ears, and back). Splenomegaly was determined by spleen weights. Kidneys were fixed in 10% formalin for 24 h at 4°C. Paraffin sections (4 μm) were stained with hematoxylin and periodic acid Schiffs (PAS) reagent. We evaluated glomerular pathology by assessing 50 glomerular cross sections (gcs) per kidney and scored each glomerulus on a semiquantitative scale: 0 = normal (35–40 cells/gcs); 1 = mild (glomeruli with few lesions showing slight proliferative changes, mild hypercellularity [41–50 cells/gcs], and/or minor exudation); 2 = moderate (glomeruli with moderate hypercellularity [50–60 cells/gcs], including segmental and/or diffuse proliferative changes, hyalinosis, and moderate exudate); and 3 = severe (glomeruli with segmental or global sclerosis and/or exhibiting severe hypercellularity [>60 cells/gcs], necrosis, crescent formation, and heavy exudation). Damaged tubules (percent; consisting of dilation and/or atrophy and/or necrosis) were determined in 400 randomly selected renal cortical tubules per kidney (×400). Perivascular cell accumulation was determined semiquantitatively by scoring the number of cell layers surrounding the majority of vessel walls (score: 0 = none; 1 = <5; 2 = 5–10; and 3 = >10). Scoring was evaluated using coded slides. The lungs were inflated and fixed with 10% formalin, and paraffin sections (4 μm) were stained with hematoxylin and PAS reagent. Perivascular and peribronchiolar infiltrates were assessed semiquantitatively in >20 vessels per section and in >20 bronchioli per section (number of cell layers surrounding the majority of vessels or bronchioli: 0 = none; 1 = 1–3; 2 = 3–6; and 3 = >6). The following primary antibodies were used for immunostaining: rat anti–mouse CD4 IgG2a clone RM4-5 (PharMingen) to detect CD4 T cells; rat anti–mouse CD8a (Ly-2) IgG2a clone 53-6.7 (PharMingen) to detect CD8 T cells; rat anti–mouse CD45R/B220 IgG2a clone RA3-6B2 (PharMingen) to detect CD4/CD8 T cells; rat anti–mouse CD21/35 IgG2b clone 7G6 (PharMingen) to detect B cells; rat anti–mouse monocyte/macrophage IgG2b clone MOMA-2 (BioSource International) and rat anti–mouse macrophage IgG2b (prepared from F4/80 hybridoma supernatant; American Type Culture Collection number HB198) to detect macrophages; rabbit anti–mouse MCP-1 IgG (Serotec Ltd.) to detect MCP-1; mouse anticytokeratin peptide 18 IgG1 clone CY-90 (Sigma Chemical Co.) to detect epithelial cells; fluorescein-conjugated mouse anti-PCNA (proliferating cell nuclear antigen) IgG1 clone 19F4 (Boehringer Mannheim) to detect proliferating cells; fluorescein-conjugated goat anti–mouse IgG (Organon Teknika) to detect mouse IgG; and fluorescein-conjugated goat anti–mouse C3 (Organon Teknika) to detect mouse complement C3. The negative isotype control antibodies for immunostaining were rat IgG2a clone R35-95, rat IgG2b clone R35-38, mouse IgG1 clone MOPC-21 (PharMingen), and normal rabbit IgG (Sigma Chemical Co.). The secondary antibodies for immunostaining were biotin-conjugated rabbit anti–rat IgG and biotin-conjugated goat anti–rabbit IgG (Vector Labs.). ELISA analysis of serum Ig (total Ig, IgM, IgG1, IgG2a, IgG2b, and IgG3) was performed using isotype-specific standards, goat anti–mouse capture antibodies, and alkaline phosphatase–conjugated goat anti–mouse detection antibodies (Southern Biotechnology Associates Inc.). To analyze kidney- and lung-infiltrating T cells and interstitial macrophages, cryostat tissue sections (4 μm) were fixed in ethanol at 4°C for 10 min and immunostained with CD4, CD8, B220, and F4/80 antibody as previously described 26 using the avidin-biotin-peroxidase detection system (Vector Labs.). Glomerular macrophages were identified in 4-μm acetone-fixed tissue sections using MOMA-2 antibody as previously reported 16 . Cells infiltrating within and around (peri) glomeruli were assessed by counting the number of labeled cells in 20 randomly selected glomeruli per section. The cells infiltrating around tubules were enumerated in 400 randomly selected tubules per section. Macrophages and T cells within glomeruli, adjacent to glomeruli, or adjacent to cortical tubules were expressed as a cell index (mean cell number/glomeruli × glomeruli/section; mean cell number/cortical tubule × cortical tubules/section). Peribronchial and perivascular macrophages and T cells (CD4, CD8, B220) in the lungs were assessed by measuring the unit area stained/unit length of bronchiole/vessel (five per animal) with a micrometer. To detect MCP-1, formalin-fixed sections were deparaffinized and incubated with 20% normal goat serum for 30 min. Tissue sections were incubated with MCP-1 antibody (10 μg/ml) in 1% BSA overnight at 4°C. Bound primary antibody was labeled with biotin-conjugated goat anti–rabbit IgG for 1 h and subsequently detected using the avidin-biotin-peroxidase system (Vector Labs.). We confirmed MCP-1 antibody specificity using two methods: (a) MCP-1 was not detectable in tissue sections from MCP-1–deficient mice (negative control), and (b) MCP-1 was expressed in Western blots of cell lysates from LPS-stimulated bone marrow macrophages and mesangial cells in MCP-1–intact but not MCP-1–deficient mice, as previously reported 21 . Glomerular MCP-1 immunostaining was assessed by the same scoring system used to detect glomerular infiltrating cells. We analyzed MCP-1 in cortical tubules in 400 tubules per cross section. We enumerated the kidney and pulmonary vessels and bronchioli that expressed MCP-1 in each section and recorded values as the percent positive. ELISA plates were coated overnight at 4°C with 5 μg/ml goat anti–mouse Ig capture antibodies (against total Ig, IgM, IgG1, IgG2a, IgG2b, and IgG3) in 0.1 M carbonate buffer, pH 9.4. Wells were blocked for 1 h with assay diluent (2% BSA in 0.1 M borate buffer, pH 8.0). We then added Ig standards to the plates (50 μl/well), starting at 1 μg/ml and performing a series of threefold dilutions, and assessed serum samples using serial (threefold) dilutions starting at 1:100 or 1:1,000. Standards and serum samples were incubated overnight at 4°C, and bound Ig was detected with goat anti–mouse detection antibodies conjugated with alkaline phosphatase and enzymatically developed by incubating with Sigma 104 phosphatase substrate in 9.6% diethanolamine and 0.1 mM MgCl 2 , pH 9.8 (Sigma Chemical Co.). Absorbance was measured at 405 nm. To examine IgG and C3 deposits in the kidney, we incubated cryostat-sectioned tissues (4 μm) with 20% normal goat or rabbit serum (30 min), followed by fluorescein-conjugated antibodies detecting mouse IgG or mouse C3 (30 min), washing, and mounting with Vectashield (Vector Labs.). We assessed immunofluorescence staining by titrating the antibodies on serial tissue sections using twofold dilution steps (1:100–1:25,600). Apoptotic cells were identified in tissue sections (4 μm) using the terminal deoxynucleotide transferase–mediated dUTP-biotin nick-end labeling (TUNEL) method and immunoperoxidase staining (In Situ Cell Death Detection Kit; Boehringer Mannheim). Proliferating cells were detected by PCNA immunostaining as previously described 27 . Sections were counterstained with PAS reagent to determine morphology. Apoptosis in glomeruli and tubules was assessed by counting the number of TUNEL-labeled cells in 50 glomeruli per section and 500 cortical tubules per section, respectively. Total RNA was extracted from the snap-frozen renal cortex of half a kidney using RNAzol B (Tel-Test Inc.) and reverse transcribed using oligo-dT and the Superscript II DNA preamplification kit (GIBCO BRL). The resulting reverse transcription product was the cDNA template for PCR analysis. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression was detected as a 500-bp product resulting from PCR with specific oligonucleotide primers (antisense, 5′-CAAAGTTGTCATGGATGACC-3′; and sense, 5′-GGTGGAGGTCGGAGTCAACG-3′; reference 21). The chemokines MCP-1, MCP-3, and MCP-5 were detected as 350-, 300-, and 370-bp PCR products, respectively, using specific oligonucleotide primers (MCP-1 antisense, 5′-GCTTGAGGTGGTTGTGGAAAA-3′; MCP-1 sense, 5′-CTCACCTGCTGCTACTCATTC-3′; MCP-3 antisense, 5′-CACATTCCTACAGACAG-CTC-3′; MCP-3 sense, 5′-AGCTACAGAAGGATCACCAG-3′; MCP-5 antisense, 5′-CTCCTTATCCAGTATGGTCC-3′; and MCP-5 sense, 5′-TCTCCCTCCACCATGCAGAG-3′; references 28 29 30 ). The cytokines IFN-γ and CSF-1 were detected as 500- and 245-bp PCR products using specific primers (IFN-γ sense, 5′-CACGGCACAGTCATTGAAAGCC-3′; IFN-γ antisense, 5′-CTTATTGGGACAATCTCTTCCC-3′; CSF-1 sense, 5′-CACATGATTGGGAATGGACA-3′; and CSF-1 antisense, 5′-CAGCTGTTCAGGTTATTGGA-3′; references 31 and 32 ). The PCR primers and conditions were chosen so that the MCP and GAPDH products were amplified with equal efficiency. Data was analyzed using the Kruskal-Wallis test for comparing survival curves, the Mann-Whitney test to compare group means, and Spearmans coefficient for correlations. MCP-1 is upregulated in MRL- Fas lpr kidneys, lungs, and lymph nodes during autoimmune disease. First, we detected an increase (threefold) in MCP-1 mRNA within the renal cortex of wild-type pure MRL- Fas lpr mice as early as 2 mo of age, as compared with the C57BL/6 strain with normal kidneys. MCP-1 mRNA increased further within the renal cortex (six- to eightfold) with advancing renal injury in these MRL- Fas lpr mice . We localized MCP-1 expression to several structures within the MRL- Fas lpr kidney and determined that the rank order of tissue expression was tubules→glomeruli→vasculature. MCP-1 in MRL- Fas lpr kidneys (5–6 mo of age) was almost exclusively localized within the TECs. More specifically, MCP-1 was within the proximal TECs (brush border histologic identification), whereas the distal TECs (histologic identification) were only weakly positive or negative. MCP-1 expression in MRL- Fas lpr glomeruli was determined to be predominantly in epithelial podocytes, based on the morphological positioning of MCP-1–stained cells in glomeruli after histologic counterstaining , whereas lesser amounts localized within the epithelial cells and macrophages within crescents (two to three cells per crescent). Finally, MCP-1 in the vasculature within endothelial and smooth muscle cells was minimal . It is noteworthy that few (1–2%) infiltrating cells in the renal interstitium express MCP-1. Thus, kidney MCP-1 expression is almost exclusively within parenchymal cells and in epithelial cells in particular, rather than infiltrating cells. MCP-1 is expressed in other tissues in MRL- Fas lpr mice during autoimmune disease. Similar to MCP-1 in the kidney, MCP-1 in the lungs is predominantly (>90%) expressed in epithelial cells and is weakly detected within vascular endothelial and interstitial cells in MRL- Fas lpr mice (5 mo of age). Massively enlarged lymph nodes caused by an influx of T cells are characteristic of MRL- Fas lpr autoimmune disease 23 . MCP-1 is readily detected in cells surrounding lymphatic vessels within these enlarged lymph nodes (inguinal, cervical) in MRL- Fas lpr mice . As anticipated, MCP-1 was not detected in MCP-1–deficient MRL- Fas lpr tissues . Taken together, these data indicate that MCP-1 is mainly expressed by parenchymal cells in multiple tissues targeted for autoimmune injury in MRL- Fas lpr mice. MCP-1–deficient MRL- Fas lpr mice have a prolonged life span as compared with MCP-1–intact MRL- Fas lpr strains. The vast majority of MCP-1–deficient MRL- Fas lpr mice (75%) remained alive at 300 d, as compared with a surviving minority of MCP-1–intact (MCP-1 +/+ , 17% and MCP-1 +/− , 44%) MRL- Fas lpr mice . Notably, the mortality (50%) of the MCP-1 +/+ MRL- Fas lpr strain third generation was 7 mo of age, which is similar to that of the pure wild-type MRL- Fas lpr strain (6 mo of age) and dissimilar to that of C57BL/6J and Sv/129 strains (>20 and 16 mo of age, respectively; reference 33 ). It is important to note that the spot analysis for protein in fresh individual urine specimens has several limitations, including the sample size (volume) and semiquantitative measurement. However, our confidence in making inferences from this method is enhanced by the large number of mice in each group and sequential monthly analysis. With these caveats in mind, we now report that MCP-1–deficient versus MCP-1–intact MRL- Fas lpr mice are protected from pathological proteinuria. From 2 to 8 mo of age, both the rate of increase and incidence of pathological proteinuria were diminished in MCP-1–deficient MRL- Fas lpr mice . For example, the vast majority (82%) of MCP-1–deficient MRL- Fas lpr mice had normal, nonpathologic proteinuria (1+), whereas the majority (62%) of MCP-1–intact MRL- Fas lpr mice were pathologically proteinuric (2–4+) at 8 mo of age. It should be noted that urinary protein in normal B6/129 wild-type mice is barely detectable (0–1+; data not shown). Thus, MCP-1–deficient MRL- Fas lpr mice are protected from proteinuria. We examined whether MCP-1 promotes autoimmune disease in the lymph nodes, spleen, and skin of MRL- Fas lpr mice from 3 to 8 mo of age. The incidence and severity of lymphadenopathy and skin lesions was reduced but not totally eliminated in MCP-1–deficient as compared with MCP-1–intact MRL- Fas lpr mice . For example, although nearly every MCP-1–intact MRL- Fas lpr mouse had palpable lymph nodes (94%), most of the MCP-1–deficient mouse lymph nodes were not palpable at 5 mo of age (69%). Similarly, the majority (84%) of MCP-1–deficient mice were spared skin lesions, whereas most (67%) of the MCP-1–intact MRL- Fas lpr mice had gross skin pathology at 8 mo of age. On the other hand, we did not detect a difference in splenomegaly in MCP-1–deficient (234 ± 64 mg) versus MCP-1–intact MRL- Fas lpr mice (242 ± 143 and 289 ± 174 mg MCP-1 +/+ and MCP-1 +/− , respectively; P = 0.2; n = 6 per group) at 5 mo of age. To determine if MCP-1 is required for kidney and lung pathology, we compared MCP-1–deficient and –intact MRL- Fas lpr mice killed at 5 mo of age. Renal (tubular, glomerular) and pulmonary pathology was reduced in MCP-1–deficient as compared with MCP-1–intact MRL- Fas lpr mice . The reduction in tubular and glomerular pathology assessed histologically was dramatic (50–70%) in MCP-1–deficient MRL- Fas lpr kidneys . In particular, MCP-1–deficient MRL- Fas lpr mice had markedly diminished peritubular infiltrate, tubular atrophy, glomerular hypercellularity, glomerulosclerosis, and crescent formation. We further evaluated the level of tubular and glomerular damage by identifying apoptotic cells. The number of apoptotic cells was reduced in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr kidneys. The majority of apoptotic TECs diminished from 4.2 ± 0.3% per section in MCP-1–intact to 2.0 ± 0.6% per section in MCP-1–deficient MRL- Fas lpr kidneys ( P < 0.05; n = 6 per group). Similarly, the number of apoptotic glomerular cells was reduced in MCP-1–deficient versus MCP-1–intact mice (0.11 ± 0.04 cells/gcs and 0.25 ± 0.05 cells/gcs, respectively; P < 0.05; n = 6 per group). Although there was a reduction in tubular and glomerular pathology in MCP-1–deficient MRL- Fas lpr kidneys, it was not totally prevented in comparison to age-matched kidneys from MRL ++ and C3H mice with normal kidneys . There was an increase in tubular and glomerular pathology in MCP-1–deficient MRL- Fas lpr kidneys. In contrast to the protection afforded by the absence of MCP-1 in renal tubules and glomeruli of MRL- Fas lpr mice, the perivascular infiltrates in MCP-1–deficient and –intact MRL- Fas lpr kidneys remained similar . Thus, MCP-1 is responsible for tubular and glomerular but not perivascular renal pathology. Pulmonary disease was reduced in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr mice. Lung pathology in MCP-1–intact MRL- Fas lpr mice consists of infiltrating cells predominantly surrounding bronchioles and vasculature. The numbers of cells surrounding bronchioles in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr strains was dramatically reduced . As in the kidney, the infiltrating cells surrounding vessels were not reduced in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr lungs . We determined if the MCP-1–deficient MRL- Fas lpr mice that died (25%; 300 d) developed renal injury that was as severe as that of the MCP-1–intact MRL- Fas lpr mice that succumbed at considerably younger ages . The MCP-1–deficient MRL- Fas lpr mice had less (40–50%) tubular and glomerular pathology compared with the MCP-1–intact MRL- Fas lpr kidneys ( P < 0.05; n = 6; data not shown). In addition, fewer (5/11) MCP-1–deficient MRL- Fas lpr mice, as compared with MCP-1–intact MRL- Fas lpr mice (12/16), died with severe proteinuria (3–4+). Thus, even in the MCP-1–deficient and MCP-1–intact MRL- Fas lpr mice that eventually die, the extent of renal injury is less severe in the MCP-1–deficient MRL- Fas lpr strain. There was a reduction in the number of macrophages and T cells (CD4 and CD8 but not B220) in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr kidneys. The majority of macrophages and T cells localized in the interstitium adjacent to tubules and glomeruli, whereas fewer were identified within glomeruli. We noted the largest (50–72%) decline in macrophages and T cells in the MCP-1–deficient MRL- Fas lpr strain surrounding tubules , a reduction that correlated with the extent of tubular injury ( r ≥ 0.80; P < 0.005). Periglomerular macrophages and CD4 but not CD8 T cells were also diminished (45–53%, P < 0.05) in MCP-1–deficient MRL- Fas lpr mice . Although intraglomerular macrophages were reduced (34%) in MCP-1–deficient MRL- Fas lpr kidneys , the numbers of intraglomerular T cells (CD4, CD8, B220) were not diminished ( P = 0.2). The reduction in glomerular macrophages correlated with diminished glomerular morphologic damage (intraglomerular, r = 0.78; periglomerular, r = 0.77; P < 0.005). The absence of MCP-1 in MRL- Fas lpr lungs reduced the number of infiltrating macrophages surrounding bronchioli but not surrounding vessels . In contrast to the reduction in the number of kidney-infiltrating T cells in MCP-1–deficient versus –intact MRL- Fas lpr mice, the number of T cells (CD4, CD8, B220) in the lungs was not reduced . We determined if the decreased accumulation of macrophages and T cells in MCP-1–deficient MRL- Fas lpr kidneys and lungs at 5 mo of age was a result of a decline in local proliferation using in situ detection of PCNA. First, few of the cells surrounding tubules (<1%), glomeruli (<1%), and bronchioli (<10%) were proliferating in the MCP-1–intact MRL- Fas lpr strain. The number of PCNA + cells was not reduced in the MCP-1–deficient MRL- Fas lpr strain ( P = 0.2; n = 6). Second, there were substantially more proliferating cells surrounding vessels (>20%) compared with proliferating cells surrounding glomeruli, tubules, and bronchioles in MCP-1–intact MRL- Fas lpr kidneys and lungs ( P < 0.002), and this number did not decrease in the MCP-1–deficient MRL- Fas lpr strain ( P = 0.1; n = 6 per group). Of course, it must be appreciated that the proliferation measurements in tissue sections are a reflection of the level of cell division at the time these tissues were removed. Thus, the reduction in kidney- and lung-infiltrating cells in MCP-1–deficient MRL- Fas lpr mice does not appear to result from decreased local proliferation. MCPs 1–5 are ligands for the MCP-1 receptor (CCR2; reference 22). To examine whether CCR2 ligands other than MCP-1 are upregulated after renal injury, we examined MCP-3 and MCP-5 in MRL- Fas lpr kidneys. Renal cortical transcripts of MCP-3 and MCP-5 in MRL- Fas lpr as compared with C57BL/6 mice were increased (twofold) in advance of overt renal pathology (2 mo of age) and rose further (fourfold) as renal pathology advanced . We probed for MCP expression in MCP-1–deficient MRL- Fas lpr kidneys. As anticipated, MCP-1 transcripts were not detected in the MCP-1–deficient MRL- Fas lpr renal cortex. On the other hand, MCP-3 and MCP-5 transcripts were detected in MCP-1–deficient MRL- Fas lpr mice . However, amounts of MCP-3 and MCP-5 transcripts were 50% lower in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr kidneys . Thus, MRL- Fas lpr mice lacking MCP-1 have reduced intrarenal productions of ligands (MCP-3, MCP-5) that bind to CCR2. We previously established that CSF-1 and IFN-γ transcripts that are upregulated with advancing renal injury in MRL- Fas lpr mice are required for autoimmune kidney disease 25 34 35 . In MCP-1–deficient MRL- Fas lpr kidneys, CSF-1 and IFN-γ transcripts were reduced as compared with the MCP-1 intact MRL- Fas lpr strain . In contrast, CSF-1 and IFN-γ transcripts in MCP-1–deficient and –intact B6/129 mice with normal kidneys were barely detectable . To determine if MCP-1 alters the antibody isotype profile in MRL- Fas lpr autoimmune disease, we evaluated serum levels of IgGs (total Ig, IgM, IgG1, IgG2a, IgG2b, IgG3) in MCP-1–deficient and –intact MRL- Fas lpr mice. We did not detect differences in serum IgGs (amount and isotype) in MCP-1–deficient and –intact MRL- Fas lpr strains . In addition, we did not detect an alteration in the amount and distribution of Igs within MCP-1–deficient and –intact MRL- Fas lpr kidneys . Similarly, complement (C3) deposition was not reduced in MCP-1–deficient as compared with MCP-1–intact MRL- Fas lpr glomeruli (data not shown). Thus, MCP-1 does not regulate circulating or kidney-depositing IgGs in MRL- Fas lpr mice. In this report, we tested the hypothesis that a specific chemokine, MCP-1, is required for autoimmune tissue injury in MRL- Fas lpr mice. We now report that MRL- Fas lpr mice genetically deficient in MCP-1 are partially protected from autoimmune disease, including injury to the kidney, lung, and skin and lymphadenopathy, resulting in a prolonged life span. Protection against tissue injury in MCP-1–deficient MRL- Fas lpr mice results from a reduced infiltration of leukocytes (macrophages and T cells) toward parenchymal cells that no longer express MCP-1 and a diminution in cytokines known to promote tissue injury. Furthermore, we determined that the accumulation of these cells is a result of recruitment, but not proliferation, toward the parenchymal cells in proportion to MCP-1 expression. Thus, MCP-1 is responsible for recruiting macrophages and T cells into multiple tissues, including the kidney, lung, and lymph nodes, in MRL- Fas lpr mice, which in turn results in autoimmune tissue destruction. During inflammation, multiple parenchymal and leukocyte cell types express MCP-1 36 . Our challenge was to identify the cell types and locations expressing MCP-1 and determine the impact of cells producing MCP-1 on disease progression in MRL- Fas lpr mice. We now report that MCP-1 is primarily expressed by parenchymal cells, and not infiltrating cells, in MRL- Fas lpr mice, beginning before and then increasing during autoimmune disease. We determined that parenchymal epithelial cells express far more MCP-1 than other parenchymal cell types (endothelial cells, smooth muscle cells, and mesangial cells). Furthermore, we established that the accumulation of macrophages and T cells was directly proportional to MCP-1 expression by these parenchymal cells. This is consistent with our previous findings in a rapid, induced form of kidney damage, NSN, consisting of glomerular and tubular pathology 21 . In NSN, we noted that MCP-1, abundantly expressed in TECs and barely detected in glomeruli, was responsible for tubular but not glomerular injury. We established that macrophages were recruited toward tubules expressing MCP-1 and after activation released molecules that induced TEC apoptosis 21 . By comparison, the importance of MCP-1 in progressive autoimmune disease in MRL- Fas lpr mice is broader than in NSN. First, MCP-1 is responsible for the accumulation of not only macrophages but also T cells within the kidney (tubules and glomeruli) and other tissues (lungs and lymph nodes) during autoimmune disease in MRL- Fas lpr mice. For example, massively enlarged lymph nodes, composed of T cells, are characteristic of MCP-1–intact MRL- Fas lpr mice 37 . The most convincing evidence for MCP-1 fostering T cell accumulation is highlighted by the substantial decrease in the incidence of lymphadenopathy and lymph node size (>50% reduced at 8 mo of age) in MCP-1–deficient MRL- Fas lpr mice. However, the numbers of T cells in the MCP-1–deficient MRL- Fas lpr lymph nodes are not reduced to normal levels. In addition, MCP-1 is not required for splenomegaly in MRL- Fas lpr spleens, as MCP-1–intact and MCP-1–deficient MRL- Fas lpr spleens were similar. Thus, MCP-1 is responsible in part for lymphadenopathy, but other molecules, perhaps chemokines, are required for splenomegaly in the MRL- Fas lpr strain. Other candidates that are likely to be involved in splenomegaly include the β-chemokines macrophage inflammatory protein (MIP)-1α and MIP-1β, reported to foster T cell trafficking (CD4, CD8) into lymph nodes after induced hypersensitivity 38 . In addition, we suggest that the broader impact of MCP-1 on kidney injury in the MRL- Fas lpr mouse versus NSN is related to the progressive accumulation of MCP-1–dependent leukocytes over a longer period of disease manifestation in the MRL- Fas lpr kidney and other tissues. Furthermore, comparison of MCP-1 immunostaining and mRNA levels in the two models indicates that tubules and glomeruli in MCP-1–intact MRL- Fas lpr mice (5–6 mo) express higher amounts of MCP-1 than in NSN (7 d). Disease in NSN is mostly limited to the kidney, whereas MRL- Fas lpr mice have multiple tissues targeted for destruction, each expressing MCP-1. Thus, by gene target–deleting MCP-1 in MRL- Fas lpr mice, we determined that MCP-1 is responsible for recruiting macrophages and T cells to numerous tissues, each undergoing autoimmune disease. The accumulation of macrophages and T cells surrounding parenchymal tissue in MRL- Fas lpr mice results from recruitment and/or proliferation of these leukocytes 26 27 34 35 39 . As MCP-1 induces IL-2 production by T cells 14 , it is possible that MCP-1 promotes T cell proliferation in the kidney, lung, and lymph nodes. However, as few infiltrating cells are proliferating (PCNA + ) at sites of MCP-1 expression within the kidney, lung, and enlarged lymph nodes in MRL- Fas lpr mice, we conclude that the primary action of MCP-1 is to recruit macrophages and T cells into tissues and thereby promote autoimmune disease. To further support the concept that MCP-1 initiates kidney injury via recruitment and not other immune events, we investigated whether MCP-1 caused an Ig isotype switch and alteration in Ig deposition in the kidney. Elevated Ig levels in MRL- Fas lpr mice are dependent on T cell 40 and B cell 41 42 events that are independent of MCP-1 in MRL- Fas lpr mice. In addition, isotypes such as IgG3 compromise glomerular function 43 . We did not detect any difference in the serum Ig isotypes nor in the amount or location of Igs in the kidney. It is interesting to note that despite the similarly high levels of serum and glomerular Igs, the glomeruli are better preserved functionally (loss of protein) and structurally in MCP-1–deficient versus MCP-1–intact MRL- Fas lpr mice. This suggests that, even when the Igs are deposited within the kidney, MCP-1 is required to attract leukocytes into the kidney to initiate glomerular and tubular/interstitial renal disease in MRL- Fas lpr mice. Protection from injury in MCP-1–deficient MRL- Fas lpr kidneys is associated with reduced number of transcripts of the nephritogenic cytokines (IFN-γ and CSF-1) and other chemokines in addition to MCP-1 that bind to the CCR2 receptor (MCP-3 and MCP-5). This could be either directly or indirectly related to the MCP-1 deletion in MRL- Fas lpr mice. For example, MCP-1 immunomodulation of CD4 cells is related to the release of IFN-γ 14 . We suggest that after MCP-1–dependent recruitment of leukocytes into the kidney, a cascade of events triggers the production of IFN-γ, CSF-1, MCP-3, and MCP-5, which each contribute to autoimmune kidney damage. For example, we have previously established that CSF-1, a macrophage growth factor, is responsible for promoting macrophage- and T cell–initiated kidney injury 35 , whereas IFN-γ, with more complex actions, either thwarts 27 or fosters 25 44 injury depending on when it is expressed during disease. In addition, MCP-3 and MCP-5 may be responsible for the migration of leukocytes into the kidney 45 . We therefore speculate that the increase in MCP-1 is a proximal stimulus responsible for recruiting macrophages and T cells, which in turn are responsible for triggering the production of cytokines and other chemokines that lead to tissue injury. Although MCP-1 depletion dampens injury in multiple tissues during autoimmune attack, these tissues are not totally protected. The kidneys, lungs, spleen, lymph nodes, and skin do not remain normal. The renal tubule/interstitium, glomeruli, lungs, and lymph nodes in MCP-1–deficient MRL- Fas lpr mice are still invaded by leukocytes, although in far lower numbers than in MCP-1–intact MRL- Fas lpr mice. Additionally, the limited number of MCP-1–deficient MRL- Fas lpr mice that do not survive kidney glomerular and tubular disease and proteinuria is far lower than younger MCP-1–intact MRL- Fas lpr mice that succumb. In contrast, vascular disease is not prevented in MCP-1–deficient kidneys and lungs. The accumulation of leukocytes around vessels, almost exclusively T cells, is just as abundant in MCP-1–deficient versus –intact MRL- Fas lpr kidneys and lungs. There are several possible explanations for this finding. Other chemokines may be responsible for recruiting these infiltrating leukocytes. For example, MCP-1 is weakly expressed in the vascular wall and perivascular leukocytes, whereas RANTES (regulated upon activation, normal T cell expressed and secreted) is abundant in both areas (data not shown). In addition, many perivascular leukocytes in the kidney and lung are proliferating (PCNA + ) and, therefore, perivascular leukocytes may accumulate because of local proliferation. It is worth noting that perivasculitis in the kidney contributes to mortality in wild-type MRL- Fas lpr mice 45 46 and, therefore, may be at least partially compromising in several tissues in MCP-1–deficient MRL- Fas lpr mice. Finally, as the accumulation of leukocytes in the renal interstitium is focal in both MCP-1–intact and –deficient MRL- Fas lpr mice, we suggest that molecules responsible for adhesion, cell activation, proliferation, and death, including integrins, selectins, and cytokines, may contribute to this process. Furthermore, we cannot rule out the possibility that other chemokines, in addition to MCP-1, enhance leukocytic recruitment. Thus, to achieve more complete protection from autoimmune disease in the MRL- Fas lpr strain, we will have to identify additional therapeutic targets. In conclusion, we suggest that MCP-1 is a therapeutic target to combat autoimmune/inflammatory diseases triggered by tissue leukocytic invasion. Our data further suggest that eliminating MCP-1 expression does not confer total protection. It is thus critical to identify the other therapeutic targets responsible for leukocyte invasion and expansion to confer total protection. | Study | biomedical | en | 0.999997 |
10601357 | Dr. Claudio Fiocchi (Case Western Reserve University School of Medicine, Cleveland, OH) provided primary cultures of HIMECs isolated from normal jejunal mucosa/submucosal tissue 26 . They were cultured in fibronectin-coated plasticware in MCDB medium (Sigma Chemical Co.) supplemented with 20% fetal bovine serum, 90 μg/ml heparin, and 50 μg/ml of endothelial cell growth factor (Sigma Chemical Co.). T-24 cells were cultured in M199 medium supplemented with 10% fetal bovine serum. PBMCs were purified by Ficoll-Paque gradient as described 27 . Vero cells were grown in RPMI medium with 5% Nu serum and infected with the Silvio X-10/4 strain of T. cruzi as described previously 25 . Epimastigotes were grown as described previously 17 23 . TS was purified by immunoaffinity chromatography as described previously 20 . In brief, supernatants from T. cruzi –infected Vero cells were passed on a protein G–Sepharose (Amersham Pharmacia Biotech) column adsorbed with mAb TCN-2 specific for the LTR domain of TS. Bound TS was eluted with TR1 peptide (DSSAHGTPSTPA) in the presence of 0.1% octyl glucopyranoside. TS was concentrated by ultrafiltration in Amicon-10 and washed extensively with PBS, pH 7.8, to remove the TR peptide. The neuraminidase from Vibrio cholera (VCNA) was purchased from Calbiochem. Penetrin (PN-1) was isolated by heparin affinity chromatography as described previously 28 . All reagents, glassware, and plasticware used in the isolation of the various proteins were LPS free. To eliminate residual LPS, the purified materials were passed through two distinct resins that remove endotoxin by different mechanisms, END-X B15 (Associates of Cape Cod, Woods Hole, MA) and AffinityPack™ Detoxi-Gel™ (Pierce Chemical Co.), following the recommendations of the manufacturers. The DNA fragment corresponding to the CD was amplified by PCR on DNA template from TS clone 19Y of the Silvio strain of T . cruzi trypomastigotes 22 . The primers for CD were 5′-GGAATTCCATATGGCACCCGGATCGAGCCGAGTT-3′ and 5′-CCGCTCGAGGCTCAAGAACAAGGTCCTGATCG-3′. The amplified fragment was cloned into pET-23b (Novagen) with a stretch of six His residues at the COOH terminus of the expressed proteins. For protein production, plasmids were used to transform the BL21 DE3 bacterial strain (Novagen) containing a chromosomal copy of the T7 RNA polymerase gene. Expression was induced by isopropyl-β- d -thiogalactopyranoside (IPTG). To isolate the recombinant CD fragment, bacterial lysates were prepared by osmotic shock in 40 mM phosphate buffer, pH 7.5, 0.3 M NaCl, 1% Triton X-100, 1 mM PMSF, followed by brief sonication. The CD peptide was purified on a Ni 2+ -nitrilotriacetic acid (NTA)/agarose column as recommended by the manufacturer (Novagen). CD was further purified by FPLC on the anion-exchange column Mono-Q HR (Amersham Pharmacia Biotech), as described previously 20 25 . All reagents were LPS free and passed through the END-X B15 resin to remove residual LPS. The full-length COOH-terminal LTR (LTR fragment) of Silvio TS, clone 7F 22 , was isolated as follows: LTR, subcloned from a pMelBac plasmid (Invitrogen) containing the TS gene, was digested with PvuII-SalI and ligated into EcoRV-SalI sites of pET-20b (Novagen). The LTR DNA was introduced into the NcoI-HindIII sites of pFASTBAC HTb vector (GIBCO BRL). The Bac-to-Bac system (GIBCO BRL) was used to generate recombinant baculovirus, which in turn was used to infect Sf 9 cells. Recombinant LTR protein was purified by Ni 2+ -NTA column (Novagen) affinity chromatography. The LTR fragment thus generated contains the full-length TR of clone 7F with 44 repeats plus TS sequences of 26 and 40 amino acids upstream and downstream of the repeat, respectively. Purified LTR was passed through AffinityPack™ Detoxi-Gel™ to remove residual LPS. Construct TS-154 is derived from enzymatically active trans -sialidase clone 154 of the Y strain of T . cruzi 29 . The NH 2 terminus of clone 154 was amplified by PCR using primers NU-17 (27 mer, 5′-GCCCATGGCACCCGATCGAGCCGAGTT-3′) and NU-18 (20 mer, 5′-CGGAATTTTCATCACCAATG-3′) with restriction sites shown in Fig. 4 A. NU-17 was designed to introduce starting ATG codon just before the NH 2 terminus of mature TS protein, and a NcoI site for subcloning. The PCR product of NU-17 and NU-18 was treated with NcoI and BglII, and subcloned into the NcoI and BamHI sites of pET-21d (Novagen). Most of the 12 amino acid repeats and hydrophobic region at the COOH terminus were removed by PCR using primers NU-19 (24 mer, 5′-GTTCCGAACGGTTTGAAGTTTGCG-3′) and NU-20 (25 mer, 5′-CTGTCGACGGGAGTTGAGGGCGTAC-3′). NU-20 corresponds to the partial sequence of the TR and, in addition, contains a SalI site. The PCR product with the minimum numbers of repeats (i.e., five repeats) was selected and used to replace the original COOH terminus of clone 154 at unique MluI site . The DNA fragment with unique BamHI-SalI sites was ligated to the pET-21d plasmid containing the NH 2 -terminal region of TS using the BamHI and XhoI sites of the vector, to yield construct TS-154 . To generate construct TS-H32, the BglII-PstI DNA fragment of pTS-154 was replaced with the corresponding fragment from the gene 121 29 . The trans -sialidase gene 121 is catalytically inactive due to a single amino acid difference in the BglII-PstI fragment, with histidine (H374) replacing tyrosine in the catalytically active TS gene 154 29 . Thus, construct TS-H32 should be enzymatically inactive, as determined experimentally . All constructs were verified by automated sequencing (ABI Perkin Elmer) using BigDye terminator. Production and purification of TS-154 and TS-H32 proteins were identical to the method described above for the CD construct. Endothelial cells and T-24 cells were plated on 24-well plates at a density of 10 5 cells/well, while PBMCs were plated in the similar wells at 10 6 /well. Cells were incubated with the various test reagents at the concentrations and for the time indicated in the figures in triplicate points. Polymyxin B was used at 10 μg/ml in all cell cultures, as it did not affect any parameter tested. Cytokines and chemokines released in the culture supernatants were measured by ELISA assay following the instructions of the manufacturer (Endogen). The cytokines tested were IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-γ, and TNF-α, and the chemokines were RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemotactic protein 1 (MCP-1). Negative control were cells incubated in medium containing polymyxin B at 10 μg/ml, and positive control always included cells incubated with bacterial LPS at the low nanogram per milliliter range (for PBMCs or HIMECs) or in the microgram per milliliter range for carcinoma T-24 cells. In some experiments, IL-1β or TNF-α was used as positive control for cytokine release. Bioassay for IL-6 was performed using IL-6–dependent human DS-1 cells 30 . In brief, 10 4 cells/well were plated in 96-well plates and incubated for 24 h in IL-6–free medium (10% FCS in RPMI) containing several dilutions of TS-conditioned medium (TS/CM) or exogenous rIL-6, pulsed for 4 h with 0.5 μCi [ 3 H]thymidine, and harvested to determine radioactivity incorporation in a microplate scintillation instrument (Packard). In some experiments, a neutralizing IL-6 rabbit IgG or normal rabbit IgG (Endogen) was added to the dilutions of TS/CM before assaying for growth stimulation of DS-1 cells. Conditioned media were prepared by incubating PBMCs or T-24 cells for 24 h in 10% FCS/RPMI without (CM) or with (TS/CM) TS at 1 μg/ml. Supernatants were centrifuged at 1,000 g to remove cell debris and were kept frozen at –20°C until use. Semiquantitative analysis of cytokine mRNA was performed by the primer-dropping method 31 . RNA of endothelial cells (10 5 ) or PBMCs (10 6 ) that had been or not stimulated with TS at 1 μg/ml for 24 h, was purified by acid guanidinium isothiocyanate-phenol-chloroform-TRI reagent (Molecular Research Center). 2 μg of total RNA was converted to cDNA in a volume of 20 μl using random hexamer primers according to the manufacturer's instructions (GIBCO BRL). Primers for human IL-6 were 5′-ATGAACTCCTTCTCCACAAGCGC and 5′-GAAGAGCCCTCAGGCTGGACTG, and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-CGGAGTCAACGGATTTGGTCGTAT and 5′-AGCCTTCTCCATGGTGGTGAAGAC. The PCR mixture contained 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl 2 , 1 mM dNTP, 200 pM primers, and 1 U of Taq polymerase in 50 μl. Thermal cycling conditions were: denaturation step, 95°C for 5 min; 35 cycles of 95°C for 30 s, 60°C for 30 s, and 75°C for 1 min, and a final step of 75°C for 5 min. At cycle 9, GAPDH primers were added. 5 μl of the PCR product was analyzed through 2% agarose gel electrophoresis in the presence of 1 μg/ml ethidium bromide. Normal human PBMCs (10 6 /well) were coincubated in 10% FCS/RPMI with either epimastigotes or trypomastigotes (10 5 /well for each stage) for 48 h at 37°C. IL-6 was measured by ELISA in cell-free supernatants (obtained by centrifugation of the cultures at 2,500 g for 5 min) at various times after the start of the cocultures. TS activity in the parasites was obtained before the start of the cultures. T . cruzi trypomastigotes expressing TS on their surface (TS + parasites) or expressing low or no TS (TS − parasites) were purified by magnetic beads coated with the mAb TCN-2, as described previously 32 . HIMECs, plated (5 × 10 4 /well) on 24-well plates for 24 h, were infected with 10 6 trypomastigotes (unfractionated, TS + , or TS − ) or with L. major promastigotes, strain V1 (gift from Dr. Stephen Beverley, Washington University, St. Louis, MO). At various times after infection, aliquots of the supernatants were collected, centrifuged at 1,000 g , filtered in 0.22 μm nitrocellulose, and assayed for IL-6 by ELISA. 200 μl of protein G–Sepharose (Amersham Pharmacia Biotech) was adsorbed with culture supernatants of the mAb TCN (IgG1 isotype) or of a control mAb IgG1 specific for p -azo-phenylarsonate (provided by Dr. Thereza Imanishi-Kari, Tufts University School of Medicine), and washed with 20 vol of LPS-free PBS, pH 7.2. 3 μg of LTR was loaded to either protein G/TCN-2 or protein G/control IgG1 column; the effluent was reapplied five times to the respective column. The last flow-through of each column was collected in 200 μl in a separate LPS-free tube, and the columns were washed with five column volumes of PBS. Elution of bound LTR was by mixing the resins with a spatula, followed by centrifugation at 250 g for 5 min at 4°C. Effluents and eluates were constituted to the original volume and added to T-24 cells. After 24 h, IL-6 in the T-24 cell supernatants was determined by ELISA. TR peptides (see Table ) were synthesized at the Tufts University Synthesis Facility. Each peptide was purified by HPLC, and the number of amino acids and molecular weight of each peptide were verified by mass spectrography. Peptides were dissolved in growth medium (10% FCS in RPMI) and added at various concentrations to 10 6 PBMCs/well in 24-well plates. After 24 h, IL-6 was assayed by ELISA in the conditioned supernatants. Endothelial cells mediate tissue inflammation, in part, through the secretion of proinflammatory cytokines and the selective expression of adhesion molecules in the vascular bed 33 . Most studies with endothelial cells rely on HUVECs, as they are readily available and easy to isolate. However, the majority of physiological and pathological events mediated by endothelial cells, in particular inflammation, wound healing, and leukocyte homing through postcapillary venules, occur at the level of microcirculation 34 . Furthermore, endothelial cell populations vary from organ to organ and in different vessels within an organ 35 . Most important, the pattern of cytokine secretion and proliferation of a microvascular endothelial cell, HIMEC, differed from that of HUVECs in response to various stimuli 36 37 . Thus, due to the relevance of the intestinal microvascular endothelium to the pathogenesis of the megacolon in Chagas's disease, and the possibility that TS subverts host immune responses by directly reacting with nonimmune cells, we attempted to determine whether HIMECs isolated from normal human mucosa would secrete cytokines in response to TS stimulation. For this purpose, we incubated HIMEC monolayers with various concentrations of purified TS for 24 h and measured the concentration of eight cytokines (IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-γ, and TNF-α) and two chemokines (RANTES and monocyte chemotactic protein 1 [MCP-1]) in the conditioned HIMEC supernatants. We found the concentration of IL-6 to be elevated in the conditioned supernatants in a manner dependent on the TS input . In contrast, the concentration of the other cytokines did not increase in response to TS stimulation under conditions in which bacterial LPS at 50 ng/ml released IL-1β, IL-8, and TNF-α (data not shown). Similar IL-6 upregulation was observed with two other primary cultures of HIMECs (data not shown). The IL-6 concentration produced by HIMECs in response to 1 μg/ml of TS corresponded to that induced by 50 ng/ml of a bacterial LPS (data not shown). Thus, HIMECs appear to be much more sensitive to produce IL-6 in response to bacterial LPS than to TS. A control VCNA corresponding to the neuraminidase activity of TS did not stimulate detectable IL-6 secretion in HIMECs , suggesting that the TS action did not depend on its intrinsic glycosidase activity. Another control protein, the T . cruzi heparin-binding penetrin (PN-1) thought to promote parasite invasion 28 , was not effective in stimulating IL-6 secretion , further emphasizing the selectivity of the TS action. TS also stimulated IL-6 release in normal PBMCs , another class of human cells relevant to the immunity against T . cruzi . The result in Fig. 1 B is for one blood donor, but a similar result was obtained with three other blood donors (data not shown). In addition, TS stimulated IL-6 secretion in the human bladder carcinoma T-24 cell line . Previous work has shown that T-24 cells express IL-6 constitutively and in response to cytokine stimuli 38 39 . TS-induced IL-6 secretion in the T-24 bladder carcinoma cells further underscores the neuraminidase action for nonimmune cells. Interestingly, the IL-6 output resulting from the 24-h stimulation of T-24 carcinoma cells with 1 μg/ml of TS corresponded to that induced by 0.75 μg/ml of bacterial LPS under the same conditions (data not shown). Thus, in this cancer cell line, TS was nearly as good as bacterial LPS in stimulating IL-6 secretion. IL-6 is produced by a large variety of cells, including endothelial cells, monocytes, fibroblasts, keratinocytes, T cells, mast cells, neutrophils, tumor cell lines, and cells of neural origin 40 . Although we found that TS stimulates IL-6 production in endothelial cells and PBMCs, it is likely that the neuraminidase will also stimulate IL-6 release in other cell types. However, normal human neutrophils, which are capable of secreting IL-6 in response to various stimuli such as TNF-α and GM-CSF 41 , did not produce detectable IL-6 when stimulated with TS under conditions in which TNF-α did (data not shown). Thus, not every human cell type may secrete IL-6 upon stimulation with TS. The kinetics of TS-induced IL-6 secretion revealed maximum effect after 24 h stimulation in both HIMECs and PBMCs . This response followed the upregulation of IL-6 transcripts, which was maximal 4–10 h after TS stimulation . These results suggest that TS triggers synthesis and then secretion of IL-6, consistent with the effect of conventional cytokine agonists in the same cell types 37 38 . The DS-1 B lymphoma cells have an intact IL-6 receptor signaling pathway, but because they cannot produce IL-6, the cells will die unless exogenous human IL-6 is added to the culture medium 30 . Therefore, this cell line is a useful probe for the biological assay of human IL-6. To determine whether TS induces secretion of biologically active IL-6, we measured the ability of conditioned media to promote growth of DS-1 cells. We prepared conditioned media by incubating PBMCs or T-24 cells for 24 h in 10% FCS/RPMI without (CM) or with (TS/CM) TS at 1 μg/ml. We found that TS/CM, but not CM, restored growth of the DS-1 lymphoma cells in a dose-dependent manner, and that a neutralizing IL-6 antibody suppressed the growth-promoting activity of TS/CM . These results indicate that TS-dependent IL-6 released by PBMCs and T-24 cells is biologically active. In addition, we also conclude that the T . cruzi neuraminidase is not an IL-6 agonist because it did not restore growth of the DS-1 cells when the enzyme was added to normal growth medium lacking exogenous IL-6 . To begin to dissect structural features of TS that elicit IL-6 release in normal human cells, we first determined whether the catalytic activity of TS mediates IL-6 secretion in immunologically naive human cells. For this, we compared the action of the catalytically inactive TS mutant TS-H32 with that of catalytically active TS-154 protein. Both TS-154 and TS-H32 constructs derive from the Y strain of T . cruzi 29 , whereas the TS used in the experiments displayed in Fig. 1 Fig. 2 Fig. 3 Fig. 4 was derived from the Silvio strain 22 . Both TS-H32 and TS-154 constructs contain five TR units . Although TS-H32 differs from TS-154 in six amino acid substitutions in the CD , the difference in enzymatic activity between the two constructs is attributed to a single amino acid, with tyrosine (Y374) in the active gene (TS-154) changed to histidine in the inactive gene (TS-H32) . To our surprise, enzymatically active TS-154 was as powerful as enzymatically inactive TS-H32 in stimulating IL-6 secretion in PBMCs and T-24 cells (data not shown). The results with the TS-154 and TS-H32 constructs suggest that the catalytic activity of TS does not mediate IL-6 release in naive cells. This was confirmed with the use of recombinant CD of TS. The NH 2 -terminal domain of TS is 663 amino acids long and contains the enzymatically active CD . In contrast, the enzymatically inactive COOH-terminal domain is made up of a 12 amino acid unit repeated 44 times in the 7F clone of TS derived from the Silvio strain of T . cruzi (LTR) ( 22 ; and data not shown). Recombinant His-tagged CD was purified from E s cherichia coli lysates by binding to and elution from Ni 2+ -NTA/agarose column and by ion-exchange chromatography on Mono-Q columns (FPLC; see Materials and Methods). Recombinant His-tagged LTR was isolated from insect cell lysates by immunoaffinity to the mAb TCN-2 and to Ni 2+ -agarose columns (see Materials and Methods). Addition of CD to T-24 cells did not promote IL-6 release, whereas addition of LTR effectively released IL-6 in a dose-dependent manner . Similar results were obtained in the IL-6 release by PBMCs, in which LTR, but not CD, upregulated IL-6 release (data not shown). To confirm that LTR is a mediator of IL-6 release, we depleted LTR from solution in a protein G–Sepharose column adsorbed with TCN-2. TCN-2 is an IgG1 mAb specific for LTR routinely used in our lab to purify TS 20 . The flow-through of such TCN-2 affinity column, which was devoid of LTR polypeptide, as determined by immunoblot analysis (data not shown), did not stimulate IL-6 release in T-24 cells . In contrast, the flow-through of a control IgG1 column contained LTR polypeptide and stimulated IL-6 secretion . Furthermore, elution of LTR from the TCN-2/protein G column restored the IL-6–secretory power of the original preparation, whereas similar elution from the IgG1 control column did not . Therefore, these results establish LTR as mediator of IL-6 secretion in naive human cells. Epimastigote, a noninvasive stage of T . cruzi , expresses TS with the same carbohydrate specificity as the trypomastigote enzyme . Yet, coculture of normal PBMCs with live epimastigotes did not result in IL-6 secretion under conditions in which coculture with trypomastigotes did . This is not a paradox, because the TS of epimastigote, with a CD 84% identical to the counterpart TS of trypomastigotes 42 , lacks LTR, as determined by immunoblot analysis with LTR-specific mAb 23 42 and by gene cloning 43 . Therefore, the failure of live epimastigotes to release IL-6 from PBMCs supports the conclusion that LTR, and not CD, is the active TS domain upregulating IL-6 in normal human cells. In addition, it emphasizes the selectivity of trypomastigote TS in stimulating cytokine secretion in normal cells. Given that LTR is the domain that mediates for IL-6 secretion, and that the LTR of TS-154 and TS-H32 contains only five COOH-terminal repeats , one would expect that LTR-based synthetic peptides up to five units should stimulate IL-6 secretion in human cells. To test the above hypothesis, we prepared synthetic peptides TR1, TR2, TR3, TR4, and TR5, which correspond to the 12, 24, 36, 48, and 60 amino acid sequence proximal to the COOH terminus of the catalytic domain of Silvio TS ( 22 ; Table ). We then tested whether these synthetic peptides could stimulate IL-6 release in PBMCs. We found that TR4 and TR5 peptides were active in promoting IL-6 release, whereas TR1, TR2, and TR3 were less active . The results with TR peptides confirm that the TR is the TS moiety that mediates IL-6 release in naive human cells. Extracellular trypomastigotes can be subdivided into two populations based on the relative abundance of TS . These two populations can be readily separated from one another by differential affinity to magnetic beads coated with LTR-specific mAb TCN-2 32 . The subset that produces TS, named TS + , represents 25% of the total trypomastigote population, whereas the subset that does not produce or produces very little TS, termed TS − , constitutes the majority of the trypomastigotes 32 . Interestingly, TS + parasites of the Silvio strain are short and stumpy (length = 9.3 ± 2.8 μm) and morphologically distinct from the TS − parasites, which are slender (length = 18.2 ± 4.3 μm) . Live TS + trypomastigotes moved slowly and sluggishly in liquid medium (10% FCS/RPMI) at room temperature, contrary to the TS − trypomastigotes, which migrated swiftly with a whipping movement (data not shown). However, the dimorphism and contrasting movements of TS + and TS − trypomastigotes were not characteristic features of two other T . cruzi strains, Tulahuen and MV-13 (data not shown). Thus, there does not appear to be a relation between the expression of TS and a particular morphological type of T . cruzi . It remains to be determined why the TS + and TS − trypomastigotes of the Silvio strain, but not of the other strains, are so morphologically distinguishable from one another. Nevertheless, the availability of purified TS + and TS − populations offered a unique opportunity to test the power of live parasites with variable TS abundance to upregulate IL-6 in normal human cells. Therefore, we challenged HIMEC monolayers with live TS + and TS − parasites and followed the time course of IL-6 released in the trypanosome-containing culture supernatants. It was remarkable to find that TS + trypomastigotes were much more effective than TS − , and somewhat better than unfractionated trypomastigotes, in inducing intestinal endothelial cells to release IL-6 . The result in Fig. 8 B is for the subpopulations of the Silvio strain, but similar results were obtained with the TS + and TS − subsets of the Tulahuen strain (data not shown). Because the TS + parasites of the Tulahuen strain are a mixture of stumpy and slender forms, the power of T . cruzi to induce IL-6 secretion in naive human cells depends on the TS content, rather than on the morphology of the parasites. Furthermore, the inability of L. major promastigotes to release IL-6 in HIMECs suggests that parasite burden per se does not suffice to induce IL-6 secretion in naive human cells. Infection of mammals by parasites and other microbes results in the release of cytokines and other mediators of the inflammatory response. The composition of the cytokines, which depends on the nature of the infecting organism and on the host genotype, may be critical for the resistance or susceptibility to microbial invasion, as best exemplified by the infection of mice with the protozoan L. major 44 and of humans with the bacterium Mycobacterium leprae 45 . It is generally accepted that cytokine networks result from antigenic stimulation of lymphocytes and macrophages. However, these antigen-driven host responses can be subverted by a group of viral and bacterial proteins, termed virokines and bacteriokines, respectively, which are capable of changing the dynamics of the cytokine networks without directly activating B and T cell receptors 46 . Virokines tend to suppress host immune responses by neutralizing inflammatory cytokines, as is the case of the protein B15R of vaccinia virus, which binds IL-1β 47 . Alternatively, virokines may downmodulate immune responses by mimicking antiinflammatory cytokines, like the protein BCRF1 of Epstein-Barr virus, which is 70% identical to IL-10 48 . On the other hand, bacteriokines, including LPS and exotoxins, are more likely to stimulate proinflammatory cytokines, thereby enhancing pathogenesis 46 . Whether protozoan parasites can alter host immune responses through molecules functionally equivalent to virokines and bacteriokines (i.e., “protokines”) remains to be determined. The findings reported in this paper identify TS as a protein that could alter the dynamics of the cytokine network by upregulating IL-6 secretion in normal human cells. As shown in Fig. 1 , Fig. 5 , Fig. 7 , and Fig. 8 , TS and its TR were active against naive microvascular endothelial cells and PBMCs. Furthermore, given that IL-6 may be produced by many other cell types, such as fibroblasts and epithelial cells, the range of cell target for IL-6 release by TS could be broader than the vascular endothelium and blood mononuclear cells. This was indicated by the ability of TS to induce IL-6 release in the T-24 bladder carcinoma cells and by parallel experiments with mouse cells, which revealed TS to be an upregulator of IL-6 secretion in naive splenocytes, bone marrow cells, and peritoneal cells from BALB/c and other strains of mice (Gao, W., and M.A. Pereira, manuscript in preparation). Therefore, TS upregulates IL-6 secretion in various cell types and animal species. The kinetics of TS-dependent IL-6 release in vitro suggests that, in vivo, TS should stimulate IL-6 release before the full development of acquired immune response against T . cruzi . This could be accomplished when T . cruzi first encounters the mammalian host, such as after an insect bite when the parasites enter the host through the mucosa, usually around the eye; or when T . cruzi enters the host during blood transfusion or congenitally, in which case the TS + parasites gain access into the circulation where they should react with endothelial cells and PBMCs to trigger IL-6 release. In addition, TS may also promote IL-6 secretion in vivo as a soluble mediator. Indeed, soluble neuraminidase was detected, before the parasites, in the blood of a T . cruzi –infected individual 24 . Copious amounts of the enzyme are released in monolayers of cells infected with T . cruzi 20 . Our findings reported here show that soluble enzyme and TS-expressing parasites are capable of inducing IL-6 secretion in normal cells. There are several ways in which TS-dependent IL-6 could alter T . cruzi invasion. IL-6 promotes polyclonal activation of B and T lymphocytes 49 50 and is an important cofactor for Th2 T cell activation, necessary for the induction of humoral immune responses 51 . Thus, the TS/IL-6 pathway could be directly relevant to the polyclonal lymphocyte responses that characterize acute Chagas's disease 52 . In addition, IL-6 is a potent inducer of collagen secretion in human fibroblasts 53 , and as such appears to underlie the pathogenesis of systemic sclerosis, a connective tissue disease characterized by fibrosis in the skin and internal organs 54 . The fibrogenic action of IL-6 could also be relevant to T . cruzi infection, as fibrosis is a prominent feature of acute and chronic Chagas's disease 55 56 . Because chronic chagasic heart contains many IL-6–containing mononuclear cells and endothelial cells 4 5 6 7 , IL-6 could in theory contribute to the fibrosis in the chronic heart, regardless of the mechanism stimulating production of the cytokine. IL-6, in addition to mediating inflammatory and immune responses, may play an important role in a variety of activities of the central and peripheral nervous systems, such as cell-to-cell signaling, protection of neurons from insult, and neuronal growth and survival 57 . Thus, TS-dependent IL-6 could be a factor in the neuroregeneration that characterizes the indeterminate phase of Chagas's disease in humans 55 and animals 58 . In support of this hypothesis, we recently found TS to be extremely potent in protecting several types of neuronal cells from undergoing apoptosis induced by growth factor deprivation (Chuenkova, M., and M.A. Pereira, manuscript submitted for publication). What's more, the TS-induced neuroprotection was synergistic with two members of the IL-6 family, ciliary neurotrophic factor and leukemia inhibitory factor (Chuenkova, M., and M.A. Pereira, manuscript submitted for publication). Thus, the neurotrophic effect of TS-dependent IL-6 could be boosted by the action of TS and by the TS synergy with ciliary neurotrophic factor or leukemia inhibitory factor on the neurons. TS itself has been implicated as a key factor in T . cruzi invasion. Experiments with cells in culture suggest that TS promotes parasite attachment to host cells through the recognition of sialyl-containing surface receptors 59 60 . Experiments in vivo are also consistent with TS being a promoter of T . cruzi invasion, as suggested by the enhancement of infection in experimental models of Chagas's disease by LTR-specific polyclonal 61 and monoclonal 62 24 antibodies, and by the inhibition of infection by CD-specific antibodies 63 64 . In addition, antibodies against TS-generated sialyl epitopes present on the trypomastigote surface block infection as well 65 . Finally, the enhancement of T . cruzi virulence in BALB/c mice sensitized with low doses of the enzyme further supports the conclusion that TS is a major factor in promoting T. cruzi invasion of mammalian hosts 25 . The novel findings reported here open up strategies to investigate an unsuspected role for TS in T . cruzi invasion and provide a new tool to study the function of IL-6 in Chagas's disease. | Study | biomedical | en | 0.999998 |
10601358 | The human B cell lines Ramos (obtained from Dr. Seth Lederman, Columbia University) and BL-41 (obtained from Dr. Riccardo Dalla-Favera, Columbia University) are EBV-negative Burkitt's lymphomas. JY (obtained from Dr. Riccardo Dalla-Favera) is an EBV-transformed lymphoblastoid B cell line. U937 (obtained from Dr. Kathryne Calame, Columbia University) is a monocytic cell line. All cells were grown in IMDM supplemented with 10% fetal calf serum (Atlanta Biologicals, Inc.). Ramos B cells (10–20 × 10 6 ) were stimulated with 0.1 μg/ml of either anti-CD40 or an isotype-matched control Ab in a final volume of 10 ml at 37°C for 24 h. IL-4 treatment (100 U/ml; PeproTech) was carried out simultaneously in separate plates in a final volume of 10 ml. Tonsillar mononuclear cells were obtained from surgical specimens after routine tonsillectomies and were isolated as described previously 47 . The rabbit polyclonal antiserum against IRF-4 used in electrophoretic mobility shift assay (EMSA) experiments was a gift of Dr. Hirai, Tokoyo Universty, Tokyo, Japan 32 . We subsequently generated our own rabbit polyclonal anti–IRF-4 antiserum using a similar glutathione S -transferase (GST)–IRF-4 (nucleotides 441–924) fusion protein as the immunogen (Babco). As previously indicated, this GST fusion protein contains a portion of the IRF-4 protein that is specific to IRF-4 and thus avoids cross-reactivity with other IRFs 32 . This antiserum was used for immunoprecipitations and immunoblot analyses. Blots were also reprobed with a commercially available anti–IRF-4 antiserum (Santa Cruz Biotechnology), which gave identical results. Rabbit polyclonal antisera against human Stat6, Stat3, p65, IRF-2, IFN consensus sequence binding protein (ICSBP), or BCL-6, were purchased from Santa Cruz Biotechnology, Inc. The hybridomas secreting the anti-CD40 mAb G28-5 (IgG1) or an isotype-matched control mAb were obtained from American Type Culture Collection. Full-length human IRF-4 cDNA cloned into pBluescript vector (pBSKS-myc-IRF-4) was a gift of Dr. Riccardo Dalla-Favera 30 . The IRF-4 expression plasmid (pCEP4-IRF-4) was constructed by cloning the entire coding region of the IRF-4 cDNA into the NotI and XhoI sites of the mammalian expression vector pCEP4 (Invitrogen). For construction of the GST–IRF-4 expression plasmid, the entire coding region of the IRF-4 cDNA was cloned, in frame, into the filled EcoRI site of pGEX-3X vector (Amersham Pharmacia Biotech). The in frame junctions in the GST–IRF-4 fusion construct were confirmed by DNA sequencing in an automated cycle sequencer (Perkin Elmer). The full-length human BCL-6 cDNA cloned into pMT2T mammalian expression vector was a gift of Dr. Riccardo Dalla-Favera 19 . The CD23a cDNA was a gift of Dr. Kikutani, Osaka University, Osaka, Japan 44 and Dr. Wang, Harvard University, Boston, MA 48 . To generate an antisense riboprobe of CD23a/b, a 600-bp 5′-EcoRI-HindIII-3′ fragment from the 5′ end of CD23a cDNA was cloned in opposite orientation, into the HindIII and EcoRI sites of pGEM1 in vitro transcription vector (Promega). Transcription of the antisense RNA was driven by SP6 RNA polymerase promoter. To prepare the CD23b GAS firefly luciferase reporter construct, a trimer of the CD23b GAS element was synthesized with flanking BamHI sites (GIBCO BRL) and then cloned into the BamHI site (immediately upstream of minimal TK promoter) of the TK200 luciferase reporter vector (a gift of Dr. Kathryne Calame). The CD23b promoter firefly luciferase reporter construct in the pGL3-enhancer vector (Promega) was a gift of Dr. Seth Lederman. The pRL-TK reporter plasmid encoding renilla luciferase to determine the transfection efficiency was purchased from Promega. The preparation and employment of DNA oligonucleotide probes for EMSAs have been described previously 49 with the following modifications: 2% glycerol was added to the polyacrylamide gel, and a reduced poly(dI-dC) concentration (1 μg/reaction) was used in the binding buffer. The CD23b GAS oligonucleotides used in these studies were as follows: CD23b GAS wild-type (wt), 5′-gatcGGGTGAATTTCTAAGAAAGGGAC-3′; CD23b GAS M1, 5′-gatcGGGTGAATTTCTAAG GTC GGGAC-3′; CD23b GAS M2, 5′-gatcGGGTG GTC TTCTAAGAAAGGGAC-3′; and CD23b GAS M3, 5′-gatcGGGTGAAT GCTG AAGAAAGGGAC-3′. Oligonucleotide competition and Ab interference assays were performed as described previously 49 . Nuclear and whole cell extracts (WCEs) were prepared as described previously 49 50 . Cell extracts were immunoprecipitated with an anti–IRF-4, anti–BCL-6, or anti-Stat6 antiserum as described previously 49 . The immunoprecipitates were resolved by 7% SDS-PAGE. The gel was transferred to a nitrocellulose membrane, and then immunoblotted with a Stat6, BCL-6, or IRF-4 Ab. The bands were visualized by ECL (Amersham Pharmacia Biotech). UV cross-linking was performed as described previously 49 . GST fusion proteins were expressed in Escherichia coli DH5α and affinity-purified on glutathione (GSH)-agarose beads (Sigma Chemical Co.), as described previously 51 . The concentration of each fusion protein was determined by SDS-PAGE/Coomassie staining. For pull-down assays, lysates (∼2 mg of total cell proteins) from control or stimulated Ramos or JY cells were incubated with ∼100 μg of GST alone or GST–IRF-4 fusion protein immobilized onto GSH-agarose beads in 400 μl of 1× lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 0.05% NP-40, 10% glycerol, 1 mM dithiothreitol, 1 mM Na 3 VO 4 , 1 mM PMSF, 1 μg/ml leupeptin, 3 μg/ml aprotinin) for 4 h at 4°C with constant agitation. The complexes were extensively washed with 1× lysis buffer (containing 0.5% NP-40). The bound proteins were eluted from the beads by boiling them in SDS-PAGE sample buffer, fractionated on a 7% SDS-polyacrylamide gel, and then blotted onto a nitrocellulose membrane. The blot was probed with either a Stat6, Stat3, or BCL-6 Ab. Total RNA was extracted by using the RNA-STAT60 kit (TelTest, Inc.). Northern blot analysis was performed with 10 μg of total RNA according to standard protocols. The blot was probed with either a human IRF-4 cDNA, a BCL-6 cDNA, or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA radiolabeled by the DNA Labeling beads (−dCTP) kit (Amersham Pharmacia Biotech). RNase protection analysis was performed as described previously 52 . 10 μg of total RNA was hybridized simultaneously to an antisense riboprobe of CD23a/b as well as of β-actin (Ambion) transcribed by SP6 RNA Polymerase in vitro Transcription System (Promega) using [α- 32 P]UTP. The annealed products were digested with ribonuclease T2 (GIBCO BRL), and then analyzed on a 6% polyacrylamide-urea denaturing gel. For the transient transfection assays, 6 × 10 6 U937 cells were cotransfected with 25 μg of a CD23b GAS wt or a CD23b GAS M2 firefly luciferase reporter plasmid and 25 μg of various expression plasmids by electroporation at 300 V and 960 μF with a BTX electroporator as described previously 53 . 5 μg of the pRL-TK reporter plasmid expressing renilla luciferase under the control of the thymidine kinase promoter was added to each transfection as a transfection efficiency control. JY cells (10 × 10 6 ) were cotransfected by the DEAE-dextran method 54 with 10 μg of the CD23b promoter firefly luciferase reporter vector and 5 μg of pRL-TK plasmid in the presence of 15 μg of a BCL-6 expression plasmid (pMT2T-BCL6) or the equivalent amount of empty pMT2T vector. After transfection, the cells were equally split into two 2-ml cultures and then incubated for 16–24 h in the presence or absence of IL-4 (10 ng/ml). The transfected cells were then harvested, lysed, and assayed for luciferase activities with the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. The firefly luciferase activity was normalized on the basis of renilla luciferase activity. To identify and characterize the downstream effectors of the IL-4 signaling cascade, we focused our attention on CD23, a B cell activation molecule whose regulation displays lineage- and stage-specific features 41 55 . Although IL-4 and CD40 can individually induce modest levels of CD23, costimulation of human tonsillar B cells or purified centroblasts with both CD40 and IL-4 leads to a synergistic induction of surface CD23 expression 42 43 . We found that Ramos, an EBV-negative Burkitt's lymphoma cell line extensively used to study the CD40 and/or IL-4 signaling pathways 56 57 58 59 , mimics the physiological regulation of CD23 in normal B cells (data not shown). Since no study had previously explored the CD23 isoform(s) induced in response to CD40 or the CD40 and IL-4 combination, we performed RNase protection assays. As shown in Fig. 1 , exposure of Ramos cells to either anti-CD40 Ab or IL-4 alone preferentially upregulated the CD23a isoform. A slight effect of IL-4 on the expression of CD23b could also be detected. Coculturing Ramos cells with both stimuli led to a strong induction of both CD23a and CD23b transcripts. Consistent with previous reports, constitutively high levels of both CD23a and CD23b were detected in an EBV-transformed B cell line (JY), whereas IL-4 stimulation of a monocytic cell line, U937, only upregulated the CD23b isoform 44 48 . β-actin levels were equivalent in all lanes (data not shown). Thus, costimulation with CD40 and IL-4 leads to a synergistic induction of both CD23 isoforms. IL-4 stimulation has been shown to lead to the rapid binding of Stat6 to a functional element within the CD23b promoter termed the CD23b GAS 13 45 . However, our RNase protection assay studies had revealed only a minimal induction of CD23b in response to IL-4 alone. To determine whether additional factors could target the CD23b GAS and modulate Stat6 activity, we first performed EMSA experiments on Ramos cells stimulated with either IL-4, anti-CD40, or a control Ab. As shown in Fig. 2 A, Ramos cells contain multiple CD23b GAS binding complexes, most noticeably a broad constitutive (C1) and a slower mobility constitutive complex (C2). CD40 stimulation of Ramos cells led to a decrease in the intensity of the C1 complex . Stimulation of Ramos cells with IL-4 activates an additional superimposed complex that contains Stat6 . In contrast to Ramos cells, the appearance of the CD23b GAS binding complexes in JY, an EBV-transformed B cell line expressing constitutively high levels of CD23b, was strikingly different. The C1 complex was absent, while the C2 complex became clearly visible . To dissect the CD23b GAS binding complexes, we then carried out EMSA experiments using a panel of mutated CD23b GAS elements as cold competitors of the radiolabeled CD23b GAS wt probe. Interestingly, a survey of the CD23b GAS had revealed that it contains two potential core sequences for IRF binding (GAAT and GAAA; Table ) 60 . We thus mutated each of these two sites (M2 and M1, respectively) as well as a region between them (M3). These oligonucleotide competition experiments revealed that the C1 and C2 complexes displayed a differential pattern of competition . In particular, the CD23b GAS M2 mutant failed to compete the C2 complex, suggesting that this complex targets the potential 5′ IRF recognition sequence. Previous studies have shown that purified BCL-6 can bind the CD23b GAS 22 . Furthermore, expression of BCL-6 is downregulated by CD40 stimulation of B cells, and undetectable in EBV-transformed B cell lines 46 61 . These observations suggested that the C1 complex, which we had detected in Ramos but not in JY cells, might contain BCL-6. Indeed, incubation of nuclear extracts from Ramos cells with a BCL-6 antiserum, but not with a control antiserum, led to the disappearance of the C1 complex . These experiments also clearly revealed the presence of the C2 complex in both untreated and IL-4–treated cells. In IL-4–stimulated cells, an additional Stat6 complex could also be observed. Our cold competition experiments suggested that binding of the C2 complex to the CD23b GAS requires the presence of a potential IRF binding site. To directly assess whether the lymphoid-specific IRF-4 participated in the CD23b GAS binding complexes, we then performed Ab interference EMSA assays with an anti–IRF-4 antiserum 32 . Incubation of extracts from untreated Ramos cells with anti–IRF-4 antiserum, but not with a control antiserum, supershifted the C2 complex . Addition of the IRF-4 antiserum completely blocked the appearance of the C2 complex in JY extracts. Since detection of the C2 complex in Ramos cells is hindered by the presence of the BCL-6 complex, we also performed Ab interference assays using as a probe the CD23b GAS M1 mutant, which has lost the ability to bind BCL-6. As shown in Fig. 2 C, this probe clearly detected the C2 complex in Ramos cells, and addition of the anti–IRF-4, but not a control, antiserum led to its disappearance. Additional Ab interference assays demonstrated that antisera against another IRF family member, IRF-2 or ICSBP, failed to affect the appearance of the CD23b GAS binding complexes in either JY or Ramos despite appropriately supershifting complexes binding to the guanylate binding protein (GBP)–IFN-stimulated regulatory element (ISRE) . Furthermore, no effect of the anti–IRF-4 antiserum on the IL-4–inducible Stat6 complex or on the IFN-γ–inducible Stat1 complex binding to the IRF-1 GAS was noted, indicating that this effect was specific for the complex binding to the CD23b GAS (data not shown). We have also detected IRF-4 binding to a cis-element adjacent to the Stat6 binding site in the CD23a promoter (data not shown). Thus, these data indicate that IRF-4, BCL-6, and Stat6 can all target the CD23b GAS element. Interestingly, these experiments also suggest that binding of IRF-4 and Stat6 to the CD23b GAS is not cooperative and can occur independently of each other. Interactions between an IRF and STATs are critical for the formation of the IFN-α–inducible complex, IFN-stimulated gene factor 3 (ISGF3), which contains Stat1 and Stat2 as well as p48, a member of the IRF family 62 . Since Stat6 and IRF-4 can bind to adjacent DNA elements, we then proceeded to test whether IRF-4 can physically interact with Stat6 by performing GST fusion protein binding assays . An association of Stat6 with IRF-4 could indeed be detected by incubating a GST–IRF-4 fusion protein with extracts from Ramos cells stimulated with anti-CD40, a control Ab, or human IL-4. No interaction of Stat6 with the GST moiety alone was observed. Furthermore, reprobing with anti-Stat3, anti-Stat1, anti-Stat2, and anti-Stat5 antisera revealed that none of these STATs were able to complex with IRF-4 . To confirm that the IRF-4–Stat6 interaction could occur in vivo, we used an anti–IRF-4 antiserum to immunoprecipitate extracts from Ramos cells stimulated with either anti-CD40 Ab, a control Ab, or IL-4. Consistent with our previous observations, presence of the Stat6 protein could be detected in the IRF-4 immunoprecipitates from both stimulated and unstimulated Ramos lysates . Similar results were also observed in JY cells. Stripping and reprobing of the filter with an anti–IRF-4 Ab ensured for equal loading of the immunoprecipitates . Consistent with recent studies, which have detected IRF-4 in both the nuclear and cytoplasmic compartments (Riccardo Dalla-Favera, personal communication), the IRF-4–Stat6 association can occur in the absence of IL-4 stimulation. Interestingly, in both GST and coimmunoprecipitation experiments the IRF-4–Stat6 interaction appears to decrease upon stimulation of Ramos cells with the CD40 Ab. Thus, our data indicate that IRF-4 is capable of specifically interacting with Stat6 both in vitro and in vivo. The preceding experiments suggested that IRF-4 could bind a functional element of the CD23b promoter. Since CD40 and IL-4 synergistically induce CD23b, we proceeded to determine whether CD23b upregulation by these stimuli was accompanied by changes in IRF-4 expression. Therefore, we cultured Ramos cells with an anti-CD40 Ab, IL-4, or a combination of anti-CD40 Ab and IL-4. Simultaneous cultures with a control Ab were also included. Northern analysis of total RNA derived from this experiment revealed that IRF-4 expression was upregulated in response to either CD40 or IL-4 stimulation . A much stronger induction was noted when the anti-CD40 Ab was used in conjunction with IL-4. Consistent with previous reports 46 61 , CD40 stimulation of Ramos cells also led to the downregulation of BCL-6 . The same pattern of IRF-4 upregulation in response to CD40 and/or IL-4 was also detected in another EBV-negative Burkitt's lymphoma cell line, BL-41 (data not shown), and by Western analysis, in human tonsillar cells . Strong induction of IRF-4 was also observed in response to transfectants expressing CD40 ligand (CD40L) but not to control transfectants, indicating that IRF-4 expression is a physiological target of the CD40–CD40L interaction ( 59 ; data not shown). Kinetic studies showed that induction of IRF-4 is first noted at 2 h and can still be detected at 24 h (data not shown). No effect on IRF-4 expression was noted by culturing Ramos cells with either IFN-γ or IFN-α (data not shown). Reprobing of this Northern blot with a probe for ICSBP, an IRF family member closely related to IRF-4 63 , revealed that the CD40 and IL-4 signaling cascades do not upregulate ICSBP expression (data not shown). Consistent with previous reports 34 , we also found that IRF-4 levels are highly increased in EBV-transformed B cell lines (data not shown). Thus, expression of IRF-4 in B cells is specifically targeted by the CD40 and IL-4 signaling cascades as well as by EBV transformation. The induction of IRF-4 expression by CD40 and IL-4 as well as by EBV suggested that IRF-4 might function as a transactivator of CD23 gene expression. However, we had detected constitutive binding of IRF-4 to the CD23b GAS in unstimulated Ramos cells, which only express low levels of CD23b. We then reasoned that, in this setting, IRF-4 function might be modulated by interaction with a repressor. Consistent with this hypothesis, our EMSA experiments had revealed binding of BCL-6 to the CD23b GAS in Ramos, but not in JY cells, which express high constitutive levels of CD23b. To assess whether IRF-4 could interact with BCL-6, we then performed UV cross-linking experiments with a bromodeoxyuridine-substituted CD23b GAS probe. This was followed by immunoprecipitations with an IRF-4 antiserum, a BCL-6 antiserum, or a Stat6 antiserum as control . The immunoprecipitates were then resolved by SDS-PAGE and Western blotted. Consistent with our EMSA results , an ∼62-kD protein was recognized by the IRF-4 antiserum. This corresponds to the molecular mass of IRF-4 plus the DNA probe. An additional cross-linked protein was also immunoprecipitated by the IRF-4 antiserum in Ramos, but not in JY cells. The size of this additional protein, after correction for the probe contribution, was ∼90 kD, and was identical to that of the BCL-6 protein cross-linked to the CD23b GAS as determined by simultaneous immunoprecipitation with an anti–BCL-6 antiserum. Indeed, reprobing of the Western blot with an anti–BCL-6 antiserum confirmed that the p90 protein could be recognized by this antiserum (data not shown). Thus, the anti–IRF-4 antiserum can coprecipitate IRF-4 and BCL-6 bound to the CD23b GAS. To determine whether interaction of IRF-4 and BCL-6 could occur independently of the presence of the CD23b GAS, we performed pull-down assays with a GST–IRF-4 fusion protein. As shown in Fig. 5 B, incubation of a GST–IRF-4 fusion protein with extracts from Ramos cells revealed a very strong association of IRF-4 with BCL-6. No interaction was observed with the GST moiety alone or upon incubation of the GST–IRF-4 with JY extracts, consistent with the lack of BCL-6 expression in EBV-transformed B cells. We also subjected extracts from Ramos cells cultured with or without IL-4 to immunoprecipitation assays with either an anti–IRF-4 or anti–BCL-6 antiserum . This experiment demonstrated that IRF-4 and BCL-6 coimmunoprecipitated. Association of BCL-6 and IRF-4 was not affected by IL-4 treatment. Surprisingly, we did not detect increased levels of IRF-4 in our immunoprecipitations of IL-4– or CD40-treated extracts , despite an induction of IRF-4 levels by these stimulations . We suspect that this may be due either to a limited ability of this antiserum to immunoprecipitate increasing amounts of IRF-4 or to the ability of IRF-4 upon IL-4–CD40 treatment to form alternative complexes that cannot be recognized by this antiserum. To further corroborate the specificity of the association of IRF-4 with BCL-6 and Stat6, we also performed coimmunoprecipitation experiments with either an anti–IRF-4 or an antiserum against a nuclear factor κB (NF-κB) family member, p65. These assays were conducted on extracts from JY cells, which constitutively express p65 in the nucleus . These experiments failed to detect an association of IRF-4 with p65. These studies thus indicate that IRF-4 can interact with the Krüppel zinc finger transcriptional repressor, BCL-6, both in vitro and in vivo. Furthermore, no such interaction is detected in cells (JY) that express high levels of CD23b, suggesting that the presence of BCL-6 affects the functional ability of IRF-4 to modulate CD23b transcription. To directly assess whether IRF-4, in the absence of BCL-6, could function as a transactivator of CD23b, we performed transient transfection assays in U937 cells, a monocytic cell line that is capable of activating Stat6 in response to IL-4, but lacks both IRF-4 and BCL-6. Cotransfection of an IRF-4 expression vector with a luciferase reporter construct driven by an oligomerized CD23b GAS wt element resulted in a threefold induction in the luciferase activity, suggesting that IRF-4 can act as a transactivator of CD23b in the absence of Stat6 activation . This level of induction was similar to that observed upon stimulation of U937 with IL-4. Interestingly, cotransfection of IRF-4 augmented, albeit not in a synergistic manner, the induction of the CD23b GAS wt reporter construct in response to IL-4. Consistent with the results of our EMSA experiments, a reporter construct driven by a mutant CD23b GAS element, which binds Stat6 but not IRF-4 , displayed a normal IL-4 inducibility but could not be activated by IRF-4 cotransfection . Furthermore, overexpression of IRF-4 was unable to enhance the IL-4–mediated activation of this CD23b GAS M2 reporter construct. Thus, these data suggest that optimal transactivation of CD23b requires the presence of both Stat6 and IRF-4. Since BCL-6 is known to repress Stat6 function, we then proceeded to determine whether BCL-6 could block IRF-4 function. Indeed, cotransfection of BCL-6, but not of an empty vector, with IRF-4 was able to repress the ability of IRF-4 to induce the activity of the CD23b GAS reporter construct . A similar inhibitory effect was also detected when BCL-6 was cotransfected with a reporter construct driven by the CD23b promoter in JY, an EBV-transformed B cell line, which constitutively expresses high levels of IRF-4 but no activated Stat6 . These studies thus indicate that IRF-4 can transactivate the CD23b GAS and that BCL-6 can block IRF-4 function independently of its inhibitory effects on Stat6. Biochemical and genetic studies have demonstrated that Stat6 plays a key role in IL-4 signaling 10 . However, the rapid activation of Stat6 cannot solely account for the complex biological activities of IL-4 1 3 . The IL-4 signaling pathway must thus use additional effector molecules. Our studies demonstrate that IRF-4 is both a target and a modulator of the IL-4 signaling pathway. This dual role of IRF-4 is thus reminiscent of that of IRF-1 in the IFN signaling cascade 26 . In contrast to the IL-4 induction of Stat6, which has been detected in a wide variety of cells 13 14 15 16 17 , IRF-4 expression is largely restricted to the lymphoid compartment 31 32 34 . Thus, the recruitment of IRF-4 by the IL-4 signaling pathway provides a potential mechanism by which this cytokine can regulate the expression of target genes in a lineage-specific manner. Similarly to IRF-1, whose induction is not restricted to the IFN pathway 64 65 , we found that the expression of IRF-4 in B cells can be independently upregulated by stimulation via the CD40 receptor. CD40 engagement is also known to lead to the rapid activation of the NF-κB/rel family of transcription factors 4 . Although other IRFs have been shown to interact with NF-κB/rel proteins 66 67 68 , we did not detect a physical interaction of IRF-4 with p65 . However, this finding does not exclude that IRF-4 may be able to associate with other NF-κB family members and/or functionally modulate the activity of CD40-inducible NF-κB complexes. Such an interaction may allow IRF-4 to participate in the regulation of genes that are activated in response to CD40 alone. Our studies indicate that costimulation of B cells with CD40 and IL-4 leads to maximal IRF-4 induction. This is accompanied by the simultaneous CD40-mediated downregulation of BCL-6 , consistent with previous reports 46 61 . Since germinal center T cells have been shown to express CD40L as well as IL-4 69 , the transcriptional events triggered by activation of B cells with both stimuli are physiologically relevant. Thus, one may predict that B cells that have been successfully selected in the germinal center and have received appropriate T cell help should express high levels of IRF-4 in addition to low or absent BCL-6. In support of this notion, a small subpopulation of centrocytes with such a phenotype has been identified in close apposition to follicular dendritic cells. These cells have been postulated to represent “surviving centrocytes” (Riccardo Dalla-Favera, personal communication). The coordinated induction of IRF-4 and downregulation of BCL-6 may thus be an important step in the progression of B cells toward the terminal stages of differentiation. Our studies demonstrate that Stat6 and IRF-4 can physically interact. Preliminary experiments suggest that association of IRF-4 with Stat6 involves the COOH-terminal region of IRF-4. This portion of IRF-4 contains a putative α-helical region, which displays strong homology to the STAT-interacting domain of p48, the IRF component of the ISGF3 complex 63 70 . As in the case of the p48–Stat2 complex 71 , tyrosine phosphorylation of Stat6 does not appear to be needed for its association with IRF-4. However, unlike the ISGF3 complex 70 72 , IRF-4 does not appear to be involved in recruiting Stat6 to the CD23b GAS. Our inability to detect a cooperative complex containing both IRF-4 and Stat6 also contrasts with the strong phosphorylation-dependent cooperative interaction between IRF-4 and the Ets protein, PU.1 29 35 36 . Our transient transfection experiments indicate that IRF-4 can act as a transactivator and augment the Stat6 inducibility of CD23b. Our studies have revealed an additive rather than a synergistic interaction between IRF-4 and Stat6, suggesting that recapitulation of the synergistic induction of CD23b in vitro may require regions flanking the CD23b GAS and/or additional components. For example, studies of the IFN-β enhanceosome, a model system for transcriptional synergism, have demonstrated that this regulatory region (−110 to −53) contains multiple IRF-1 binding sites 73 . Consistent with this notion, our survey of the CD23b promoter has revealed that the regions flanking the CD23b GAS may contain additional IRF-4 binding sites. Our inability to detect enhanced IRF-4 DNA binding in EMSAs upon IL-4 treatment may thus be due to the lack of these additional sites in the probes used. Although, in the CD23 system, IRF-4 acts as a positive regulator, IRF-4 contains multiple regions with transactivation and/or repressing potential 74 75 76 77 . Therefore, the precise outcome of the Stat6–IRF-4 interaction is likely to be dictated by the specific arrangement of their DNA binding sites as well as by the presence of additional cofactors. Indeed, our studies suggest that the function of IRF-4 can be profoundly affected by the presence of the Krüppel zinc finger transcriptional repressor, BCL-6 19 20 21 78 79 . BCL-6 is able to repress IRF-4 function in the absence of Stat6. This may allow BCL-6 to modulate the expression of Stat6-independent target genes. Indeed, we have found that BCL-6 binds to the IRF-4 binding site present in the Igκ 3′ enhancer ( 35 36 ; data not shown). In contrast to the CD23b GAS, this DNA element does not bind Stat6. Thus, the BCL-6–IRF-4 interaction may underlie some of the defects exhibited by the BCL-6–deficient mice that are not corrected by the lack of Stat6 24 . Various mechanisms may account for the repressive effects of BCL-6 on IRF-4 function. In addition to the known ability of BCL-6 to recruit the corepressor machinery 80 81 82 83 , BCL-6 may prevent high-affinity DNA binding by IRF-4 as suggested by our UV cross-linking experiments, which detected stronger CD23b GAS–IRF-4 complexes in the absence of BCL-6. Furthermore, previous studies have indicated that IRF-4 contains an inhibitory region (amino acids 207–300) that can mask its own transactivation domain 84 . Thus, BCL-6 may maintain IRF-4 in an autoinhibitory state. IRFs have previously been shown to be critical components of the virally induced IFN-β enhanceosome 73 . Interestingly, another member of the Krüppel zinc finger family of transcription factors, PRDI-BF/Blimp1 85 86 87 , can bind to one of the IRF binding sites within the IFN-β enhanceosome and act as a repressor of IFN-β gene expression 85 . Thus, interaction between IRFs and Krüppel proteins may be a conserved feature of the transcriptional regulation of a variety of genes. The ability of the CD23b GAS to bind an IRF family member as well as its organizational features are indeed reminiscent of regulatory DNA elements targeted by enhanceosomes 88 . This suggests that the CD23b GAS and its flanking regions may function in the assembly of higher-order transcriptional complexes. The synergistic induction of CD23b by the CD40 and IL-4 signaling pathways may thus result from their ability to simultaneously target the expression/function of Stat6, IRF-4, and BCL-6, leading to a profound remodeling of the architecture of this nucleoprotein complex. Although we have been unable to clearly demonstrate formation of a trimolecular complex consisting of Stat6, BCL-6, and IRF-4, prolonged exposures of our UV cross-linking experiments have revealed that the IRF-4 antiserum can immunoprecipitate, in addition to IRF-4 and BCL-6, a faint band of mobility identical to that of Stat6. Assembly of this complex may thus require specific three-dimensional contacts, which we are unable to fully reproduce with the techniques available to us. Alternatively, some of the interactions between Stat6, IRF-4, and BCL-6 could be mediated by additional cofactors. These findings thus lend support to the notion that assembly of these enhanceosome-like complexes may represent ideal targets for the final integration of signaling pathways 73 . Furthermore, presence of lineage-specific components like IRF-4 and stage-specific repressors like BCL-6 within these complexes would endow cells with the ability to regulate gene expression in response to signals such as CD40 and IL-4 in a context-appropriate manner. | Study | biomedical | en | 0.999997 |
10601359 | Full-length dynamin 2 (aa isoform) with a single amino acid mutation that changed the lysine at position 44 to an alanine, dyn K44A , was cloned into the pTIGZ2 vector. In this vector, expression of dyn K44A is under the control of a tetracycline-repressible promoter. Removal of tetracycline from the media results in a bicistronic mRNA that concomitantly directs translation of the dominant-negative dynamin protein and green fluorescent protein (GFP). pTIGZ2 consists of pcDNA3.1/Zeo (Invitrogen) in which the CMV promoter was replaced by the tetracycline-regulated promoter from pTetSplice (XhoI-HindIII fragment; GIBCO BRL) followed by a multiple cloning site, the cap-independent translational enhancer region of pCITE (amplified using the 5′ primer, GTGGATCCGTTATTTTCCACCATATT, and the 3′ reverse primer, GGGAGCTCCCATATTATCATCGTGTT; Novagen) and the coding region for enhanced GFP (eGFP) from peGFP-N1 (EcoRI-NotI fragment; Clontech). V5 epitope–tagged dynamin 2 and dyn K44A were constructed by TA cloning into the pcDNA3.1/V5/HisTOPO vector (Invitrogen). pNeo/Tak was constructed to direct expression of the tetracycline transactivator under neomycin selection. The plasmid uses a tetracycline-regulated promoter to direct expression of the tetracycline transactivator (both from pTet-Tak; GIBCO BRL). The neomycin resistance marker was from pcDNA3 (Invitrogen), and the remainder of the plasmid was derived from pBluescript SK (Stratagene). Murine resident peritoneal (RP) macrophages were isolated and cultured as described previously 1 . Synchronized phagosomes were created by centrifuging particles onto the cells at 1,600 rpm and 4°C for 1 min. (Before exposure to C3b i -opsonized particles, cells were treated with 200 nM PMA for 30 min.) After washing with PBS, the cells were incubated in media at 37°C for the times indicated in the Results. The cells were fixed in formalin (10 min, room temperature), permeabilized in 0.25% Triton X-100 in PBS (10 min, room temperature), washed twice in PBS, and incubated with primary antibody (Dyn 2, an affinity-purified anti-dynamin 2 antibody generated as described 25 ; MC63, an anti-pan dynamin antibody generated as described 26 ; or the anti-V5 antibody [Invitrogen], and anti–human RBC antibody purchased from Jackson ImmunoResearch Labs) for 1 h at room temperature. The coverslips were washed in PBS and incubated with the appropriate secondary antibodies (all FITC- and TxR-conjugated antibodies were from Cappel, Cy5 conjugates from Jackson ImmunoResearch Labs). Actin was stained with rhodamine-phalloidin (Molecular Probes). After a 1-h incubation, the slides were washed in PBS, rinsed briefly in distilled water, and mounted in a polyvinyl alcohol–based mounting medium (Harlow and Lane). All confocal images were obtained on a Zeiss Axiophot microscope equipped with Bio-Rad confocal optics. IgG-coated RBCs were prepared by incubating fresh human RBCs, diluted in PBS, with anti–human glycophorin IgG (Jackson ImmunoResearch Labs) at room temperature for 60 min. Complement-coated RBCs were prepared by incubating human red cells with anti–human glycophorin IgM (supernatant from the NN3 hybridoma; American Type Culture Collection) at room temperature for 30 min, and the cells were then washed and resuspended in RPMI with 10% C5-depleted human serum (Sigma Chemical Co.), and incubated at 37°C for 1 h. Zymosan (Molecular Probes) was prepared as described previously 27 . Phosphoinositide 3-kinase (PI3K) was inhibited by incubating RP macrophages with 100 nM wortmannin (Sigma Chemical Co.) for 1 h. These cells were incubated with zymosan for 10 min, then prepared and examined as above. Macrophages, either RP or RAW 264.7 cells (American Type Culture Collection), were lysed on ice into lysis buffer (20 mM Tris, pH 7.4, 120 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM dithiothreitol, and 1% SDS, 0.09 trypsin inhibitor units [TIU] aprotinin, 0.5 mg/ml leupeptin, 1 mM PMSF). Total murine brain homogenate was lysed into brain lysis buffer (10 mM Hepes, 150 mM NaCl, 10 mM benzamidine, 1% Triton X-100 with the same protease inhibitors as above), incubated at 4°C with agitation for 1 h, and insoluble material was removed by centrifugation at 40,000 g for 1 h at 4°C. Samples were quantified by the BCA assay (Pierce Chemical Co.), and 20 μg of protein/well was run on a 10% SDS-PAGE acrylamide gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) and blocked overnight at 4°C in 10% nonfat dried milk in PBS. Membranes were incubated for 1 h at room temperature with one of the anti-dynamin antibodies (either Dyn 2 or the anti-dynamin 1 rabbit polyclonal antibody DG2, provided by Pietro De Camilli, Yale University School of Medicine, New Haven, CT), washed three times for 15 min each in TBS/Tween and incubated in a 1:10,000 dilution of peroxidase-conjugated secondary antibody (Cappel). After three 15-min washes in TBS/Tween, specific binding was detected using chemiluminescence (Amersham Pharmacia Biotech). Cells to be stained for FcγRII and FcγRIII were resuspended into FACS ® buffer (PBS, 2% FCS, 0.5 mM azide) while cells to be stained for Mac-1 were resuspended into 2.4G2 supernatant (American Type Culture Collection), then incubated for 15 min on ice. Primary antibody (biotinylated 2.4G2 for FcR staining or biotinylated anti–mouse CD11b antibody, both from PharMingen) was added, and the cells were incubated on ice for 20 min. Cells were washed in FACS ® buffer, resuspended in diluted streptavidin-PE (Caltag), and incubated on ice for 15 min. The cells were washed, resuspended in FACS ® buffer with 1% paraformaldehyde, and analyzed on a FACScan™ (Becton Dickinson). A tetracycline transactivator–expressing RAW cell line was generated by transfecting RAW 264.7 cells with pNeo/Tak, and stable cell lines were selected using 400 μg/ml G418 (GIBCO BRL). After 10 d of selection, the cells were cloned by limiting dilution, and one cell line (designated RAW-TT10) that demonstrated good tetracycline-regulated expression from a subsequently transfected reporter plasmid was used for all experiments. In the experiments reported here, tetracycline was always absent from the media, resulting in strong activity of the tetracycline-regulated promoter. RAW-TT10 cells were transiently transfected by electroporation. 10 μg of DNA was added to 5 × 10 6 RAW-TT10 cells in 250 μl of RPMI (JRH Biosciences) with 10% heat-inactivated FCS (Hyclone). The cells were electroporated at 280 V, capacitance 960 μF, and immediately washed in 5 ml of RPMI with 10% FCS. The cells were plated and analyzed 18–24 h later by FACS ® or confocal microscopy. Tetramethyl rhodamine isothiocyanate (TRITC)-zymosan was purchased from Molecular Probes. TRITC-labeled SRBC “ghosts” were prepared by incubating SRBCs (ICN/Cappel) in hypotonic lysis buffer (1 mM MgCl 2 , 100 μM EGTA in 0.02× PBS) with TRITC-BSA (Molecular Probes) on ice for 1 h. Isotonicity was restored to the cells with 5× PBS, and the ghosts were resealed at 37°C for 1 h. Unincorporated TRITC-BSA was removed by washing in PBS, and the ghosts were opsonized as described above (anti-SRBC IgG and IgM were purchased from Intercell). The specified particles were centrifuged onto the transiently transfected RAW-TT10 cells at 1,600 rpm and 4°C for 1 min. (Before exposure to C3b i -opsonized particles, cells were treated with 200 nM PMA for 30 min.) The cells were incubated at 37°C for 10 min. The extracellular particles were removed, ghosts were lysed with a 20-s water wash, and the TRITC-zymosan was digested for 10 min with 100 U/ml lyticase (Sigma Chemical Co.). The cells were resuspended in PBS/EDTA, fixed with 1% formalin, and analyzed by FACS ® . The effect of dynamin 1 on phagocytosis was assessed with three dominant-negative mutants of dynamin 1 expressed in pCMV5: DI ΔPH (deletion of amino acids 541–618), DI K535M, and DI N272 (deletion of amino acids 1–272) 28 . Each of these constructs was cotransfected with pSFFV-eGFP (eGFP under the control of the spleen focus–forming virus LTR) into RAW-TT10 cells, and phagocytosis was assayed as described above. RAW-TT10 cells were transfected with dyn K44A -pTIGZ2 18 h before the assay. The cells were incubated with the indicated particles at 37°C for 30 min, at which time 5 μM brefeldin A was added to the media. The zymosan particles were endotoxin free as assessed by the limulus amebocyte lysate assay . The cells were incubated for an additional 2.5 h, then collected for staining. The cells were blocked in 2.4G2 hybridoma supernatant (American Type Culture Collection) for 15 min on ice, fixed in 4% paraformaldehyde for 15 min at room temperature, washed, and stained with anti–TNF-α antibody (PharMingen) conjugated to PE in permeabilization buffer (1% FCS, 0.1% [wt/vol] sodium azide, 0.1% [wt/vol] saponin, in PBS, pH 7.4) at room temperature for 30 min, washed, and analyzed on a FACScan™ (with CELLQuest™ software; both from Becton Dickinson). RAW-TT10 cells were transfected with dyn K44A -pTIGZ and plated on 35-mm glass-bottomed microwell dishes (MatTek Corp.). 18 h later, the dish was mounted on a Zeiss Axiophot microscope equipped with a cooled CCD camera (Princeton Instruments) and Metamorph digital imaging software system (Universal Imaging), and differential interference contrast (DIC) images were collected every 30 s. 18 h after transfection, cells expressing high levels of the indicated proteins were sorted onto Thermonox coverslips (Nunc) using a FACStar PLUS™ (Becton Dickinson). Cells were adhered for 3 h at 37°C, then incubated with IgG-opsonized SRBCs for the indicated amount of time. Cells were fixed in 3% glutaraldehyde in EM buffer (0.1 M cacodylate, 0.1 M sucrose) at room temperature for 1 h, then washed with EM buffer. The cells were postfixed in 1% OsO 4 in 0.1 M cacodylate, 4 mM CaCl, pH 7.3, for 30 min at room temperature. The cells were dehydrated through serial changes in ethanol (35 and 50%) for 5 min each, en bloc stained in 3% uranyl acetate, 70% ethanol for 30 min, and ethanol dehydration was then completed (80, 90, 95, 100, and 100%, each for 5 min). The coverslips were critical point dried, mounted onto scanning stubs, and air dried overnight. Cells were sputter coated with 30 nm gold/palladium and examined on a Jeol JSM-6300F scanning electron microscope. Dynamin 2 was found to be expressed in murine RP macrophages and the RAW-TT10 macrophage cell line, whereas dynamin 1 was not detected in macrophages . Interestingly, dynamin 2 was recruited to early phagosomes, as demonstrated by staining with two independent antibodies to dynamin as well as by the localization of the epitope-tagged protein . The phagocytosis of different particles is mediated by different receptors. For example, FcRs mediate the uptake of IgG-coated particles, complement receptors (CRs) mediate the uptake of C3b i -opsonized particles, and the mannose receptor (among others) mediates the uptake of zymosan (yeast cell wall particles) 2 . Phagocytosis stimulated by these receptors has common features, such as a reliance on the actin cytoskeleton, and distinct features, such as their different requirement for the cytoskeletal proteins vinculin and paxillin 1 . Immunofluorescence microscopy of murine RP macrophages using two independent antibodies demonstrated that dynamin 2 was enriched on early phagosomes containing IgG-opsonized RBCs, C3b i -opsonized RBCs, and zymosan . In addition, an epitope-tagged version of the aa isoform of dynamin 2 transiently expressed in RAW-TT10 macrophages also localized to phagosomes, demonstrating that this macrophage-expressed isoform contains the domain responsible for targeting to phagosomes . The kinetics of association of dynamin 2 with phagosomes precisely mirrored that of F-actin: both were recruited to the forming phagocytic cup and the early phagosome , and both were concomitantly lost from the phagosome after particle internalization (data not shown). To examine the role of dynamin 2 in phagocytosis, RAW-TT10 macrophages were transfected with a dominant-negative mutant form of the aa isoform of dynamin 2, dyn K44A , which is unable to bind GTP 11 . The mutant dynamin 2 gene was expressed in a bicistronic vector with GFP (pTIGZ2 vector), allowing transiently transfected cells to be identified by their green fluorescence. Phagocytosis of either TRITC-labeled zymosan, IgG-opsonized RBCs, or C3b i -opsonized RBCs was assessed as a function of the level of expression of the GFP/dominant-negative protein by two-color FACS ® . In all cases, dyn K44A inhibited phagocytosis in a dose-dependent manner ; a typical FACS ® profile is shown in Fig. 2 B. Dyn K44A inhibited FcR-mediated phagocytosis by 85%, CR-mediated phagocytosis by 63%, and zymosan phagocytosis by 65% . As expected, dyn K44A also inhibited receptor-mediated endocytosis as determined by the uptake of DiI-labeled acetylated low-density lipoprotein (LDL) in macrophages . V5 epitope–tagged dyn K44A colocalized with actin on nascent phagocytic cups, demonstrating that the mutant is also recruited to the site of particle-induced signaling and establishing that it is correctly localized to inhibit the function of dynamin on phagosomes . This construct also inhibited phagocytosis (data not shown), and its effects were indistinguishable from those seen with untagged dyn K44A . These effects were specific for dynamin 2, since dominant-negative mutants of dynamin 1 did not inhibit phagocytosis or receptor-mediated endocytosis in macrophages (data not shown). The defect in phagocytosis was not due to an effect of dyn K44A on the level of phagocytic receptors, since transfected cells expressed normal cell surface levels of FcRs (CD16 and CD32) and CRs (Mac-1) (data not shown), and particle binding was unimpaired . Cell viability and other actin-based processes were unaffected by the expression of dyn K44A as demonstrated by the observations that RAW-TT10 cells expressing dyn K44A were able to migrate, polarize, extend and retract ruffles, and spread in response to phorbol esters (data not shown). Further, engagement of phagocytic receptors stimulated actin polymerization at the site of particle binding . In addition, mutant dynamin had no effect on particle-induced TNF-α production , demonstrating that one arm (internalization) of a bifurcating signaling pathway was selectively inhibited. Our initial hypothesis was that dynamin would serve a similar role in phagocytosis as it serves in endocytosis and therefore would be required only for scission of the nascent phagosome from the plasma membrane. However, examination of dyn K44A -expressing RAW-TT10 cells attempting to internalize particles revealed that dynamin was exerting a role earlier in the process. Cells expressing dyn K44A were able to bind particles, and phalloidin staining demonstrated that this was accompanied by localized actin polymerization; however, actin extended only partially around the particles . To determine the stage at which phagocytosis was arrested, dyn K44A -expressing RAW-TT10 cells were studied by scanning electron microscopy. 10 min after contacting IgG-coated SRBCs, control cells (expressing pTIGZ2 alone) were identified at many different stages of particle internalization , while very few of the dyn K44A -expressing cells extended membrane more than halfway around the SRBCs . This indicated that mutant dynamin arrested particle internalization at an intermediate stage. Indeed, after unbound SRBCs were washed away and phagocytosis was allowed to proceed for an additional 50 min at 37°C, the pTIGZ2 control cells had internalized >90% of the particles . In contrast, <30% of the particles associated with the dyn K44A -expressing cells were internalized . PI3K is a key regulator of macrophage phagocytosis 29 30 . Inhibition of PI3K causes incomplete phagosome closure 29 30 , a very similar phenotype to that observed in cells expressing mutant dynamin. Inhibition of PI3K with wortmannin prevented the recruitment of dynamin 2 to the site of particle binding and actin polymerization . Thus, it is possible that PI3K might act upstream of dynamin in phagocytosis. Macrophage phagocytosis of IgG-coated particles and zymosan results in several signaling events, including the production of inflammatory mediators such as TNF-α 2 . Cells expressing dyn K44A generated normal amounts of TNF-α upon interaction with particles ; thus, mutant dynamin uncoupled particle internalization from particle-dependent cytokine production. In this study, we have demonstrated that dynamin 2 is essential to the formation of macrophage phagosomes, and that it functions at the stage of membrane extension around the particle. This role for dynamin is conserved in all of the phagocytic receptor systems examined. Dominant-negative mutant dynamin did not impinge on the cell's capacity to polymerize actin beneath the particle, or to produce inflammatory mediators such as TNF-α in response to particle binding. The phagocytic defect induced by dyn K44A resembles that seen when PI3K is inhibited in macrophages 29 30 . This is of interest, since dynamin interacts with the p85 regulatory subunit of PI3K and this interaction stimulates dynamin's GTPase activity 31 . We report here that inhibition of PI3K prevents the recruitment of dynamin 2 to the site of particle binding, suggesting that the activation of PI3K is upstream of dynamin in mediating phagocytosis. PI3K supports phagocytosis in macrophages, in part, by facilitating the insertion of membrane into forming phagosomes 30 . The scanning electron micrographs shown here suggest that membrane extension may also be the stage of arrest in the cells expressing dyn K44A . Membrane extension is known to require the fusion of vesicles with the plasma membrane 30 32 33 34 35 ; thus, it is tempting to speculate that dynamin's role in phagocytosis is related to its capacity to recruit membrane to nascent phagosomes. In support of this, the yeast homologue of dynamin, Vps1p, is required for bidirectional trafficking between endosomes and the vacuole 19 . Although our data suggest a role for dynamin 2 in extending membrane around the nascent phagosome, it does not rule out other mechanisms for dynamin's effect on phagocytosis. For example, dynamin might have a direct effect on actin during phagosome formation, since it has been demonstrated to interact with profilin, an actin-binding protein 36 . It remains possible that dynamin is also involved in the scission of the neck behind the phagosome, similar to its known role in endocytosis. However, we have not observed any enrichment of dynamin at the scission site of the phagosome. The phagocytosis of pathogens by macrophages is tightly coupled to the elaboration of inflammatory cytokines that, in turn, orchestrate an appropriate immune response. It has long been known that particle binding by macrophages induces actin-mediated internalization and inflammatory mediator production through a bifurcating signaling cascade 37 38 . Dynamin clearly regulates the particle internalization limb of this pathway while it has no role in the production of inflammatory cytokines. | Study | biomedical | en | 0.999998 |
10601360 | BCBL-1 cells at a concentration of 5 × 10 5 cells/ml were induced with 20 ng/ml TPA (Calbiochem) for 12 h, reseeded in fresh medium , and cultured for an additional 4 d in 5% CO 2 at 37°C. This induction protocol results in ∼10 6 DNA genome equivalents per milliliter of culture 22 . Debris was removed by centrifugation for 10 min at 5,000 rpm in a GS4 rotor, and virus was pelleted by centrifugation for 2 h at 14,000 rpm in a SS-38 rotor. Virus from 180 ml supernatant was pelleted, and the pellet was resuspended in 1,000 μl PBS, of which 50 μl was injected per implant. HIV-1 NL4-3 was prepared as previously described 41 . SCID-hu Thy/Liv mice were generated by coimplantation of fragments of human second trimester fetal liver and thymus under the kidney capsule of male homozygous C.B-17 scid/scid (SCID) mice 31 . Implants were infected 4–7 mo after implantation by direct inoculation as previously described 42 . In brief, mice were anesthetized, the left kidney was surgically exposed, and the implant was inoculated with 50 μl of virus stock. The abdominal wall incision was sutured, and the skin was closed with staples. Where indicated, ganciclovir sodium salt (Cytovene; Hoffman-La Roche) was administered intraperitoneally twice daily at 50 mg/kg/d from the day of virus inoculation. At indicated times after inoculation, mice were killed and their implants surgically removed and processed for PCR. For flow cytometry, cell sorting, and quantitative PCR, single-cell suspensions were obtained by grinding the implants between glass plates. The cells were counted in a Coulter counter and divided in aliquots for the different assays. Qualitative reverse transcriptase (RT)-PCR and subsequent Southern hybridization for lytic messages was performed as described by Renne et al. 27 . Qualitative RT-PCR was performed using the same procedure with plasmid pDD4 17 as hybridization probe. A description of the primers used in these experiments is shown in Table . In brief, RNA was isolated using RNAzol (Tel-Test, Inc.) according to the supplier's protocol. 500 ng total RNA was reverse transcribed using 200 U of Mo-MuLV RT (GIBCO BRL) in a total volume of 20 μl containing 125 μM dATP, dGTP, and dTTP, 20 U RNasin (Promega Corp.), and 120 pmol random hexanucleotide primers (Boehringer Mannheim). After incubation at 42°C for 35 min, and the reaction was stopped by heating to 95°C for 5 min, and then 80 μl of a PCR mix containing 10× PCR Mg-Buffer (Perkin-Elmer Corp.), 100 pmol of each primer, and 5 U of Taq polymerase (Perkin-Elmer Corp.) was added and amplified over 30 cycles (30 s at 94°C, 1 min at 58°C, and 1 min 30 s at 72°C). Quantitative DNA and RT-PCR was carried out in duplicate using Taqman ® RT and Taqman ® PCR with Amplitaq Gold ® reagents (PE Biosystems). Reverse transcription was carried out using 2.5 μM random hexamers at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. PCR was carried out using universal cycle conditions (2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C) on an ABI PRIZM 7000™ sequence detector 43 . To prevent contamination, all PCR reactions were assembled in the segregated space in which neither KSHV virions nor cloned KSHV DNA was handled. Carryover of the amplification product was avoided using positive displacement pipettes and UNG glycosylase in the amplification reaction 44 . To determine the sensitivity of the assay, RNA derived from BCBL-1 cells was used (data not shown). Quantitative analysis revealed that the signal for the lytic orf29 message was on average 50–100-fold lower than the signal for spliced latent messages in latently infected BCBL-1 cells, consistent with prior observations 22 that only 1–5% of BCBL-1 cells undergo spontaneous KSHV replication at any given time. The assay was linearly dependent on input cell number in the range of 1–10 5 infected cells per reaction). We found that KSHV could be most sensitively detected using primers that detect both spliced and nonspliced latent transcripts (primers lat-273F, lat-335R, and lat-294T; see Table ). With such primers, we were consistently able to detect 1 infected cell in 10 5 uninfected cells. Single-cell suspensions were incubated with biotinylated mAbs directed against human CD4, CD8, and CD3–biotin (all from Becton Dickinson) for 0.5 h in PBS and 2% FBS on ice and depleted using Dynabeads (Dynal Inc.) according to recommendations from the manufacturer. Pre- and postdepletion cell populations were stained with streptavidin–FITC and PE-conjugated mAbs directed against human CD19 (Becton Dickinson) and separated on a FACSVantage™ (Becton Dickinson) cell sorter to isolate CD19 + B cells, CD4 + , CD8 + , and CD3 + T cells, and cells negative for all four markers. Sort-purified cells were frozen as pellets and stored at −80°C before processing. To test whether KSHV would replicate in the human Thy/Liv implants of SCID-hu mice, we prepared concentrated KSHV virions from induced BCBL-1 cell supernatant 22 and inoculated 50 μl of virus suspension (containing ∼10 7 genome equivalents) directly into each implant, according to established procedures 32 . As an average implant contains ∼10 8 human cells, the maximal multiplicity of infection is an estimated 0.1 genome equivalents per cell. However, it is likely that this substantially overestimates the true multiplicity of infection, because it is unlikely that all of the input virions are infectious (in many viruses, the particle/pfu ratio can be as high as 10 2 –10 4 ). Each cohort included animals injected with UV-inactivated KSHV to serve as negative control; UV irradiation should completely abolish infectivity by cross-linking the double-stranded DNA genome 45 46 47 . At various times after inoculation, the mice were killed. To have all cell types represented, the implants were removed in toto, and 2.5 μg of total implant DNA (corresponding to ∼4.5 × 10 5 cells) was assayed for the presence of viral DNA by quantitative real-time DNA PCR using primers lat-273F, lat-335R, and lat-294T ( Table ). Fig. 1 A shows a standard curve for the real-time quantitative DNA PCR to demonstrate the linear range of the assay, and Fig. 1 B shows a time course of infection. KSHV-specific DNA was low or undetectable in most implants 7 d after inoculation, peaked with a mean signal of ∼100 times background at day 14, and then receded to lower levels at days 21 and 28. This increase in KSHV genome copy number between days 7 and 14 after inoculation and subsequent reduction provides evidence for initial viral replication in the implant, as would be expected in a de novo infection. Exposure of KSHV virions to UV irradiation blocked infectivity. Assuming that, as in cultured PEL cell lines, infected cells harbor ∼50 copies of episomal KSHV DNA per cell, we estimate that this level of viral DNA would correspond to infection of ∼1 cell in 1,000 in the implant . As CD19 + B cells represent between 0.2 and 2% of the total number of cells in the implant 31 , the actual density of infection in this permissive subpopulation is likely to be higher. No difference in the levels of KSHV-specific DNA or transcripts was found between implants derived from different fetal sources ( n = 4), indicating that susceptibility to KSHV infection is independent of the donor background. Lytic herpesvirus replication proceeds in a regulated cascade of differential gene expression. Late lytic gene transcription in particular is dependent on viral replication and an array of viral transacting factors, whereas latent gene transcription occurs in the absence of lytic viral transactivators. If the initial increase in viral DNA noted in the SCID-hu Thy/Liv implants was indeed due to bona fide lytic replication, it should have been accompanied by late lytic gene transcription. Therefore, evidence for mRNA from orf29, a well characterized late viral gene product whose production occurs only in cells undergoing viral replication, was sought by RT-PCR. Following our previously established protocol 27 , total implant RNA was isolated, reverse transcribed using random hexamers, and amplified using primers specific for lytic messages (primers 290A and 290B; Table ). Amplified products were separated on a 2% agarose gel and hybridized with 32 P-labeled probes specific for either product. In this assay, a cutoff was established, and only implants yielding detectable amplification products were scored positive. Primers for the amplification of the lytic orf29 message were located on opposite sides of intron #1; hence, amplification products of the proper size can derive only from late messages and not from contaminating viral DNA. We also developed similar qualitative techniques to detect evidence for latent transcription , using primers specific for either the latent nonspliced orf73 message or spliced orf72 message . As for orf29, correct amplification products using orf72-specific primers can only derive from properly processed mRNA. Amplification products with orf73 primers could derive from either DNA or RNA; however, we estimate that >90% of the signal is derived from amplification of mRNA. Fig. 2 shows the location of the primers and the result of a control amplification from BCBL-1 RNA. Lanes 7–9 show evidence of latent message as judged by the presence of amplification products of the expected size . Using primers specific for spliced orf72 message, no signal was generated in the absence of RT (compare lanes 7 and 8 to 4 and 5). Using primers for unspliced orf73 message, a faint band was detected in the absence of RT, presumably resulting from residual viral DNA (lane 6), but was strongly augmented in the presence of RT (lane 9). Using the RT-PCR assays outlined above, evidence for productive KSHV infection could be detected in human Thy/Liv implants injected with KSHV ( Table ). At day 14 after inoculation, KSHV-latent transcripts were detectable in all infected implants but not in any of the implants that were injected with UV-inactivated KSHV ( P ≤ 0.005 by X 2 ). Human GAPDH (glyceraldehyde-3-phosphate-dehydrogenase)-specific message could be amplified in each case. This demonstrates that infection was dependent on intact, functional virions. Up to 50% of implants harbored levels of lytic transcripts above the cutoff detectable in this assay ( P ≤ 0.1 by X 2 ), suggesting that lytic KSHV replication was supported in these instances (summarized in Table ). To demonstrate that these results were not specific to the BCBL-1 isolate of KSHV, the experiments were repeated with KSHV purified from the equivalent number of induced BC-3 cells (data not shown). As in previous experiments, KSHV latency–specific messages could be amplified from all implants injected with BC-3–derived KSHV but not from those injected with UV-inactivated virus. These results establish that both PEL-derived isolates are functional in this assay. We next determined the time course of latent and lytic gene transcription. Latent transcripts were detectable in almost all animals (seven to eight of eight) up to 4 wk after inoculation and in two of four animals 16 wk after inoculation. Moreover, one of two infected mice showed evidence of latent transcripts 6 mo after inoculation. However, the fraction of implants with detectable lytic transcripts peaked at day 14 and then declined . The peak of lytic gene transcription coincided with the peak of viral DNA load , supporting the hypothesis that the increase in genome copy number resulted from authentic viral replication. Furthermore, no lytic transcripts were detected 21 d after inoculation, suggesting that productive infection was dramatically diminished or had ceased by that time. As before, UV inactivation of the virus abolished both latent and lytic signals. In the SCID-hu Thy/Liv model, human B cells do not migrate outside the implant. Consistent with this observation, no KSHV-specific signal could be detected in the spleens of infected animals (data not shown), suggesting that the infection was species specific and did not spread to murine tissue. As ganciclovir inhibits KSHV replication in vitro 48 49 , we investigated whether the drug would also inhibit KSHV infection in the SCID-hu Thy/Liv model. Six infected animals were treated with twice-daily intraperitoneal injection of 50 mg/kg/d ganciclovir beginning at the time of KSHV inoculation. To concentrate our analysis specifically on productively infected cells, we quantitatively analyzed spliced transcript levels rather than viral DNA. ABI Taqman ® technology was used for real-time quantitative RT-PCR analysis 43 . RNA was prepared and amplified in duplicate using primers specific for either spliced lytic (primers tac29-5F, tac29-68R, and tac29-22T) or spliced latent messages . Primers for amplification of the lytic orf29 message were located on opposite sites of intron #1, and the fluorescently labeled oligonucleotide probe spanned the splice junction. Hence, only correctly spliced late messages for orf29 were detected . For the amplification of latent messages, primers specific for the spliced orf72 message were used . As these primers differed in sequence from those employed for qualitative RT-PCR, RNA from each implant was analyzed twice for each transcript (qualitatively and quantitatively) using two different primer pairs. Implants that yielded significant signal using real-time quantitative RT-PCR always scored positive in the qualitative PCR, and samples that did not result in an amplified product using the qualitative RT-PCR and radioactive hybridization never showed a signal using real-time quantitative PCR and fluorescent detection. Only a single amplification product was produced in the quantitative RT-PCR reaction (data not shown). The amount of product was quantified at each cycle using a third specific, fluorescently labeled oligonucleotide present during the reaction. Hence, the number of cycles (ct) needed for the fluorescence intensity to reach a threshold value is a direct measure of the amount of amplified product. Based on control reactions using RNA from latently infected BCBL-1 cells, sensitivity limits were established to be 1–10 cells per reaction for the spliced latent orf72 assay and 100–1,000 cells per reaction for the lytic orf29 assays , consistent with the observation that <3% of the population undergoes spontaneous lytic reactivation at any given time and the levels of orf29 mRNA in this population are much lower. The assay was linearly dependent on input RNA over three orders of magnitude. Fig. 5 shows relative transcript levels in infected implants at day 14 after inoculation. Total RNA was isolated from individual implants in each group (implants injected with KSHV, implants injected with UV-inactivated KSHV, and implants injected with KSHV and treated with ganciclovir) and assayed in duplicate. To normalize for the amount of RNA in each reaction, rRNA was coamplified and quantified using a differently labeled probe (primers riboF, riboR, and riboP; Table ). Ct values for KSHV-specific probes were subtracted from corresponding Ct values for rRNA and expressed as the number of latent or lytic cells per 10 6 cells (based on BCBL-1 control). As expected, implants injected with live virus exhibited significant levels of spliced latent ( P < 0.005) and lytic ( P < 0.01) transcripts compared with implants injected with UV-inactivated virus. Many animals displayed transcript levels that would correspond to 1 infected cell in 1,000 (assuming that transcript levels per cell are similar to those in BCBL-1 cells). Animals treated with ganciclovir showed a reduction in both latent ( P < 0.1) and lytic ( P < 0.05) transcript levels compared with untreated implants. RNA derived from latent BCBL-1 cells served as positive control. These data suggest that ganciclovir affected both the initial replication and subsequent spread of KSHV as well as establishment of latent infection, resulting in a decrease for both messages. To determine the cell tropism of KSHV in the Thy/Liv organ, we purified relevant cell populations from infected implants using flow cytometry and examined them for the presence of KSHV-specific transcripts . B cells were identified by staining with anti-CD19–PE antibodies and T cells by staining with a mixture of FITC-coupled anti-CD4, anti-CD8, and anti-CD3 antibodies. As reported previously 31 , an average of 0.5–2.5% of all cells in the implant were committed to the B cell lineage, as indicated by CD19 expression, whereas >90% of the implant cells were thymocytes, as indicated by CD3 expression . A fraction of the implant was used to isolate 10 6 CD3 + thymocytes directly using flow cytometry. The remainder of the implant was depleted of thymocytes using biotinylated anti-CD3 and anti-CD8 antibodies coupled to streptavidin-linked magnetic beads . The depleted population was stained with CD19–PE and avidin–FITC, and CD19 single-positive cells were isolated by flow cytometry. Although only 40,000 CD19 + cells could be obtained for analysis, KSHV-specific latent and lytic transcripts could be detected in this cell population. This is consistent with previous observations, which detected KSHV in peripheral blood CD19 + B cells 27 47 50 . Low levels of KSHV-specific latent but never lytic transcripts were also detected in the cells negative for CD3, CD4, CD8, and CD19. We would expect to find stromal cells and macrophages in this population, which harbors KSHV in KS tumors 51 . Experiments to determine the exact nature of these cells as well as the level of KSHV gene expression in them are currently underway. Even lower levels of latent transcript were detected in cells positive for CD3, i.e., T cells. Although we cannot exclude the possibility that the signal originates from contaminating B cells, reanalysis of the sorted CD3, CD4, and CD8 fractions determined them to be ≥99% CD3 + (data not shown). Histological (hematoxylin and eosin stain) and flow cytometric analysis (using mAbs directed against CD19, CD20, and CD45) showed no evidence of B cell depletion during the first 4 wk of infection. During this time frame, in fact, KSHV infection of the Thy/Liv implant was not correlated with any histopathologic changes (data not shown). We are currently following a cohort of infected mice to assess long-term consequences of infection. To test whether an interaction between KSHV and HIV-1 might occur in coinfected implants, cohorts of SCID-hu Thy/Liv mice were injected with a mixture of HIV-1 and KSHV or with each virus alone. The pathogenic, syncytium-inducing HIV-1 isolate NL4-3 was used at an input inoculum of 10 3 tissue culture (TC)ID 50 . This strain induces massive T cell depletion starting at day 14 after inoculation, with <85% of implant thymocytes being destroyed by day 28–35 42 . Accordingly, we chose to examine the coinfected implants on day 14, when HIV-1 infection is well established but before the onset of T cell death complicates the analysis of effects on viral load. KSHV viral load and HIV-1 p24 and RNA levels were quantitated at this time point . Relative to animals infected with KSHV alone, no difference was noted in the levels of KSHV DNA in animals coinfected with HIV-1 and KSHV. The mean KSHV-specific DNA PCR signal in these two cohorts was 10–100 times the signal obtained from animals infected with UV-inactivated virus or HIV-1 alone, suggesting that KSHV replicated as in previous experiments and that its level of replication was unaffected by HIV-1. Correspondingly, the level of HIV-1 p24 in the implant was reduced only minimally in animals coinfected with KSHV as compared with animals infected with HIV-1 alone. We conclude that KSHV did not significantly influence HIV-1 replication, at least in the short period of acute coinfection. The absence of an appropriate animal model to study de novo infection has severely limited our understanding of the biology of KSHV. This paper reports the first small animal model for de novo infection by KSHV. It demonstrates that BCBL-1–derived KSHV preparations can infect SCID-hu Thy/Liv mice, resulting in transient lytic replication and persistent latent infection, as judged by (a) the increase in genome copy number and (b) the presence of latent and lytic viral transcripts in infected animals. Flow cytometric analysis demonstrates that KSHV replicates primarily in CD19 + B cell populations of the implant, which is consistent with the tropism described for KSHV in humans 47 51 52 53 . As KSHV does not replicate in SCID mice 30 that have received human peripheral blood leukocytes (a model that does not support hematopoiesis; reference 29), our observations suggest that immature or developing cell populations may serve as the predominant target of KSHV infection. Alternatively, it is possible that transmission to human blood cells requires a lymphoid microenvironment not reproduced by the implant. Dissecting the tissue tropism of KSHV in detail and mapping KSHV gene expression in different host cells will be one of the major uses of the SCID-hu Thy/Liv system. The presence of lytic replication during the early phase of infection suggests another potential application of this model: to assess the impact of antivirals and biological response modifiers on KSHV replication in vivo, similar to its established use in the development of anti-HIV drugs. The susceptibility of KSHV infection to ganciclovir in vivo faithfully mirrors prior in vitro findings and affirms that this model, once optimized, may become a useful part of preclinical drug development. Coinfection with HIV-1 did not enhance or interfere with KSHV replication, and the presence of KSHV did not influence the replication kinetics or cytopathogenicity induced by HIV-1. KSHV encodes viral chemokines that block interactions between HIV-1 and CCR5/CCR3 in vitro 54 55 56 , raising the speculation that this mechanism might also be operational in vivo (for review see reference 57 ). The absence of a significant effect of KSHV on HIV-1 replication argues against this, although the small number of KSHV-infected cells in the implant does not allow us to entirely exclude the possibility of interactions in vivo. HIV-1 itself is known to induce a number of cytokines (IFN-γ, TNF-α, IL-1, and IL-6), and these as well as HIV-1 Tat itself have been proposed to be involved in the development of KS (for review see reference 58 ). Although analogous changes in cytokine expression may occur in HIV-1–infected SCID-hu Thy/Liv implants compared with infected PBMCs, no effect of HIV-1 on KSHV replication was observed, suggesting that HIV-1–induced cofactors did not have an impact on KSHV replication in this context. Ongoing experiments are now testing the possibility that interactions between HIV-1 and KSHV might be observed after coinfection with different isolates of HIV-1 and/or after longer periods of time. KSHV infection of the Thy/Liv implant did not produce detectable histological phenotypes reminiscent of known KSHV-related human diseases. Certainly the model was not expected to reproduce KS, a neoplasm involving poorly understood cells of endothelial lineage 5 . Although some CD34 + cells are doubtless present in the implant, it is unlikely that the full range of endothelial precursors, stromal components, and growth factors needed to support KS development would be present in this system. Rather, the model was designed to examine infection of the lymphoid compartment. The model successfully produces transient lytic infection and more sustained viral persistence and appears to mirror the correct tissue tropism of infection. However, it did not give evidence for lymphoid depletion or lymphoproliferation. Although the small number of B cells present in the implant severely constrained our ability to score for B cell depletion, the proportion of CD19 + cells did not appear to diminish over time. Similarly, we did not observe expansion of the B cell population and did not detect features reminiscent of Castleman's disease or PEL. However, it is important to remember that both of these phenotypes are exceedingly rare manifestations of human KSHV infection. Judging from the fact that ∼5–7% of healthy subjects are KSHV seropositive, it is likely that most primary infections with this agent are subclinical or asymptomatic. Thus, it is not clear that the relatively bland histologic picture generated here is not representative of many human primary infections. We are continuing to study the model to see if disease appears at late times after inoculation or if the system can be experimentally modified to allow induction of cell injury or proliferation. In the meantime, in addition to its potential uses in drug screening, the model offers an excellent opportunity to better define in detail which subsets of CD19 + cells are most permissive for KSHV and whether and how efficiently T cells and macrophages can be targets of infection. | Study | biomedical | en | 0.999996 |
10601361 | The following oligonucleotides were used in reverse transcription PCR on cDNA from clone CN.8 28 : Vα10cass 5′-AACGTCGCAGCTCTTTGCAC-3′, Vβ5.2cass 5′-AAGGTGGAGAGAGACAAAGGATTC-3′ 32 , Alcon 5′-GATGTTTTACTGGTACACAG-3′, and Cβreverse 5′-TGTGCTTGGCCAGGGGTTCTT-3′. For cloning of the full VDJ regions from D7 hybridoma genomic DNA, the following primer pairs were used: Vα10lead-XmaI 5′-GACCCGGGCTTCTCACTGCCTAGCCATGAAGAGCCTGCTGAGCTCTCTG-3′ and JαD7intron-NotI 5′-CTTACGGCCGAGGAAGTACTGTCCTGAG-3′, and Vβ5.2lead-XhoI 5′-CCAGCATCTCGAGAAGAAGCATGTCTAAC-3′ and Jβ2.2intron-SacII 5′-GATGCCGCGGAGCTGTCCTGCTCTGAATATCTTC-3′. The amplified D7 TCR-α and -β DNA fragments were sequenced and cloned into the XmaI and NotI sites in TCR-αcass, and into the XhoI and SacII sites in TCR-βcass (TCR cassette vectors were provided by Drs. C. Benoist and D. Mathis, Harvard Medical School, Boston, MA ). The α and β TCR transgenic constructs were cotransfected with the neomycin resistance plasmid (pPNT-neo) into R1 embryonic stem (ES) cells (strain 129) by electroporation, as described previously 34 . Transfected cells were selected by G418, and the TCR-α and -β transgenes were detected by PCR using Vα10lead-XmaI and JαD7intron-NotI, and Vβ5.2cas and Jβ2.2intron-SacII. Presence of both transgenes was confirmed by Southern blot with probes for the constant regions of TCR-α 35 and TCR-β 36 on HindIII– or EcoRI-digested genomic DNA, respectively. ES cells containing both TCR-α and -β transgenes were injected into C57BL/6 (B6) blastocysts to generate chimeric mice at the Gwen Knapp Center Transgenic Facility. Chimeric mice that contained high levels of Vβ5 + Ly9.1 + cells in PBL were chosen as founders and bred with B6 mice to produce transgene-positive offspring. Three lines of transgenic mice were established. The development of the transgenic T cells in these three lines of mice is quite similar. Therefore, only one transgenic line was used for further breeding. B6, B10.D2, β2m −/− , TAP −/− , and TCR-α 2/− mice were purchased from The Jackson Laboratory. B6.CAS3(R9) (B6.R9)—carrying a haplotype with H2-K through H2-D from B6 and the rest of H2 , including M3, from Mus musculus castaneus (cas3)—was provided by Dr. Kirsten Fischer Lindahl (University of Texas, Southwestern Medical Center, Dallas, TX). Mice were housed in the conventional animal facility or in the barrier facility of the University of Chicago. During backcrosses, animals were typed for transgene by expression of Vβ5 on CD8 + PBLs. Where Vβ5 expression was minimal, PCR of D7 α and β chains was performed as stated for ES cell typing. Changes in haplotype, as well as loss of functional β2m or TAP, were detected by loss of staining with FITC–anti-K b . TCR-α 2/− status was assessed by expression of endogenous Vα2 on TCR-α/β 1 PBLs, and confirmed by Southern blot analysis of HindIII-digested genomic DNA with a probe for the TCR Cα region. To type D7 + B6.R9 animals, PBLs were incubated with 10 μM of LemA peptide for 4 h at 37°C, and stained for M3 surface expression by anti-M3 mAb (mAb130). Fetal thymi were cultured according to procedures described by Ashton-Rickardt et al. 1 . In brief, thymic lobes from gestational day 16.5 fetal mice were placed onto nitrocellulose filters (Millipore Corp.). Filters were placed in 2-cm-diameter dishes, then incubated for 10 d at 37°C in RPMI 1640 medium containing 10% fetal bovine serum (HyClone), 2 mM l -glutamine, 20 mM Hepes, 50 μM 2-ME, nonessential amino acids, sodium pyruvate, penicillin, and streptomycin (RPMI-10). The peptide and media were replenished every day where indicated. The following mAbs were purchased from PharMingen: FITC–anti-CD8α, PE–anti-CD8α, PE–anti-Vα2, CyChrome–anti-CD4, FITC–anti-Vβ5, FITC–anti-K b , FITC–anti-IA b , biotin–anti-Ly9.1, and biotin–mouse anti–hamster IgG. Y3, anti-K b ; and B22, anti-D b were purchased from American Type Culture Collection. PE–M3-LemA tetramer and allophycocyanin–M3-LemA tetramer were prepared as described previously 37 . mAb130 was purified as described previously 38 . Synthetic peptides were purchased from Research Genetics. Peptide sequences were as follows: LemA, fMIGWII; LemA 5I→A , fMIGW A I; LemA 6I→A , fMIGW A I; Fr38, fMIVIL; ND1, fMFFINIL; ND4, fMLKIILP; and COI, fMFINRWLFS. All peptides were >90% pure as determined by mass spectrometry. Peptides were dissolved in DMSO at concentrations of 1–20 mM. Thymocyte suspensions were prepared from cultured fetal thymi by mechanical desegregation in RPMI-10 media, and stained in immunofluorescence (IF) buffer (HBSS containing 2% fetal bovine serum and 0.1% NaN 3 ) using combinations of fluorescent-conjugated Abs for 30 min at 4°C. When staining involved M3-LemA tetramer, incubation time was extended to 1 h. The stained cells were analyzed by flow cytometry using a FACSCalibur™ (Becton Dickinson) with the CELLQuest™ software. Thymic stromal cell suspensions were prepared by digesting fetal thymi in 0.1% trypsin, 0.5 mM EDTA for 40 min at 37°C. Digestion was stopped by addition of IF buffer. After mechanical disruption of the lobe, cells were harvested and washed two times with IF buffer before cell surface staining experiments 10 . Cells were stained with anti-M3 mAb (130) followed by biotinylated mouse anti–hamster IgG, and a third incubation with streptavidin-conjugated PE and FITC-anti–I-A b . Staining with each reagent was performed for 30 min on ice in IF, followed by washing with the same buffer. The expression of M3 on I-A b –positive thymic stromal cells was analyzed by FACS ® . CTL effectors were established by culturing splenocytes of D7 + TCR-α 2/− mice at 5 × 10 6 cells/ml in RPMI-10 with 5 μM of LemA peptide for 3 d at 37°C. 1 × 10 6 target cells were incubated in RPMI-10 with or without 1 μM of LemA peptides for 18–20 h. Cells were washed free of excess peptide and labeled with 100 μCi [ 51 Cr]sodium chromate for 1 h at 37°C. Target cells (1 × 10 4 cells) were added to round-bottomed microtiter wells containing effector cells. After 4 h incubation at 37°C, 100 μl of supernatant from each well was removed and assayed for 51 Cr release. Percent specific lysis = (experimental − spontaneous release) × 100/(maximal release − spontaneous release). Limiting dilution analysis for CTL precursor frequency was performed on fetal thymic lobes after 10 d of culture. Four or five lobes from each treatment were pooled, and thymocytes were diluted to concentrations between 10,000 and 3 cells per well and incubated with 1 × 10 6 irradiated B6 stimulators per well, 10 U/ml IL-2, and 5 μM LemA peptide. Peptide was replenished at days 3 and 6. On day 7, 51 Cr-labeled LemA-coated L929 targets were added at 1 × 10 4 per well, and 51 Cr release was measured after 4 h. Splenocytes from D7 + mice (5 × 10 5 cells per well) were cultured in round-bottomed microtiter wells in a final volume of 200 μl of RPMI-10, with 10 μM of various peptides. After 48 h, the culture supernatants were harvested and the levels of IFN-γ were quantitated by sandwich ELISA (PharMingen). The D7 hybridoma generated from CTL clone CN.8 was chosen for the construction of TCR transgenic mice 25 28 . This CTL is CD8 + and specific for a listerial peptide, LemA (f-MIGWII), in the context of M3, and can confer protection from infection by adoptive transfer. Leader and J-C intron primers were designed and used to clone the entire coding sequence (Vα10.2JαD7 and Vβ5.2Jβ2.2) from D7 hybridoma DNA into the TCR cassette vectors 33 . Three lines of D7 transgenic mice were generated and bred onto the B6 background. Lymphocytes isolated from thymus, spleen, and lymph nodes of D7 transgenic mice were stained with FITC–anti-CD8, PE–anti-CD4, and APC–M3-LemA tetramer to examine the development of D7 + T cells. FACS ® analysis showed that D7 + T cells (M3-LemA tetramer positive) are highly enriched in the CD8 lineage . Compared with nontransgenic controls, the transgenic mice showed an increase in percentage of D7 + CD8 + cells (4-fold in thymus, 20-fold in spleen, and 18-fold in lymph node). Splenocytes from transgenic animals were evaluated against nontransgenic littermates for antigen-specific responses against the hexameric LemA peptide. Nontransgenic splenocytes showed no reactivity to LemA peptide (data not shown). Splenocytes from D7 animals developed into M3-restricted, LemA-specific CTLs after 3 d in culture with LemA peptide. D7 CTLs specifically lysed LemA-coated L929 targets bearing the wild-type M3 allele ( M3 wt ) . B10.CAS2 fibroblasts express a mutant castaneus allele of M3 ( M3 cas ) that does not present N -formylated peptides efficiently. LemA-coated B10.CAS2 targets were only slightly susceptible to lysis, whereas an M3 wt transfectant of B10.CAS2 (TR8.4a) was recognized as efficiently as the L929 targets. These data suggest that D7 transgenic T cells preserve functional capacities and antigenic specificity of the original T cell clone. Because M3 is nonpolymorphic and expressed at low levels, it is possible that M3-restricted T cells are selected on the more abundant class Ia molecules H-2K, D, or L. We bred the D7 transgenes onto the B10.D2 background which bears the H-2 d class Ia molecules to see if the selection and development of M3-restricted T cells was dependent on a particular class Ia allele. Staining with M3-LemA tetramers showed that D7 + CD8 + T cell development is unimpaired in the B10.D2 background . To eliminate the possibility that class Ia interaction with endogenous α chains contributes to positive selection, we bred the transgene onto the TCR-α 2/− background. The CD8 + population in D7 + TCR-α 2/− mice is exclusively positive for M3-LemA tetramer staining , showing that the D7 TCR is sufficient for selection. The development of transgenic T cells is impaired in TCR-α 2/− TAP −/− and β2m −/− mice . These data suggest that the selection and development of D7 + T cells requires the presentation of peptide on a class I molecule. Both TAP and β2m have been shown to be important for the expression of M3 on the cell surface 31 39 40 . The lack of H-2 restriction and the TAP and β2m dependence of D7 + T cell development suggested that M3 expression might be required for positive selection. We analyzed the selection of the D7 + T cells in FTOC in order to manipulate TCR access to M3 with an mAb (mAb130) that blocks M3-restricted CTL responses 31 . Fetal thymic lobes were harvested from gestational day 16.5 D7 + TCR-α 2/− animals, and incubated either with anti-M3 or a control hamster Ab at 50 μg/ml for 10 d. Additional lobes were incubated with anti–class Ia Abs or a control mouse Ab. Fig. 3 shows representative reduction in the percentage of CD8 + single positive (CD8 sp ) thymocytes in anti-M3–treated lobes. The average percent reduction was ∼80% (from average 20% reduced to 4%). Treatment with Ab to class Ia molecules had no effect, in agreement with our data for D7 + T cell development in different H-2 haplotypes. CD8 sp thymocytes expressed Vβ5 at high levels (data not shown), which is indicative of antigen specificity in the TCR-α 2/− background. We bred the D7 transgene onto an H-2 recombinant background B6.R9, which expresses M3 cas , to detect any effect of reduced M3 expression on thymic development. The castaneus allele of M3 contains an amino acid substitution (Leu 95 →Gln) that results in reduced recognition by M3-specific CTLs 30 . This substitution might affect M3 surface expression, as we were unable to detect M3 by IF staining on the surface of B6.R9 splenocytes (data not shown). Fig. 4 shows the relative efficacy of M3 wt (B6) and M3 cas (B6.R9) in selection of D7 + T cells. In FTOC of D7 + B6 lobes, antigen-specific CD8 sp thymocytes account for 13.4% of total thymocytes (57.8% of 23.2%). In comparison, D7 + B6.R9 lobes have a reduced total percentage of CD8 sp cells (11.5%), and only a minor proportion of these cells are antigen specific (15.9%). Thus, the total percentage of D7 + CD8 sp thymocytes in the B6.R9 background is reduced by ∼85% (from 13.4 to 1.8%). The percentage of D7 + CD8 sp thymocytes in the B6.R9 thymic lobes is reduced when anti-M3 Ab is present during FTOC (66 ± 16% reduction, n = 6; data not shown). This indicates that the small percentage of D7 + CD8 sp cells in the B6.R9 thymus develop in an M3-dependent manner, despite the low level of expression of M3 cas . M3 binds N -formylated peptides with 100–1,000-fold greater affinity than nonformylated peptides 12 . Mitochondria are the only source of these peptides in mammalian cells. In a previous study, we showed that the mitochondrial peptides ND1 and COI bind M3 with higher affinity than the 11 remaining mitochondrial peptides 31 . We also found that Fr38, a peptide from Listeria monocytogenes , which is not recognized by the D7 hybridoma, binds M3 with high affinity. To determine whether these peptides can induce increased surface expression of M3 on TAP −/− thymic stromal cells, we cultured fetal thymic lobes from TAP −/− mice with or without 20 μM peptide for 10 d, and harvested the cells to stain with anti–class II and anti-M3 Abs. Fig. 5 shows that ND1, COI, and Fr38 increase surface expression of M3 on I-A b+ thymic stromal cells from TAP −/− mice. M3 expression is low on thymic stromal cells from B6 lobes as well (data not shown), staining at a similar level as cells from DMSO-treated TAP −/− thymi in the absence of exogenous N -formylated peptide. The induced increase in expression of M3 is similar for these three peptides. Because of the ability of these N -formylated peptides to stabilize surface expression of M3 on thymic stromal cells, we analyzed the role of each peptide in positive selection. Fig. 6 A shows representative staining of thymocytes developed in FTOC of D7 + TCR-α 2/− TAP −/− thymic lobes incubated with or without 20 μM peptide for 10 d. Although each peptide was able to stabilize surface expression of M3, the increase in positive selection of CD8 sp thymocytes varied between peptides. Fig. 6 B displays the percentage increase in CD8 sp thymocytes between pairs of lobes from each animal. The average increase in CD8 sp cells was approximately twofold for ND1 (1.85 ± 0.65, n = 14) and COI (1.78 ± 0.60, n = 20), and approximately threefold for Fr38 (2.86 ± 1.15, n = 16). ND4 is a mitochondrial peptide that binds to M3 weakly and is very inefficient at stabilization of M3 on the cell surface. Incubation with ND4 caused no increase in positive selection of transgenic CD8 sp thymocytes (1.05 ± 0.31, n = 6). The ability of N -formylated peptides to increase development of functional CTL precursors was assessed by a limiting dilution assay. Thymocytes from peptide-treated lobes from D7 + TCR-α 2/− TAP −/− animals were compared with the control partner lobes. The pooled lobes from each group were diluted to concentrations between 10,000 and 3 per well and incubated with LemA peptide and irradiated stimulators to allow the development of CTLs. Each peptide induced at least a threefold increase in functional CTL precursors , indicating effective positive selection by two mitochondrial peptides and one bacterial sequence. Because of the low level of expression of M3 compared with class Ia molecules, we sought to determine if negative selection was effectively mediated by M3. Thymic lobes from D7 + TCR-α 2/− mice were incubated with or without 20 μM LemA peptide for 10 d, and cells were stained for CD4, CD8, and either anti-Vβ5 or M3-LemA binding. Both the percentage of CD4 + CD8 + double positive cells and the percentage of CD8 sp cells were significantly reduced . Double positive cells were reduced by 70–85%, and CD8 sp cells were reduced by 40–60%. The remaining CD8 sp cells in peptide-treated lobes had reduced TCR expression as assessed by either Vβ5 staining or M3-LemA staining (data not shown). In addition, total cell counts were also reduced in peptide-treated lobes (2.4 ± 0.23 × 10 5 cells/lobe without peptide, 1.2 ± 0.3 × 10 5 cells/lobe with 20 μM LemA). A similar effect was observed when lobes were incubated with a LemA variant (LemA 6I→A , fMIGWI A ), a weak agonist which stimulates D7 + T cells minimally. Percentage reduction in LemA 6I→A treated lobes was ∼80% for CD4 + CD8 + double positive, and 20% for CD8 sp (data not shown). In contrast to LemA and LemA 6I→A , the peptides that promote positive selection (ND1, COI, and Fr38) elicit no response from D7 + T cells, even at 10 μM concentrations . Thus, M3 is capable of effective negative selection even with weak agonist peptides. To determine whether MHC class Ib molecules can contribute to thymic education in the same manner as class Ia molecules, we analyzed positive and negative selection in TCR transgenic animals bearing the D7 TCR-α/β specific for the listerial peptide LemA in the context of M3. M3 plays the dominant role in positive selection of the D7 TCR, as shown by the impaired development of transgenic thymocytes in the presence of anti-M3 blocking Ab, or in the presence of the castaneus mutant M3 allele. Although selection was significantly diminished in the B6.R9 (M3 cas ) background, incubation with anti-M3 Ab further reduced the percentage of D7 + CD8 sp thymocytes, suggesting that M3 cas is capable of selecting some D7 + T cells. This is consistent with the ability of B6.R9 animals to develop allogeneic responses against M3 wt molecules 41 42 . No apparent contribution is required from class Ia molecules, as suggested by the development of transgenic T cells in an MHC-unrestricted fashion. Peptide is required for positive selection of D7 TCR, as shown by the differential rescue of CD8 sp development in TAP −/− fetal thymic lobes by four peptides from mitochondria and bacteria. The concentration of peptide (20 μM) used in these FTOCs increased the surface expression of M3 on thymic stromal cells significantly over background levels. Because of the hydrophobicity of M3-binding peptides, higher concentrations of peptide caused precipitation. Negative selection is also mediated by addition of the antigenic peptide to TCR-α 2/− FTOCs. Thus, M3 is responsible for both positive and negative selection of M3-restricted T cells. It is of interest that M3 is a potent positive selecting element, despite surface expression levels in the thymus that are undetectable by IF staining in wild-type animals 31 . Because M3 can bind only two mitochondrial peptides with high affinity 31 42 , it is likely that the majority of M3 molecules on the cell surface are occupied by these ligands. The remaining mitochondrial ligands may also contribute, depending on the balance of affinity and abundance, which is currently unexplored. By comparison, the pool of 10 5 –10 6 class Ia molecules on each cell presents as many as 10 3 –10 4 different self-peptides 43 . Although the expression level of M3 is about one to two orders of magnitude lower than that of class Ia molecules, the total number of identical M3 self-peptide complexes such as M3–ND1 or M3–COI complexes may be comparable to the number of any given class Ia–peptide complex, because of the greater numbers of ligands competing for presentation on class Ia molecules. A similar argument can be made in comparing low levels of M3 in the wild-type animal to low levels of class Ia in TAP −/− animals. We have shown previously that M3 behaves in a wild-type background as a class Ia molecule does in a TAP −/− background because of a limited supply of endogenous peptides. However, class Ia molecules expressed at low levels in TAP −/− animals are not sufficient to support positive selection 1 . It is possible that even in the absence of TAP, the supply of peptides available to class Ia molecules in the endoplasmic reticulum still has sufficient diversity to dilute any particular MHC–peptide combination below the minimum level required for positive selection. Additionally, we have shown that M3–peptide complexes on the cell surface are more long-lived than class Ia complexes 31 . The combined effect of limited ligand diversity and surface stability may explain the ability of M3 to select M3-restricted T cells. The peptides used in these experiments differed in their ability to promote positive selection. Incubation with listerial Fr38 peptide increased CD8 sp development more effectively than the self-derived mitochondrial peptides. Correspondingly, the percentage of CD8 sp cells in Fr38-treated D7 + TCR-α 2/− TAP −/− lobes was, on average, greater than that in D7 + TCR-α 2/− lobes . Because the ability to bind and stabilize M3 is not significantly different between ND1 or COI and Fr38, this suggests that peptide sequence can impact M3-restricted selection. An explanation for the efficiency of Fr38 at enhancing LemA-specific TCR selection may be provided by the nature of the M3 peptide binding groove. In the crystal structure of M3, the second residue is partially exposed to the TCR contact surface, while the side chains of residues three and four are buried. The position five side chain points up, and then a position six residue would be buried 13 . Fr38 and LemA share the second amino acid (Ile) and a similar fifth residue (Ile in LemA, Leu in Fr38), whereas ND1 and COI share phenylalanine as the second residue, and have a fifth residue which is dissimilar to LemA (Asn in ND1 and Arg in COI). Notably, we found that a LemA 5I→A variant with a substitution at position five is a nonagonist (Chiu, N.M., and C.-R. Wang, unpublished results), indicating that this residue may be an important contact residue for the D7 TCR. However, as is the case for class Ia–restricted selection 9 10 , sequences lacking similarity to the antigenic peptide, i.e., ND1 and COI, are also able to promote positive selection, although not to wild-type levels. Although M3 has 100–1,000-fold greater affinity for N -formylated over unformylated peptides 12 , a potential role of unformylated peptide–M3 complex in thymic selection cannot be ruled out. Unformylated peptides can stimulate CTLs when present at high concentrations 12 . However, even at high concentrations, unformylated peptides do not stabilize M3 on the cell surface. It remains possible that some unformylated peptide(s) with low affinity for M3 is present in such high abundance that it can contribute to positive selection. Positive selection of class Ia-restricted CD8 sp thymocytes has been shown to be effective even with low affinity self-peptides 10 . We are currently attempting to elute naturally processed self-ligand from M3 by immunoaffinity chromatography. This may provide insight into the proportion of M3 molecules on the cell surface, which may contain nonmitochondrial peptides. The limitation in diversity of peptide bound to M3 has implications for the breadth of M3's role in selection. A diverse peptide supply has been shown to be important for the restoration both of normal amounts of positive selection and of a broad repertoire of class I–restricted responses 1 2 . This has also been shown to be the case in H-2M −/− animals and “single peptide” MHC transgenic mice for class II–restricted responses 44 45 . Since M3 has such a small repertoire of self-ligands, the diversity and total number of M3-restricted T cells may be reduced as a consequence. That the contribution of class Ib molecules to positive selection has a limit is demonstrated by the reduced numbers of CD8 + cells in K b D b double-knockout animals 46 . It has been proposed that the selective advantage of presenting numerous foreign peptides has driven the polymorphism of class Ia molecules, but that the mechanisms for self-tolerance provide pressure of an opposite type. After thymocytes rearrange the antigen receptor genes to randomly generate diversity, a large proportion of thymocytes is negatively selected. An increase in numbers of polymorphic class Ia molecules, while expanding the range and diversity of foreign peptides that can be presented, may eventually decrease the responding TCR repertoire by a greater factor 47 . This would occur if the range of self-peptides presented also increases and removes an additional subset of the TCR repertoire. A less polymorphic molecule may evolve a specialized function, for instance, presentation of a commonly encountered pathogenic epitope. The conservation of such a sequence serves to present the pathogen efficiently, while the limited polymorphism allows a reliably small set to be removed from the T cell repertoire. This strategy is particularly effective if the pathogenic epitope is never, or rarely, found in the repertoire of self-antigens. Human CD1 molecules exemplify this strategy by presenting bacterial lipids to T cells 48 49 . M3 also follows this strategy by presenting low amounts of a small set of self-peptides, allowing positive selection, while responding to a class of bacterial N -formylated peptide antigens that are present in large amounts exclusively during an infection. Therefore, selection of M3–restricted T cells can serve as a model for other nonpolymorphic class Ib molecules with restricted ligand specificity. | Study | biomedical | en | 0.999995 |
10601362 | The broad spectrum benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD), the caspase-3–like Cbz-Asp-Glu-Val-Asp(OMe)-fluoromethylketone (zDEVD) or caspase-8–like Cbz-Ileu-Glu-Thr-Asp(OMe)-fluoromethylketone (zIETD) caspase inhibitors, and the biotinylated zVAD were purchased from Enzyme Systems Products. IL-2 was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, and PHA from Murex Diagnostics. Staphylococcus aureus Cowan fixed cells (SAC) were purchased from Calbiochem. Rabbit antiserum against caspase-3 was generated as described previously 5 . The anti–caspase-8 and anti-DFF45 antisera are rabbit polyclonal antibodies generated in the laboratory against the p18 of caspase-8 and the full-length DFF45, respectively. The anti-PARP antiserum, a second anti–caspase-8 serum, the anti-lamin B, and anti-Fas (M3) mAbs were gifts from Dr. G. Poirier (Centre Hospitalier de l'Université Laval, Québec, Canada), Dr. M.E. Peter (German Cancer Research Center, Heidelberg, Germany), Dr. R. Bertrand (Centre de Recherche du Centre Hospitalier de l'Université de Montréal), and Dr. D. Lynch (Immunex Corp., Seattle, WA), respectively. Dr. D.W. Nicholson (Merck Frosst, Kirkland, Canada) provided antisera against caspase-1, -6, -7, and -9. The mAbs against caspase-2, caspase-4, and Wee1 were purchased from Transduction Laboratories, Santa Cruz Biotechnology, and StressGen Biotechnologies Corp., respectively. The anti-CD3 was produced and purified from the OKT3 clone (American Type Culture Collection). PBMCs were purified by Ficoll-Hypaque and resuspended in 10% FCS-RPMI medium (GIBCO BRL). To monitor proliferation, 10 5 cells were cultured for 1–4 d at 37°C in 96-well plates in the absence or presence of 1 μg/ml anti-CD3, anti-TCR (BMA031; Immunotech), or PHA and 20 U/ml recombinant human IL-2. For B cell stimulation, PBMCs were cultured in the presence of SAC (1:10 4 vol/vol). 3 H-labeled thymidine (1 μCi/well) was added during the last 6 h for each time point. Cells were harvested, and DNA-associated radioactivity was counted by liquid scintillation (Betaplate™; Wallac) and expressed as a mean cpm ± SEM of triplicate cultures. Cell staining for T cell activation markers was performed on PBMCs activated as described for 4 d, and using anti-CD69, anti-CD25 (Becton Dickinson), or anti–HLA-DR (Caltag Laboratories) antibodies. Cell sorting was performed after 4 d of stimulation with 1 μg/ml anti-CD3 and 20 U/ml IL-2, using PBMCs stained with annexin V (AV)-FITC (BIODESIGN International). Living (AV − ) and dying or dead cells (AV + ) were sorted using a FACStar™ (Becton Dickinson) after gating on lymphocytes on the basis of forward/side scatter. Purified lymphocyte subsets from treated PBMCs were obtained by cell sorting after staining with anti-CD4, anti-CD8, anti-CD19 (Becton Dickinson), or anti-CD45RO and anti-CD45RA (Serotec Ltd.) antibodies. For T cell subset sorting, dead cells were gated out on the basis of forward/side scatter, and the purity of sorted T cell subsets was >99%. For each sample, 10 4 events were collected using the FACScan™ flow cytometer. Staining for in vivo caspase activity was performed using the cell-permeable substrate Phiphilux-G 1 D 2 (OncoImmunin, Inc.) containing the consensus sequence DEVDG. In brief, resting or activated cells were washed, then incubated with the substrate (10 μM final) for 1 h at 37°C, followed by another wash with the dilution buffer, according to the manufacturer's instructions. Fluorescence was monitored using the FL1 detector of an EPICS ® XL flow cytometer (Coulter Corp.), and gates were set on living and either resting or blastic cells before analysis. For each sample, 3 × 10 4 events were collected. Treated cells were washed with PBS, and pellets were resuspended in sample buffer and boiled for 3 min. Proteins from 0.5–1 × 10 6 cells were separated on SDS-PAGE and transferred to nitrocellulose membrane (Hybond C Super; Amersham). Blots were blocked for 1 h at room temperature in PBS, 0.05% Tween, containing 5% nonfat dried milk, washed, and incubated with the different antibodies. Detection was achieved with the appropriate secondary antibodies coupled to horseradish peroxidase (HRP), followed by enhanced chemiluminescence ECL Western blotting kit (NEN), and visualized by autoradiography. Proteins from PBMCs (5 × 10 6 cells), stimulated with 1 μg/ml PHA and 20 U/ml IL-2 for 4 d, were extracted in lysis buffer (2% NP-40, 0.5% deoxycholic acid, 50 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, 10 mM NaH 2 PO 4 , 2 mM EGTA, 10 mM EDTA, 0.5 mM Na 3 VO 4 ). Cell lysates were incubated with 5 μM of biotinylated zVAD-fmk and subjected or not to immunoprecipitation using the caspase-3 antiserum. Protein G–Sepharose-coupled beads (Amersham Pharmacia Biotech) were preincubated for 30 min at 4°C with 10 μl of anti–caspase-3 antiserum. After three washes with lysis buffer, the beads were added to total cell lysates for a 2-h incubation at 4°C with gentle agitation. The beads were then washed three times with lysis buffer and boiled for 5 min in 50 μl of Laemmli buffer. Whole cell lysates or immunoprecipitated proteins were loaded on polyacrylamide gels for electrophoresis and transferred to Hybond C membranes. Blots were blocked overnight at 4°C with PBS, Tween 0.05% supplemented with BSA (3%) and nonfat dry milk (2%), and revealed after incubation with HRP-conjugated extravidin (Extra-HRP, 1:2,000; Sigma Chemical Co.) for 1 h at room temperature for affinity blot, or with anti–caspase-3 antiserum for Western blot as described above. Jurkat cells or PBMCs stimulated for 4 d with anti-CD3 antibody and IL-2 were incubated at 10 6 cells/well in 24-well plates coated or not with anti-Fas (M3) mAb. Coating was performed for 2 h at 37°C using 20 μg/ml of antibody in 0.05 M Tris, pH 9.3. After 4 h (Jurkat cells) or 6 h (PBMCs) of incubation on coated plates, cells were washed with PBS, and the pellet was kept at −80°C until Western blot analysis. An aliquot of stimulated cells was used for AV staining as described previously 11 . Several reports have demonstrated a costimulatory function of TNF receptor (TNFR) family members, such as Fas and TNFR, which are also associated with apoptosis through activation of the caspase cascade 25 26 27 . Whether caspases play a role in the costimulatory function of TNFR family members has yet to be demonstrated, although caspase-3 processing has been reported after T cell stimulation 17 . To determine whether caspase-3 activation was involved in the response to TCR triggering, we used the broad spectrum caspase inhibitor zVAD 4 5 11 28 . Stimulation of PBMCs with anti-CD3 antibody and IL-2 in the presence of zVAD resulted in a dose-dependent and reproducible inhibition of T cell proliferation and a 2.6-fold decrease in the percentage of cells in S phase after 4 d of stimulation, as assessed by DNA content analysis using propidium iodide (PI) staining (data not shown). Similar results were obtained using zIETD, an inhibitor of caspase-8–like members, or the caspase-3–like protease inhibitor zDEVD (inhibition of >50% of the proliferation with 100 μM in three independent experiments; data not shown). To exclude the possibility that zVAD exerts a general cytostatic effect, the caspase inhibitor was added at 1–3 d after stimulation of PBMCs, and DNA synthesis was assessed after 4 d of culture. Inhibition of T cell proliferation by zVAD was much less significant when added 2 or 3 d after T cell activation , indicating that it did not inhibit a component of the cell cycle machinery, and was not endowed with a nonspecific cytostatic activity. A toxic effect of zVAD was also excluded, since after 4 d of stimulation, zVAD could block up to 73% of the proliferation without affecting cell viability, as assessed at day 2 or 4 by AV/PI staining (% AV − cells was 56.7% with zVAD vs. 57.3% in the absence of zVAD). On the contrary, in the same experiment the serine protease inhibitor N - p -tosyl- l -lysine chloromethyl ketone (TLCK) also blocked DNA synthesis, but induced a high level of cell death (up to 90% AV + cells; data not shown). Anti-CD3–induced blastic transformation was also abolished by zVAD, as assessed by light scatter properties in flow cytometry , confirming that inhibition of caspase activity blocked an early and critical step of T cell activation. Addition of zVAD also resulted in the accumulation of cells expressing the early and transient activation marker CD69, whereas surface expression of the late activation marker HLA-DR was inhibited . Induction of CD25 (the α chain of the high-affinity IL-2 receptor) and CD95 upregulation remained comparable in the presence or absence of zVAD . Since an excess of IL-2 is added at the onset of T cell activation, and since the expression of CD25 is maintained in the presence of zVAD, it appears that the caspase inhibitor does not block T cell proliferation by interfering with the IL-2–IL-2R autocrine loop. As illustrated in Fig. 1 D, the lack of effect of zVAD on the de novo expression of CD69 and CD25, and the fact that Jurkat cell proliferation was not affected by addition of zVAD (data not shown) further confirm that zVAD effect on T cell proliferation cannot be attributed to a general cytostatic effect. Engagement of CD3 on PBMCs induces the production of cytokines by monocytes such as IL-1β and IFN-γ inducible factor (IGIF)/IL-18, which in turn will enhance T cell activation 29 30 . Since production of these cytokines is dependent on caspase-1 (ICE) activity 31 32 , inhibition of T cell proliferation by zVAD could be due to a lower level of costimulatory cytokines produced by monocytes. To exclude this possibility, purified T cells (>95% TCR + ) were stimulated with anti-CD3 and IL-2 in the presence of zVAD. Proliferation of these T cells triggered by anti-CD3 and IL-2 was also blocked by the caspase inhibitor , confirming that zVAD was directly inhibiting T cell activation, and not accessory cell function. Taken together, these results show that inhibition of caspase activity resulted in defective T cell activation and impaired proliferation after TCR triggering of resting T cells. To rule out the possibility that caspase-3 processing induced by TCR triggering resulted from a lysis artifact 19 , anti-CD3–activated PBMCs were boiled directly in Laemmli buffer containing 2% SDS, 5% mercaptoethanol, and 8 M urea, and cell lysates were subjected to Western blot analysis. Even under these denaturing conditions, caspase-3 proenzyme was reproducibly found ( n = 10) completely processed into protein species ranging from 24 to 17 kD after TCR triggering of PBMCs . These species included the 17-kD form previously associated to the induction of apoptosis 2 3 4 5 and a doublet migrating at ∼20 kD. Processing of caspase-3 occurred as early as day 1 after TCR stimulation and before the detection of DNA synthesis . None of these bands was observed in resting T cells. The same caspase-3 pattern was also obtained after anti-TCR or PHA/IL-2 stimulation (data not shown), confirming previous results 17 . A role for granzyme B in the processing of caspase-3 during cell lysis was excluded by using the same denaturing lysis buffer in all experiments. To determine which of these bands constituted the active enzyme, their ability to bind to a peptidic substrate was assessed by affinity blot. The affinity blot technique takes advantage of the ability of peptides mimicking caspase substrates to bind irreversibly to the active caspases, but not to their precursors 33 . Fresh PBMCs were stimulated for 4 d with anti-CD3, anti-TCR antibody, or PHA in the presence of IL-2, and cell lysates were incubated with a biotinylated zVAD peptide. Proteins were separated by SDS-PAGE, and the blot was probed using Extra-HRP. Three forms ranging from 17 to 21 kD were detected in lysates of activated, but not resting cells . To verify whether these proteins originated from caspase-3, the caspase was immunoprecipitated from lysates of PHA-stimulated PBMCs using the antiserum, and whole lysates or immunoprecipitated proteins were separated on SDS-PAGE. Western blot analysis shows that the immunoprecipitate contained a doublet at 20 kD, as well as the 17-kD subunit and a 15-kD band sometimes observed in late apoptotic Jurkat cells using this antiserum (data not shown). When the membrane was probed with Extra-HRP, only the 17-kD band and the upper band from the doublet were detected , showing that both caspase-3 forms were able to bind to the synthetic substrate. Activation of caspase-3 was also assessed in intact cells using the cell-permeable caspase substrate Phiphilux, containing the caspase-3 consensus sequence GDEVDGI 34 . Fluorescence of resting or stimulated PBMCs was compared by flow cytometry after staining in the presence of the substrate. Fig. 2 E shows that Phiphilux was cleaved in T cells 4 d after anti-CD3 stimulation. Similar results were also obtained as early as 2 d of stimulation, in which a 4.7-fold increase in mean fluorescence intensity was observed (mean fluorescence intensity of 27.3 vs. 5.8 for resting cells). Interestingly, induction of apoptosis by anti-Fas antibody did not result in increased Phiphilux cleavage in activated PBMCs , suggesting that caspase activity was already elevated in proliferating cells. Therefore, caspase-3 cleavage in stimulated lymphocytes corresponded to activation of the proenzyme. The complex pattern of caspase-3 subunits suggested that they could originate from several proenzyme species observed in nonstimulated cells , or from different cell subsets. To distinguish between these two possibilities, sorting of CD4 + , CD8 + , CD45RO + , and CD45RA + cells was performed on viable, blastic cells. Processing of caspase-3 was similar in both CD4 and CD8 T cell subsets and occurred not only in CD45RO + , but also in the remaining CD45RA + /CD25 + cells , further confirming that caspase activation was an early event after TCR triggering. When PBMCs were stimulated for 4 d with the B cell mitogen SAC, and B lymphocytes were purified by CD19 + cell sorting, caspase-3 cleavage was observed in sorted, viable cells and resulted in the production of the 20-kD doublet and the 17-kD large subunit as in activated T lymphocytes . These results indicate that caspase-3 processing occurs in several T cell subsets and in activated B cells. Having confirmed caspase-3 activation in stimulated T lymphocytes, we next considered whether other caspases could also be activated under the same conditions. Western blot analysis was performed on lysates of unstimulated or activated PBMCs, using a panel of antibodies recognizing several caspases. Fig. 4 A shows that caspase-6 and caspase-7 were also cleaved into their active subunits after TCR triggering. A decrease in levels of both proenzymes (p34 for caspase-6 and p35 for caspase-7) already occurred after 24 h of stimulation, and their cleaved subunits (p20 and p17) were clearly detected 24 h later. However, caspase processing was selective since caspase-1, caspase-2, and caspase-4 remained as proenzymes under these same conditions . These results further exclude a role for granzyme B since one of its substrates, caspase-2 35 , remained as a proenzyme in activated cell lysates. Activation of the downstream caspases during apoptosis is usually triggered by either caspase-8 (associated to the cytoplasmic tail of death receptors) or caspase-9 (activated through cytochrome c release from mitochondria), depending on the apoptotic stimulus 36 37 38 39 . To identify the pathway involved in caspase-3, -6, and -7 activation during T lymphocyte stimulation, Western blot analysis was performed using antibodies against caspase-8 or caspase-9. Fig. 5 shows that after stimulation of PBMCs, caspase-8 was fragmented into a 43–45-kD doublet corresponding to the large subunit of caspase-8 linked to the propeptide 40 , whereas the 18-kD large subunit was detected only at day 3 (observed in at least three independent experiments), when levels of DNA synthesis were already high . This result was confirmed using another anti–caspase-8 serum also specific for the p18 (data not shown). In the same extracts, caspase-9, which was upregulated after TCR triggering, remained as a proenzyme , whereas caspase-8 and -3 were processed as early as 16 h after T cell activation. Therefore, these results suggest that caspase-8–mediated processing of the downstream caspases was triggered through upregulation of death receptor ligands and FADD recruitment after T cell activation. Since several downstream caspases were activated after TCR triggering, we next determined whether caspase substrates were processed in stimulated lymphocytes. PARP was the first caspase-3 substrate identified during apoptosis 2 9 . Stimulation with anti-CD3 and IL-2 increased PARP protein levels within 24 h , confirming previous reports showing an upregulation of PARP message and activity after PHA stimulation of primary T lymphocytes 41 42 . Concomitant with its induction, >90% of PARP protein was found cleaved in activated cell lysates . PARP fragment comigrated in the same gel with the 85-kD form found in apoptotic Jurkat cell extracts, further confirming that caspase activity was induced after primary T cell activation. Another recently identified caspase substrate, the Cdc2 tyrosine kinase Wee1 13 , was also upregulated in stimulated PBMCs . Interestingly, the 97-kD full-length Wee1 that appeared 48 h after stimulation was always detected along with 65-, 34-, and 32-kD bands that result from caspase-mediated cleavage during Fas-mediated apoptosis in Jurkat cells . Therefore, two caspase substrates, PARP and Wee1, were processed in activated T cells in a pattern expected from their cleavage by caspases. Caspase-mediated cleavage of substrates such as DFF45 or RFC140 in activated T lymphocytes would not be compatible with the maintenance of DNA integrity and the proliferation of T cells, since they play a critical role in DNA fragmentation and DNA synthesis, respectively. Thus, it was of interest to determine whether caspase activation in stimulated lymphocytes resulted in cleavage of these substrates. The same extracts analyzed for PARP and Wee1 cleavage were subjected to Western blot analysis using an anti-DFF45 antiserum generated against the full-length DFF45. Results shown in Fig. 6 demonstrate that DFF45 was not cleaved in these lysates, although caspase-3 was almost completely processed . The cleavage of RFC140 was analyzed by Western blot, and results confirmed that this substrate was also resistant to caspase-mediated cleavage in activated lymphocytes, as we have observed previously ( 11 ; data not shown). To ensure that Wee1 and PARP cleavage resulted from caspase activity, fresh PBMCs were stimulated with anti-CD3 antibody and IL-2 in the absence or presence of 100 μM of zVAD. Western blot analysis of cell lysates shows that Wee1 cleavage to a 60-kD fragment and the appearance of the 85-kD form of PARP were both inhibited by zVAD . Interestingly, addition of zVAD, which inhibited T cell proliferation, also reduced caspase-3 processing , further supporting a correlation between caspase-3 activation and cell cycle entry. The caspase-6 substrate lamin B 7 was also analyzed by Western blot in lysates of activated lymphocytes, and a zVAD-sensitive cleavage generating a 28- and a 35-kD fragment was observed in stimulated cells , further confirming that caspase-6 was active in these cells. As published previously, cleavage of RFC140 was detectable in activated PBMCs only after anti-Fas treatment 11 . To ensure that this was also the case for DFF45, anti-CD3–stimulated PBMCs were incubated for 6 h on anti-Fas–coated plates at 37°C and subjected to Western blot analysis using anti-DFF45 antiserum. This treatment resulted in a decrease of living, AV − cells from 65 to 20% . Western blot analysis demonstrated that the 14-kD fragment of DFF45 was only detectable in lysates of apoptotic cells. Therefore, caspase activity observed in proliferating T cells resulted in selective substrate specificity, different from the one observed during T cell apoptosis. To rule out a potential contribution of dying cells for cleaved forms of caspases or for their substrates, Western blot analysis was performed on highly purified living cells. PBMCs were stimulated for 4 d using anti-CD3 antibody in the presence of IL-2, and stained with AV-FITC. Purified living (AV − ) or dying (AV + ) cells were obtained by flow cytometric cell sorting. The sorted populations were restained with AV to ensure that sorting did not affect cell viability, and >92% of the cells were found to be AV − . Lysates of sorted cells were subjected to caspase-3 or PARP Western blot analysis. Results show that all of the processed forms of caspase-3, as well as the 85-kD fragment of PARP, were present in AV − cells , further confirming that the presence of caspase-3 cleavage products was not due to contamination with dead cells. In nonapoptotic sorted cells, the caspase-3 substrate PARP was almost completely cleaved, showing that caspase-3 processing correlated with caspase activity. Strikingly, caspase-3–processed forms were almost absent in lysates of apoptotic (AV + ) cells, suggesting that their half-life could be very short. Confirming this hypothesis, we have observed that caspase-3–cleaved forms progressively disappear from activated lymphocytes after IL-2 withdrawal, concomitant with an increase in mortality (up to 70%; data not shown). To ensure that cleavage of other caspases or substrates also occurred in nonapoptotic cells, PBMCs stimulated for 2 d (to further limit the proportion of apoptotic cells) with anti-CD3 antibody were stained with AV-FITC, and after gating on blasts, AV − cells were sorted by flow cytometry. Extracts from 5 × 10 5 cells were prepared from resting, unsorted PBMCs or activated, AV − sorted cells, and proteins were analyzed by Western blot using antibodies against caspase-6, caspase-7, PARP, and Wee1. As a control for dead cell contamination, these extracts were also analyzed for DFF45 cleavage. Fig. 8 B shows that both caspases, as well as PARP and DFF45, were not cleaved in resting cells (lane 1), whereas Wee1 was not detected. After TCR cross-linking, caspase-6– and caspase-7–cleaved subunits were detected in AV − sorted cells (lane 2), as well as the PARP 85-kD cleaved form and the 66-, 32-, and 34-kD fragments of Wee1. In these extracts, DFF45 was not processed, excluding a potential contamination by dead cells. Altogether, these results clearly indicate that a selective caspase substrate cleavage occurs in viable T lymphocytes activated through the TCR. Results presented in this report show that caspase activation after TCR triggering is a physiological, tightly regulated, and early response that appears to be required for efficient T cell activation. Indeed, the selective processing of caspase-3, -6, -7, and -8 was detected within 24 h after anti-CD3 stimulation of peripheral blood lymphocytes. Caspase processing occurred in various T and B cell subsets, and was found in proliferating and nonapoptotic lymphocytes. Activation of caspases was confirmed through binding of caspase-3–processed forms to a specific substrate, and by showing that a cell-permeable substrate was cleaved in intact, activated lymphocytes. Importantly, activation of the caspase cascade was associated to restricted substrate specificity, with cleavage of PARP and Wee1 being observed while two other substrates, DFF45 and RFC140, remained unaffected. Caspase processing after T cell stimulation correlated with a defective lymphocyte activation in the presence of the caspase inhibitor zVAD, suggesting that caspase activity could be involved in some early steps of lymphocyte activation. Death receptors such as TNFR and Fas can act as costimulatory molecules and enhance T cell proliferation 25 26 27 . We have also observed that a soluble form of TNFR significantly inhibited T cell proliferation induced by anti-CD3 antibody (our unpublished results). Cross-linking of these death receptors triggers the FADD-dependent recruitment of caspase-8, which is processed and can directly mediate the activation of caspase-3, -6, and -7 by proteolytic cleavage 36 37 . Our results show that caspase-8 is processed after T cell stimulation (as early as 16 h after TCR cross-linking), and activates caspase-3, -6, and –7, leading to a selective cleavage of their substrates. Interestingly, addition of the caspase-8 inhibitor zIETD during T cell stimulation inhibited >60% of the proliferation (data not shown), which further confirms that caspase-8 activity is involved in cell cycle entry of activated lymphocytes. The fact that activation of the whole caspase cascade occurs in proliferating, viable (AV − ) cells indicates that caspases could be the executioners in the costimulatory function of members of the TNFR family. In support of this hypothesis, impaired T cell proliferation is also observed in FADD-deficient and in FADD dominant negative transgenic mice 20 21 22 , suggesting a potential role for caspases in the early events leading to cell division. Caspase processing in stimulated cells appears to be mediated mostly through the activation of caspase-8 associated to death receptor. The other pathway of downstream caspase activation involves caspase-9, which is triggered after the release in the cytosol of mitochondrial cytochrome c 38 39 . However, we have found that in stimulated T cells this caspase remains as a proenzyme. Bcl-2 and other antiapoptotic members of the family have been shown to block caspase activation after several apoptotic stimuli 43 . However, Bcl-2 is also endowed with the ability to influence cell cycle progression. Indeed, Bcl-2 overexpression in transgenic mice was reported to decrease cell cycle entry of primary lymphocytes 23 . Transfection of Bcl-2 into NIH 3T3 cells also delayed their reentry into cell cycle after serum withdrawal, and a tyrosine 28 mutant in the NH 2 -terminal BH4 domain exhibited dominant negative effects over the wild-type Bcl-2 24 . Whether the effect of Bcl-2 on cell cycle progression also involves regulation of caspase activity remains to be addressed. However, these data associated with our results showing the inhibitory effect of zVAD on DNA synthesis suggest that caspases (along with their upstream regulators FADD and Bcl-2) may be involved in cell cycle entry during lymphocyte activation. Results presented in this study clearly demonstrate a selective processing of caspase substrates. PARP, which is involved in DNA repair 44 , is probably processed in apoptotic cells to allow for DNA fragmentation after DFF45 cleavage. Of note, PARP expression and activity increase after T cell stimulation 41 . Therefore, the 85-kD fragment of PARP probably conserves an enzymatic activity that mediates an unknown function in proliferating cells. Our results also demonstrate the selective processing of Wee1. This tyrosine kinase is a negative regulator of Cdc2 45 , a cyclin-dependent kinase required for the G 2 /M transition during cell cycle, as well as for Fas-mediated apoptosis in immortalized cell lines 13 . Wee1 is a critical component of the G 2 /M cell cycle checkpoint machinery, and mediates cell cycle arrest after DNA damage by phosphorylation of Cdc2 45 . Therefore, cleavage of Wee1 in proliferating lymphocytes could lead to its inactivation, thus allowing cell cycle progression. Of note, Wee1 processing by caspases during apoptosis in Jurkat cells correlates with a 20-fold decrease in Wee1 activity and an increase in Cdc2 activity 13 . The nuclear protein lamin B is considered as a caspase-6 substrate 7 . Our results show that in fresh PBMCs, two forms of 66 and 45 kD can be identified by Western blot using an anti–lamin B antibody, and at least two fragments of 35 and 28 kD are produced after T cell activation. Appearance of these products correlates with a decrease in the amount of the 45-kD form of lamin B, and is inhibited by addition of zVAD . Although the significance of this cleavage in activated lymphocytes is unclear, it confirms the activation of caspase-6, which generates the 28-kD fragment after a cleavage at the consensus sequence VEID 7 . In our experiments, DFF45 is present in resting as well as in proliferating cells, whereas RFC140 is upregulated after T cell stimulation. Since caspase-mediated DFF45 processing leads to DNA fragmentation, one could easily predict that it would not be cleaved in activated living cells. Indeed, we were unable to detect DFF45 cleavage in AV − cells after T cell activation, although the 14-kD fragment generated by caspase-3 cleavage at the second site was observed in lysate from activated lymphocytes after anti-Fas treatment. The lack of RFC140 cleavage in proliferating T cells was also critical, since processing of RFC140 by caspases leads to inactivation of the DNA replication machinery and cell cycle arrest at the G 2 /M boundary 11 . This selective substrate processing could explain why T lymphocytes survive and proliferate although the caspase cascade including caspase-8, -3, -6, and -7 is activated after TCR triggering. To explain this selective caspase specificity in stimulated lymphocytes, several hypotheses can be proposed. Endogenous caspase inhibitors, such as the inhibitor of apoptosis (IAP) family members, could selectively inhibit the cleavage of specific substrates in stimulated cells 46 . Supporting this hypothesis, the expression of thymic IAP, a murine homologue of survivin, is upregulated within 24 h of stimulation in splenic T cells 47 , and this member of the IAP family was shown to inhibit already processed caspase-3, -7, and -9 46 . Association of thymic IAP to caspases could have a differential effect on the cleavage of their substrates, depending on the affinity of the caspase for these substrates or their accessibility. Alternatively, some caspase substrates involved in cell survival could be protected against cleavage by phosphorylation near the caspase cleavage site. This mechanism was recently demonstrated for presenilin-2. Caspase-mediated cleavage of this molecule was abrogated by Ser 327 and Ser 330 phosphorylation inside the consensus cleavage site DSYD↓S 48 . In summary, activation of the caspase cascade in nonapoptotic lymphocytes, together with the impairment of T cell activation by caspase inhibitors, indicates that these proteases may play an important role during T cell stimulation, and caspases may be added to the growing list of molecules that are involved in cell death and proliferation. However, elucidation of the function of caspase processing in activated lymphocytes will require further experiments and will provide new insights into the biological function of caspases, as well as the regulation of T cell activation. | Study | biomedical | en | 0.999996 |
10601363 | Purified human T cells were prepared by Ficoll-Hypaque centrifugation followed by rosetting with sheep erythrocytes. Positively rosetted lymphocytes were at least 98% CD3 + by flow cytometry. Purified T cells were cultured in 96-well plates at 5 × 10 4 cells per well and preincubated for 30 min with the indicated concentrations of caspase peptide blockers Ile-Glu-Thr-Asp fluoromethyl ketone (IETD-fmk), benzyloxycarbonyl-Val-Ala-Asp (zVAD)-fmk, Asp-Glu-Val-Asp (DEVD)-fmk, and Tyr-Val-Ala-Asp (YVAD)-fmk (Enzyme Systems Products), or a similar dilution of the stock solvent DMSO. Cells were then stimulated with the indicated concentrations of immobilized anti-CD3 antibody TR66 at either an optimal concentration of 3 μg/ml or suboptimally at 0.5 μg/ml. To some cultures containing suboptimal anti-CD3 was added either soluble recombinant fluoresceinated antigen (FLAG)-tagged FasL at the concentrations shown (Alexis Corp.), with or without cross-linking by 1 μg/ml of anti-FLAG antibody (M2; Sigma Chemical Co.); with soluble IgM anti-CD28 antibody 28/34 at 5 μg/ml; or with immobilized Fas-Fc (Alexis Corp.); or human IgG at the concentrations shown. Proliferation was measured by tritiated thymidine ([ 3 H]TdR) incorporation during the final 18 h of a 4-d culture. Supernatants for IL-2 production were taken from PBLs (10 6 /ml) that were stimulated for 24 h with immobilized anti-CD3 (3 μg/ml), with or without each caspase blocker (50 μM), or with cross-linked FasL (50 ng/ml). IL-2 levels were assayed using the CTLL bioassay. Cells were washed once with PBS, and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5), 1% Triton X-100, 2 mM dithiothreitol, 2 mM sodium vanadate, and protease inhibitor cocktail (Complete™; Boehringer Mannheim), followed by centrifugation. Postnuclear lysates from 2 × 10 6 cells per lane were separated by SDS-PAGE, and analyzed by Western blotting using antibodies to caspase-3 (Transduction Laboratories) or caspase-8 (PharMingen). Cells were stimulated by immobilized anti-CD3 (0.5 μg/ml), anti-CD3/FasL (50 ng/ml plus anti-FLAG, 1 μg/ml), anti-CD3/anti-CD28 (28/34, IgM soluble at 10 μg/ml), or medium control. Samples were taken on each day for 5 d, washed in PBS, and then stained in 250 μl using 50 μg/ml propidium iodide (PI) in 0.1% Triton X-100, 4 mM sodium citrate, and 360 U/ml RNase, pH 7.2. Cells were incubated for 30 min at 37°C, and then 250 μl of salt solution was added (50 μg/ml PI, 0.1% Triton X-100, 0.4 M NaCl, pH 7.2). Samples were stored in the dark at 4°C for at least 1 h, and then analyzed within 24 h by flow cytometry. Stimulation of purified resting human T lymphocytes by anti-CD3 antibody was extensively blocked by the caspase inhibitors IETD-fmk and zVAD-fmk over a dose range of 12.5–50 μM . By contrast, two other caspase blockers, YVAD-fmk and DEVD-fmk, showed no inhibition of T cell growth at similar concentrations. Although differences in the specificity of these caspase blockers has been suggested, they are actually irreversible blockers, with the ability to titrate all accessible caspases. Thus, the different degrees of inhibition by the various blockers should not be viewed as implicating a particular caspase. An alternative explanation for the differences would be varying degrees of permeability by the caspase blockers in primary T cells. We also considered the possibility of nonspecific toxicity selectively by IETD-fmk and zVAD-fmk. However, the addition of exogenous IL-2 to cultures containing IETD-fmk or zVAD-fmk could largely overcome the block in proliferation (data not shown). Furthermore, to block proliferation, the addition of IETD-fmk had to occur within the first 24 h of CD3 stimulation, after which it conferred no inhibition even at relatively high concentrations of 50 μM . This observation also underscores the view that a caspase is involved in early T cell activation events, and that evidence of its processing should therefore be examined early, before secondary activation-induced cell death (AICD) complicates the picture, as cycling T cells become sensitive to FasL-induced apoptosis. A possible sequence by which TCR stimulation could activate the caspase cascade would be via the known TCR-induced upregulation of FasL 10 that would in turn engage surface Fas. Consistent with this view, immobilized Fas-Fc, but not control human IgG, largely blocked suboptimal (0.5 μg/ml) anti-CD3 activation of T cell growth in a dose-dependent manner . This finding does not exclude the additional possibility that Fas-Fc might also confer a negative signal directly retrograde through FasL 11 . However, in agreement with the former model, the addition of soluble FasL to suboptimal doses of anti-CD3 promoted a dose-dependent increase of T cell growth by as much as threefold . At higher concentrations of anti-CD3 (3.0 μg/ml), there was less block of proliferation by Fas-Fc, and less augmentation of proliferation by FasL (data not shown). This is in agreement with similar findings using anti-Fas antibodies ( 12 ; our unpublished observations). The magnitude of this costimulation approached that seen with the same concentration of anti-CD3 and an optimal dose of anti-CD28. FasL costimulation was observed only when FasL was oligomerized through cross-linking of its FLAG tag using anti-FLAG, which mimics active membrane-bound FasL 13 . As with the caspase blockers, costimulation with FasL needed to be delivered within the first 24 h of CD3 activation, otherwise no effect was observed . Similar to the findings with CD3 stimulation, the costimulation of T cell growth conferred by FasL was also blocked by the same two caspase inhibitors . Evidence for activation of caspase-8 and caspase-3 was investigated by Western blot within 2–4 h of T cell activation by anti-CD3, with or without FasL . Purified resting T cells were treated with immobilized suboptimal anti-CD3 (0.5 μg/ml) antibody in the absence or presence of cross-linked FasL or with cross-linked FasL alone, and caspase processing was analyzed during the first 4 h. Given the need to block caspase activity within the first 24 h, we elected to examine early time intervals after activation. It was also reasoned that evidence of caspase processing after this time might reflect FasL-induced apoptosis of actively dividing T cells. Caspase-8 processing in primary T cells is detectable by cleavage of its full-length 55-kD form between the procaspase and caspase domains to release a cleaved 28-kD fragment. As shown in Fig. 2 A, no evidence of caspase-8 cleavage was detectable in unstimulated T cells for up to 4 h, or even after overnight culture (data not shown). However, cleavage of caspase-8 became apparent by 4 h after suboptimal anti-CD3 stimulation (0.5 μg/ml) alone, or within 2 h using anti-CD3 plus FasL . This was particularly visible by longer exposure of the blot . Similar to the proliferation findings, the amount of the cleaved p28 fragment was more intense at high doses of anti-CD3 (3.0 μg/ml), but less augmentation by FasL was observed (data not shown). Thus, FasL costimulation was best observed for both proliferation and caspase-8 processing at suboptimal doses of anti-CD3. The cleavage of caspase-8 was not secondary to apoptosis, as <1% cell death was detectable by PI staining in either the unstimulated controls or stimulated cultures up to 24 h later (data not shown). Furthermore, IETD-fmk, but not DEVD-fmk or YVAD-fmk, at the same dose (50 μM) that inhibited T cell growth also blocked cleavage of caspase-8 during anti-CD3/FasL costimulation over 6 h . Although caspase-3 cleavage was readily detectable in FasL-treated Jurkat T cells undergoing apoptosis , no indication of caspase-3 cleavage products could be found in primary T cells costimulated with anti-CD3/FasL over 4 h, even with longer exposure of the blot , or with costimulation for as long as 22 h (data not shown). Consequently, cleavage of poly(ADP-ribose) polymerase (PARP), a substrate for caspase-3, was also not observed (data not shown). This indicated that processed caspase-8 did not lead to detectable processing of caspase-3 under these conditions of CD3/Fas costimulation. The augmented proliferation conferred by anti-CD3/FasL was paralleled by increased production of IL-2, which was also blocked by IETD-fmk and zVAD-fmk . As IL-2 promotes entry of T cells into the S phase of the cell cycle, the effect of FasL costimulation on cell cycling was examined. As shown for day 4 in Fig. 3 B, and summarized for all days in Table , FasL costimulation with CD3 increased the percentage of cells in S+G2+M over CD3 alone, to levels at least as high as observed with CD3 plus CD28 activation. Furthermore, the increased proliferation with FasL could be at least partly blocked by addition of anti-CD25 , suggesting that FasL costimulation may occur partly through upregulation of cytokines necessary for T cell proliferation. Combined with the reported T cell activation defect in FADD-deficient mice and FADD-dominant negative mice 6 7 8 9 , the current findings suggest a model whereby T cell activation via TCR upregulates surface FasL, which engages Fas and leads to recruitment of FADD and caspase activation. However, in resting T cells, this does not result in apoptosis, but rather augments IL-2 gene activation and cell proliferation. The notion that FasL can augment proliferation of resting T cells may be more broadly applicable to other death receptors such as TNF-related apoptosis-inducing ligand receptor (TRAIL-R) and TNFR-related apoptosis-mediated receptor/death receptor 3 (TRAMP/DR3) 1 , as well as to other cell types. These other death receptors may be particularly important in initiating proliferation of primary T cells lacking Fas. In addition to T cells, fibroblasts have also been observed to manifest increased proliferation with anti-Fas antibodies 12 14 , as well as with FasL (our unpublished observations). The exact caspase(s) required for proliferation by primary T cells and its substrate cannot be concluded at present, given the broad specificity of the caspase blockers. However, one might speculate that a proximal caspase in the Fas signal cascade is required for activation of primary T cells, given our findings of cleavage of caspase-8, but not of caspase-3, under conditions of FasL costimulation. A likely substrate for caspase-8 by which a death signal could be diverted to a proliferative response would be cellular FADD-like IL-1β–converting enzyme (FLICE) inhibitory protein (c-FLIP), the natural inhibitor of Fas-induced cell death. c-FLIP is homologous to caspase-8, but bears a nonfunctional caspase domain. c-FLIP contains two potential caspase cleavage sites, and as such functions as a substrate trap for caspase-8, while also competing for binding to FADD 15 . In further studies we have observed that c-FLIP is cleaved during T cell activation, and its overexpression in Jurkat T cells or in transgenic mouse T cells increases the activities of the mitogen-activated protein (MAP) kinase, extracellular signal–regulated kinase (ERK), and nuclear factor (NF)-κB after CD3 stimulation, leading to augmented IL-2 production. In addition, Fas costimulation of normal T cells with CD3 also augments the activator protein 1 (AP-1) and NF-κB pathways (our manuscript in preparation). It is apparent from these findings that inhibitors of caspases could prove valuable in therapeutic maneuvers to diminish T cell activation. | Study | biomedical | en | 0.999995 |
10601364 | Der p 1 was isolated from house dust mite fecal pellets (Allergon) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti–Der p 1 mAb (clone 4C1; Indoor Biotechnologies), removal of contaminating serine proteases on immobilized soybean trypsin inhibitor (Sigma Chemical Co.), and finally fast protein liquid chromatography (FPLC) to remove low-molecular-mass contaminants. The purity of the preparation was confirmed by NH 2 -terminal sequencing on an automatic amino acid sequencer (Applied Biosystems, Inc.), SDS-PAGE analysis (15% gel), and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by E-64 ( l -trans-epoxysuccinyl-leucylamido [4-guanidino]butane). Protein concentration was determined using a bicinchoninic acid (BCA) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for Der p 1 of E 1% (280 nm) = 16.4. Before use, Der p 1 was preactivated with 5 mM cysteine (Sigma Chemical Co.) to regenerate its thiol group, which becomes oxidized during purification. The catalytic activity of Der p 1 was ascertained in a continuous rate (kinetic) assay using the fluorogenic peptide substrate N -tert-butoxy-carbonyl (Boc)-Gln-Ala-Arg–7-amino-4-methyl-coumarin (AMC; reference 6). To block the proteolytic activity of cysteine-activated Der p 1, 1,000-fold molar excess of E-64 (Sigma Chemical Co.) was used; a similar molar ratio of the cysteine protease inhibitor iodoacetamide (Sigma Chemical Co.) was used as another sulfhydryl reactive agent. Spleen T cells were obtained from C57BL/6J mice using standard procedures. The cells (2 × 10 6 ) were suspended in RPMI (GIBCO Life Technologies) and stimulated for 3 d at 37°C with Con A (5 μg/ml final concentration) in the presence of IL-2 (100 U/ml) in a humidified atmosphere of 5% CO 2 . CD25 cleavage was performed by incubating 10 5 cells with up to 10 μg/ml Der p 1 (preactivated with 5 mM cysteine) for 1 h at 37°C in a total volume of 200 μl serum-free AIM V medium (GIBCO Life Technologies). The cells were then resuspended in RPMI containing 2% FCS, stained for 45 min at room temperature in the dark with FITC-labeled anti–mouse CD25 mAb (clone AMT-13; Sigma Chemical Co.), and fixed with 5% formaldehyde. Cells were analyzed on a FACScan™ (Becton Dickinson) as described 4 . The expression of other T cell surface markers, namely CD3, CD4, and CD8, was monitored in the same way using appropriate PE- or FITC-labeled antibodies (clone KT3, Beckman Coulter; and clones YTS191.1 and KT15 [Serotec Ltd.], respectively). Five groups of 10 female CBA/J mice were given six weekly intraperitoneal injections of 10 μg of proteolytically active Der p 1, 10 μg of E-64–blocked Der p 1, 10 μg of iodoacetamide-blocked Der p 1, 10 μg of OVA (as proteolytically inactive antigen; Sigma Chemical Co.), or 10 μg of OVA with E-64, respectively. All immunizations were given in 200 μg of Al(OH) 3 as adjuvant. A tail bleed was obtained 1 wk before the start of immunization (prebleed), and a total bleed was obtained by cardiac puncture 1 wk after the last injection (final bleed). The proteolytic activity of Der p 1 and its inhibition with E-64 or iodoacetamide in the immunization mixture were ascertained as described above. Serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass. The optimal dilutions used here were 1/10 for detecting total IgE, Der p 1–specific IgE, and OVA-specific IgE and 1/20,000, 1/40,000, and 1/250 for detecting Der p 1–specific IgG, IgG1, and IgG2b, respectively. Total IgE was detected by a sandwich ELISA using one monoclonal anti–mouse IgE (clone R35-72; PharMingen) as capture antibody and a second biotinylated monoclonal anti–mouse IgE (clone R35-118; PharMingen) as a detection antibody. Der p 1–specific IgE, OVA-specific IgE (measured using samples that have been depleted of IgG on a protein G column [Pharmacia]), and Der p 1–specific IgG, IgG1, and IgG2b were detected on microtiter plates coated with a 4 μg/ml solution of either Der p 1 or OVA and developed with biotinylated (for IgE clone R35-118 and IgG1 clone A85-1 [PharMingen] and for IgG2b clone AB275 [The Binding Site]) or alkaline phosphatase–conjugated isotype-specific antibodies (for IgG; Sigma Chemical Co.). Alkaline phosphatase–conjugated Extravidine (Sigma Chemical Co.) was used in conjunction with biotinylated antibodies. Unpaired Student's t test was used to compare levels of antibody responses between the different immunization groups; P < 0.05 was considered significant. Der p 1 is a 25-kD cysteine protease whose structure has been modeled 7 on the crystal structure of papain, with which it shows considerable sequence similarities, most notably for residues involved in the enzyme active site 8 . The proteolytic activity of Der p 1 can be inhibited by E-64, the class-specific inhibitor of microbial origin 9 . This inhibition is brought about when cysteine within the Der p 1 active site forms a thioether covalent bond with the epoxy group of E-64. This is an irreversible process that does not lead to significant structural changes, as evidenced by crystallographic studies of a papain–E-64 complex 10 . We have purified Der p 1 from fecal pellets using a multistep procedure and confirmed its purity by NH 2 -terminal sequencing, SDS-PAGE analysis, and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and inhibited by E-64 and iodoacetamide . We have recently shown that Der p 1 selectively cleaves human CD25 from the surfaces of peripheral blood T cells 4 . Here we demonstrate that Der p 1 also selectively cleaves CD25 from cultured mouse spleen T cells , which is not surprising given the high degree of sequence homology that exists between human 11 and mouse 12 CD25. This observation has therefore provided the justification for using this animal species for testing our hypothesis, namely that the proteolytic activity of Der p 1 is a major contributor to its allergenicity. Intraperitoneal immunization of groups of 10 CBA/J mice with either proteolytically active or inactive (E-64–blocked) Der p 1 over a 6-wk period showed a statistically significant enhancement in total IgE ( P < 0.01) and Der p 1–specific IgE ( P < 0.02) responses in animals immunized with proteolytically active Der p 1. This effect was IgE specific, as Der p 1–specific IgG, IgG1, and IgG2b responses increased to the same extent with proteolytically active or inactive Der p 1 . We are not sure why the IgG1 response did not follow that of IgE, as these two isotypes are considered to be coregulated in the mouse. However, the IgE-restricted enhancement seen in response to immunization with proteolytically active Der p 1 does suggest a mechanism that is unique to IgE isotype switching/synthesis. Furthermore, our control experiments clearly show that the IgE-specific effect observed here is not due to E-64 exerting a suppressive influence on IgE production by a mechanism that is independent of its binding to the Der p 1 enzyme active site . First, suppression of total IgE ( P < 0.04) and Der p 1–specific IgE ( P < 0.05) productions was also obtained when the proteolytic activity of Der p 1 was blocked with iodoacetamide, another sulfhydryl reactive agent. Second, the IgE antibody response to OVA, a proteolytically inactive antigen, was not suppressed when the animals were immunized with OVA plus E-64. Our results are direct evidence that the cysteine protease activity of Der p 1 induces a significant increase in IgE responses. Such an effect is clearly consistent with the ability of Der p 1 to proteolytically cleave mouse CD25 and induce a Th2 response by modulating the balance between IL-4 and IFN-γ 13 . The recent demonstration in mice that Leishmania mexicana cysteine proteinase–deficient mutants potentiate a Th1 response, compared with the Th2 response normally seen in response to infection with wild-type parasite 14 , is also of great relevance here. These findings suggest that immune deviation toward Th1 in dust mite–allergic individuals could potentially be achieved by administering Der p 1 in a catalytically inactive (mutant) form. On the other hand, exploring the potential Th2 adjuvant property of the proteolytic activity of Der p 1 would have important implications in defining principles for modulation of Th1-mediated pathological conditions. Our demonstration that the cysteine protease activity of Der p 1 enhances total IgE production, apart from increasing Der p 1-specific IgE, suggests that this allergen may play a central role in destabilizing the microenvironment within target tissues to one that is proallergic and thus aids in the initiation and propagation of the allergic cascade. In other words, the proteolytic activity of Der p 1 may exert an IgE-specific adjuvant effect. The in vivo relevance of the proteolytic activity of Der p 1 is further highlighted by reports demonstrating that it increases the permeability of the human respiratory epithelium to macromolecules 15 16 . Such observations, together with our current findings showing a direct effect on the immune system, indicate that the proteolytic activity of Der p 1 is a major contributor to its allergenicity. | Study | biomedical | en | 0.999997 |
10601365 | A subclone of DO11.10 T hybridoma cells with a low survival rate in PMA plus ionomycin was used for expression cloning. φNX-Ampho is a retrovirus packaging cell line 15 . DO11.10 cell line expressing a tailless version of human CD2 (DO11.10hCD2) as control was described previously 16 . DO11.10 cells expressing high levels of CD43 were generated by transduction of mouse CD43 cDNA using the pMX retrovirus vector, followed by FACS ® sorting for CD43 expression. Cells were cultured in DMEM containing 10% FCS, 2 mM glutamine, 25 mM Hepes, 50 μM β-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin. Anti–human CD2 mAb 35.1 (American Type Culture Collection) was purified and labeled in our laboratory. The following mAbs were purchased from PharMingen: purified or PE–anti-CD3 (145-2C 11 ), FITC– or PE–anti-CD4 (GK1.5), FITC–, PE–, or biotin–anti-CD8α (53-6.7), FITC– or PE–anti-CD43 (S7), PE–anti-CD69 (H1.2F3), biotin–anti-FasL (Kay-10), PE–anti-Fas (Jo2), biotin–anti-CD62L (MEL-14), biotin–anti-CD45RB (16A), and biotin–anti-CD44. Ceramide and staurosporine were purchased from Calbiochem Corp. Recombinant human IL-2 was purchased from Chiron Corp. Expression cloning was performed as described 16 . In brief, a thymocyte cDNA library in the retroviral vector pMX 17 was transfected into the φNX-Ampho packaging cell line using CaPO 4 precipitation, and retrovirus-containing supernatant was harvested 2 d later. DO11.10 cells were infected with the retrovirus supernatant. A total of 5 × 10 6 DO11.10 cells were infected, as assessed using control pMX-hCD2 retrovirus infection. 2 d after the infection, DO11.10 cells were placed into 96-well plates (5 × 10 4 cells/well) and selected for survival and growth in the presence of PMA (10 ng/ml) plus ionomycin (0.2 μg/ml). Resistant clones were identified after 2–4 wk in culture. cDNA inserts were amplified by reverse transcriptase–PCR and sequenced. Before staining, cells were incubated with a mAb to FcR (2.4G2) and then sequentially stained with an excess of biotinylated mAb, PE–streptavidin, or FITC– and PE–conjugated mAbs on ice for 30 min and washed with PBS containing 0.1% BSA. Data were collected on 1–5 × 10 4 cells on a FACScan™ flow cytometer (Becton Dickinson) using CELLQuest™ software. 24- or 96-well tissue culture plates were precoated with rabbit anti–hamster antiserum (50 μg/ml) overnight at 37°C, washed with HBSS, and coated with anti-CD3 mAb (145-2C 11 ) at the amount indicated in figure legends. Hybridoma cells were added to the plates for the indicated time , harvested, and analyzed. Alternatively, PMA plus ionomycin was added to the culture instead of anti-CD3 mAb. For the thymidine incorporation assay, 2 × 10 4 hybridoma cells per well were added to 96-well plates, cultured for 20 h, and labeled with [ 3 H]thymidine (1 μCi/well, 25 Ci/mmol; New England Nuclear) for a further 4 h. For TCR-induced apoptosis of CD4 + cells, splenocytes were activated with Con A (5 μg/ml) for 2 d, treated with methyl-α- d -mannopyranoside (10 mg/ml) at 37°C for 30 min, washed twice with PBS, and then cultured in 100 U/ml human IL-2 for an additional 2 d. The cells were then harvested and plated on an anti-CD3–coated plate. 16–24 h later, cells were quantitated by trypan blue exclusion and FACS ® analysis. We employed an expression cloning strategy to identify genes that regulate AICD in a T cell hybridoma. We infected DO11.10 cells with a thymocyte cDNA library packaged in a retrovirus and selected for cells that could grow in the presence of PMA plus ionomycin. cDNA inserts from resistant clones were sequenced, revealing that one of the inserts encoded a full length CD43. To confirm the antiapoptotic effect of CD43, we transduced DO11.10 cells with either the full length CD43 or a tailless human CD2 as control. DO11.10 cell lines expressing high levels of CD43 were established by fluorescence-activated cell sorting. As shown in Fig. 1 A, DO11.10hCD2 control cells express a low level of endogenous CD43 on their surfaces. After transduction with pMXCD43, we isolated DO11.10 cell lines expressing ∼6–20-fold higher surface levels of CD43 compared with control cells . Interestingly, DO11.10CD43 high cells expressed two- to threefold lower levels of CD3 on their surfaces compared with DO11.10hCD2 control cells . The lower CD3 surface staining was not due to nonspecific blockade of cell surface accessibility, as the expression levels of other surface molecules such as CD2 and CD5 were not changed (data not shown). To examine the effect of high level expression of CD43 on activation-induced apoptosis, we measured [ 3 H]thymidine uptake of DO11.10hCD2 control and DO11.10CD43 high cells in the absence or presence of anti-CD3 mAb or PMA plus ionomycin. [ 3 H]thymidine incorporation by these cells closely correlated with their viability, measured by either propidium iodide uptake or trypan blue exclusion 16 . As shown in Fig. 1 B, DO11.10 cells expressing high levels of CD43 were protected from anti-CD3–induced death. In contrast, control cells were readily induced to undergo apoptosis. We further determined whether the antiapoptotic effect in DO11.10CD43 high cells could be due to a lowered TCR/CD3 surface expression by using PMA plus ionomycin as a stimulus for AICD, which bypasses the TCR. DO11.10CD43 high cells were also refractory to apoptosis induced by PMA plus ionomycin . The protection from AICD by CD43 correlated with its expression level, and the dosage effect was more pronounced when these cells were stimulated with anti-CD3, possibly due to some steric hindrance of CD43 imposed on the interaction of TCR/CD3 with the plate-bound anti-CD3 mAb. Next, we tested the effect of high level expression of CD43 on apoptosis induced by other stimuli. DO11.10 cells expressing high levels of CD43 were protected from apoptosis only in the presence of low amounts of staurosporine and were not protected from ceramide-induced killing . Previous studies have demonstrated that CD43 acts as a negative regulator of T cell activation, presumably due to its highly charged nature and its large size 9 . To examine the effect of high level expression of CD43 on T cell hybridoma activation, we stimulated control DO11.10hCD2 and DO11.10CD43 high cells with either plate-bound anti-CD3 mAb or PMA plus ionomycin for 6 h and monitored the upregulation of Fas, FasL, and CD69 by FACS ® analysis. Before TCR stimulation, the expression of Fas on DO11.10CD43 high cells was lower compared with control cells . However, both Fas and FasL were upregulated by anti-CD3 stimulation of DO11.10CD43 high cells , even though these cells express lower levels of CD3 . The T cell activation marker CD69 was also upregulated to a similar level in both cell types . Similarly, PMA plus ionomycin upregulated expression of these three markers in control and CD43 high cells (data not shown). These data indicate that high levels of CD43 expression did not obviously affect the activation of these T hybridoma cells and the upregulation of Fas and FasL and suggest that high level expression of CD43 may interfere with the death signal initiated through the Fas molecule. Previous studies have shown that TCR/CD3-mediated apoptosis of CD4 + T cells in vitro is primarily induced through Fas, whereas CD8 + T cell death is primarily induced through TNFR 18 . Given the antiapoptotic effect exhibited by high level expression of CD43 on T hybridoma cells, we examined its expression on peripheral T lymphocytes. As shown in Fig. 3 A, splenic CD4 + T cells of unimmunized, young adult C57BL/6 mice can be divided into two subsets based on CD43 expression. CD4 + CD43 high cells consist of 10–15% of the total CD4 + T cells, and the surface expression of CD43 on these cells is six- to eightfold higher than that on CD4 + CD43 low cells . Interestingly, splenic CD8 + T cells exhibit uniformly high levels of CD43 on their surfaces . To further determine the phenotype of CD4 + CD43 high and CD4 + CD43 low T cells, we analyzed these cells with three-color staining using mAbs directed against CD62L, CD45RB, and CD44. Surprisingly, the two CD4 + subsets expressing high or low levels of CD43 exhibited a characteristic phenotype of memory or naive T cells, respectively . CD4 + CD43 high T cells express high levels of CD44 and low levels of CD62L and CD45RB, whereas CD4 + CD43 low T cells express low levels of CD44 and high levels of CD62L and CD45RB. Peripheral CD4 + and CD8 + T cells from lymph nodes exhibited a similar phenotype to splenic T cells in the above analysis (data not shown). These results suggest that CD43 may be used as a marker to define memory CD4 + T cells. Recent studies have demonstrated that naive CD4 + T cells are more susceptible to TCR-mediated, Fas-dependent cell death compared with memory phenotype CD4 + T cells 2 3 4 . To test whether the CD4 + CD43 high primary cells are resistant to TCR/CD3-mediated cell death, we used an in vitro assay for AICD of CD4 + T cells. Splenocytes from normal C57BL/6 mice were activated with Con A for 2 d, followed by culture in a high concentration of IL-2 for another 2 d. The activated cells were then restimulated with plate-bound anti-CD3, and cells that survived the restimulation were analyzed by FACS ® . After restimulation with plate-bound anti-CD3, a majority of the CD4 + T cells were readily induced to undergo apoptosis, whereas CD8 + T cells did not suffer obvious cell loss in 16–24-h assay. When live cells from the restimulation plates were stained with CD4 and CD43, it was observed that only those CD4 + cells expressing high levels of CD43 survived anti-CD3 stimulation . These results demonstrate that CD4 + CD43 high T cells are resistant to TCR/CD3-mediated cell death and imply that CD43 may determine this property of memory CD4 + T cells. Several recent studies have demonstrated that CD4 + memory T cells are resistant to TCR-mediated, Fas-dependent cell death, whereas CD4 + naive T cells succumb 2 3 4 . In this report, we show that high level expression of CD43 protects T cell hybridomas from AICD. Peripheral CD4 + CD43 low T cells exhibit a naive phenotype, whereas CD4 + CD43 high exhibit a memory phenotype, suggesting that the surface expression levels of CD43 on CD4 + naive and memory T cells determine their susceptibility to TCR-mediated, Fas-dependent cell death. How is the inhibition of TCR/CD3-mediated cell death by high level expression of CD43 achieved? CD43 has been shown to have multiple, sometimes contradictory functions. This may be due to its structural features and the systems that have been used to study its function. CD43 is arguably the most abundant protein on the T cell surface 19 . It extends 45 nm from the cell surface, at least six times the height of the TCR. It is also highly O -glycosylated and bears numerous sialic acid residues. The antiadhesive and antiproliferative effects mediated by CD43 are thought to result from a physical barrier formed by this highly negatively charged and rigid rod–like structure. However, this functional model of CD43 has been challenged by recent experiments showing that the cytoplasmic domain of CD43 plays an important role in its function 20 21 . Moreover, the extracellular portion of CD43 may also be highly dynamic and actively interact with other surface structures. In support of this notion, a recent study showed that CD43 moves away from the contact sites of T cells and APCs, whereas CD45, similarly large in size and strongly negatively charged, does not 22 . In addition, CD43 was reported to be physically associated with the TCR/CD3 complex 23 . Several mechanisms can be envisioned for the anti-apoptotic effect caused by high level expression of CD43. First, highly expressed CD43 might block Fas signaling by physically preventing the interaction between FasL and Fas. Alternatively, it may prevent Fas from forming trimers. The fact that high level expression of CD43 on DO11.10 specifically decreases the expression of TCR/CD3 and Fas indicates that they may physically interact or form surface complexes with each other. Furthermore, high level expression of CD43 may interfere with the recruiting of death signaling molecule by Fas or inhibit the activation of caspases. Overexpression of Toso, a surface protein that contains an Ig domain, was shown to inhibit Fas signaling by upregulating the caspase-8 inhibitor cFLIP (cellular FLICE [FADD-like IL-1β–converting enzyme]-inhibitory protein; reference 15). We have not observed differences in the expression of cFLIP in DO11.10 control cells and CD43 high –expressing cells (our unpublished observation). It is interesting to note that although CD43 high CD4 + T cells from spleen and lymph nodes express six- to eightfold higher levels of CD43 on their surfaces than CD43 low CD4 + cells, these cells are much easier to activate by either anti-CD3 or Con A than CD43 low CD4 + cells (our unpublished data), in agreement with previous studies showing that memory T cells have a lower threshold for activation 1 . These data further argue against the model that CD43 negatively regulates T cell activation by increasing the stimulation threshold or by physically hindering the interaction between T cells and APCs. Our attempt to investigate whether the cytoplasmic tail of CD43 is required for the inhibition of AICD was inconclusive. A tailless CD43 retaining two amino acids of the cytoplasmic tail can be expressed at only two- to threefold higher levels than the endogenous CD43 on DO11.10 cells, and this tailless version of CD43 had no obvious antiapoptotic effect (our unpublished data). We propose that high level expression of CD43 be used as a surface marker to define CD4 + memory T cells in C57BL/6 mice. Our results further suggest that CD43 may protect activated cells from AICD. In addition, we note that, correlating with their resistance to Fas-mediated killing, CD8 + T cells uniformly express high levels of CD43. The protection against Fas-mediated killing by high level expression of CD43 may provide a novel mechanism for its role in tumorigenesis. CD43 has been shown to be overexpressed in Friend erythroleukemia cells 24 and abnormally expressed in nonhematopoietic tumor lines such as colon carcinoma and adenoma cells 25 26 27 . Surface expression of CD43 diminishes susceptibility of target cells to T cell–mediated cytolysis 28 , and the obvious implication is that CD43 expression on tumor cells protects them against lymphoid effectors bearing FasL. | Study | biomedical | en | 0.999997 |
10601366 | OVA peptide 323–339 was synthesized by Bio-Synthesis. IFN-γ was obtained from Genzyme Corp. All other reagents were obtained from Sigma Chemical Co. except where indicated. The I-A d –restricted OVA-specific murine T cell hybridoma DO11.10, the I-A b –restricted Eα-specific hybridoma 1H3.1, the H-2 b murine macrophage line BM12 (provided by Dr. K. Rock, University of Massachusetts Medical School, Worcester, MA), and the H-2 d murine macrophage line RAW 264.7 (no. TIB-71; American Type Culture Collection) were grown in RPMI containing 10% fetal bovine serum, 1% l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified incubator with a 5% CO 2 atmosphere. The vector pNFATeGFP contains the Neo r marker of pcDNA3 and drives enhanced GFP (eGFP) expression under control of an NFAT-induced promoter. The CMV promoter of pcDNA3 (Invitrogen) was replaced with the 0.2-kb ClaI-HindIII fragment of NFATZH ( 17 ; provided by Steve Fiering, Fred Hutchinson Cancer Research Center, Seattle, WA) containing a promoter consisting of three copies of the NFAT binding site linked to the IL-2 promoter (−72 to +47 of IL-2). The 0.7-kb NcoI-NotI fragment of peGFP (Clontech) containing the coding region for eGFP was then cloned downstream of the NFAT-induced promoter. 3 × 10 6 DO11.10 cells were placed in 0.5 ml sterile PBS in a 0.4-cm gap cuvette (Bio-Rad) together with 20 μg DNA. The sample was electroporated with a Gene Pulser (Bio-Rad) set at 250 μF and 300 V. The cells were grown for 24 h before adding G418 (GIBCO BRL) to 1 mg/ml final concentration. After 10 d selection, the surviving cells were cloned by limiting dilution. Clones were then screened for OVA-specific induction of GFP by APCs. One responding clone, 5.6, was designated DO11-GFP and used for the experiments reported here. For presentation by RAW 264.7 cells, the cells were plated with 20 U/ml IFN-γ at 3 × 10 4 /well in 96-well plates or 2 × 10 5 /well in 24-well plates and incubated 48 h before use. The cells were antigen loaded overnight with OVA added from a 100 mg/ml sterile stock, or for 2 h with OVA 323–339 peptide. The cells were then rinsed once with medium, DO11-GFP cells were added at 8 × 10 4 /well in 96-well plates or 2 × 10 5 in 24-well plates, the plates were centrifuged at 450 g to initiate contact between the cells, and the cells were incubated together at 37°C in a CO 2 incubator for the indicated times. Loose cells were transferred by vigorous pipetting into a fresh tube, and adherent cells were released from the dish with PBS containing 1 mM EDTA and added to the loose cells. The cells were analyzed by flow cytometry using a FACScan™ and CELLQuest™ software (Becton Dickinson). To rapidly identify the different cell populations so as to gate on just the APCs or on the T cells, RAW 264.7 cells were prelabeled at the beginning of the experiment with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) or the DO11-GFP cells were prelabeled with CellTracker™ Orange (CTO; Molecular Probes) as follows. For DiI labeling, RAW 264.7 cells were collected by centrifugation, washed one time with labeling buffer (300 mM glucose, 10 mM Hepes, pH 7.1), resuspended in labeling buffer containing 1 μg/ml DiI, and incubated for 15 min at 37°C. The cells were then washed twice with labeling buffer and once with medium before use. For CTO labeling, DO11 cells were collected by centrifugation and resuspended in medium containing 500 nM CTO, incubated 15 min at 37°C, washed twice with medium, and incubated for 30 min at 37°C before use. Separate experiments determined that neither of these treatments had any measurable effect on the data presented. RAW 264.7 cells were plated 48 h before use on 35-mm glass-bottomed microwell dishes (MatTek Corp.) with 20 U/ml IFN-γ, and OVA was added to 10 mg/ml for the final 16 h. DO11-GFP cells were added to the dish at fivefold excess, centrifuged onto the RAW 264.7 cells to initiate contact, and the dish was immediately mounted on a Axiophot microscope (Carl Zeiss, Inc.) equipped with a cooled CCD camera (Princeton Instruments) and Metamorph™ digital imaging software system for image collection (Universal Imaging). Differential interference contrast (DIC) images were collected at 2-min intervals for the periods indicated in the text, and GFP images were collected before the first frame and after the last frame of the series. To visualize antigen-specific interactions of macrophages with T cells by continuous video microscopy, we transfected T cells with a reporter construct that causes them to express GFP upon productive engagement of an APC. In this construct, GFP expression is regulated by a promoter, comprising three copies of the NFAT binding site linked to a minimal IL-2 promoter, that has been demonstrated to faithfully report NFAT activity in T cells when driving chloramphenicol acetyltransferase (CAT) or LacZ expression 15 16 17 . We transfected the OVA-specific T cell hybridoma DO11.10 with this vector, selected stable clones, and found that >60% (12/19) of the clones showed GFP expression upon NFAT activation. In this paper, we report our results with a clone designated DO11-GFP. Identical results were obtained with additional clones. The mouse macrophage cell line RAW 264.7 was stimulated with IFN-γ to upregulate MHC class II expression and incubated overnight with OVA. When DO11-GFP cells were allowed to interact for 6 h with these macrophages, cells in direct contact with the macrophages expressed GFP , whereas those physically separated from the APC did not . By contrast, if the IFN-γ–stimulated RAW 264.7 cells were not first incubated with OVA, the DO11-GFP cells expressed no GFP, even when contacting the APC . Examination of >100 such pairs confirmed that cell–cell contact was required for T cell stimulation as reported by GFP expression; few GFP-positive DO11-GFP cells were observed not in contact with a macrophage. We next examined the antigen concentration dependence of the response of DO11-GFP cells to RAW 264.7 cells. IFN-γ–stimulated RAW 264.7 cells were loaded with OVA 323–339 peptide at the indicated concentrations for 2 h before DO11-GFP cells were added. After 6 h of interaction, the cells were harvested and analyzed by flow cytometry for GFP expression. As the peptide dose increased, both the number of T cells expressing GFP and the relative fluorescence intensity of the GFP-positive cells increased . At intermediate antigen concentrations, a bimodal distribution of cells was observed, with some cells responding to stimulation while others did not respond. At higher antigen concentrations, the cells became more uniformly GFP-positive. Translated into a dose–response curve , the data show that the stimulation is dose dependent and saturable. Interestingly, the antigen dose that resulted in the maximum number of cells becoming GFP-positive was an order of magnitude lower than the dose required to stimulate maximal GFP expression. This suggests that even under conditions when all of the DO11-GFP cells were productively interacting with antigen-presenting macrophages, more antigen still resulted in a stronger NFAT-mediated response in the hybridoma. This result was also obtained if OVA was used as the antigen instead of synthetic peptide, or if primary peritoneal macrophages were used as APCs (data not shown). Similar data were obtained by transfecting the NFAT-GFP reporter construct into the T cell hybridoma 1H3.1 18 , which recognizes a peptide derived from the I-E α chain, and then stimulating the cells with the I-A b macrophage cell line BM12 or by primary macrophages (data not shown). Immunoblot analysis demonstrated that GFP fluorescence accurately reflected the level of GFP expression in the T cell hybridoma (data not shown). These data are at variance with previous observations using the same promoter driving a β-galactosidase reporter, which showed that T cell activation occurs by a binary mechanism in which the cells are either on or off 19 20 . A possible explanation for this discrepancy might be the differences in stability of GFP (long-lived) and β-galactosidase (short-lived). We next examined the temporal induction of GFP expression during antigen presentation. IFN-γ–stimulated RAW 264.7 cells were loaded with the indicated dose of OVA, and DO11-GFP cells were allowed to interact with them for the indicated times. The T cells were subsequently harvested, and the average fluorescence of the GFP-positive cells was measured . GFP expression was detectable after 2 h and leveled off after ∼12 h. The rate and extent of GFP accumulation differed for different doses of OVA; higher doses of antigen resulted in more rapid accumulation of GFP to higher final levels in individual stimulated T cells. The rapid, antigen-specific expression of GFP in DO11-GFP cells allowed real-time visualization of productive APC–T cell interactions by video microscopy: we observed that macrophage–DO11-GFP cell interaction is dynamic. IFN-γ–stimulated RAW 264.7 cells were preloaded overnight with OVA, DO11-GFP cells were added, and the cells were mounted on a temperature-controlled microscope stage and observed for 3 h. At the beginning of the experiment, no cells were fluorescent, as shown in Fig. 4 . After 3 h, many T cells in the field were expressing GFP. Fig. 4 illustrates several points about the dynamic interaction between the RAW 264.7 and DO11-GFP cells. First, T cells that were bound to macrophages for the entire 3 h (cells B, C, and E) expressed more GFP than cells that bound to macrophages later in the experiment (cells F, H, and I), consistent with the dose and time dependence illustrated above. Cells A, D, and G moved out of the field of vision during the experiment and could not be followed. Generally, 1.5–2 h of interaction was required for detection of GFP. In separate experiments using populations of cells analyzed by flow cytometry, we confirmed that separating macrophages from T cells within 1.5 h of initiating the interaction prevented the induction of GFP in T cells (data not shown). Second, when rapidly moving T cells encountered antigen-presenting macrophages, the T cells became attached to the surface of the macrophage, but not restricted to one region of the presenting cell. For example, cell F moved rapidly between 0.5 and 1.25 h across the field of view and attached to a macrophage. It then wandered up and down one side of the macrophage, being activated to the same extent as cells that interacted with the macrophage for the same amount of time (cell H). Analysis of a large number of T cells ( n = 50) demonstrated that 90% of them moved around on the surface of macrophages during antigen presentation. Third, macrophages are clearly capable of interacting simultaneously with multiple T cells. Cells B, C, D, and E all bound to the macrophage in the top center of the field at some point in the series, whereas cells B, C, F, and H all interacted with the macrophage in the center of the field. In other experiments, as many as 10–12 T cells could be observed interacting simultaneously with a single macrophage (data not shown). Fourth, nearly 50% of T cells ( n = 50) that were bound to one macrophage migrated to neighboring macrophages during the 3-h observation period. Thus, cells B and C began the series on one macrophage and ended on another. The fact that GFP expression was very high in cells B and C after 3 h indicated that the interactions with both macrophages were productive: cell C spent 0.5 h on the first macrophage and 2.5 h on the second macrophage, whereas cell B spent 1.25 h on the first macrophage and 1.75 h on the second macrophage. Comparison of the high GFP expression levels of cells B and C to the low expression level of cell F (which only interacted with a macrophage for 1.75 h) further supported the notion that the GFP expression levels in cells B and C were the result of the cumulative interactions of these cells with both macrophages. The data also indicate that the time of interaction between the macrophage and T cell is the predominant determinant of GFP expression, rather than one macrophage being a better APC than another macrophage. Similarly dynamic interactions were observed between the NFAT-GFP–transfected 1H3.1 hybridoma and macrophages (data not shown). We have used a novel system for examining the interaction of macrophages with T cell hybridomas by video and immunofluorescence microscopy and FACS ® analysis. Due to the difficulty in transfecting primary T cells and T cell lines, our studies were restricted to T cell hybridomas, which may behave differently. Although the stimulation of the NFAT pathway only reflects one aspect of T cell activation, the rapidity of the response allows real-time analysis of productive APC–T cell interactions. It has been known for a long time that macrophages and dendritic cells can bind many T cells at the same time; our system has permitted us to directly observe these interactions and to conclude that a single macrophage is capable of simultaneously activating multiple T cells. Most interestingly, these interactions are dynamic, and the T cells move both from contact site to contact site on individual macrophages and from APC to APC while being activated. We were surprised to observe that the T cell–macrophage interactions were so dynamic, because the observations of Dustin and colleagues demonstrated that T cells stop migrating upon interaction with ICAM-1 and MHC class II–peptide complex embedded in a lipid bilayer 14 21 . It is possible that this inhibition of migration reflects an initial event that permits the formation of a productive contact zone, and that subsequent movement of T cells could not be detected because the full repertoire of signals required for T cell activation was not present. This view is supported by the fact that the T cells do stop for some minutes at particular sites on the macrophage, perhaps allowing the establishment of signaling junctions. Indeed, when individual macrophage–T cell pairs were observed by laser scanning confocal microscopy, we noticed the recruitment of cytoskeletal and signaling molecules such as actin, talin, and tyrosine-phosphorylated proteins to the site of cell–cell contact (data not shown), as has been described in other studies of APC–T cell interactions 3 22 23 . Interestingly, T cells immobilized on a lipid bilayer containing ICAM-1 and the MHC class II–peptide complex can be stimulated to move upon activation of PKC by phorbol esters 14 . It is well known that PKC is activated when T cells interact productively with APCs, and PKC-θ translocates to the sites of APC–T cell interaction 11 . Thus, it is possible that the stop signal is reversed by PKC upon T cell activation. The data also imply that the contact zone need not be stable for hours to mediate T cell activation, although T cells have to interact continuously with macrophages for ∼2 h to be activated. This notion is further supported by the observation that T cells that interacted continuously with antigen-presenting macrophages for 3 h, albeit dynamically, were considerably brighter than cells that interacted with the APCs for shorter periods of time. In contrast to macrophages, previous studies have demonstrated that B cells make stable 1:1 pairs with T cells during antigen presentation 24 25 , and we also observed that B cells invariably interact with DO11-GFP cells in a one to one fashion (data not shown). These observations make teleological sense, since macrophages need to activate many T cells to establish a concerted Th1 response, whereas a single B cell clone has more chance of being expanded by the focused and sustained delivery of cytokines. T cells need to interact with APCs for a sufficient length of time to allow the TCR to sample the large array of MHC class II–peptide complexes present on the surface of the APC. This requires that the motile T cell rearranges its cytoskeleton and becomes static. Our data demonstrate that this stasis is transient, and that the T cell–APC interaction is quite dynamic during antigen presentation. Our data indicate that T cells can be stimulated by summing up signals received during multiple sequential interactions with an antigen-presenting macrophage. | Other | biomedical | en | 0.999998 |
10613894 | The dorsal skin of 7–9-wk-old female C57BL/6J mice (BRL) or GR dim mice was shaved 4 d before experimentation. Mice were treated topically with 1–10 nmol TPA or with 50 μg dexamethasone (Sigma Chemical Co.) dissolved in 200 μl acetone. The animals were killed 0–6 h after application. Skin tissues were homogenized and RNA was prepared as described previously and analyzed by Northern blot . The probes for 18 S rRNA, HSP-27, and plasma glutathione peroxidase-3 (PGX-3) were obtained by reverse transcriptase PCR using a mouse skin RNA preparation. 6-μm paraffin sections from skin biopsies were subjected to in situ hybridization using 35 S-UTP–labeled sense and antisense probes of MMP-13 and MMP-3 as described in Gack et al. 1995 . Immunohistochemistry was performed using a polyclonal rabbit anti–mouse GR antibody (M20; Santa Cruz), followed by an ABC staining procedure (ABC Rabbit IgG Kit; Vector Labs, Inc.) according to the manufacturer's instructions. Atlas™ mouse cDNA expression array I filters were hybridized with radioactively labeled first strand cDNA following the specifications of the manufacturer (CLONTECH). Differences in expression patterns were analyzed using AIS software and Array Vision software module (Imaging Research). To establish an in vivo experimental system suitable to measure AP-1–dependent gene expression and to study mutual interference between AP-1 and GR in the animal, we first determined expression of MMP-3 and MMP-13 in mouse skin after treatment with TPA. In mock-treated animals, transcription levels of MMP-3 and MMP-13 were barely detectable . Upon application of TPA, an up to 100-fold induction of both genes was observed within four to six hours. Induction of both genes is dose-dependent, reaching maximal levels at 10 nmol, whereas 1 nmol of TPA was not sufficient for potent induction . Furthermore, upregulation was preceded by transcriptional activation of the main regulators of MMP-3 and MMP-13 gene expression, c-jun and c-fos . Induction of these immediate early genes was already detectable within one hour and declined at four hours in the case of c-jun , and between four and six hours for c-fos . To identify the specific cell type responsible for enhanced MMP-13 and MMP-3 gene expression in response to TPA, in situ hybridization analysis of parallel transversal sections of skin biopsies was performed. Whereas in untreated skin, no significant signals for the expression of MMP-13 and MMP-3 could be detected (data not shown), both genes were highly induced in a cell type-specific manner upon phorbol ester treatment . MMP-13 transcripts were observed in the epidermis only in a subset of basal keratinocytes . MMP-3 expression was found exclusively in monocytic cells in the dermal compartment . Interestingly, in contrast to primary and immortalized skin fibroblasts showing enhanced expression of both genes in response to TPA , neither MMP-13 nor MMP-3 transcripts were found in fibroblasts of the dermis . We next wanted to investigate whether GR could interfere with AP-1 activity under these conditions. Since the expression pattern of GR in murine skin has not yet been described, we first wanted to confirm the presence of GR in MMP-3– and MMP-13–expressing cells in the skin by immunohistochemistry. Strong expression of GR protein was observed throughout the epidermis and in mononuclear cells in the dermis . Weaker signals for GR protein were observed in dermal fibroblasts . These data show that GR is expressed by a broad range of cells in the skin, including MMP-13 and MMP-3 positive cells. Therefore, TPA-induced expression of MMP-13 and MMP-3 in skin in the presence or absence of GC is an appropriate system to measure GR-specific inhibition of gene expression in vivo. Consequently, we asked whether TPA-induced expression of MMP-13 and MMP-3 is repressed by dexamethasone in these cells. We found a complete inhibition of induction of both genes in skin after concomitant treatment with TPA and dexamethasone in skin . To analyze in more detail the molecular mechanism responsible for the repression of AP-1–dependent genes by GC in skin, we took advantage of GR dim mice. These mice carry a DNA binding-defective GR. Previously, transient transfection studies in tissue culture cells suggested that the DNA binding function of the GR is not required for transrepression of AP-1–mediated transcription . However, in light of the lack of induction of MMP-3 and MMP-13 expression by TPA in dermal fibroblasts in vivo and the complex regulatory processes present in skin, it is still possible that both DNA binding-dependent and -independent functions of GR are required for repression of TPA-induced expression of MMP-3 and MMP-13 in skin cells. To determine the importance of either one of these functions of GR, we analyzed the expression of both genes in GR dim mice. Expression of both MMP-3 and MMP-13 is dramatically induced upon TPA treatment in GR dim mice, similar to wild-type mice. In situ hybridization analysis confirmed that induction of these genes in GR dim mice originates from basal keratinocytes and monocytic cells (data not shown), resembling the pattern of expression in wild-type mice . Interestingly, induction was completely repressed by dexamethasone in both wild-type and GR dim mice , which strongly suggests that the DNA binding-dependent function of the GR is not required for repression of TPA-induced MMP gene expression in vivo. GR has been reported to either activate or repress c-jun expression in tissue culture cells, depending on the cell type. To confirm that GR-dependent repression of MMP-3 and MMP-13 cannot be explained by a loss of c-jun and/or c-fos expression upon hormone treatment, we measured the level of c-fo s and c-jun transcripts in unstimulated and stimulated mouse skin. Significant basal level of c-jun transcripts can be detected in wild-type and GR dim mice, which became further enhanced upon TPA treatment . Induction of c-fos was even more pronounced. Expression of c- jun , but not c-fos , was significantly induced by dexamethasone in both wild-type and GR dim mice, which might be mediated by the DNA binding-independent, positive function of GR on Jun/Jun homodimers binding to the c-jun promoter. Most importantly, dexamethasone reduced TPA-induced expression of c-fos and c-jun only slightly . The presence of c-jun and c-fos transcripts in TPA- and dexamethasone-treated animals confirmed that repression of phorbol ester-induced MMP-3 and MMP-13 expression is most likely due to inhibition of AP-1 activity by GR and cannot be explained by a loss of expression of the critical AP-1 components c-Jun and c-Fos. To prove abrogation of GR-mediated transactivation function in GR dim mice, we aimed to detect differences in GC-dependent regulation of GR target genes in skin cells of wild-type and GR dim mice. Therefore, we performed gene expression profiling on a mouse Atlas™ cDNA expression array. Filters containing spotted DNA from 588 known genes were hybridized in parallel with radiolabeled cDNA derived from RNA of skin from untreated and dexamethasone-treated mice. Among the differentially expressed genes (data not shown), two examples, PGX-3 and HSP-27, were analyzed by Northern blot analysis . In the skin of wild-type mice, expression of both PGX-3 and HSP-27 was significantly upregulated (4.3- and 2.9-fold, respectively) six hours after dexamethasone treatment. Enhanced levels of PGX-3, but not HSP-27, already were detectable after 1.5 hours (data not shown). Importantly, in GR dim mice, almost no elevation in mRNA levels of PGX-3 and HSP-27 is detectable . Taken together, negative interference of AP-1 by GR does not require the DNA binding function in vivo, since in wild-type and in GR dim mice, MMP-13 and MMP-3 are equally repressed, whereas genes positively regulated are activated by GC only in wild-type mice, but not in GR dim mice. We established an experimental system to induce transcription of the AP-1–dependent genes MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in skin by the application of TPA. In line with the critical role of de novo synthesis of c-Jun and c-Fos for full transcriptional activation of interstitial collagenases , we found a rapid upregulation of c-jun and c-fos mRNA upon TPA treatment in skin . In contrast to the coordinate induction of both genes in tissue culture cells , the expression pattern of MMP-13 and MMP-3 genes in the skin was restricted to distinct cell types. MMP-13 was expressed upon TPA treatment in basal keratinocytes, which are the sites of expression of MMP-13 observed during wound healing in mice . TPA-induced MMP-3 expression was mainly found in monocytic cells in the dermal compartment. However, we could not detect expression of MMP-3 in the basal layer of the epidermis by in situ hybridization, although others have observed MMP-3 expression in fractionated keratinocytes derived from TPA-treated epidermis . During experimental wound healing, induction of MMP-3 has been found in some cells of the mesenchymal compartment of the skin and in basal keratinocytes after 24 hours . Thus, most likely MMP-3 induced by TPA treatment only partially reflects the expression pattern observed during wounding. Surprisingly, in contrast to fibroblasts in tissue culture , we found no induction of either MMP-13 or MMP-3 in dermal fibroblasts upon TPA treatment of skin. The specific microenvironment composed of soluble factors and components of the extracellular matrix obviously does not allow the responsiveness of these cells to TPA by induced expression of MMPs. The molecular mechanism controlling cell type-specific upregulation of MMP-13 and MMP-3 transcripts in the skin remains to be elucidated. Importantly, the phorbol ester-induced expression of MMP-13 and MMP-3 was efficiently inhibited by GCs in a cell type-specific manner. To analyze if DNA binding of the GR is involved in this downregulation of MMP-13 and MMP-3 in vivo, we made use of GR dim mice. To address the question whether gene activation by GCs is indeed impaired in GR dim mice, we identified GC-induced genes in the skin using a high-density filter screening approach. PGX-3 and HSP27 were among the strongest induced genes and as yet have not been described to be responsive to GCs. For both PGX-3 and HSP27, we could show a failure of GC-mediated upregulation in GR dim mice. In contrast to loss of GC-dependent gene activation GR dim mice, TPA-mediated expression of AP-1 target genes, such as MMP-13, MMP-3, and MMP-9 (gelatinase B; data not shown) is efficiently downregulated by dexamethasone. Obviously, the DNA binding function of GR and subsequent transcriptional activation of GRE-dependent genes is not required for transrepression of AP-1 in vivo. The presence of c-fos and c-jun transcripts upon cotreatment with TPA and dexamethasone suggests that the de novo synthesis of both AP-1 components is not abolished by GCs. Specifically, induction of c-jun by TPA, which requires the activity of MAP kinases, such as JNK, to hyperphosphorylate preexisting c-Jun protein, was not repressed by GCs. Thus, inhibition of the JNK pathway by GR, which was described in tissue culture cells , may not play a major role in vivo, at least in skin. In transgenic mice, overexpression of c-jun and c-fos , as well as tissue-specific expression of the AP-1 target genes, human collagenase and stromelysin, induces, or at least enhances, tumorigenesis . Downregulation of AP-1 target gene expression has been proposed to be a crucial event in GC-mediated inhibition of tumor formation in the multiple stage model of carcinogenesis in skin . Here, we demonstrate that TPA, one of the most potent and best characterized tumor promoters in mouse skin carcinogenesis, induces MMP-13 and MMP-3 very rapidly at the same dosage, which is, when applied periodically, optimal to mediate tumor formation . The critical role of AP-1 in MMP-13 and MMP-3 expression and the downmodulation of expression of both genes by GCs strongly suggest that transrepression of AP-1 activity is a key feature of antitumor promoting activity of GCs. The finding that this activity of GR does not require the DNA binding function might be highly valuable in the search for better therapeutical strategies of GC application in skin carcinogenesis and related diseases. | Study | biomedical | en | 0.999996 |
10613895 | Embryos were fixed (37% formaldehyde) and stained with purified polyclonal anti–γ-tubulin (made against a peptide present in the maternal isoform of γ-tubulin [QIDYPQESPAVEASKAG]), anti–β-tubulin antibody (1:100 dilution; Amersham). Fluorescent secondaries were used at 1:300 (Jackson ImmunoResearch Laboratories, Inc.). DNA was stained with 10 μg/ml of bisbenzamid . Fluorescence was observed on a Leica DMRD or an Olympus 1X70 microscope. Photographs were taken on the Leica using a Photometrics CH250 CCD and 35-mm cameras. Three-dimensional data sets of centrosomes were collected using Deltavision software (Applied Precision, Inc.), based on an Olympus 1X-70 microscope with a Photometrics PXL-cooled CCD camera. Data sets were projected onto a single plane and exported to Adobe Photoshop for further processing. Embryos were collected for 2 h from a stock carrying transgenes encoding nondegradable versions of the Drosophila mitotic cyclins, cyclin A and cyclin B (homozygous for both transgenes) . After aging the collection for 2 h at 25°C, stable cyclins were induced by floating the agar collection plates on 37°C water for 30 min (heat shock). The embryos were allowed to recover for 1–2 h and fixed. string7B and fizzy1 are amorphic alleles. Embryos were prepared for EM by high pressure freezing, followed by freeze substitution , or by slight modifications of a glutaraldehyde fixation protocol ( string and fizzy mutants). Thin serial sections (80–90 nm) cut on a Leica ultramicrotome were stained and examined on a Philips EM 400 at 80 kV, and the images were recorded on film. Though only one section is shown, all centrioles were reconstructed from thin serial sections to assess separation and orientation of centrioles. Embryos were stained with Hoechst and homozygous fizzy mutant embryos were identified based on their abnormally high mitotic index during late stage 11 and stage 12. string embryos were identified by a low cell density and the absence of mitotic figures. Cell cycle stages in wild-type embryos, prepared for EM, were identified based on the morphology of the associated nuclei. Cells with condensing/condensed chromatin not fully aligned at the metaphase plate were identified as prophase; cells with a mitotic spindle and aligned chromosomes as metaphase; cells containing decondensed chromatin in nuclei that are separated away from each other as telophase; and cells with intact nuclear membranes containing decondensed chromatin as interphase cells. Despite differences in lengths of G2, cells enter mitosis with only two centrosomes, suggesting that centrosome replication is coupled to cell cycle progress, or that centrosome replication is independently, yet appropriately, regulated (e.g., by a developmental timer) . To distinguish between these possibilities, we monitored centrosome duplication in string mutant embryos that arrest in G2 but continue to develop. Arrested cells can be driven into mitosis by expression of Cdc25 string transcribed from a heat shock–inducible promoter on a transgene . After delaying cells in G2 beyond their normal time of mitosis, we induced expression of Cdc25 string . β-Tubulin staining of the resultant mitotic cells revealed no significant spindle abnormalities (99.4% bipolar, n = 1,350) (data not shown). Immunostaining for γ-tubulin revealed two foci of staining, one at each pole of 42 preanaphase spindles . In the absence of coupling, we would expect to see four centrosomes in cells that had remained in G2 beyond their normal time of entry into mitosis. We conclude that centrosome duplication does not continue during a G2 arrest. To assess the ultrastructure of the centrioles during the G2 arrest, we examined string mutant embryos by EM. In 21 out of 22 centriole pairs ( Table ), the daughters were significantly shorter than the mothers and their tubular fine structure was not obvious . We conclude that Cdc25 string is required, directly or indirectly, for the completion of daughter centriole assembly. We investigated for a role of the APC in the centrosome cycle by examining centrioles in embryos mutant for the APC activator, Cdc20 fizzy . fizzy embryos exhibit mitotically arrested cells in the ventral epidermis (VE) during cell cycle 16 . However, morphogenesis continues, providing a timer to estimate the duration of the mitotic arrest. We scored centriole pairs as engaged, if they were at right angles to each other and closely spaced (48–56 nm). In late stage 11 fizzy embryos, >80% of cells in the VE are arrested at metaphase and 62% of the metaphase cells ( n = 21, from two embryos) contained engaged centrioles . If centriole disengagement was unperturbed in fizzy mutants, only cells traversing metaphase should have engaged centrioles. Since fewer than 5% of the VE cells are normally in metaphase at any time (in wild type), <5% of the cells in fizzy mutant embryos would be traversing metaphase. Since ∼50% of cells in the VE contained orthogonal centrioles (62% of the metaphase cells), we conclude that centriole disengagement is delayed in metaphase cells arrested as a result of Cdc20 fizzy depletion. Older fizzy embryos exhibited disengaged centrioles. In early stage 12 embryos, corresponding to ∼30 min of arrest, all observed mother and daughter centrioles appeared disengaged but not widely separated . During midstage 12, corresponding to ∼1 h of arrest, all observed centriole pairs had disengaged and separated . Thus, disengagement and separation of centrioles, events that normally occur within a couple of minutes after metaphase, occur more slowly in the fizzy mutant. We conclude that Cdc20 fizzy is required, directly or indirectly, for timely centriole disengagement. We monitored the centrosome cycle upon expression of stable cyclins, which blocks some aspects of mitosis. Destruction box and NH 2 terminally deleted forms of both cyclins A and B were ectopically expressed when transgenic embryos were exposed to heat shock. Entry into mitosis occurred at the normal time in these embryos, but cells failed to exit mitosis . Immunostaining for γ-tubulin revealed no more than two centrosomes at each pole (50% had two centrosomes, 23% had one, and 27% had one elongated centrosome, n = 51 from two embryos) . To explore the status of centrioles, we examined stable cyclin-expressing embryos by EM. Centrioles at 17 spindle poles had disengaged with varying degrees of separation (between 80 nm to 1 μm), consistent with the distribution of γ-tubulin foci. Importantly, all observed centrioles ( n = 39; Table ), were present as singlets, i.e., were not associated with daughter centrioles . Possible heat shock effects independent of transgene induction were investigated by monitoring events in the centrosome cycle upon heat shock of nontransgenic embryos. Both cell cycle progression and procentriole formation continued after heat shock (data not shown). We conclude that stabilization of mitotic cyclins can, directly or indirectly, inhibit production of procentrioles. Cell doubling requires the coordination of many cellular events including DNA replication and centrosome duplication. We show that the activities of mitotic regulators influence progress of the centrosome cycle in addition to the nuclear cycle . Cdc25 string , which induces entry into mitosis, is required for the completion of daughter centriole assembly. Cdc20 fizzy triggers chromosome separation by APC-dependent degradation of inhibitors and is required for timely centriole disengagement. Destruction of cyclins at mitosis is required for synthesis of new daughter DNA strands and elaboration of a new daughter centriole. By genetically arresting cell cycle progress at specific points and identifying a correspondingly specific arrest of the centrosome cycle, we have resolved multiple points of coupling between the cell cycle and the centrosome cycle. Each point of coupling might represent direct action of the cell cycle regulator on the centrosome cycle, or a checkpoint induced as a consequence of the cell cycle arrest. Ultimately, available genetic and molecular tools ought to allow identification of the specific events responsible for the observed coupling. In Drosophila embryos, a procentriole appears during S phase, but its assembly is completed during mitosis . We found that Cdc25 string function, the expression of which drives cells into mitosis, is required for the completion of daughter centriole assembly. To our knowledge, this is the first indication that a G2 arrest does not support daughter centriole maturation. However, it is possible that centriole maturation continues at a slow rate in the absence of Cdc25 string function and is accelerated upon its expression. The centriole maturation induced by Cdc25 string might be secondary to the activation of mitotic cyclin/Cdk1 and subsequent mitotic events. For example, since several centrosomal proteins are recruited to the nucleus during interphase, nuclear membrane breakdown at mitosis might stimulate centriolar growth by releasing previously sequestered components. The EM analysis of fizzy embryos reveals that disengagement and separation of mother and daughter centrioles are delayed: disengagement for ∼10 times the length of metaphase. Requirement for Cdc20 fizzy in timely centriole disengagement could reflect a corresponding requirement for degradation of an APC substrate that inhibits centriole disengagement. The block to centriole disengagement might be incomplete because residual maternal Cdc20 fizzy could slowly promote centriole disengagement in mitotically arrested cells. Alternatively, centriole disengagement might be timed by activation of Cdc20 fizzy , while not absolutely requiring its function. For example, slow Cdc20 fizzy -independent separation might reflect APC-independent turnover of an inhibitor of centriole disengagement. The mitotic cyclins are degraded in a fizzy -dependent fashion at the mitotic exit , and could be candidates for the inhibitors of centriole disengagement. However, cyclins A and B persist in fizzy mutant embryos at times when we observe disengagement . Additionally, ectopic expression of stabilized cyclins A and B does not block centriole disengagement but inhibits assembly of new procentrioles. Similar results have been obtained upon injection of stable cyclin B into sea urchin embryos . Inhibition of procentriole formation during mitosis may reflect a requirement for nuclear envelope formation (say for nuclear sequestration of inhibitors) or dephosphorylation of certain key proteins upon downregulation of mitotic cyclin/Cdk1. We infer that in an unperturbed cell, downregulation of mitotic cyclin/Cdk1 is essential for procentriole formation. How might these observed points of coupling be enforced? Cell cycle regulators could contribute rather indirectly to progression through the centrosome cycle. In the yeast Saccharomyces cerevisiae , spindle pole body duplication is coordinated with the nuclear cycle by cell cycle–specific transcription of spindle pole body components . Two of the three points of coupling that we have uncovered in Drosophila operate within mitosis, during which there is no transcription . Hence, mitotic maturation of daughter centrioles and disengagement of centriole pairs are apparently regulated posttranscriptionally. In contrast, procentriole formation might require new gene expression and would be deferred until transcription resumes upon mitotic exit. While such a mechanism is possible, it would be short circuited by the persistence of maternal gene products, which appears to play a significant role in the progression of the postblastoderm cell cycles. The present work demonstrates coupling of three steps in the centrosome duplication cycle to transitions in the cell cycle. However, uncoupled centrosome duplication has been observed in early embryonic systems and tissue culture cell lines in the absence of visible cell cycle progression . These disparate observations could be explained if coupling is conserved, but unseen local oscillations of activities of cell cycle regulators (maybe even at the centrosome) drive the centrosome cycle. Alternatively, perhaps the coupling itself is differentially regulated in various species and at different stages of development. Thus, the syncytial cell cycles (that precede the postblastoderm cell cycles) may exhibit regulation of the centrosome cycle similar to sea urchin and Xenopus . One mechanism that could account for regulated coupling is regulated expression of inhibitors that block specific steps in the centrosome cycle which, in turn, are then inhibited by cell cycle regulators. Cyclin E has been shown recently to be required for centrosome duplication . During the postblastoderm cell cycles, cyclin E/Cdk2 activity is continually high . Our data do not address roles for cyclin E in Drosophila , but indicate that its continuous activity is not sufficient for continued centrosome duplication in the absence of the requirements that we have uncovered. | Study | biomedical | en | 0.999999 |
10613896 | pDM128, pDM138, pDM121 , pCEVEps15, and pCEVEps15R have been previously described. pMTHrb and pMTHrbl were generated by subcloning the open reading frame of the human cDNAs into the pMT2 vector. pCEVαEps15 harbors the Eps15 cDNA in the anti-sense orientation in the LTR-based pCEV vector. pMTHA-HrbNPV was generated by changing the sequences coding for the four NPF motifs to sequences coding for NPV in the pMTHA-Hrb vector . pCEVEps15ΔEH, encoding a Eps15 protein devoid of its EH domains (and encompassing amino acids [aa] 342–897), was engineered by PCR and subcloned into pCEV. All constructs were sequenced in the regions that underwent genetic manipulations. CV-1 cells were transfected by the calcium phosphate method, with the CAT (chloramphenycol acetyltransferase) reporter plasmid pDM128 (100 ng) together with the Rev expression vector pDM121 (10 ng) and the pCMVβgal reporter plasmid (Clontech, 100 ng). Various combinations of pMT-Hrb (0.25 μg), pMT-Hrbl (0.25 μg), pCEVEps15 (1 μg), and pCEVEps15R (1 μg) were also transfected. 48 h after transfection, cellular lysates were tested for β-galactosidase reporter activity using a commercial kit (Promega). Cellular lysates, normalized for β-galactosidase activity, were tested for CAT activity using a commercial kit (Promega). Immunoprecipitation, subcellular fractionization, and immunoblotting were performed as previously described . Antibodies used were: anti-Eps15 and anti-Eps15R sera; polyclonal anti-Hrb IgGs directed against the last 19 amino acids of Hrb (Santa Cruz Biotechnology); a polyclonal anti-Hrbl serum recognizing amino acids 280–481 of the protein. Routinely, immunoblots were stripped and reprobed with an anti-tubulin antibody, to ensure equal protein loading in the various lanes (not shown). To gain insight into the contribution of Eps15 and Eps15R to the nucleocytoplasmic export pathway used by Rev, we used a previously described assay based on cotransfection of Rev with the reporter plasmid pDM128 . Transcripts from this latter construct contain a CAT coding sequence within an intron from the env region of HIV-1, which includes an RRE. Upon cotransfection, Rev binds to the RRE, thus allowing cytoplasmic translocation and expression of the unspliced transcripts . In preliminary experiments (not shown), we transiently cotransfected CV-1 cells with pDM128 and increasing amounts of a Rev expression vector pDM121. Transactivation of CAT by Rev was linear in a range from 4–100-fold activation. For all subsequent experiments we used an amount of pDM121 yielding ∼20% of the maximal transactivation. When expression vectors for Eps15 or Eps15R were cotransfected with Rev, an increase in CAT activity, ∼50% greater than the value obtained with Rev alone, was reproducibly detected . Expression vectors for Hrb, a known cofactor of Rev, and for Hrbl yielded a comparable 50% increase in Rev activity . All responses were Rev dependent, since they could be abolished by replacing pDM128 with pDM138, a variant construct lacking the RRE sequence , or by omitting the pDM121 construct (not shown). The effects were also not due to influence of Eps15, Eps15R, Hrb, or Hrbl on transcription, since none of the corresponding vectors affected expression of an exonic CAT gene under the control of an SV-40 or RSV promoter in the absence of the RRE (not shown). To prove the physiological relevance of Eps15 to the Rev export pathways, we used an antisense Eps15 construct (pCEVαEps15). Transfection of pCEVαEps15 into CV-1 cells significantly reduced the steady state levels of Eps15, as revealed by both immunoblotting and immunofluorescence analyses . When pCEVαEps15 was cotransfected with pDM128 and the Rev expression vector, a dose-dependent reduction in the activity of Rev was observed . The finding that Eps15 and Eps15R can function as cofactors for the Rev export pathway prompted us to investigate whether they also synergize with Hrb and Hrbl. We cotransfected, together with Rev and the reporter pDM128, various combinations of the four molecules . The transfected cells expressed three- to fivefold higher levels of these proteins than mock-transfected cells . A synergistic effect of coexpressing Eps15 or Eps15R with either Hrb or Hrbl, was readily observable . Whereas transfection of the individual plasmids (Eps15, Eps15R, Hrb, and Hrbl) brought about an ∼1.5-fold increase in CAT activity, compared with Rev alone, various combinations (Eps15/Hrb, Eps15/Hrbl, Eps15R/Hrb, and Eps15R/Hrbl) caused an ∼3.0–3.5-fold increase. Maximal activation was thus significantly superior to the sum of the individual effects, suggesting that Eps15 (or Eps15R) and Hrb (or Hrbl) can synergistically activate the Rev export pathway. Binding of Hrb to the EH domains of Eps15 depends on the presence of four NPF (asparagine-proline-phenylalanine)-containing motifs in its COOH terminus . Mutagenesis of any of the residues in the NPF consensus abolishes the binding of EH domains to their targets . We engineered an HA-tagged mutant of Hrb (HA - Hrb-NPV) in which the phenylalanines of its four NPF motifs were substituted with valines. The HA-Hrb-NPV mutant and a wild-type HA-tagged Hrb (HA-Hrb) could be expressed at comparable levels . However, HA-Hrb-NPV could no longer be coimmunoprecipitated with Eps15 . HA-Hrb-NPV was also unable to increase CAT expression, when compared with Rev alone, and, most importantly, was unable to synergize with Eps15 . We also engineered an Eps15 mutant devoid of its EH domains (Eps15ΔEH). We have previously shown that the EH domains are necessary and sufficient for binding of Eps15 to Hrb . In the Rev-dependent CAT assay, Eps15ΔEH was not able to potentiate Rev activity, nor was it able to synergize with wild-type Hrb . Thus the EH-mediated interaction between Eps15 and Hrb is required for their synergistic effect in the Rev export pathway. Hrb has been reported to have a nuclear localization, with either nucleolar accumulation or a nucleoporin-like staining pattern . The existence of a cytosolic fraction has also been reported . Such a fraction should in principle be the sole one available for interaction with Eps15, which is exclusively localized to the cytoplasm . We determined colocalization of Eps15 and Hrb by confocal microscopy. An affinity purified anti-Hrb peptide serum was used which recognized a single band in Western blot . As previously described, Eps15 showed a punctate cytosolic distribution . Hrb was present both in the nucleus and in extranuclear compartments . The extranuclear fraction of Hrb displayed a punctate morphology. The majority of these punctate structures were also stained specifically by the anti-Eps15 antibody . We do not know the nature of these structures, which do not contain AP2, AP1 and various markers for the ER and Golgi compartments (not show). The nuclear fraction of Hrb was homogeneously dispersed and did not show nucleolar accumulation or a clear nucleoporin-like pattern. A biochemical subcellular fractionization confirmed the nuclear and cytosolic localization of Hrb . Of note, by both methods, the majority of Hrb appeared to be present in the cytosolic compartment. The above results suggest that the action of a Eps15–Hrb complex must somehow be exerted at the cytosolic level. In search of a possible mechanism, we measured the steady state levels of Rev in the presence of Eps15 alone or in combination with Hrb/Hrbl. Transfection of Eps15 or Hrb or Hrbl, together with Rev, caused a modest increase in the steady state levels of Rev . However, when Rev was cotransfected with Eps15 with either Hrb or Hrbl, a four- to sixfold increase in Rev levels was detected . These differences were not due to variable transfection efficiency, since all cellular lysates displayed the same amount of β-galactosidase activity, which was expressed by the cotransfected plasmid pCMVβ-gal (see Materials and Methods), and used as an internal standard. We have previously shown that Eps15 (and Eps15R) is associated in vivo with Hrb through its EH domain . In this study, we showed, by an antisense approach, that Eps15 is physiologically required in the Rev export pathway. Likewise, overexpression of Eps15 (or of Eps15R) led to a small, yet reproducible, increase in Rev activity. The difference in the magnitude of the effects in the two types of experiments suggests that availability of endogenous Eps15 is not a limiting factor in the Rev export pathway. We further showed that Eps15 and Eps15R synergize with Hrb (and Hrbl) in the control of Rev activity and that they do so through their EH-mediated physical interaction. Since Hrb has been directly implicated in the control of the Rev export pathway, our results indicate that an Eps15–Hrb complex is involved in this pathway. Hrb is thought to be a recruiter of Rev to the nuclear pore, either directly or, more likely, through interaction with Crm1 . Hrb might, therefore, function as a nucleoporin, as also suggested by the presence of FG nucleoporin-like repeats. However, Bogerd et al. 1995 failed to detect a clear localization of Hrb in the nuclear membrane, suggesting that Rev might sequentially interact with Hrb and then with authentic nucleoporins. We also failed to observe a nucleoporin-like localization of Hrb. Our results instead point to an unexpected site of action of Hrb, i.e., in an extranuclear compartment where the majority of Hrb localizes to punctate structures where also Eps15 is present. Together with the findings of a physical interaction between Eps15 and Hrb and of its role in Rev activation (this paper), these results indicate that an important role of Hrb is exerted on the cytosolic side of nucleocytoplasmic transport. The subcellular localization of Hrb is, therefore, critical to the understanding of its function. Discordant findings have been reported in literature, which, therefore, deserve further comment. Bogerd et al. 1995 reported significant levels of Hrb in the cytosolic fraction of HeLa cells, whereas Fritz et al. 1995 reported exclusive partitioning in the nuclear fraction in the same cells. Our results agree with those of Bogerd et al., except that the magnitude of the cytosolic pool appears relatively greater under our conditions of analysis. Our morphological analysis, however, diverges from previous literature in which Hrb was found predominantly in the nucleus by immunofluorescence . We note that in previous studies polyclonal anti-Hrb antibodies were used that were generated against portions of Hrb containing multiple FG repeats and displaying homology to other nucleoporins. One might speculate that those sera were cross-reactive with authentic nucleoporins. In our case, we used an anti-peptide serum, which was generated against a portion of Hrb without significant homologies. The mechanisms by which a cytosolic Eps15–Hrb complex participates to the regulation of the Rev export pathway remain to be elucidated. A direct effect on the rate of nucleocytoplasmic export of Rev might result in altered partitioning of Rev. However, attempts to verify whether overexpression of Eps15 and Hrb could alter the nuclear vs. cytosolic partitioning of Rev did not reveal any significant effect (not shown). However, we noticed, increased levels of immunoreactive Rev (not shown). This prompted us to perform a quantitative biochemical analysis of the steady state levels of Rev under conditions of overexpression of Eps15 and Hrb. Our finding of a significant increase in Rev levels upon concomitant overexpression of the two proteins provides a mechanistic basis for their synergistic effect on Rev activity. Increased Rev levels might reflect stabilization of the protein, or of its mRNA, or an increased transcription/translation rate. The possibility that Eps15–Hrb affects Rev stability is an intriguing one. Rev is less stable in the cytosol than in the nucleus , thus predicting the existence in the cytosol of a molecular machinery able to divert it from its metabolic destiny, to allow reimport into the nucleus. The Eps15–Hrb complex might be part of this machinery. The Rev export pathway has recently come into focus as a major mechanism involved in the regulation of levels of important proteins such as hdm2, p53, and NUMB, by targeting them to cellular compartments (nucleus or cytosol) appropriate for their degradation by the 26S proteasome . It is thus possible that the Eps15–Hrb complex is involved in the regulation of the trafficking of cellular proteins whose localization and stability are controlled through the Rev export pathway. The EH network is implicated in the control of endocytosis and actin cytoskeleton organization . Our results indicate additional levels of involvement, i.e., modulation of nucleocytoplasmic shuttling and possibly of protein degradation. We note that both Eps15 and Eps15R possess FG repeats in their COOH termini. In addition, another component of the clathrin coat, AP-180 also displays FG repeats. It will be of interest, therefore, to analyze whether endocytic proteins interact with members of the importin family. If true, this would unveil an unexpected convergence of molecular machineries governing vesicle sorting and nucleocytoplasmic shuttling. | Study | biomedical | en | 0.999997 |
10613897 | Recombinant baculovirus was isolated by conventional protocols . For HMM expression, Sf9 cells in suspension culture were coinfected with two recombinant viral stocks, one coding for the heavy chain and one coding for both the RLC and essential light chains (ELCs). The heavy chain was cloned with a FLAG-tag at the COOH terminus to facilitate purification (DYKDDDDK). The cells were harvested at 65–75 h, and the recombinant proteins isolated on an anti-FLAG affinity column (Sigma Chemical Co.). The expressed HMM yielded a homogeneous product with intact heavy and light chains. Thiophosphorylated HMM was prepared by incubation of unphosphorylated HMM with ATP-γ-S, CaCl 2 , myosin light chain kinase, and calmodulin. Crystallization of both unphosphorylated and thiophosphorylated HMM was achieved using a positively charged lipid monolayer, essentially as described previously . Crystals were found over a range of conditions in a phosphate buffer containing NaCl, MgCl 2 , EGTA, and ATP in combination with polyethylene glycol. The arrays typically appeared within 1–3 d, and degraded quickly thereafter. Specimens for EM were made by transferring the crystals to 200–300-mesh copper grids with reticulated carbon support film . Crystals were negatively stained with 2% uranyl acetate. After drying, the specimens were stabilized by vacuum deposition of a thin layer of carbon before examination. Micrographs where recorded on a Philips CM120 electron microscope at 120 kV acceleration voltage and a magnification of 28,000 and 35,000, respectively, under standard conditions. Electron micrographs initially were screened by optical diffraction and then digitized on a Perkin Elmer PDS1010M microdensitometer with a step size of 0.46 or 0.37 nm, with respect to the original object. Correction for lattice distortions was performed using SPECTRA before calculating structure factors. The defocus was determined using ICE and corrected using CTFAPPPLY from the MRC package . Structure factor data from the different images were merged using a modified version of an origin refinement program originally written by S.D. Fuller (EMBL, Heidelberg, Germany) and averaged in plane group P2. Maps were calculated using the CCP4 software and then imported into O for model building. The ten micrographs of unphosphorylated HMM that were analyzed had average unit cell dimensions of a = 11.95 ± 0.25 nm, b = 28.20 ± 0.89 nm, and γ = 90.6° ± 0.8°. Structure factors were refined to a common phase origin and symmetrized in the plane group P2. This yielded a total of 76 averaged structure factors with a diffraction spot quality of IQ ≤ 4, as defined by Henderson et al. 1986 . The averaged phase residual was 10°, extending to a resolution of 2.3 nm. The 21 images of thiophosphorylated HMM that were analyzed had averaged unit cell dimensions of a = 15.53 ± 0.49 nm, b = 18.38 ± 0.24 nm, and γ = 80.2° ± 1.7°. A total of 65 unique averaged structure factors were obtained (IQ ≤ 4) with an average phase residual of 11°, extending to a resolution of 2.3 nm. We constructed an S1 model based on the high resolution X-ray structure of the smooth muscle motor domain plus ELC (MDE) with bound MgADP·AlF 4 − , a transition state analogue . It should be noted that this structure was indistinguishable from one obtained with an ATP analogue, MgADP·BeF x . Thus, the MDE·MgADP·AlF 4 − crystal structure should mimic the head conformation in our 2-D crystals produced with MgATP. The skeletal muscle RLC and the heavy chain associated with it were then modeled onto the smooth MDE crystal structure by aligning homologous regions of the light chain binding domain. Figures of the model were prepared using Bobscript 2.3 . 2-D crystalline arrays of inhibited, unphosphorylated HMM on lipid monolayers were obtained in the presence of MgATP (see Materials and Methods). The arrangement of heads in unphosphorylated HMM is highly asymmetric. To further interpret the projection maps , an atomic model for smooth muscle S1 was docked into the electron density. Even in projection, the docking is relatively unambiguous. All of the density seen in projection can be accounted for by the two heads. No obvious feature corresponding to the S2 fragment of the rod is visible. Without modifying the S1 model, a relatively good alignment can be achieved . In this alignment, the long axis of one of the motor domains is oriented nearly perpendicular to the plane of the crystal, and the other is oriented nearly within the plane. The highest density in the projection occurs where the motor domain is oriented perpendicular to the crystal plane. However, the COOH-terminal ends of the heads are relatively far apart, and some density is unaccounted for by this model. To move the COOH termini closer together and achieve a better fit, it was necessary to modify the position of one of the light chain binding domains. An ∼30° rotation of the light chain binding domain about gly779 brings the light chain binding domain, in particular the region where the RLC is located, into density that had previously been unaccounted for, and at the same time brings the COOH-terminal residues closer together to form a vertex at the head–rod junction . The quality of the model was assessed by projecting the rebuilt X-ray coordinates into a 2-D image for comparison with the initial electron density map . The phase residual for this model when compared with the original data is 25°. This is reasonably good considering that solvent and negative stain effects have not been incorporated into the model. The important molecular contacts within the crystal occur between the motor domains of the same unphosphorylated HMM. The actin-binding interface of one head (the blocked head) is located in close proximity to the converter domain of the companion head (the free head). The unit cell is smaller in the crystals of thiophosphorylated HMM, but the projected density and twofold symmetry requires incorporation of two HMM molecules into the unit cell. Thus, the protein packing is much denser in these crystals than in the unphosphorylated HMM crystals. The docking of the smooth muscle S1 model into the thiophosphorylated HMM projection shown in Fig. 2 , a–c, is the only way we have found to fit two HMM molecules into the unit cell and account for the mass in the electron density map, with the reasonable assumption that the two HMM molecules within the unit cell are the same. As was found for the unphosphorylated HMM crystals, the long dimension of the motor domain of one of the heads extends out of the plane of the crystal, whereas the other lies more within the plane. As with the unphosphorylated HMM, it was necessary to rebuild the light chain binding domain of one of the heads to obtain a model where the two light chain binding domains connect to form the beginning of the S2 region . The rod region is not identifiable in the projection. The agreement between the model and the reconstruction was evaluated by projecting the model coordinates for comparison with the experimental electron density . The phase residual for the model when cross-correlated with the original reconstruction is 31°. In contrast to the model for the unphosphorylated HMM, the contacts between heads appear to be intermolecular, rather than intramolecular. Moreover, it is possible to build a head-to-head interaction in the thiophosphorylated HMM crystals roughly similar to that constructed in the unphosphorylated HMM crystals, but the interacting heads come from different HMM molecules. This is the first report of 2-D crystalline arrays of unphosphorylated and thiophosphorylated smooth muscle HMM, which provides the highest resolution structure of a double-headed myosin to date. Both 2-D arrays were formed in the presence of ATP, a state that cannot be analyzed using decorated F-actin because of its weak binding properties. The major new finding is the direct observation of interactions between the heads of one HMM molecule in the inhibited, unphosphorylated state. Such contacts have long been inferred from biochemical, structural, and mutagenic data. In contrast, only contacts between different HMM molecules are observed in the active thiophosphorylated HMM. The thiophosphorylated HMM structure is probably one of many conformations; the particular conformation obtained here is stabilized in the crystal by packing forces. Mobility of the two heads in the active state is a key feature of myosin's ability to search for actin monomers in adjacent thin filaments. The docking of an atomic model for the myosin head into our 2-D projections allows a model for the disposition of the heads in the active and inhibited states to be proposed. The crystal structure used for docking into the electron density is expected to be structurally similar to that formed in the 2-D crystals in the presence of MgATP. Consistent with this idea, one head of the HMM molecule in both the unphosphorylated and the thiophosphorylated HMM was fitted with this model without modification. The light chain binding domain of the second head required modification; this region of the molecule undergoes large rigid body movements during the power stroke. The arrangement of heads in both the unphosphorylated and the thiophosphorylated HMM is highly asymmetric. This differentiates our data from the only previous structural model for HMM that was obtained by modeling and energy-minimizing the head–tail junction of scallop myosin. In that model, the myosin heads are related by a molecular twofold rotation axis. The present results, though not excluding such a possibility under some conditions, favors asymmetric models and suggests that the heads are relatively independent of each other. Significantly, in the unphosphorylated HMM, the actin-binding interface on one head is positioned in the middle of the other head near the converter domain. This arrangement leaves the actin-binding domain of one head free while the other is blocked. The interaction between heads seen with the unphosphorylated HMM can explain several existing pieces of experimental data. One is that the binding constant of unphosphorylated and phosphorylated HMM to actin is only three- to fourfold different, although phosphate release is regulated several 100-fold . The model proposed here shows that only one of the two heads of unphosphorylated HMM would be capable of interacting with actin, and thus the difference in binding constant could reflect the difference between single-headed binding in the inhibited state versus double-headed binding in the active state. More importantly, the interaction of one head with the converter region of the adjacent head could prevent the domain motions that are required to open myosin's back door for phosphate release from that head . A large rotation (70°) of the converter region has been shown to occur as myosin goes from the closed to the open conformation . Mutagenesis studies also showed that conversion of the converter domain from a smooth to a skeletal muscle sequence produced a chimera that was not regulated by phosphorylation . Although these experiments were interpreted as suggesting that sequences near the neck/motor domain interface are important for properly positioning the RLCs in an inhibitory position, the data could also accommodate the idea that there is a direct interaction between heads that involves the converter region. The 2-D projection map is currently being extended into the third dimension, which will enable us to determine the interaction sites leading to the inhibited state more precisely. Nonetheless, this study has already provided a general mechanism by which product release could be inhibited from both heads (direct blocking of the actin-binding interface in one head, inhibition of converter rotation in the other), while maintaining one head's ability to bind to actin in the presence of MgATP. | Study | biomedical | en | 0.999995 |
10613898 | Mouse myoblast cells C2C12 and African green monkey COS-7 cells were from American Type Culture Collection. Cells were maintained in DME supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Rabbit anti–β1 integrin antibody MC231 was kindly provided by Dr. John A. McDonald (Mayo Clinic, Scottsdale, AZ). Mouse monoclonal antimyogenin F5D was obtained from the Developmental Studies Hybridoma Bank. A cDNA fragment encoding the cytoplasmic domain of human integrin β1D (residues 749–801) was amplified by PCR and inserted into the EcoRI/XhoI site in the pLexA vector (pLexA/β1D). The bait construct was introduced into EGY48 (p8op-lacZ) yeast cells by transformation. The transformants were used to screen a human heart MATCHMAKER LexA cDNA library (>3 × 10 6 independent clones) as described previously . To analyze protein–protein interaction between MIBP and β1 mutants, yeast cells were cotransformed with pB42AD and pLexA expression vectors encoding MIBP and the β1 sequences. The transformants were plated and growth of blue colonies in the leucine -deficient medium indicates a positive interaction . A MIBP cDNA probe was prepared by labeling the full-length human MIBP cDNA using an AlkPhos-direct labeling-detection system (Amersham Pharmacia Biotech). A blot containing equal amounts of polyA + RNA (2 μg/lane) from different human tissues (Clontech Laboratories, Inc.) was hybridized with the MIBP probe. The hybridized mRNA bands were detected with CDP- Star Detection System (Amersham Pharmacia Biotech). Human fetal tissues were washed twice with PBS, homogenized in lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 0.2 mM 4-(2-aminoethyl)benzenesulfonylfluoride, HCl, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, and 5 μg/ml leupeptin), and analyzed by immunoblotting with monoclonal anti-MIBP antibody 5B4.7. DNA constructs encoding maltose binding protein (MBP)–MIBP (pMAL-c2/MIBP), glutathione S -transferase (GST)–β1D (pGEX-5x-1/β1D), and His–MIBP (pET-15b/MIBP) fusion proteins were generated by inserting cDNAs encoding full-length or partial sequences of human MIBP and β1D integrin into the corresponding vectors. The recombinant vectors were used to transform Escherichia coli cells, and the recombinant proteins were purified with glutathione–Sepharose 4B beads, amylose-agarose beads, and His-Bind R Resin (Novagen), respectively. Mouse monoclonal anti-MIBP antibody was generated using purified His–MIBP recombinant protein as an antigen . Hybridoma supernatants were screened for anti-MIBP antibody activity by ELISA and immunoblotting. One mAb (clone 5B4.7) recognized MBP–MIBP and His–MIBP but not MBP or irrelevant His-tagged proteins. For direct binding assays, glutathione–Sepharose 4B beads were preincubated with affinity-purified GST fusion protein containing the β1D cytoplasmic domain (GST–β1D) or GST as a control (5 μg/30 μl beads), and then mixed with His–MIBP (5 μg) and incubated at 4°C for 1 h. After washing, His–MIBP coprecipitated with GST–β1D was detected by immunoblotting with anti-MIBP antibody 5B4.7. To perform GST fusion protein pull down assays using cell lysates, C2C12 cells were washed once with cold PBS and lysed with the lysis buffer (PBS, 1% Triton X-100, 0.2 mM 4-(2-aminoethyl)benzenesulfonylfluoride, HCl, 10 μg/ml aprotinin, 1 μg/ml pepstatin A and 5 μg/ml leupeptin). The cell lysates (500 μg) were incubated with equal amounts (10 μg) of GST–β1D, or GST alone as a negative control, and the GST fusion proteins were precipitated with glutathione–Sepharose 4B beads. MIBP in the precipitates was detected by immunoblotting with anti-MIBP antibody 5B4.7. The full-length MIBP cDNA was inserted into the HindIII/SalI site of pFLAG-CMV2 vector (Kodak). COS-7 cells were transfected with pFLAG-MIBP, or pFLAG-CMV2 as a control, using Lipofectamine Plus (Life Technologies, Inc.). 48 h after transfection, the cells were lysed using lysis buffer. Cell lysates (500 μg protein) were incubated with agarose beads conjugated with mouse monoclonal anti-FLAG antibody M2 (50 μl) or protein A–agarose beads coupled with an irrelevant mouse IgG (50 μl) at 4°C for 1 h. The beads were washed and FLAG-MIBP and β1 integrin were detected in precipitates by immunoblotting with anti-FLAG antibody M5 and anti–β1 integrin antibody MC231, respectively. C2C12 cells were cotransfected with pFLAG-MIBP (or FLAG-CMV2 as a control) and a vector containing a neomycin-resistant marker pEGFP-c2; Clontech Laboratories Inc., using Lipofectamine Plus. The transfectants were selected with 0.5 mg/ml G418 and cloned. Five clones (E3.11, D9.8, B3, C4, and D4) that stably express FLAG-MIBP were obtained. The expression of FLAG-MIBP by the transfectants was analyzed by immunofluorescence staining and immunoblotting with anti-FLAG antibody M5. To analyze the effect of MIBP overexpression on myogenic differentiation, C2C12 cells stably expressing FLAG-MIBP, FLAG control transfectants, and the parental C2C12 cells were grown in DME containing 10% FBS in 24-well collagen-coated plates (Becton Dickinson) until confluence was reached. Myogenic differentiation was induced by switching the medium to DME containing 2% horse serum. Myogenin was detected by immunoblotting with monoclonal antimyogenin antibody F5D. We have used yeast two-hybrid screens to identify β1 integrin cytoplasmic binding proteins. A bait construct (pLexA/β1D) encoding the β1D integrin cytoplasmic domain (residues 749–801) was used to screen a human heart LexA cDNA library (>3 × 10 6 independent clones). 15 positive clones were obtained. DNA sequencing showed that plasmids from 3 out of the 15 positive clones contained an open reading frame encoding a novel protein that we termed muscle integrin binding protein or MIBP . The binding of MIBP to the β1D cytoplasmic domain was confirmed by yeast two-hybrid binding assays using purified pB42AD encoding MIBP ( Table ). In control experiments, elimination of either the β1D or MIBP sequence failed to activate the reporter genes. In addition, replacement of the integrin cytoplasmic sequences with those of irrelevant proteins (e.g., lamin C) abolished the interaction ( Table ), further confirming the specificity of the interaction. We also found that MIBP interacts with the β1A cytoplasmic domain ( Table ). Northern blot analysis of human tissues revealed that MIBP mRNA is predominantly expressed in skeletal and cardiac muscle . No expression was detected in brain, placenta, lung, liver, kidney, or pancreas . To facilitate studies on MIBP, we generated a monoclonal anti-MIBP antibody (5B4.7) using His–MIBP fusion protein as an antigen. mAb 5B4.7 recognizes both His–MIBP and MBP–MIBP , but not MBP or an irrelevant His-tagged protein . Moreover, analyses of mAb 5B4.7 with a series of MIBP deletion mutants revealed that it recognizes an epitope located within the NH 2 -terminal region (residues 1–109) of MIBP . To test whether mAb 5B4.7 recognizes endogenous MIBP expressed by mammalian cells, we probed mouse C2C12 myoblast lysates. The results showed that it recognizes a single protein band with an apparent molecular mass of ∼19 kD, which is similar to the predicated mass of MIBP . Furthermore, binding of the antibody to endogenous 19-kD protein was completely inhibited by an excess of MBP–MIBP , but not of MBP . We conclude that mAb 5B4.7 specifically recognizes mammalian MIBP as well as recombinant MIBP proteins. Next, we analyzed the expression of MIBP protein in different human tissues using the monoclonal anti-MIBP antibody. Consistent with the results from Northern blotting , MIBP protein was detected in skeletal muscle and heart, but not in other tissues . However, although abundant MIBP mRNA was detected in the heart , the level of MIBP protein in the heart was significantly lower than in skeletal muscle , suggesting that the tissue-specific expression of MIBP may be controlled at the translational as well as the transcriptional level. To test whether MIBP can directly bind to the β1 integrin cytoplasmic domain, we expressed and purified a GST fusion protein containing the β1D cytoplasmic domain. GST–β1D , but not GST , readily interacted with the purified recombinant His-tagged MIBP, indicating that the two proteins can directly interact with each other in the absence of other proteins. In addition, mammalian MIBP protein expressed by the C2C12 myoblasts was coprecipitated with GST–β1D fusion protein but not GST . Thus, both mammalian and recombinant MIBP proteins interact with the β1 integrin cytoplasmic domain in vitro. To test whether MIBP associates with β1 integrins in vivo, we expressed a FLAG-tagged MIBP in mammalian cells . Coimmunoprecipitation experiments with a monoclonal anti-FLAG antibody showed that the β1 integrins were specifically coprecipitated with FLAG-MIBP from the lysate of the FLAG-MIBP transfectants, but not from that of the control transfectants . In additional control experiments, no β1 integrins were precipitated from the FLAG-MIBP lysates with a control mouse IgG . Thus, MIBP forms a complex with the β1 integrins in mammalian cells as well as in vitro. The cytoplasmic domains of β1D and β1A share a common membrane-proximal region. Since MIBP binds to both β1D and β1A cytoplasmic domains ( Table ), it most likely recognizes a site located within this region. To test this, we generated a series of β1D/β1A mutants and analyzed their ability to interact with MIBP in yeast two-hybrid binding assays. The results showed that MIBP specifically interacts with the membrane-proximal region of the β1D or β1A cytoplasmic domain ( Table ). To begin to investigate the role of MIBP in myogenic differentiation, we analyzed MIBP expression during myogenic differentiation using the mouse C2C12 myoblast line as a model system. The results showed that abundant MIBP protein is expressed before terminal differentiation of C2C12 myoblasts . Myogenic differentiation was induced by switching the culture medium to DME containing 2% horse serum. Myotubes were observed within the first 2 d of induction, and >80% of the cells were fused into multinucleated myotubes on day 4. The MIBP expression level was decreased upon induction of myogenic differentiation . Less than 10% of MIBP was expressed 4 d after the induction of differentiation , and the expression of MIBP was further decreased beyond detection after day 5. In control experiments, the same membrane was stripped and reprobed with an anti–β1 integrin antibody (MC231, which recognizes both β1A and β1D integrins). The β1 integrins (β1A and/or β1D) were detected at all stages of C2C12 differentiation . The striking downregulation of MIBP during myogenic differentiation suggests that a higher MIBP expression level may prevent myoblasts from undergoing terminal differentiation. To test this, we overexpressed FLAG-tagged MIBP in C2C12 myoblasts. The expression of FLAG-tagged MIBP in the FLAG-MIBP transfectants, but not C2C12 cells transfected with a vector lacking the MIBP sequence, was confirmed by immunoblotting with an anti-FLAG antibody and the anti-MIBP antibody 5B4.7 . Two independently isolated C2C12 clones (E3.11 and D9.8) that express FLAG-MIBP at a level comparable to that of endogenous MIBP in the proliferating myoblasts were selected for further analysis. As expected, myogenin and abundant multinucleated myotubes were detected in both the parental C2C12 cells and the vector control transfectants after induction of myoblast differentiation. In contrast, no multinucleated myotubes were detected in E3.11 and D9.8 cells overexpressing FLAG-MIBP under identical experimental conditions . Similar results were obtained with all other FLAG-MIBP–overexpressing C2C12 clones that were analyzed . Consistent with the suppression of myotube formation, no myogenin was detected in the cells overexpressing FLAG-MIBP . The expression of FLAG-MIBP in the FLAG-MIBP transfectants but not the parental C2C12 or the vector control transfectants, before and after the induction of myoblast differentiation was confirmed by immunoblotting with an anti-FLAG antibody . We conclude from these experiments that overexpression of FLAG-tagged MIBP in C2C12 myoblasts suppresses terminal myogenic differentiation. In this study, we have identified and cloned a novel muscle β1 integrin binding protein, MIBP, and provided strong evidence for an important role of MIBP in the regulation of terminal myogenesis. Using C2C12 myogenic cells as a model system, we show that MIBP is abundantly expressed in proliferating myogenic cells. The expression level of MIBP decreases upon induction of terminal myogenic differentiation, and becomes undetectable after the majority of the myoblasts have fused to form multinucleated myotubes. This striking downregulation of MIBP suggests that the amount of MIBP may be a crucial element in the decision-making process of fusion versus proliferation during myogenic differentiation. In support of this, overexpression of an epitope-tagged MIBP under a promoter that is not subject to regulation in muscle cells resulted in a complete suppression of the terminal differentiation of C2C12 cells. MIBP is shown to bind to the β1 integrin cytoplasmic domain, which is known to play a key role in controlling myoblast proliferation and differentiation . In addition to suppression of myogenic differentiation, our preliminary results indicate that after switching to differentiation medium, expression of FLAG-MIBP in C2C12 myoblasts enhances cell proliferation (Li, J., R. Mayne, and C. Wu, unpublished observations). Taken together, our results suggest that MIBP most likely functions in the regulation of myogenesis via its interaction with the β1 integrin cytoplasmic domain. In this regard, it is particularly interesting to note that the MIBP-binding site is located within the membrane-proximal region of the β1 integrin cytoplasmic domain, a region likely to play an important role in integrin activation . It has been shown that the ligand binding affinity of α5β1 integrin in myoblasts is downregulated during myogenesis . Moreover, this inactivation of α5β1 integrin is functionally important to myogenesis and could potentially contribute to the matrix switch (from a fibronectin-rich matrix to a laminin-rich matrix) that accompanies myogenic differentiation . Thus, one mechanism whereby MIBP potentially functions is by regulating integrin activation, and consequently, matrix deposition and cell adhesion. In addition, MIBP could modulate signal transduction from integrins to other downstream targets such as focal adhesion kinase and paxillin , and thereby influence the decision of myoblasts to fuse and undergo terminal differentiation. | Study | biomedical | en | 0.999997 |
10613899 | Mouse somatic cell lines used in this study were C127, XX mammary gland tumor cells (provided by Dr. B.M. Turner, University of Birmingham Medical School, Birmingham, UK); Is1Ct and T37H, XX primary fibroblasts ; FSPE, XX female Mus spretus primary ear fibroblasts (provided by Dr. T.B. Nesterova, Medical Research Council, Hammersmith Hospital, London, UK); and MSPL, XY male M. spretus primary lung fibroblasts (provided by Dr. S.M. Duthie, Medical Research Council Hammersmith Hospital). Cells were maintained as described previously . Mouse ES cell lines used were, PGK12.1, XX ; LF2, XX and Efc-1, XY (both provided by Dr. A.G. Smith, Centre for Genome Research, Edinburgh, UK); and 129/1, XY (derived in-house by G.F. Kay). All but 129/1 ES lines were cultured in the absence of feeder cells on 0.1% gelatin-coated tissue culture plates. Cells were maintained in DME containing murine leukemia inhibitory factor (LIF) as described by Penny et al. 1996 . Differentiated cultures were generated by withdrawal of LIF from the ES media . Details of this protocol and alternative methods of differentiation used are described in the legend of Fig. 6 . Cells were harvested by trypsinization at selected timepoints during a 1–22-d differentiation period and analyzed by indirect immunofluorescence and Western blotting. To confirm that progression of X-inactivation was occurring in XX cells, we assessed the H4 histone acetylation status of the X chromosome. Previously, an underacetylated X chromosome was detected from day 4 of differentiation onwards, frequencies increasing from 9% of metaphases to a maximum of 25% at day 7 . We obtained similar results, with 28% ( n = 105) of metaphases showing an underacetylated Xi at day 5 of differentiation. Indirect immunofluorescence labeling using an affinity-purified rabbit polyclonal antiserum against the nonhistone region of macroH2A1.2 was carried out as described by Costanzi and Pehrson, 1998. The incubation period with the primary antibody was reduced to 1 h at 37°C. Data using formaldehyde fixation (4%) are presented in all figures. Identical results were obtained using different fixation (0.5% glutaraldehyde or methanol/acetone) and permeabilization procedures . In protein and RNA double-labeling experiments, FISH preceded macroH2A1.2 immunostaining. Trypsinized cells were pipetted onto Superfrost Plus glass slides (BDH) and allowed to attach for 3–5 h. Slides were subsequently washed in PBS and fixed in 4% formaldehyde, 5% acetic acid, 0.9% NaCl for 30 min at room temperature, rinsed three times in PBS, dehydrated through a 70-90-100% ethanol series, air-dried, washed in 100% Xylene for 5 min, rehydrated to PBS, incubated with 0.01% pepsin in 0.01 M HCl for 30 s at 37°C, washed in PBS for 5 min, postfixed in 1% formaldehyde in PBS for 2.5 min at room temperature, washed in PBS, dehydrated through an ethanol series, and air-dried before applying 50–100 ng of nick-translated DNA probe. Biotinylated GPT16, a 6-kb probe spanning most of murine Xist exon 1, was used. Hybridization and wash conditions were as described previously . After probe detection with avidin–Texas red (TR) followed by biotinylated antiavidin and a final layer of avidin-TR, slides were washed twice for 3 min in 4× SSC, 0.1% Tween 20 at 37°C, followed by twice for 3 min in PBS, 0.1% Tween 20. After blocking in 5% nonfat dry milk at room temperature, slides were incubated for 1 h at 37°C with the primary antibody, anti-macroH2A1.2 rabbit polyclonal antiserum . After three washes in PBS, 0.1% Tween 20 slides were incubated with FITC-labeled mouse anti–rabbit IgG for 30 min at 37°C, washed, and mounted in Vectashield antifade (Vector Labs, Inc.) containing 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescently labeled secondary antibodies, avidin-TR, and biotinylated antiavidin were from Vector Labs, Inc. Images were acquired on a Leica DMRB fluorescence microscope equipped with a Photometrics CCD camera using Smartcapture Software (Vysis Ltd.). Metaphase chromosome spreads were prepared as described by Keohane et al. 1996 . Mitotic C127 cells and undifferentiated ES cells were harvested by mechanical shake-off, and differentiated ES cells were trypsinized after collection. C127 cells and ES cells were swollen in 0.075 and 0.1 M KCl, respectively. To assess the histone H4 acetylation status, immunolabeling was performed on unfixed metaphases with an antibody to acetylated lysine 8 of histone H4 (R232; gift of Dr. B.M. Turner, University of Birmingham Medical School) as described previously . For the detection of macroH2A1.2, metaphases were fixed in 4% formaldehyde after swelling in KCl, followed by indirect immunofluorescence as described above, but without further permeabilization. Total cell extracts were prepared by washing harvested cells twice with cold TB buffer (20 mM Hepes, pH 7.3, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 1 mM EGTA, 2 mM DTT, protease inhibitors). They were then incubated for 40 min at 37°C in TB buffer with 0.1% NP-40, 10 mM MnCl 2 , and 20 μg/ml DNaseI followed by addition of SDS-PAGE loading buffer. NP-40 permeabilization and salt extraction of cells was performed by washing harvested cells twice with cold TB buffer and incubating them on ice for 4 min in TB buffer containing 0.1% NP-40. The supernatant was recovered (S1) and pellets were resuspended in TB without (Ctrl) or with 0.5, 1.2, or 2 M NaCl and incubated for 20 min on a rotating wheel at 4°C. After centrifugation at 100,000 g for 40 min, supernatants were recovered (S2). Cell pellets were digested with 20 μg/ml DNaseI for 40 min at 37°C in TB buffer with 0.1% NP-40 and 10 mM MnCl 2 . SDS-PAGE loading buffer was added to both supernatants and cell pellets and samples were analyzed by SDS-PAGE and Western blotting. For immunoblot analysis, protein was transferred to polyvinylidene difluoride membranes and incubated with antibodies as described by Pehrson et al. 1997 . Antibodies were an affinity-purified rabbit polyclonal serum against the nonhistone region of macroH2A1.2 , and a donkey anti–rabbit IgG conjugated to alkaline phosphatase (Pierce). For lactate dehydrogenase (LDH) detection, the goat primary antibody (Rockland) was followed by incubation with a rabbit anti–goat Ig conjugated with alkaline phosphatase (Sigma Chemical Co.). Alkaline phosphatase was detected using the 1-step NBT/BCIP kit (Pierce). To analyze MCB localization during X-inactivation, it is essential to distinguish between the Xi and Xa. Previously, macroH2A1.2 association with the Xi was shown indirectly using combined immunofluorescence and chromosome painting . Therefore, we developed a protocol for double-labeling of macroH2A1.2 and Xist RNA (immuno-RNA FISH) that allows us to directly correlate an MCB with the Xi. Initially four XX and one XY murine somatic cell lines were analyzed using this approach. In all XX cell lines, an antibody specific to the nonhistone region of macroH2A1.2 revealed diffuse staining throughout the nucleus in addition to an MCB. The MCB was detected in >86% of cells in every line using several different methods of fixation and permeabilization . It localized to DAPI-bright regions in the nucleus, except in the case of C127 cells. Consistent with previous studies, the XY cell line exhibited diffuse macroH2A1.2 nuclear staining with nucleolar exclusion, and only infrequently did cells have an MCB (data not shown) . A simultaneous analysis of Xist RNA localization in the XX cell lines revealed a large Xist RNA signal that reflects accumulation of late Xist transcripts in cis . In three primary fibroblast cell lines (FSPE, Is1Ct, and T37H) Xist RNA and the MCB colocalized in 99–100% of the cells as indicated by the overlap of the two signals . The degree of overlap was not always complete in all cells . Colocalization was not observed in all cell types. In C127 cells, a mammary gland tumor cell line, the Xist RNA, and the MCB signals did not overlap; rather, they occupied separate territories . The presence of accumulated Xist RNA suggests that C127 cells have retained an Xi. In addition, C127 cells have an underacetylated chromosome corresponding to the Xi (data not shown) . In summary, we show that immuno-RNA FISH can be used to analyze association of macroH2A1.2 with the Xi. We went on to use this methodology to examine the formation of the MCB and its localization with respect to Xist RNA before and during ES cell differentiation. We anticipated that an MCB would not be evident in undifferentiated XX and XY ES cells since their X chromosomes are active. Surprisingly, in two independent XX ES cell lines we found that a single macroH2A1.2-rich domain was readily detectable in the majority of interphase cells . The appearance of this macroH2A1.2-rich domain was similar to the MCB characterized in somatic cells, and thus we refer to it also as an MCB. In the two XY ES cell lines we analyzed, an MCB was also detected . Thus, the presence of a single MCB appears to be an intrinsic property of undifferentiated ES cells, regardless of X chromosome constitution. This contrasts with somatic cells, in which the MCB is found exclusively in XX cells despite macroH2A1.2 expression in both XX and XY cells . The single MCB was frequently found at the periphery of the nucleus , close to the nuclear membrane as assessed by double-label immunofluorescence with an antibody against the nuclear pore complex (data not shown). Interestingly the MCB appeared to lie in a region that stained poorly with DAPI . In addition to the MCB we detected diffuse nuclear staining in XX and XY ES cells . Thus, as is the case of XX and XY somatic cells , the MCB is unlikely to represent the total macroH2A1.2 protein content. We confirmed that the presence of an MCB in undifferentiated ES cells can be attributed to macroH2A1.2 protein using the same antibody as used for immunofluorescence detection in Western blots. A single protein with an expected electrophoretic mobility corresponding to that seen previously in somatic cells was detected in both XX and XY ES cells. The relative macroH2A1.2 protein content was similar not only between undifferentiated XX and XY ES cells but also between undifferentiated and differentiated ES cells . To investigate the characteristics of macroH2A1.2 in ES cells at the biochemical level, we permeabilized undifferentiated XX PGK12.1 cells with NP-40 to disrupt membranes. As reported previously , this treatment brought about the complete release of LDH . However, we found that macroH2A1.2 was quantitatively retained in the pellet . Similarly, macroH2A1.2 protein remained associated with the insoluble fraction when either undifferentiated XY ES cells or XX ES cells differentiated for 10 d were treated with NP-40 (data not shown). Thus, macroH2A1.2 in undifferentiated ES cells is not present in a pool of soluble protein. Next, the pellet fraction produced by NP-40 treatment was extracted with different concentrations of sodium chloride. Most nonhistone chromosomal proteins and histone H1 are extracted by 0.5 M sodium chloride, but not the core histones . These conditions did not release macroH2A1.2 protein from the NP-40 pellet . Similar results have been described for macroH2A1.2 in somatic cells . Treatment of the NP-40 pellet fraction with 1.2 M sodium chloride released ∼50% of histones H2A and H2B but only a small fraction of macroH2A1.2. Complete extraction of all the core histones, including H3 and H4, is brought about with 2 M sodium chloride . This treatment also resulted in extraction of the vast majority of macroH2A1.2 . The small fraction of the histones not extracted are probably trapped within the pellet . Taken together, these results indicate that the association of macroH2A1.2 with the NP-40 pellet has the biochemical properties associated with a core histone and is consistent with the bulk of the protein being chromatin associated. The presence of an MCB before X-inactivation in undifferentiated cells raised the question of its localization relative to the X chromosome. Immuno-RNA FISH was performed on both XX and XY ES cells. In XX cells, early Xist transcripts were detected as two punctate dots, representing expression from both X chromosomes . The MCBs observed lie in different focal planes with respect to Xist transcripts, clearly occupying different territories . Identical results were obtained with another XX ES cell line, LF2 . Thus the MCB in XX ES cells does not colocalize with Xist RNA expressed from either of the two active X chromosomes. Similarly, in XY ES cells, early Xist transcripts expressed from the single X chromosome were not associated with the MCB . Although this does not rule out that the MCB is on the X chromosome but at a different locus, analysis of early differentiation stages indicate that this is not the case (see below). Results presented above show that >80% of undifferentiated XY ES cells have a single MCB . However, few or no XY somatic cells show an MCB . This suggests that the number of cells that contain an MCB declines during the process of differentiation. To determine whether this is the case, the number of XY Efc-1 cells displaying an MCB was scored over a 1–16-d differentiation time-course . Early in differentiation (day 1–7), 88–95% of cells contained a single MCB. At progressively later timepoints (day 8–16), a clear decline in the number of cells containing an MCB was observed, reaching a minimum of 3% at day 14. This decline showed first order kinetics with a half-life of ∼1.7 d. For comparison, an identical analysis of XX ES cells was carried out. Fig. 4 shows that the percentages of cells containing an MCB (average 86%) remained unchanged during differentiation of PGK12.1 cells. Diffuse nuclear macroH2A1.2 staining was observed throughout differentiation and is consistent with Western blot analysis showing that macroH2A1.2 protein is expressed not only in differentiating XX cells but also in differentiating XY cells . To address the key issue of when macroH2A1.2 accumulation on the Xi occurs, we carried out immuno-RNA FISH analysis on differentiating PGK12.1 XX ES cells. During day 1–3 Xist RNA undergoes a transition from a punctate to an accumulated signal. This reflects the switch from early transcripts to late transcripts that coat the inactivating X chromosome . At early stages of differentiation, up to day 6, we observed no colocalization of the MCB with Xist RNA . Because Xist RNA coats the entire Xi from day 3 onwards , our results indicate that the MCB is not associated with any part of the Xi. Similarly, in XY ES cells differentiated for 3 d, no colocalization of punctate Xist RNA signal and the MCB was observed ( n = 105, Efc-1). Between day 7 and 9, we observed a striking change in localization patterns in XX ES cells. At day 7, Xist RNA and MCB signals were separate from one another in the majority of cells . However, 48 h later colocalization was observed in most cell nuclei . Within this transition period, cells frequently exhibited patterns of Xist RNA and MCB distribution with both signals close together or showing a slight overlap . We never observed nuclei with two MCBs during this period. At later timepoints (day 12, 16, and 22) the number of cells exhibiting colocalization did not change significantly . All the patterns described and their timing of appearance were highly reproducible. Importantly, a second XX cell line (LF2) gave identical results. A quantitative analysis of the data is presented in Fig. 5 j. This clearly demonstrates that colocalization occurs in most cells between day 7 and 9 of differentiation. To address whether this colocalization at interphase represents stable association of macroH2A1.2 with the chromatin of Xi, we analyzed metaphases of PGK12.1 cells differentiated for 10 d. We found that macroH2A1.2 is preferentially enriched on a single chromosome in each chromosome spread . From day 12 of differentiation onwards we observed an increasing number of nuclei with more than one MCB . At day 16 up to eight MCBs could be detected in ∼50% of nuclei (PGK12.1 49%, n = 172; LF2 47%, n = 125). Xist RNA always colocalized to one of the MCBs. Less than 5% of nuclei had more than one Xist RNA signal, indicating that multiple MCBs are not attributable to polyploidy. Additional MCBs correlate with DAPI-dense staining regions in the nucleus. Given the diversity of differentiating cells in an embryonic body (EB) , the precise and reproducible formation of the MCB on the Xi between day 7 and 9 indicates that the process is independent of the developmental fate of individual cells. The occurrence of these changes coincides with the replating of EBs into tissue culture plates at day 7. To eliminate the possibility that these events are a consequence of the method of differentiation we tested two other differentiation procedures in which a replating step is avoided . We found that the timing and extent of colocalization were identical, and thus independent of the method of differentiation used . We have shown that a single MCB is present before X-inactivation in both undifferentiated XX and XY ES cells; however, in neither case does it colocalize with Xist RNA. Only after the initiation and propagation of X-inactivation in differentiating XX ES cells do the MCB and Xist RNA colocalize. Concurrent with colocalization taking place in XX cells, the number of cells with an MCB declines significantly in differentiating XY cells. The timing of MCB and Xist RNA colocalization suggests that the accumulation of macroH2A1.2 on the Xi is unlikely to play a role in the primary events of X-inactivation. Previous analysis of the random X-inactivation process has shown that in differentiating XX ES cells the events leading to a fully inactivated X chromosome occur sequentially over a period of several days . The earliest detectable changes (expression of late Xist transcripts, late replication of the inactivating X, and a progressive increase in silencing X-linked genes) occur around day 2 of differentiation. These events are followed by overall histone H4 deacetylation on the Xi, which is essentially completed by day 7. We now have placed accumulation of macroH2A1.2 on the Xi in this pathway. We demonstrate that colocalization of the MCB with Xist RNA takes place after day 7 of differentiation in a highly synchronized wave. It occurs in all nuclei, is independent of the method of differentiation used or cell line studied, and is essentially completed within a 48-h period. Our analysis of H4 deacetylation (see Materials and Methods) and Xist expression during differentiation show that the timing of events in this study is identical to previous studies . Thus, MCB formation on Xi occurs subsequent to other steps in the X-inactivation process, with the exception of CpG island methylation. Therefore, we conclude that the MCB on the Xi cannot play a major role in initiation or propagation of random X-inactivation. Whether early events before day 7 are themselves necessary preconditions for MCB formation on the Xi is currently unknown. Our data do not exclude a function for the Xi-MCB in maintenance of the inactive state, similar to CpG island methylation or global H4 underacetylation . Consistent with such a possible role, an MCB is observed on the Xi in somatic cells and in a number of cell lines . However, the MCB is not absolutely required for maintenance. In C127 cells, no colocalization between Xist RNA and the MCB was observed, yet the X chromosome is underacetylated and coated with Xist RNA . Furthermore, loss of the MCB did not affect the inactive state in embryonic fibroblasts . Whether this simply reflects redundancy in maintenance mechanisms remains to be established. In this respect, it may resemble cases in which X-inactivation can be maintained in the absence of CpG island methylation in marsupials and in murine extra-embryonic lineages . These observations have previously led to the suggestion that there are a number of mechanisms that ensure maintenance of X-inactivation . An alternative hypothesis is that localization of the MCB on the Xi is required only at a certain developmental time window, for example, to mark nucleosomes for subsequent event(s). The presence of a single MCB in undifferentiated XX ES cells before X-inactivation raises the possibility that it is involved in marking the X chromosome to be inactivated. Two results argue against this. Firstly, the MCB does not colocalize with Xist RNA in undifferentiated cells or in differentiating cells up to day 7. Xist RNA coats the entire Xi after day 3 of differentiation , hence lack of colocalization in these cells indicates that the MCB is not on the Xi. Secondly, we show that undifferentiated XY cells also have an MCB, even though they never inactivate their single X chromosome. In a previous study, macroH2A1.2 protein was readily detected in differentiated but not in undifferentiated ES cells . We have detected both macroH2A1.2 protein and an MCB in undifferentiated ES cells by using a significantly more sensitive antibody and a different method of extract preparation. The MCB was usually peripheral and did not correspond to DAPI-dense areas, suggesting that it is not associated with heterochromatin. Cell fractionation studies indicated that macroH2A1.2 in undifferentiated ES cells is not released by detergent (NP-40) lysis and is not in a pool of soluble cellular protein. However it is extracted by high concentrations of salt. In these respects, its behavior is similar to the core histones and is consistent with macroH2A1.2 protein being chromatin-associated in undifferentiated cells. However, the data do not rule out the possibility that the protein is associated with some other subcellular structure present in the NP-40 pellet. A more detailed analysis will now be required to understand the nature of the ES cell–MCB and whether its role at this stage of development is related to X-inactivation. A single Xi-independent MCB was also observed in the mammary tumor cell line C127. It is not clear whether this structure, which does not correlate with obvious heterochromatic regions, is equivalent to that seen in undifferentiated and early differentiating ES cells. Presumably the MCB either never associated with the Xi or a previous association has been reversed. If the latter were true, it could reflect epigenetic remodelling processes associated with transformation. Investigation of other transformed and primary cell lines will be of interest in light of this discovery. A number of possible mechanisms may be postulated for the developmentally regulated formation of an MCB on the Xi. One mechanism involves the disassembly of the non-Xi MCB and import of newly synthesized macroH2A1.2 protein into the nucleus that is targeted to the Xi. Another possibility is that the non-Xi MCB disassembles, and macroH2A1.2 protein disperses and is reassembled at the Xi to form an MCB. A third possibility involves reorganization in the nucleus to bring the non-Xi MCB and the Xi in close proximity, allowing dynamic repositioning of macroH2A1.2 protein from the non-Xi MCB onto the Xi. Although we cannot yet distinguish between these and other possible mechanisms, we favor the latter model for the following reasons: The MCB is detectable throughout XX ES cell differentiation . During the period when the MCB appears on the Xi, we never see more than one MCB per cell, arguing that the non-Xi MCB cannot be slowly disassembled concurrently with the Xi–MCB being formed. We do, however, observe an increase in the frequency of cells in which the non-Xi MCB is close to the Xi . Furthermore, we detect cells in which Xist RNA and MCB signals are slightly overlapping. To investigate the repositioning hypothesis further, the precise dynamics of colocalization could be observed in living cells, for example, by using GFP-tagged macroH2A1.2. Evolutionary sequence conservation suggests that the function of macroH2A1.2 in X-inactivation may be an adaptation of a more general role of the protein in gene silencing . Thus, similar mechanisms and components might be used for silencing of autosomal genes and X-inactivation . One example of an involvement of the MCB in silencing of autosomal genes could be the multiple MCBs observed in some cells from day 12 of differentiation onwards. They coincide with DAPI-dense regions of the nucleus, which may reflect incorporation of macroH2A1.2 into constitutive heterochromatin such as centromeres. Alternatively, these structures may represent clusters of loci with macroH2A1.2-enriched chromatin. Clustering of silenced genes with constitutive heterochromatin has been observed in recent studies on lymphocytes . Whether multiple MCBs are temporary structures or are related to the lineage or differentiated cell type is currently unknown. Interestingly, out of four murine somatic cell lines analyzed, only one displayed multiple MCBs (T37H; data not shown), whereas the other cell lines never had more than one MCB per diploid genome. | Study | biomedical | en | 0.999997 |
10613900 | Human lymphocytes were separated according to standard procedures. The separated lymphocytes were washed twice in PBS and embedded in low temperature-melting agarose (type VII; Sigma Chemical Co.) according to the protocol of Weipoltshammer et al. 1996b . One part of the embedded cells was subjected to the nucleoskeleton preparation procedure. The other part was incubated in RPMI medium (supplemented with 20% fetal calf serum and phytohemagglutinin; Abbott Laboratories) at 37°C for 72 h. Stimulated lymphocytes were then washed twice in PBS and subsequently nucleoskeletons were produced. All solutions were prepared with diethylpyrocarbonate (DEPC)-treated distilled water (0.1%, inactivated) to minimize RNase content. Agarose-embedded cells were transferred into 0.1 M Soerensen buffer (SB; 70 mM Na 2 HPO 4 , 30 mM KH 2 PO 4 , pH 7.4), and then incubated in 0.05 M SB for 5 min at 37°C to disperse the granular component of nucleoli . Afterwards, cells were permeabilized in two changes of lysis buffer (70% PB diluted with DEPC-water, supplemented with 0.2% Triton X-100, 0.1 mM PMSF, and 2.5 U/ml RNase inhibitor; Amersham International), 5 min each on ice, and washed in “physiological buffer” (PB; 70 mM K-acetate, 30 mM KCl, 10 mM Na 2 HPO 4 , 1 mM MgCl 2 , 1 mM Na 2 ATP, 1 mM DTT, 0.1 mM PMSF, pH 7.4 adjusted with KH 2 PO 4 , 2.5 U/ml RNase inhibitor; Amersham International) 4 × 10 min on ice. Chromatin in permeabilized cells was cut using 340 U/ml HaeIII (Roche Laboratories) and 1,650 U/ml EcoRI (Roche Laboratories) in the presence of 25 U/ml RNase inhibitor (Amersham International); the reaction was carried out in a 3-ml vol bead suspension in PB for 20 min at 33°C. For the telomeric repeat, we cross-checked our results by cutting one sample of stimulated lymphocytes with RsaI (1,000 U/ml; Sigma Chemical Co.) and HinfI (1,000 U/ml; Roche Laboratories) that cut within the subtelomeric DNA sequences . No difference between the two samples was found. Subsequently, the bead suspension was introduced into the slots of an agarose gel (0.8% in tetraethylammonium [TAE] buffer, supplemented with 2.5 U/ml RNase inhibitor). Electrophoresis was run for 30 min at 20 V, and then for 2 h and 30 min at 35 V. For electrophoresis, PB diluted with DEPC-water to 70% and containing 0.1 mM PMSF and 0.5 U/ml RNase inhibitor was used as a running buffer. Cells were retrieved from the slots and fixed in 4% formaldehyde in PBS for 30 min. Control samples were fixed with 4% formaldehyde in PBS for 30 min without any enzymatic digestion of DNA. All samples were washed twice in PBS, dehydrated in graded series of methanol, and stored in 100% methanol at −20°C for further analysis. After recovering agarose beads from the gel slots, the gel was treated with RNAse A (0.5 μg/ml, 1 h, 30°C), proteinase K (5 μg/ml, 1 h, 20°C), and stained with ethidium bromide as described . The gel was photographed and blotted onto a Nylon membrane (Roche Laboratories) for hybridization. As an internal control, DNA was isolated from three fractions of cells by phenol extraction: (a) before cutting with restriction enzymes, (b) after cutting with EcoRI and HaeIII before electrophoresis, and (c) after electrophoresis. With these three samples of isolated DNA, gel electrophoresis (1.7% agarose in TAE buffer) was performed. The agarose-embedded cells were spread over aminoalkylsilane-coated slides (Polysciences Inc.) and immobilized by incubating the slides at 65°C for 3 d. Fluorescence DNA in situ hybridization on nucleoskeleton preparations and control cells was performed according to Wachtler et al. 1991 . Instead of proteinase K, 0.1% pepsin in 0.01 N HCl (pH < 2) was used . To optimize hybridization conditions for the embedded cells, the formamide content of the hybridization mix was reduced to 30%. Sample and probe were denatured simultaneously on the slide for 10 min at 95°C. The hybridization took place at 37°C overnight in a moist chamber. Stringency washes were carried out at 42°C with 0.6× standard sodium citrate containing 20% formamide (three changes, 10 min each). For the detection of telomeres and centromeres, commercially available probes were used: the digoxigenin-labeled “all human telomere DNA-probe” consisting of the TTAGGG repeat (Vysis), and the biotin-labeled “all human centromere probe” containing centromeric alpha-satellite sequences (Vysis). Alternatively, we used a Cy3-labeled PNA-telomere probe (DAKO SA) that has a higher sensitivity but a lower staining intensity. The probes for the detection of the ribosomal gene repeat were the EcoRI A fragment (part of the transcribed unit; stretching from the 3′ end of 18S to the 3′ end of 28S subunit, thereby including both internal transcribed spacers and the 5.8S subunit of the ribosomal gene; the A fragment measures 7.1 kb), and the HindIII D HH fragment (located within the noncoding intergenic spacer; it is part of the EcoRI-defined D fragment; the D HH fragment measures 9 kb). These probes were kindly donated by Prof. James Sylvester. They were labeled by nick-translation (nick-translation kit; Roche Laboratories) with either digoxigenin or biotin. For simultaneous detection of telomeres and rDNA, the digoxigenin-labeled “all human telomere probe” in combination with either A or D HH fragment, both biotin-labeled, was used. Simultaneous detection of centromeres and rDNA was carried out with the biotin-labeled “all human centromere” probe plus the digoxigenin-labeled rDNA fragments. Detection of DNA probes was performed with commercially available antibodies against digoxigenin (rhodamine conjugated), or using FITC-conjugated avidin in the biotin/avidin detection system as in Weipoltshammer et al. 1996a . After electroelution, the agarose gel containing the eluted fraction of chromatin was blotted onto a Nylon membrane using 0.4 N NaOH overnight. DNA was fixed on the membrane by baking for 30 min at 110°C. The total extracted DNA was detected by hybridization with human genomic DNA as a probe (Sigma Chemical Co.). Specific sequences (telomeres, centromeres, and the ribosomal gene segments A and D HH ) were detected with the same probes used for in situ hybridization. All DNA probes were digoxigenin-labeled, and then detected with antidigoxigenin antibodies conjugated with alkaline phosphatase (Roche Laboratories); NBT/X-phosphate solution (Roche Laboratories) was used as a substrate. Optical sections of nuclei were recorded with a confocal laser scanning microscope (LSM 410; Carl Zeiss, Inc.). The nuclei displayed in Fig. 2 Fig. 3 Fig. 4 Fig. 5 are projections of stacks of optical sections. Control and eluted cells were densitometrically evaluated for several criteria. For all in situ evaluations, confocal series of nuclei were gained under identical conditions (pinhole, resolution, z distance, gain, contrast, etc.). First of all, the elution efficiency for DNA was checked. Control and eluted cells of unstimulated and stimulated lymphocytes were stained with quinacrin mustard. Samples were recorded and densitometric quantification of the sections was carried out with KS400 software (Kontron). The samples were segmented with a fixed threshold. The total signal density per nucleus was calculated from the optical sections and the mean signal density per nucleus was determined for each sample. Elution efficiency was expressed as the relation of mean signal density per nucleus of eluted cells to the control cells. The same method was used to compare hybridization signals between control and eluted cells. For all sequences hybridized (telomeric repeat, alpha-satellite repeat, and A and D HH fragments) the relations of mean signal density per nucleus of eluted cells to the corresponding control cells were calculated. The hybridized membranes were digitized with a flat-bed scanner. The signal was densitometrically evaluated and normalized against the membrane background with KS400 software. Linearity of the detection system was checked with a dilution series of the alkaline phosphatase-labeled antibody. For comparison of A and D HH fragments, the densities of the hybridization signals were measured and given as relative value. To correct for differences in probe characteristics, a dilution series of total human DNA was blotted and hybridized with the A as well as the D HH fragment. The relation of the signal intensity of A and D HH fragments was the same for all DNA concentrations. The obtained ratio of signal intensity of A versus D HH fragment was used to normalize the hybridization signal of the electroeluted DNA fraction. The amount of extracted A and D HH fragments was compared within the sample of unstimulated lymphocytes and within the sample of stimulated lymphocytes densitometrically. The extracted total DNA was visualized on blots of the same elution experiments after hybridization with a total genomic probe. Nuclear morphology after the electroelution was well preserved as checked at the electron microscopic level (data not shown). In both unstimulated and stimulated lymphocytes, the DNA was generally extracted in equal amounts (>80% of DNA) as assessed with densitometric evaluation after quinacrin mustard staining ( Table ). The amount of extracted DNA is in line with data from the literature . The permeabilization conditions used in our experiments represent a good balance between preservation of nuclear morphology and a high yield of DNA extracted. The reliability of the electroelution procedure was checked on the DNA level at several points. DNA was isolated from the agarose-embedded cells after restriction enzyme cutting (EcoRI, HaeIII) and directly after electroelution. Both samples were subjected to electrophoresis . Total DNA after restriction enzyme cutting showed a DNA smear plus nucleosomal bands in the lower weight range (lane 2). The DNA fraction remaining in the nucleus after electroelution also shows a smear, the nucleosomal bands are no longer found (lane 3). This pattern is well in line with published pictures of extracted DNA from nucleoskeleton preparations . The extraction of chromatin from nuclei results in a smear in the agarose gel . Nucleosomal bands are not clearly visible and are likely to be obscured by other extracted charged molecules . The corresponding blot hybridization with a total genomic probe is shown in Fig. 1 c. A prominent portion of DNA from eluted chromatin is present in the high–molecular weight range. In this respect, it should be added that it has been shown that chromatin containing DNA fragments larger than 125 kb can be removed from nuclei during preparation of nucleoskeletons . In metaphase cells, the “all human centromere DNA-probe” hybridized to all centromeres, resulting in bright foci, including the pericentromeric regions (data not shown). In the agarose-embedded control interphase nuclei, the hybridization signal was highly clustered and located preferentially at the nuclear periphery and around the nucleolus in unstimulated as well as stimulated lymphocytes . In nucleoskeleton preparations, the hybridization signal patterns were dramatically changed. The overall signal intensity was reduced and distinct signal clusters were no longer clearly visible in both unstimulated and stimulated lymphocytes . Quantification revealed that <2% of signal specific for centromeric DNA remained in the nucleus after electroelution ( Table ). The blot hybridization of the extracted chromatin fraction (i.e., the unattached chromatin fragments) proved that a considerable amount of centromeric alpha-satellite sequences was removed during the extraction procedure regardless of the overall transcriptional activity of the cells used for elution . In chromosome spreads, the all human telomere DNA-probe produced bright dots at the chromosomal ends (data not shown). In interphase control nuclei, the hybridization signal was visible as bright foci distributed throughout the entire volume of the nucleus, in both unstimulated and stimulated cells. Hybridization with the PNA-telomere probe generated identical results (data not shown). In nucleoskeleton preparations of unstimulated and stimulated cells, no apparent differences in signal intensity and distribution were found when compared with the control cells . Densitometric evaluation showed that ∼90% of telomeric DNA remained in the nucleus after nuclear extraction ( Table ). Southern blot hybridization of extracted chromatin fragments with the all human telomere probe produced only a very faint signal . This faint signal is most likely attributed to extra-telomeric TTAGGG repeats . In metaphase spreads, both rDNA probes marked the nucleolus organizer regions on all five acrocentric chromosome pairs (data not shown). In interphase control nuclei, both probes hybridized to the nucleolus. In unstimulated cells, this signal was usually roundish or ring-shaped . In addition to the typical nucleolar signal, one to several small dot-like signals were observed in ∼50% of the unstimulated lymphocytes. These dots correspond to the silent ribosomal gene clusters located outside of the active nucleolus . In stimulated lymphocytes, the nucleolar signal was much more prominent and extended; the extra-nucleolar signals were absent . When in situ hybridization was performed on chromatin-depleted nuclei of unstimulated lymphocytes, the remaining nucleolar signal consisted of one or occasionally a cluster of several dots for both rDNA fragments. The small extranucleolar signals representing silent gene repeats were completely removed from nuclei. An overall loss of signal intensity was seen for the A as well as the D HH fragment . However, densitometric quantification revealed that significantly more (factor 1.8) of the A than the D HH fragment remained in the nucleus after electroelution ( Table ). Some of the cells had no hybridization signals at all; the percentage of such cells was different for the two rDNA fragments (34.6% for the D HH fragment and 14.6% for the A fragment). In the case of in situ hybridizations to stimulated lymphocytes after chromatin depletion, the signal consisted of one to several dots for both probes . The signal intensity was also considerably lower than in control cells. In this case, however, densitometry showed that equal amounts of both fragments were removed from nuclei ( Table ). The percentage of cells lacking any signal was practically identical for both probes hybridized (22.1% for A fragment and 23.4% for D HH fragment). The results of double in situ hybridization with telomeric and rDNA probes (or with centromeric and rDNA probes) proved that the differences in attachments of the various repetitive genome elements were reproducible on a single cell level ; thus, these differences cannot result from cell-to-cell variations in the efficiency of chromatin depletion. The eluted chromatin fragments were blotted and hybridized with A and D HH probes. The hybridization intensities were semiquantitatively compared under normalized conditions. In unstimulated lymphocytes, significantly more of the D HH than the A fragment was removed from the cells by the extraction procedure . In stimulated lymphocytes, however, no difference between the amount of eluted A and D HH fragments was detected . The fraction of stimulated lymphocytes contained ∼2% of mitotic cells with potentially different parameters for detaching chromatin fragments, as indicated by Gerdes et al. 1994 and Craig et al. 1997 . The low abundance of mitotic cells allows us to neglect the possible effect on the quantified hybridization values. The principal aim of this paper was to compare the attachment of three functionally different repetitive genome elements: centromeres, telomeres, and ribosomal genes to the nucleoskeleton. The experimental model, human lymphocytes during their metabolic activation, was chosen for two reasons. Firstly, the model provides an excellent system to study possible changes in chromatin/nucleoskeleton attachment during transcriptional activation. Secondly, the model allows us to use native cells without the possible effects of long-term cultivation. Our approach is based on a gentle and controlled removal of unattached chromatin fragments from permeabilized cells , followed by a combination of detection of the DNA sequences remaining in the nucleus by in situ hybridization, and that of the extracted DNA fraction by Southern blot hybridization. The extraction method used is known to preserve not only the basic morphological characteristics of the nucleoskeleton, but also the synthetic activities of cell nuclei . The in situ approach allowed us to investigate signal intensity as well as signal distribution at the single cell level. Double in situ hybridization experiments served as a reliable control to describe attachment characteristics of the repetitive genome elements simultaneously in a single cell, thus eliminating possible errors when comparing parallel experiments. We found that centromeric and telomeric DNA sequences show great differences in their interactions with the nucleoskeleton. The majority of centromeric alpha-satellite repeats are removed during the extraction procedure; i.e., they cannot be anchored by a massive number of attachment sites to the nucleoskeleton . The general attachment pattern observed was not connected with the activity of cell metabolism as the results were identical in both unstimulated and stimulated lymphocytes. However, it is possible that a minority of the centromeric sequences is still attached as Strissel et al. 1996 mapped SARs on chromosomes 1 and 16, and were able to hybridize the SAR fraction to the centromeres of the other mitotic chromosomes. In contrast, telomeric chromatin is tightly attached to the nucleoskeleton in unstimulated as well as stimulated human lymphocytes. This observation is in agreement with the outcome of studies on nuclear matrix preparations of several established cell lines . In two studies on further characterization of the sequences retained in nuclei after nucleoskeleton preparation, telomeres or telomeric repeats were not described . The reason for this difference to the findings presented here might be found in different methods of evaluation used after the electroelution step. Our results on nucleoskeleton attachment of alpha-satellite repeats and telomeric repeats prove that two genomic elements can vary dramatically in their attachment properties despite of the fact that they are both transcriptionally silent. Moreover, it seems that the attachment of telomeric and alpha-satellite repeats is similar throughout interphase. It is known that unstimulated lymphocytes are in G0 phase of the cell cycle . In our samples of stimulated lymphocytes, we counted that ∼58% of the cells were in G1 and 40% were in S and G2 phase . Nevertheless, the amount of electroeluted alpha-satellite and telomeric sequences was similar in both samples of lymphocytes. It has been reported that the different chromatin-depletion protocols preferentially retain either structural (nuclear matrix, nuclear scaffold) or functional (nucleoskeleton) sequences . This may explain discrepancies of our findings to reports about nuclear scaffold attachment of centromeres . However, in the case of telomeres, our results are comparable to the study of Ludérus et al. 1996 , although different preparation techniques were used (nucleoskeleton, nuclear scaffold). The nature of the attachments of centromeres and telomeres to the nucleoskeleton is not yet fully understood. As both centromeres and telomeres are transcriptionally inactive, the attachment sites cannot be attributed to anchoring via transcriptional complexes. de Lange 1992 suggested that a nucleoprotein complex containing TTAGGG repeats could be the element responsible for nuclear scaffold attachments as TTAGGG repeats introduced by DNA transfection did not behave as matrix-attached loci. The exact nature of these interactions still remains to be defined. However, we can conclude that telomeres rather than centromeres contribute to the formation of intranuclear order by being anchored to the nucleoskeleton. The interaction of ribosomal genes with the nucleoskeleton is of a more complex nature. (a) Clusters of nontranscribed ribosomal genes, which are found outside the nucleolus in unstimulated human lymphocytes , are completely removed during the extraction procedure. (b) When comparing the attachment pattern of the transcription unit (A fragment) and the intergenic, nontranscribed spacer (D HH fragment), considerable differences between unstimulated and activated lymphocytes can be observed. In unstimulated lymphocytes, the amount of D HH fragment removed during extraction is significantly higher than the amount of A fragment. Blot hybridization confirmed the results obtained by in situ analysis. A fraction of cells exists with weaker signal than seen in control cells, or with no signal at all for both the A and D HH fragments. The cell fraction without hybridization signal is larger for the D HH fragments. In activated lymphocytes, the amount of A and D HH fragments eluted is roughly the same. The results on unstimulated lymphocytes can be partly explained by an attachment via the transcription complexes . Within the extranucleolar ribosomal gene clusters, no transcription takes place . As expected, the transcription unit (A) and the intergenic spacer (D HH ) are extracted completely. Within the nucleolus, a preferential extraction of the D HH fragment is observed. Nevertheless, a certain amount of D HH fragment remains within the nucleolus. Furthermore, in stimulated lymphocytes, the amount of A and D HH fragment removed during the extraction procedure is roughly the same. These results imply that additional mechanisms of attachment of rDNA to the nucleoskeleton must be of importance. It could, for instance, be assumed that the permanent attachment sites (matrix attachment regions, MARs) that organize the DNA into loops are responsible for at least part of the attachments. MARs have been found preferentially within the intergenic spacers by several authors . One also has to bear in mind that unstimulated lymphocytes are in G0 phase of the cell cycle, whereas stimulated lymphocytes consist of cells in various phases of cell cycle. This could explain the somewhat unexpected finding that the rDNA fragments of stimulated lymphocytes are not significantly more retained in nuclei than those of unstimulated cells. Therefore, in contrast to telomeric and alpha-satellite repeats, a cell-cycle dependence of the rDNA attachment to the nucleoskeleton cannot be ruled out at this point. In addition, differences might exist in nucleoskeleton attachment of ribosomal genes between different cell types studied. As we have shown in Weipoltshammer et al. 1996b , in nucleoskeleton preparations of HeLa cells, the A fragment remains in the nucleus, whereas the D HH fragment is almost completely removed. All these nucleoskeleton preparations were performed several times with each cell type (unstimulated/stimulated lymphocytes, HeLa cells growing in suspension), and the results were highly reproducible. The reasons for these differences are unknown. However, one can speculate that the differences are due to the cell types studied. In contrast to lymphocytes of the peripheral blood, HeLa cells are malignant, long-term cultured cells. They show alterations in genome (more rDNA gene repeats present and presumably different transcriptional activity of ribosomal genes). Thus, the nucleoskeleton attachment characteristics of ribosomal genes are dependent on the level of transcriptional activation of ribosomal genes. The results obtained by our study cannot be explained only by one model of nucleoskeleton attachment. Concerning the nature of the interaction of ribosomal genes with the nucleoskeleton, it is most probable that the attachment characteristics we observe result from a combination of sequence-specific DNA/nucleoskeleton attachments (attached either directly or via a mediating protein complex), and of functional attachments, mediated above all by RNA polymerase I transcriptional complexes. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments with the nucleoskeleton, and (c) the activation of nucleolar transcription is connected with a spatial rearrangement of specific rDNA elements relative to the nucleoskeleton. In conclusion, we can observe that very stable DNA/nucleoskeleton attachment sites exist that seem to be independent of cell type and activation state of the cell such as the telomeres . It can be speculated that telomeres play an important role in the formation of chromosome order in interphase nuclei. Vice versa, the majority of the centromeric alpha-satellite sequences can be removed from the nucleus independent of the activation state of the cell. There also exist, however, DNA stretches, like the ribosomal gene repeats, where an attachment to a nucleoskeleton is not only dependent on the activation state of the DNA fragment/gene in question (actively transcribed versus silent genes), but probably also on the cell type. Furthermore, our results on ribosomal genes indicate that more than one attachment mechanism has to be taken into account. Additional studies will be necessary to understand properly the nature of these attachment mechanisms and their functional significance. | Study | biomedical | en | 0.999995 |
10613901 | All chemicals and reagents were from Sigma Chemical Co. unless indicated otherwise. Initial studies were carried out with wild-type megakaryocytes derived from BALB/C mice. p45 NF-E2–deficient mice (a generous gift from Drs. Ramesh Shivdasani and Stuart Orkin, Harvard Medical School, Boston, MA) are in a mixed C57Bl/6-129/Sv background . When their megakaryocytes were studied, wild-type mice with a similarly mixed background were used as controls. There was no obvious difference between wild-type megakaryocytes from these different strains with respect to the αIIbβ3 functions studied. Bone marrow cells were harvested from 8–9-wk-old mice by flushing both sets of femurs and tibias with PBS containing 2% bovine serum albumin, 0.38% trisodium citrate, and 1 U/ml DNAse. Mononuclear cells were isolated over Ficoll Hypaque (1.080 g/ml; Pharmacia) after a 30-min centrifugation at 400 g . Low-density mononuclear cells were cultured for up to 9 d at a starting density of 10 6 cells/ml in Iscove's Modified Dulbecco's medium (Irvine Scientific) supplemented as described , and including 10 ng/ml murine TPO (a gift from Kirin Brewery Ltd., Japan), murine IL-6 and human IL-11 (Biosource International, Inc.). Cytospins of 20,000–100,000 cells were stained with Wright-Giemsa to analyze cell morphology. Cell size, antigen expression and DNA ploidy were assessed by flow cytometry. After 0–9 d in culture, bone marrow-derived cells were centrifuged at 200 g for 10 min, washed and resuspended in PBS at 4 × 10 6 /ml. Then 50-μl aliquots were incubated for 30 min at 4°C with one of the following antibodies: 10 μg/ml FITC-conjugated anti-αIIb (rat anti–mouse CD41; PharMingen); biotin anti-mouse GP V (murine clone C24.25.1; a gift from V. Ramakrishnan, Cor Therapeutics, Inc., South San Francisco, CA); FITC-anti-Gr-1, a granulocyte marker (rat anti–mouse; PharMingen); phycoerythrin-anti-Sca-1, a progenitor cell marker (rat anti–mouse; PharMingen); or a 1:1,000 dilution of rabbit anti–mouse GP Ibα (from V. Ramakrishnan). Negative controls included an irrelevant isotype-matched rat or mouse antibody (PharMingen) or normal rabbit serum, as needed. When the primary antibodies were biotin-anti–GP V or rabbit anti-GP Ibα, the cells were washed and subsequently incubated for 15 min at 4°C with 20 μg/ml FITC-streptavidin or 14 μg/ml FITC anti-rabbit immunoglobulin (heavy plus light chains), respectively (Biosource International). Cells were finally resuspended in PBS containing 1 μg/ml propidium iodide and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). DNA ploidy was assessed as described . Purified human fibrinogen (Enzyme Research Laboratories) was labeled with FITC . Human fibrinogen was used as a ligand instead of murine fibrinogen because of its wider availability and its capacity to bind to and support the aggregation of activated murine platelets . Furthermore, in preliminary studies, murine and human fibrinogen were found to be equivalent inhibitors of FITC-human fibrinogen binding to agonist-stimulated murine and human platelets. After 6 d in culture, bone marrow cells were sedimented by gravity for 60 min at 37°C in a 50-ml conical polypropylene tube. In addition to achieving modest enrichment of megakaryocytes, gravity sedimentation limited any functional damage that might occur to these fragile cells during centrifugation. When the effects of certain inhibitors were studied (see Results), they or appropriate vehicle control buffer were added at this stage for the final 20 min. Cells were then gently resuspended to 4 × 10 6 /ml in modified Tyrode's buffer containing 1 mM CaCl 2 and 1 mM MgCl 2 and then incubated for 30 min at room temperature in the presence of FITC-fibrinogen (250 μg/ml), specific agonists and inhibitors, 10 μg/ml of the non–function-blocking anti-αIIb antibody, and 20 μg/ml phycoerythrin-streptavidin (Molecular Probes). After a 10-fold dilution with buffer containing 1 μg/ml propidium iodide, fibrinogen binding was quantified by flow cytometry . FITC-fibrinogen binding was monitored in the FL1 channel of the flow cytometer on the gated subset of viable cells (e.g., negative for propidium iodide, FL3) that expressed αIIbβ3 (FL2). Preliminary studies indicated that fibrinogen binding was inhibited ≥90% by either 10 mM EDTA , 5 μM kistrin , or 20 μg/ml 1B5, a function-blocking hamster monoclonal antibody specific for murine αIIbβ3 . Therefore, specific fibrinogen binding was defined as binding that was inhibited by kistrin or EDTA. When it was necessary to compare the binding data between subpopulations of cells that were heterogeneous in size and αIIbβ3 density, specific FITC-fibrinogen binding was expressed as a percent of maximal binding obtained in the presence of 1 mM MnCl 2 , an activator of integrins . To determine the effect of isolated integrin cytoplasmic tails on fibrinogen binding to megakaryocytes, cDNA encoding chimeric proteins consisting of the extracellular and transmembrane domains of the Tac subunit of the human IL-2 receptor (CD25) and the human β3 cytoplasmic tail (Tac-β3) or the α5 cytoplasmic tail were cloned into a Sindbis virus vector (pSinRep5; Invitrogen). As a further control, a tailess Tac construct was also prepared. Linear, capped viral RNA was prepared and pseudovirions were produced in BHK cells using a Sindbis Expression System (Invitrogen). Pseudovirion titers were such that a 1:3,000 dilution of BHK cell supernatant infected virtually all BHK cells in a 100-mm dish, as determined by surface expression of Tac using a phycoerythrin-conjugated antibody to human CD25 (PharMingen). To infect megakaryocytes, day 5 bone marrow cells were suspended in a 1:1.5 dilution of virus for 1 hand then diluted with 7 vol of serum-free medium and cultured for another 24 h. The cells were stained with FITC-fibrinogen, phycoerythrin-anti–human CD25 (PharMingen), and propidium iodide, and fibrinogen binding to large, viable megakaryocytes was quantified by flow cytometry. Bone marrow-derived cells were cultured for 5 or 6 d, allowed to settle by gravity for 1 h and resuspended to 2 × 10 5 /ml in buffer containing 137 mM NaCl, 2.7 mM KCl, 3.3 mM NaH 2 PO 4 , 1 mM MgCl 2 , and 3.8 mM Hepes, pH 7.35. Cells were then incubated on fibrinogen-coated coverslips for 45 min at 37°C in the presence or absence of 100 nM phorbol myristate acetate, the latter added to activate protein kinase C and enhance cell spreading . After removing nonadherent cells, the adherent cells were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and stained with monoclonal anti-vinculin antibody, FITC-anti-mouse IgG and rhodamine-phalloidin. Cells were then analyzed by confocal laser scanning microscopy and photomicrographs were prepared using Adobe Photoshop 5.0 . In some experiments, megakaryocytes from day 5 cultures were infected with recombinant Sindbis virus expressing either C3 exoenzyme, an inhibitor of Rho GTPase , or chloramphenicol acyl transferase (Sindbis/CAT; kind gifts from Drs. C.S. Hahn, University of Virginia and M.A. Schwartz, Scripps Research Institute). Viral stocks were produced and titered in BHK cells as described . For infection, megakaryocytes were allowed to adhere to fibrinogen-coated coverslips for 30 min at 37°C and incubated with Sindbis/C3 or Sindbis/CAT for 1 h at 37°C at a multiplicity of infection of 50. After washing, the cells were incubated another 2.5 h in buffer, and 100 nM phorbol myristate acetate was added for the final 30 min to enhance spreading. After washing, adherent cells were fixed, permeabilized, stained, and examined by confocal microscopy. In parallel, some cells were infected on fibrinogen-coated plastic dishes, scraped into SDS sample buffer, and 50-μg aliquots were subjected to SDS-PAGE under reducing conditions . After transfer to nitrocellulose, blots were probed with a rabbit polyclonal antibody specific for Sindbis E1 protein (a gift from J.H. Strauss, California Institute of Technology, Pasadena, CA) or with a murine monoclonal antibody to Rho (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed for 0.5–2 min by chemiluminescence (ECL; Amersham Corp.). To obtain primary murine megakaryocytes for studies of αIIbβ3 signaling, a low-density fraction of bone marrow was cultured in serum-free medium containing 10 ng/ml TPO, IL-6, and IL-11. Total cell number decreased progressively by 45, 73, and 89% on days 3, 6, and 9, respectively. On the other hand, Wright-Giemsa staining showed a progressive increase in the proportion of large, polyploid cells, such that by day 6 both small, immature megakaryocytes with basophilic cytoplasm and larger, mature megakaryocytes with granular, eosinophilic cytoplasm were readily apparent . The vast majority of cells on day zero were small, as assessed by flow cytometric light scatter . However, by day 3 a subpopulation of larger cells of intermediate size was evident, and by day 6 a subpopulation of large cells was present, typically amounting to 5–10% of the total. When the large cells were FACS-sorted and stained, they consisted of mature megakaryocytes . The relative proportion of large megakaryocytes decreased slightly by day 9 in culture, presumably the result of programmed cell death . On day 6, the DNA ploidy of the small cells was 2N, while that of the intermediate-size and large cells ranged from 2-32N and 8-128N, respectively . Surface antigen expression was characterized by flow cytometry ( Table ). The proportion of αIIbβ3-expressing cells increased progressively with time and by day 6 it represented 21% of all small cells, 98% of the intermediate-size cells and >99% of the large cells. On day zero, 15% of the small cells were positive for αIIbβ3, due in part to the presence of platelets. Throughout the culture period, a significant minority of the cells expressed Sca-1 ( Table ), and some coexpressed αIIbβ3, suggesting that they were committed megakaryocyte progenitors. Expression of components of the platelet GP Ib-V-IX complex appeared to lag behind expression of αIIbβ3 because not every αIIbβ3-expressing cell was positive for GP Ib or GP V ( Table ). Moreover, some cells expressed GP Ib but not GP V, suggesting that GP V is not required for GP Ib expression. On day 6, most of the small cells were positive for the myeloid marker, Gr-1, as were 53% of the intermediate-size cells and 2% of the large cells. Taken together, these results indicate that murine megakaryocytes can undergo progressive expansion and maturation in culture, permitting a detailed analysis of αIIbβ3 signaling during megakaryocyte development. Inside out signaling in αIIbβ3-expressing megakaryocyte progenitors and developing megakaryocytes was assessed on day 6 of culture by measuring the agonist-dependent binding of a saturating concentration of FITC-fibrinogen. Large megakaryocytes bound little or no fibrinogen in the absence of an agonist . On the other hand, specific fibrinogen binding was observed after a 30-min incubation of the cells with 10 ng/ml SDF-1α, a CXCR4 receptor agonist or 100 ng/ml TPO, the agonist for c-Mpl receptors . Fibrinogen binding was even greater in response to typical platelet agonists, including 10–100 μM ADP plus epinephrine, 0.5 U/ml thrombin, or 2 mM PAR4 thrombin receptor-activating peptide . Although higher concentrations of these agonists did not stimulate additional fibrinogen binding, combinations of three or more agonists markedly increased fibrinogen binding, in some cases equivalent to that obtained with MnCl 2 . None of the agonists increased surface expression of αIIbβ3. Fibrinogen binding induced by the combination of PAR4-activating peptide, ADP, and epinephrine was inhibited >80% by preincubation of the cells with 2 μM PGI 2 or PGE 1 , compounds that inhibit platelet aggregation through receptor-mediated stimulation of adenylyl cyclase . In addition, fibrinogen binding was inhibited 48% by 100 nM wortmannin, an inhibitor of certain PI 3-kinases, 40% by 12 μM bisindolylmaleimide, a protein kinase C inhibitor, and 57% by 100 μM BAPTA-AM, a chelator of cytoplasmic free Ca 2+ . In contrast, preincubation of cells with 1 mM aspirin to inhibit cyclooxygenase had no effect on fibrinogen binding, although it did inhibit the response to arachidonic acid as expected. Collectively, these results indicate that excitatory agonists can stimulate and certain inhibitory agonists can block the ligand binding function of αIIbβ3 in large megakaryocytes, just as in platelets. Furthermore, activation of αIIbβ3 is regulated in a positive fashion by signaling pathways that likely involve PI 3-kinase, protein kinase C, and free Ca 2+ , and in a negative fashion by cyclic AMP. Fig. 3 B shows that up to 60% of large megakaryocytes bound fibrinogen in response to agonists. To place this in perspective, ≥90% of agonist-activated blood platelets obtained from the same donor mice bound fibrinogen (not shown). The lack of responsiveness of some large megakaryocytes appeared to be due to impaired signaling to αIIbβ3 because FITC-fibrinogen binding to all of the large cells could be induced with MnCl 2 . Furthermore, very few intermediate-size megakaryocytes and no small αIIbβ3-expressing cells bound fibrinogen in response to the agonists depicted in Fig. 3 or in response to as much as 500 nM phorbol myristate acetate to activate protein kinase C. The results were similar if fetal hematopoietic tissue from the livers of day 14.5 murine fetuses was used instead of bone marrow as the source of megakaryocyte progenitors . Overall, these results indicate that inside out signaling pathways responsible for activation of αIIbβ3 become fully developed only late in megakaryocyte development. Platelet adhesion to an immobilized αIIbβ3 ligand triggers the reorganization of actin filaments, filopodial and lamellipodial extension, and cell spreading . To determine if megakaryocytes can engage in such outside in signaling, day 6 bone marrow cultures were incubated on fibrinogen-coated coverslips for 45 min. By this time, most of the large megakaryocytes had become adherent, and this response was αIIbβ3-dependent because it could be blocked by 5 μM kistrin. Adherent megakaryocytes ranged in diameter from 10 to >50 μm, and many exhibited filopodia and lamellipodia . When 100 nM phorbol myristate acetate was present, the megakaryocytes spread more extensively and most now exhibited focal adhesions and stress fibers . Actin filaments were often circumferential and parallel to the cell margins, rather than perpendicular as in fibroblasts, an observation made previously with guinea pig megakaryocytes . These results indicate that ligand binding to αIIbβ3 can promote cytoskeletal reorganization in megakaryocytes, accompanied by changes in cell shape. Studies of mice deficient in the p45 subunit of NF-E2 have revealed that this transcription factor is necessary for the final phases of megakaryocyte development and platelet production . Since inside out signaling to αIIbβ3 was fully developed only in mature megakaryocytes , we considered whether genes regulated by NF-E2 might be required for αIIbβ3 activation. Accordingly, bone marrow from NF-E2 −/− mice was cultured for 6 days with TPO, IL-6, and IL-11, yielding a subpopulation of large megakaryocytes similar to wild-type and NF-E2 +/− controls. However, unlike cells from wild-type or NF-E2 +/− mice, large NF-E2 −/− megakaryocytes displayed a virtual absence of FITC-fibrinogen binding in response to the combination of PAR4 thrombin receptor-activating peptide, epinephrine, and ADP . Similar results were obtained when the cells were stimulated with as much as 500 nM phorbol myristate acetate. This impairment of fibrinogen binding was due to a defect in inside out signaling because NF-E2 −/− megakaryocytes expressed normal amounts of αIIbβ3 on their surfaces, and they bound FITC-fibrinogen as well as wild-type or NF-E2 +/− megakaryocytes in response to MnCl 2 . These results indicate that NF-E2, or more likely genes regulated by NF-E2, are required for agonist-induced activation of αIIbβ3. In fibroblastic cell lines, the heterologous expression of membrane-tethered β3 integrin cytoplasmic tails (in the form of a human Tac-β3 fusion protein) can reverse the high-affinity state of a constitutively active mutant of αIIbβ3. In contrast, Tac-α5 tails have no such effect . To determine if chimeric β3 tails could block agonist-induced activation of αIIbβ3, day 5 bone marrow cultures were incubated for 1 h with Sindbis pseudovirions expressing Tac-β3 RNA or, as a control, pseudovirions expressing Tac-α5. 24 h later, flow cytometry showed that the Tac chimeras had been expressed in 5–20% of large megakaryocytes. Under these conditions, Sindbis virus infection did not affect either αIIbβ3 surface expression or megakaryocyte viability. Large megakaryocytes expressing relatively low levels of Tac-β3 or Tac-α5 bound fibrinogen in response to agonists almost as well as noninfected cells. In contrast, megakaryocytes expressing relatively high levels of Tac-β3 bound very little fibrinogen, and significantly less than cells expressing high levels of Tac-α5 . The more modest reduction in fibrinogen binding in cells expressing high levels of Tac-α5 was also observed with a tailless Tac construct (not shown), suggesting that it might have been due to viral infection, per se. Since Tac-β3 cannot form a heterodimer with αIIb but may nevertheless interact with cytoplasmic proteins that normally bind to αIIbβ3 , these results suggest that one or more β3 cytoplasmic tail-binding proteins mediates physiological activation of αIIbβ3. To explore mechanisms of outside in signaling in megakaryocytes, cells adherent to fibrinogen were exposed for 1 h to Sindbis virus encoding C3 exoenzyme, which ADP-ribosylates and inactivates the small GTPase, Rho . After subsequent incubation in the presence of phorbol myristate acetate to enhance cell spreading, cytoskeletal organization was analyzed. Both Sindbis/C3 and a control virus, Sindbis/CAT, infected adherent cells, as evidenced by the time-dependent expression of the 61-kD viral E1 protein on Western blots of cell lysates (not shown). Infection with Sindbis/C3 was associated with a reduction in the percent of megakaryocytes that contained focal adhesions and stress fibers . Whereas 70% of adherent megakaryocytes incubated with Sindbis/CAT displayed focal adhesions and stress fibers, only 30% of cells incubated with Sindbis/C3 did so . Furthermore, the remaining stress fibers in Sindbis/C3-treated megakaryocytes were often thinner and less densely packed than in the control cells. The effect of Sindbis/C3 on the megakaryocyte cytoskeleton could reasonably be attributed to ADP-ribosylation of Rho, because ∼50% of this protein in cell lysates displayed a retarded electrophoretic mobility characteristic of ribosylation . In contrast to its effect on focal adhesions and stress fibers, Sindbis/C3 did not appear to impair megakaryocyte spreading or the formation of filopodia and lamellipodia. Thus, Rho is responsible for a subset of cytoskeletal responses in megakaryocytes that is triggered, in part, by outside in signaling through αIIbβ3. A common strategy to characterize intracellular signaling pathways is to express wild-type and mutant gene products and observe the effects on cell function. Unfortunately, this approach cannot be used to characterize integrin signaling in platelets because of inherent difficulties in expressing recombinant proteins in these anucleate cells. Consequently, we hypothesized that primary murine megakaryocytes might serve as a relevant and tractable model system for studies of αIIbβ3 signaling. Culture of murine bone marrow in the presence of TPO, IL-6, and IL-11 led to megakaryocyte expansion and maturation, allowing functional studies of αIIbβ3 and the following conclusions to be drawn. (a) As with platelets, megakaryocytes are capable of inside out signaling to control agonist-induced fibrinogen binding to αIIbβ3. This process becomes fully functional late in megakaryocyte development, requires a gene or genes regulated by transcription factor NF-E2, and converges on the β3 cytoplasmic tail. (b) As with platelets, the adhesion of megakaryocytes to immobilized fibrinogen triggers outside in signals through αIIbβ3 that lead to cell spreading and cytoskeletal reorganization. Adherent megakaryocytes exhibit filopodia, lamellipodia, and Rho-dependent focal adhesions and stress fibers. (c) Sindbis virus vectors can be used to express heterologous proteins in megakaryocytes, enabling αIIbβ3 signaling to be studied in a physiological context in ways not possible with platelets. In both human and murine platelets, ligand binding to αIIbβ3 is regulated by signaling events that modulate integrin affinity and/or avidity . In platelets, the use of selective enzyme inhibitors has implicated protein and lipid kinases, such as PI 3-kinase and protein kinase C, in receptor-mediated activation of αIIbβ3. However, the mechanism by which effectors modulate αIIbβ3 function is unknown. A recent report concluded that human bone marrow-derived megakaryocytes stimulated with TPO adhere to immobilized fibrinogen in a manner dependent on αIIbβ3 and PI 3-kinase . The present studies establish that the binding of soluble fibrinogen to αIIbβ3 is regulated in murine megakaryocytes. Unstimulated megakaryocytes failed to bind soluble fibrinogen, indicating that αIIbβ3 is in a default low affinity/avidity state. Stimulation with agonists, including TPO and substances that interact with G protein-linked receptors (SDF-1α, thrombin, PAR4 receptor-activating peptide, ADP, epinephrine), caused rapid activation of αIIbβ3 and fibrinogen binding to a majority of mature megakaryocytes . In fact, a combination of agonists caused as much fibrinogen binding as did MnCl 2 , an activator of integrins . Platelet aggregation inhibitors, such as PGI 2 and PGE 1 as well as inhibitors of PI 3-kinase and protein kinase C, blocked agonist-induced fibrinogen binding. Thus, αIIbβ3 is likely subject to similar regulatory mechanisms in mature megakaryocytes and in platelets. A consistent finding in this study was that few intermediate-size megakaryocytes and no small αIIbβ3-expressing progenitors bound fibrinogen in response to agonists . This failure could not be explained by a lack of αIIbβ3 expression, by prior binding of ligands to αIIbβ3 during culture, or by structural alterations in the integrin, since these cells could bind fibrinogen in response to MnCl 2 . The most likely explanation for these observations is that inside out signaling pathways to αIIbβ3 become fully developed relatively late in megakaryocytopoiesis. Developmental regulation of αIIbβ3 affinity/avidity, as opposed to regulation of αIIbβ3 expression, could have beneficial in vivo consequences by limiting the binding of soluble adhesive ligands to αIIbβ3 on progenitors and young megakaryocytes, thereby preventing their unnecessary aggregation. At the same time, the presence of αIIbβ3 on the surface of these cells, even in a low affinity/avidity state, might enable them to adhere to fibrinogen or other ligands in the extracellular matrix , and to initiate outside in signals . The most striking finding in this study was an inability of p45 NF-E2 −/− megakaryocytes to bind fibrinogen in response to agonists . NF-E2 contains a 45-kD subunit that is restricted to hematopoietic cells and a ubiquitous 18-kD subunit . Since p45 and p18 hetero-oligomers are required for transcriptional activation, the results with p45-deficient megakaryocytes suggest that one or more genes regulated by NF-E2 are required for full inside out signaling. This discovery would have been difficult to validate and explore in platelets because NF-E2 −/− mice have so few circulating platelets . In addition to highlighting the value of the megakaryocyte model, these results suggest that the identification and characterization of NF-E2–regulated genes should advance our understanding of the mechanisms of inside out regulation of αIIbβ3. Recently, an in vivo immunoselection strategy was used to identify thromboxane synthase as a p45 NF-E2 target gene in the HEL cell megakaryoblastic cell line . In platelets, this enzyme is responsible for the conversion of prostaglandin endoperoxides to thromboxane A 2 . Since thromboxane A 2 is a platelet agonist, a deficiency of thromboxane synthase in NF-E2 −/− megakaryocytes could, in theory, help to explain the inside out signaling defect in these cells. However, this is unlikely for several reasons. First, most investigators including ourselves have found that agonists such as thrombin, ADP, and epinephrine stimulate little or no fibrinogen binding to HEL cells or to other megakaryoblastic cell lines. Thus, although these cells express thromboxane synthase, they appear to lack critical intermediates required for inside out signaling . Second, we found that large megakaryocytes from wild-type mice bound fibrinogen in response to PAR4 peptide, ADP, and epinephrine even after treatment with aspirin, which blocks cyclooxygenase and subsequent production of thromboxane A 2 . Third, thromboxane A 2 is not involved in the initial activation of αIIbβ3 in platelets, but rather reinforces the responses to other agonists . It is now feasible, and may be preferable, to use primary megakaryocytes instead of megakaryoblastic cell lines to identify NF-E2–regulated genes involved in αIIbβ3 signaling. Sindbis virus-mediated expression of Tac-β3 integrin cytoplasmic tails in megakaryocytes caused dose-dependent inhibition of agonist-induced fibrinogen binding to αIIbβ3 . Although expressed to the same extent as Tac-β3, Tac-α5 caused significantly less inhibition of agonist-induced fibrinogen binding to megakaryocytes ( P < 0.01), and the extent of inhibition was identical to that observed with a tailless Tac construct. The marked inhibitory effect of Tac-β3 is consistent with the suggestion that overexpressed β3 tails may bind and titrate cytoplasmic proteins that otherwise would interact with αIIbβ3 to regulate receptor affinity or avidity . Several proteins have been shown to interact with the β3 tail in model systems and by affinity chromatography, including cytoskeletal proteins such as talin, α-actinin, filamin, and myosin, a 14-kD polypeptide called β3-endonexin, and the cytoplasmic tail of αIIb . In principle, any or all of these interactions with the β3 tail might influence the conformation or oligomerization state of αIIbβ3 and, therefore, its ligand binding properties . The adhesion of platelets to surfaces coated with fibrinogen or von Willebrand factor triggers tyrosine phosphorylation of numerous proteins, cell spreading, and actin rearrangements . In the current study, we found that murine megakaryocytes also adhere to fibrinogen via αIIbβ3 and undergo spreading and cytoskeletal reorganization in an αIIbβ3-dependent fashion, particularly in the presence of a costimulus like phorbol myristate acetate . This response is similar in some respects to the morphological changes and actin rearrangements in fibroblasts that are triggered by the combination of cell adhesion via β1 integrins and stimulation by growth factors . β1 integrins (α4β1 and α5β1) have been demonstrated to play key roles in hematopoietic cell development, presumably by mediating outside in signaling . In addition, β1 integrins in mature human or guinea pig megakaryocytes can support attachment and spreading on fibronectin and focal adhesion formation . In contrast, other than regulation of fibrinogen uptake into α-granules, very little is known about the function of αIIbβ3 in megakaryocytes . αIIbβ3 signaling cannot be essential for megakaryocytopoiesis or thrombopoiesis because platelet numbers are normal in mice and humans deficient in αIIbβ3 . This is not to say, however, that αIIbβ3 plays no role in these processes. For example, proplatelet and platelet formation may involve a series of membrane protrusive events which could involve integrin-triggered cytoskeletal reorganization . In support of this, certain anti-αIIbβ3 antibodies can inhibit proplatelet formation by cultured megakaryocytes . The formation of filopodia, lamellipodia, and focal adhesions in fibrinogen-adherent megakaryocytes suggests that signals from αIIbβ3 may activate Rho family GTPases, including cdc42, Rac, and Rho . Rho in particular is implicated by the reduction in focal adhesions and stress fibers in megakaryocytes transduced with C3 exoenzyme . Since Rho GTPases may become activated during integrin-mediated cell adhesion and signal to the cytoskeleton and nucleus , we suggest that signals downstream of αIIbβ3 may play some adjunctive role in megakaryocyte or platelet development. In this context, antibody or peptide blockade of ligand binding to αIIbβ3 has been shown to impair human megakaryocyte colony formation in a fibrin gel , and treatment of a human megakaryoblastic cell line with C3 exoenzyme caused an increase in DNA ploidy . Further studies of the role of αIIbβ3 signaling in megakaryocytes are warranted. Retroviruses and lentiviruses are being evaluated as transfer vectors for gene therapy, and they also hold promise for studies of megakaryocyte function . The present experiments show that Sindbis virus vectors provide an alternative way to achieve transient expression of recombinant proteins in terminally differentiated hematopoietic cells like megakaryocytes. Transient expression may be particularly useful in situations where long-term expression of a recombinant protein might interfere with cell growth or differentiation, as with C3 exoenzyme and Tac-β3 . Taken together with new strategies to achieve megakaryocyte-specific gene expression in vivo , several complementary approaches are now available to manipulate gene expression in megakaryocytes, establishing these cells as an ideal system for determining the molecular basis of αIIbβ3 signaling. | Study | biomedical | en | 0.999997 |
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