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1 Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan
2 Department of Microbiology, Kansai Medical University, Osaka 570-8506, Japan
3 Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany
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Abstract |
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B did not, suppressed Fas-mediated apoptosis by inducing c-FLIP expression. Furthermore, expression of the dominant negative mutant of either NF-B or IB
canceled not only c-FLIP expression but also inhibitory activity against Fas-mediated apoptosis by Tax. Inactivation of NFAT, however, did not decrease the expression of c-FLIP in HTLV-I-infected T-cells. Taken together, Tax inhibits Fas-mediated apoptosis by up-regulating c-FLIP expression in HTLV-I-infected cells, and NF-B activity plays an essential role in the up-regulation of c-FLIP. |
Introduction |
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Stimulation of Fas on the cell surface leads to binding of the adaptor protein, FADD, via death domain (DD)-DD interaction, and pro-caspase-8/FLICE was subsequently recruited to Fas-associated FADD via death effector domain (DED)-DED interaction. In a complex containing at least Fas, FADD and pro-caspase-8, designated the death-inducing signaling complex (DISC), the proximity of pro-caspase-8 induces its proteolytical autoactivation. Then, activated caspase-8 cleaves and activates the downstream effector caspases, such as caspase-3, 6 and 7, followed by cleavage of many kinds of death substrates that play central roles in apoptotic events (Krammer 2000).
A number of recent reports have indicated that an anti-apoptotic protein, cellular FLICE/caspase-8-inhibitory protein (c-FLIP), which is the mammalian homolog of viral-FLIP encoded by several Herpes and Pox viruses, intensively inhibits Fas-mediated apoptosis during DISC formation (Thome & Tschopp 2001). Multiple splice variants of c-FLIP were reported at the mRNA level, but only two endogenous forms at the protein level, a long form (c-FLIPL; 55 kDa) and a short form (c-FLIPS; 25 kDa). c-FLIPL is structurally similar to pro-caspase-8 containing two DEDs, yet lacks catalytic protease activity, and c-FLIPS has only two DEDs. Both forms have been shown to suppress the activation of caspase-8 after Fas stimulation through blocking DISC formation by directly binding FADD or pro-caspase-8 via homophilic DED-DED interaction.
Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) (Matsuoka 2005). HTLV-I was shown to be involved in several autoimmune diseases, such as HTLV-I-associated myelopathy, HTLV-I-associated uveitis, and HTLV-I-associated arthropathy (Watanabe 1997). The virus-encoded, regulatory protein Tax is known to play a central role in cellular transformation and immortalization during viral infection (Matsuoka 2005). The main function of Tax is the potent activation of several transcription factors including CREB (cyclic AMP-responsive element binding protein)/ATF (activating transcriptional factor)-1 and NF-B. These transcriptional activations would affect the transcriptional regulation in host cells, resulting in dysregulation of cellular proliferation. In addition, Tax was also reported to stimulate transcriptional activity of NFAT, which is involved in Tax-induced expression of IL-2 and FasL in host cells (Good et al. 1996; Rivera et al. 1998).
Transgenic mice carrying the tax gene were indicated to develop autoimmune arthropathy at a high incidence, showing marked synovial and periarticular inflammation with articular erosion caused by the invasion of granulated and mononuclear cells, which resembles human rheumatoid arthritis (Iwakura et al. 1991). Interestingly, splenic T cells derived from tax transgenic mice were resistant to Fas-induced apoptosis and this resistance was correlated with the development of arthropathy in these mice (Kishi et al. 1997). Moreover, tax transgenic mice carrying the lpr mutation developed a more serious arthropathy at higher frequency, while tax transgenic mice with the exogenous fas transgene developed milder arthopathy with less frequency, than tax transgenic mice with only the endogenous fas gene (Iwakura et al. 1998). Thus, accumulating evidences show that the evasion of HTLV-I-infected T-cells from Fas-mediated apoptosis via the function of Tax is involved in the persistent infection of HTLV-I followed by the development of autoimmune arthropathy as well as the onset of HTLV-I-induced leukemogenicity. However, the precise molecular mechanisms of Tax-induced suppression of Fas-mediated apoptosis are not well understood.
In this study, we show that c-FLIP is highly expressed in Tax-expressing HTLV-I-infected T-cells, and Tax-induced constitutive expression of c-FLIP is sufficient to inhibit Fas-mediated apoptosis in HTLV-I-infected T-cells. Tax-induced transcriptional activation of NF-B was shown to be necessary for the expression of c-FLIP without the support of CREB/ATF1 or NFAT. Thus, Tax-induced up-regulation of c-FLIP in HTLV-I-infected T-cells would play a crucial role in the evasion of T-cells from elimination by the host immune surveillance.
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Results |
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To analyze the sensitivity of HTLV-I-infected cells to Fas-mediated apoptosis, we chose seven HTLV-I-infected T-cell lines, MT-2, MT-4, HUT-102, ATL-2, MT-1, TL-Om1 and ATL-43Tb(-). All the HTLV-I-infected cell lines were previously shown to include HTLV-I proviral genome (Mori et al. 2001; Yoshida et al. 2004) while only MT-2, MT-4, HUT-102 and ATL-2 cells produced HTLV-I mRNA and expressed high levels of Tax protein (Fig. 1A). MT-2 cells were reported to produce HTLV-I and express high levels of 40 kDa Tax protein together with gp68, known to be a fusion protein comprising Tax and envelope protein (Tanaka et al. 1992). We examined the sensitivity of these HTLV-I-infected cell lines to Fas-mediated apoptosis by TUNEL assay. MT-1, TL-Om1 and ATL-43Tb(-) cells, which did not express Tax protein, were sensitive to Fas-induced apoptosis as were HTLV-I-negative T lymphoblast CEM-C7 cells, while MT-2, MT-4, HUT-102 and ATL-2, which express high levels of Tax protein, were eminently resistant to Fas-mediated apoptosis (Fig. 1B). We then analyzed the processing of endogenous caspases after Fas-stimulation in these cells (Fig. 1C). In caspase-8, the cleaved active subunit p18 as well as the intermediate fragment p43/41 were observed after stimulation of Fas in CEM-C7, MT-1, TL-Om1 and ATL-43Tb(-) cells. In MT-2 cells, the p18 subunit was never detected, whereas the intermediate fragment p43/41 was slightly observed. In MT-4, HUT-102 and ATL-2 cells, neither the p18 subunit nor p43/41 intermediates were detected. The processed form p19/17 of caspase-3 was observed in CEM-C7, MT-1, TL-Om1 and ATL-43Tb(-) cells, but not in MT-2, MT-4, HUT-102 and ATL-2 cells (Fig. 1C). In all the cells, endogenous Fas was highly expressed on the cell surface, whether Tax protein was expressed or not (Fig. 1D). These results indicate that Fas-mediated apoptosis is inhibited mainly upstream of caspase-8 activation in HTLV-I-infected cell lines expressing Tax.
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Tax was previously reported to induce anti-apoptotic proteins, such as Bcl-XL and c-IAP (inhibitor of apoptosis protein) 2, in both mouse and human T-cells, while Bcl-2 was reported not to be induced by Tax (Tsukahara et al. 1999; Mori et al. 2001). We confirmed that Tax induced expression of Bcl-XL and c-IAP2, but not of Bcl-2, c-IAP1 or XIAP, in CEM-C7 cells transfected with Tax (data not shown). However, Bcl-XL and c-IAP2 were considered to play hardly any role in the insensitivity of the Tax-expressing cells, because they can not function upstream of caspase-8 activation.
Since c-FLIP is the only molecule that has been shown to inhibit Fas-mediated apoptosis upstream of caspase-8 activation, we examined the expression level of c-FLIP in HTLV-I-infected cell lines. Western blot analyses revealed that expression of both c-FLIPL and c-FLIPS was highly and moderately up-regulated in Tax-positive HTLV-I-infected three cell lines (MT-2, HUT-102 and ATL-2) and another Tax-positive cell line, MT4, respectively, while both c-FLIPL and c-FLIPS were hardly detected in Tax-negative cell lines (MT-1, TL-Om1 and ATL-43Tb(-)) (Fig. 2A). Northern blot analyses were performed for c-FLIPL and c-FLIPS by using cDNAs covering the C-terminal and N-terminal sequences of c-FLIPL mRNA, respectively, as probes (Fig. 2B). c-FLIPL mRNA could be quantitatively detected by using the C-terminal sequence but not the N-terminal sequence. Expression of mRNA for both c-FLIPL and c-FLIPS was up-regulated in Tax-expressing MT-2, HUT-102 and ATL-2 cells. Although expression of c-FLIPS mRNA in another Tax-positive cell line, MT4, was low, that of c-FLIPL mRNA was significantly higher than other Tax-negative cell lines. In addition, over-expression of Tax in CEM-C7 cells was indicated to strikingly induce expression of both c-FLIPL and c-FLIPS (see Figs 5 and 7). Taken together, it suggested that Tax induces an accumulation of c-FLIP in both mRNA and protein levels.
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We then analyzed the processing of endogenous caspase-8, -3, and -7 and c-FLIPL after Fas stimulation in CEM-C7, MT-2 and ATL-43Tb(-) cells. Both of the p43/41 intermediates and the active subunit p18 of caspase-8 were induced by the stimulation of Fas in CEM-C7 and ATL-43Tb(-) cells (Fig. 3A). In MT-2 cells, however, the p43/41 intermediates were slightly detectable while the p18 subunit was not observed even 4 h after Fas-stimulation (Fig. 3A). In MT-2 cells, up-regulated c-FLIPL was cleaved to the p43 form 30 min after Fas stimulation, probably by caspase-8 as previously described (Thome & Tschopp 2001). Active forms of caspase-3 and caspase-7 (p19/17 and p20, respectively) were observed in CEM-C7 and ATL-43Tb(-) cells after Fas-stimulation, whereas they were not generated in MT-2 cells.
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Suppression of c-FLIP expression sensitizes MT-2 and HUT-102 cells to Fas-mediated apoptosis
To further analyze the relevance of up-regulated c-FLIP to the resistant phenotype against Fas-induced apoptosis in HTLV-I-infected T-cells, we performed the RNA interference (RNAi) experiment using a lentivirus-based vector which expresses c-FLIP-specific short hairpin RNA (shRNA). MT-2 and HUT-102 cells were infected with lentiviruses expressing c-FLIP-specific shRNA (shFLIP) or no shRNA, and the efficiency of viral infection was quantified by measuring the expression of enhanced green fluorescence protein (EGFP) by flow cytometer, finding that more than 97% of cells express virus-encoded EGFP (Fig. 4A and data not shown). The viral vectors could generate high-titer infectious lentiviruses. Then, we analyzed whether the expression of shFLIP could silence c-FLIP expression, clearly indicating that infection with the lentiviruses expressing shFLIP could dramatically reduce c-FLIP expression in MT-2 and HUT-102 cells (Fig. 4B). As we expected, infection with lentiviruses expressing shFLIP did not affect the expression of other anti-apoptotic proteins, Bcl-XL, Bcl-2, XIAP and c-IAP2 (Fig. 4B).
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We further analyzed DISC formation in MT-2 cells expressing shFLIP (Fig. 4E). In MT-2 cells expressing shFLIP, c-FLIPL/S were scarcely co-immunoprecipitated with Fas, compared with MT-2 cells infected with control virus producing no shRNA. Expression of shFLIP caused pro-caspase-8 to be significantly recruited into DISC after Fas-stimulation, resulting in auto-cleavage of caspase-8 in DISC and activation of the downstream caspases. Therefore, Tax-induced expression of c-FLIP was shown to be enough to suppress Fas-mediated apoptosis in HTLV-I-infected T-cells.
Exogenously expressed Tax inhibits Fas-mediated apoptosis by up-regulating c-FLIP expression
To analyze effects of exogenously expressed Tax on Fas-mediated apoptosis in HTLV-I-negative T-cells, it would be desirable to express it in an inducible fashion, since prolonged expression of Tax conceivably alters various characters of the cells. We then applied a conditional Cre-loxP-mediated induction system for the expression of Tax. An expression vector encoding a chimeric Cre recombinase (MerCreMer) that is flanked on both sides (N- and C-terminal) by a mutated hormone-binding domain of the murine estrogen receptor (Mer) (Zhang et al. 1996), was introduced into CEM-C7 cells, and two stable transfectants expressing MerCreMer protein were selected (clones 14 and 22). These clones were infected with a Lex-Puro/Tax lentiviral vector, which carries a puromycin resistance (puror) gene flanked on both sides by a loxP site and a Tax gene (Fig. 5A). Because of the presence of a poly(A) site connecting to the puror gene, the Tax gene cannot be expressed until the puror-poly(A) sequence is deleted by the function of Cre. MerCreMer, which usually remains in the cytoplasm, can translocate into the nucleus and exert its enzymatic activity in the presence of the synthetic ligand to Mer, 4-hydroxytamoxifen (4-OHT). After treatment with 4-OHT, Cre-loxP-mediated recombination leads to delete the floxed puror-poly(A) cassette, thereby allowing expression of the Tax gene (Fig. 5A). As we expected, clones 14 and 22 infected with Lex-Puro/Tax viruses (hereafter called Lex14 and Lex22, respectively) were potently induced to express not only Tax but also c-FLIP when stimulated with 4-OHT for 36 h (Fig. 5B). Lex14 and Lex22, which were sensitive to Fas-mediated apoptosis in the absence of 4-OHT, became resistant to Fas-mediated apoptosis 36 h after treatment with 4-OHT (Fig. 5C,D). These data indicate that transiently over-expressed Tax can induce the protective activity against Fas-mediated apoptosis in CEM-C7 cells.
To analyze whether the resistance phenotype of CEM-C7 cells expressing Tax is induced by c-FLIP expression, Lex22 cells were further infected with lentiviruses expressing shFLIP or no shRNA and their sensitivity to Fas-mediated apoptosis was examined. Expression of shFLIP was shown not only to inhibit induction of c-FLIP expression directed by 4-OHT-induced Tax (Fig. 5E), but also to specifically sensitize Lex22 cells to Fas-mediated cell death with associated activation of caspase-3 (Fig. 5F,G). These results indicate that transiently expressed Tax intensively inhibits Fas-mediated apoptosis through induction of c-FLIP expression.
Minute analyses of the activation of caspase-3 in Lex22 cells showed that the expression of shFLIP induced caspase-3 activation in transiently Tax-expressing Lex22 cells without the stimulation of Fas, while the expression of green fluorescence protein (GFP)-specific shRNA (shGFP) never induced caspase-3 activation (Figs 5G and 6A). In addition, the expression of shFLIP was shown to weakly induce apoptosis in Tax-expressing HUT102 and MT2 cells without the stimulation of Fas (Fig. 4C,D). To clarify whether down-regulation of c-FLIP expression induces apoptosis in Tax-expressing cells, or expression of shFLIP is toxic independently of the down-regulation of c-FLIP expression, we examined the effects of transient expression of viral FLIP (v-FLIP) E8, v-FLIP MC159, mouse c-FLIPL, and mouse c-FLIPS on apoptosis in Lex22 cells expressing Tax and shFLIP. All the FLIP molecules were clearly shown to inhibit not only Fas-mediated caspase-3 activation in Lex22 cells without expressing Tax (Fig. 6B) but also the caspase-3 activation induced by shFLIP in Tax-expressing Lex22 cells expressing shFLIP (Fig. 6A). Thus, over-expressed Tax can induce apoptosis by itself when expression of c-FLIP was inhibited.
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B is required for Tax-induced up-regulation of c-FLIP
We then examined the molecular mechanism of the Tax-induced expression of c-FLIP by using Tax mutants (Fig. 7). Tax mutant M22 activates the transcriptional activity of CREB/ATF1 but not NF-B, while another Tax mutant d3 can activate NF-B but exert little affects on CREB/ATF1 (Fig. 7C,D) (Shimizu et al. 2003). CEM-C7 cells were transiently transfected with the expression vectors encoding Tax or its mutants together with that of GFP and then GFP-positive cells were enriched using a cell sorter. Expression of both c-FLIPL and c-FLIPS was up-regulated by Tax, which is correlated with the expression level of Tax protein (Fig. 7A, lanes 1–5). Since wild-type Tax enhances promoter activity of the expression vector (CMV promoter), whereas its mutants do not, higher expression was introduced in wild-type Tax than in its mutants, M22 and d3 (lane 5, 6 and 7). We then compared the effect of wild-type Tax to those of its mutants at the same expression level (lane 2, 3, 6 and 7). Tax mutant d3 was shown to induce comparable level of c-FLIP to wild-type Tax (lane 2, 3 and 7), while another mutant M22 could hardly induce c-FLIP (lane 2, 3 and 6).
We next analyzed whether Tax mutants can inhibit Fas-mediated apoptosis in CEM-C7 cells (Fig. 7B). Fas-mediated apoptosis was strongly inhibited by not only wild-type Tax but also d3 mutant, whereas M22 mutant could not inhibit Fas-mediated apoptosis. Induced levels of c-FLIP by Tax and its mutant are correlated with their inhibitory activities against Fas-mediated apoptosis, suggesting that d3 mutant-induced activation of NF-B activity plays a role in the up-regulation of c-FLIP expression enough to inhibit Fas-mediated apoptosis.
To further confirm the involvement of NF-B activity in the up-regulation of c-FLIP expression by Tax in CEM-C7 cells, we examined the effects of the dominant negative mutant of NF-B (DNp50) and the dominant negative mutant of IB (IB DN). Since both mutants fractionally suppressed promoter activity of the expression vector of Tax (CMV promoter), we used two or three times more amounts of the expression vector of Tax when co-expressed with these mutants. Expression of either DNp50 or IB DN was shown to strongly inhibit Tax-induced expression of c-FLIPL in cells expressing the same level of Tax protein (Fig. 8A, lane 3-5) and to simultaneously cancel the protective activity of Tax against Fas-mediated apoptosis (Fig. 8B). In this experiment, expression level of Tax was too low for expression of c-FLIPS to be detected. Luciferase assay showed that DNp50 and IB DN suppressed Tax-induced transactivation of NF-B, but not that of CREB/ATF1 (Fig. 8C,D). Taken together, transcriptional activity of NF-B was shown to be necessary for the efficient induction of c-FLIP by Tax.
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Previous studies suggested that Tax can induce transcriptional activity of NFAT, which is involved in induction of IL-2 and FasL genes through a NFAT-specific-binding element, termed as CD28-responsive element (CD28RE), in Tax-expressing T-cells (Good et al. 1996; Rivera et al. 1998). Additionally, expression of c-FLIP is induced in CEM-C7 cells by treatment with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin, which induces transcriptional activity of NFAT (data not shown). We then examined whether activation of NFAT was involved in Tax-induced expression of c-FLIP by using the calcineurin inhibitor cyclosporinee A (Cys A) (Hogan et al. 2003). The constitutive expression of c-FLIP in MT-2 cells was not affected by treatment with Cys A for 4, 6 or 16 h (Fig. 8E). Although the expression of full-length c-FLIPL was reduced after 16 h treatment with Cys A, total amounts of c-FLIPL seemed to be unaffected because the quantity of the cleaved intermediate of c-FLIPL increased. To examine whether NFAT activity was blocked by treatment with Cys A, electrophoretic mobility shift assay (EMSA) was performed using the radiolabelled CD28RE-probe containing the NFAT binding element (Fig. 8F). While two major bands of DNA-protein complexes (C1 and C2) were detected in Cys A-untreated MT-2 cells, only C1 complex was observed even after treatment with Cys A. Moreover, C2 complex was shown to specifically immunoreact to an NFAT1-specific antibody in supershift assays, indicating that NFAT1 was involved in the formation of C2 complex. C1 complex was shown to be composed of cRel and p50 subunits of NF-B because not only anti-cRel Ab but also anti-p50 Ab abolished the band of C1 complex in supershift assays. Thus, Cys A blocked DNA-binding activity of NFAT1 but not NF-B in MT-2 cells. All the results indicated that activation of NF-B is required for Tax-induced up-regulation of c-FLIP expression, whereas NFAT was not involved in Tax-induced c-FLIP expression.
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Discussion |
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We showed transcriptional activity of NF-B to be necessary for the efficient induction of c-FLIP by Tax, because expression of either DNp50 or IB DN inhibited Tax-induced c-FLIP expression (Fig. 8A). In addition, Tax mutant d3, which is capable of activating NF-B but not CREB/ATF1, induced expression of c-FLIP enough to inhibit Fas-mediated apoptosis in CEM-C7 cells (Fig. 7A,B). Although M22 mutant which can activate CREB/ATF1 was shown to faintly induce c-FLIP expression, the amount of M22-induced up-regulated c-FLIP is insufficient to inhibit Fas-mediated apoptosis (Fig. 7A,B). It is important that c-flip promoter was found to contain binding sites for not only transcription factor NF-B but also NFAT(Ghosh 1993; Wingender et al. 2001). TCR-mediated activation of NFAT was reported to up-regulate c-FLIP expression (Thome & Tschopp 2001). We also found that stimulation of CEM-C7 cells with PMA and ionomycin which mimics the effect of TCR-mediated activation of NFAT significantly up-regulated c-FLIP expression, and the expression was inhibited by the treatment with Cys A (data not shown). Additionally, Tax was reported to be able to induce transcriptional activity of NFAT, resulting in induction of IL-2 and FasL through an NFAT-binding element, CD28RE, in HTLV-I-infected T-cells (Good et al. 1996; Rivera et al. 1998). We could, however, exclude the possibility that NFAT is involved in Tax-induced c-FLIP expression in HTLV-I-infected T-cells, because Cys A could not inhibit constitutive expression of c-FLIP in MT-2 (Fig. 8E,F) and HUT-102 cells (data not shown). The c-flip promoter also contains binding sites for AP-1 (Ghosh 1993; Wingender et al. 2001), which is a complex of transcription factors composed of members of the Fos and Jun families. AP-1 was reported to be activated by Tax, while the molecular mechanism of this activation is still unclear (Jeang 2001). We found that expression of dominant negative mutant of AP-1, A-Fos (Olive et al. 1997) or TAM67 (Brown et al. 1993) could not cancel the Tax-induced up-regulation of c-FLIP (data not shown). Taken together, transcriptional activities of NFAT and AP-1 were dispensable in Tax-induced expression of c-FLIP.
Although transcriptional activity of NF-B was shown to be necessary for the efficient induction of c-FLIP by Tax, activation of only NF-B must be insufficient to induce c-FLIP expression, because: (1) NF-B was previously reported to be activated in some Tax-negative ATL cell lines, including MT-1 and TL-Om1 where expression level of c-FLIP is as low as those in HTLV-1-negative T-cell line, CEM-C7 (Fig. 2); (2) transient expression of RelA or c-Rel could not induce c-FLIP expression in CEM-C7 cells (data not shown). Tax-expressing cell-specific unidentified factor(s) other than NF-B might play a role in Tax-induced c-FLIP expression in cooperation with NF-B.
In this study, T-cells were shown to acquire the resistibility to Fas-mediated apoptosis by expressing Tax. HTLV-I-infected T-cells might be able to escape the host immune surveillance by the protective function of Tax against Fas-mediated apoptosis utilized by killer T-cells and so on. In addition, Tax might be also involved in protection of HTLV-I-infected T-cells from self-destruction mediated by continuous interactions of their expressing death ligands and death receptors including FasL and Fas. We confirmed Tax-induced expression of both Fas and FasL, and both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor DR5 by RT-PCR (data not shown) as partly described in previous reports (Rivera et al. 1998; Rivera-Walsh et al. 2001). It is interesting that activation of caspase-3 was observed in Lex22 cells expressing both Tax and shFLIP even in the absence of Fas stimulation (Fig. 5G), and this activation of caspase-3 was completely inhibited by the expression of exogenous v-FLIP and mouse c-FLIP (Fig. 6A). In acutely HTLV-I-infected T-cells expressing Tax, Tax-induced c-FLIP expression must play an important role in protection of the cells from self-destruction mediated by interaction of their expressing Fas/FasL and TRAIL/DR5.
A long latent period of about 60 years was reported to precede the onset of ATL, and ATL cells were shown to frequently lose the expression of Tax by several mechanisms (Matsuoka 2005). Tax promotes the proliferation and survival of HTLV-I-infected cells by its pleiotropic actions; however, Tax is the major target of HTLV-I-infected cell-specific cytotoxic T-cells utilizing perforin and granzyme system. Thus, the presence of Tax in HTLV-I-infected cells provides both advantages and disadvantages for the survival of HTLV-I-infected cells. We speculate that Tax plays an important role for HTLV-I-infected cells to persistently proliferate and to escape Fas-mediated apoptosis during the acute and carrier states, and then genetic and epigenetic changes accumulate in the host genome mediated by a mutator phenotype of Tax, which finally leads to Tax-independent proliferation and survival of HTLV-I-infected T-cells. Interestingly, 2–5% of ATL cells were reported to exhibit mutations in the Fas gene with associated Fas-negative phenotype (Tamiya et al. 1998). HTLV-I-infected T-cells in the early stage might be resistant to Fas-mediated apoptosis by not only Tax-induced expression of c-FLIP but also shutdown of the Fas expression in some cases associated with the mutation of Fas gene.
In this report, we clarified that Tax can inhibit Fas-induced apoptosis by up-regulating c-FLIP mainly through activation of NF-B, which would be critically involved in the evasion of HTLV-I-infected T-cells from either host immune surveillance or self-destruction mediated by their expressed death receptors and death ligands at the acute and carrier stages of HTLV-I infection, resulting in the onset of HTLV-I-induced leukemogenicity and autoimmune arthropathy, development of which is closely related to the persistent infection of HTLV-I.
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Experimental procedures |
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CEM-C7 and MT-2 cells were kindly provided by Y. Komada (Mie University, Tsu, Japan) and K. Shimotohno (Kyoto University, Kyoto, Japan), respectively. MT-4, HUT-102, ATL-2, MT-1, TL-Om1, and ATL-43Tb(-) cells were kindly provided by M. Matsuoka and M. Maeda (Kyoto University, Kyoto, Japan). MT-2 and MT-4 were established by in vitro transformation of normal T-cells with HTLV-I. MT-1, HUT-102, ATL-2, TL-Om1 and ATL-43Tb(-) were leukemic T-cell lines derived from a patient with ATL (Mori et al. 2001; Yoshida et al. 2004).
Plasmid constructs
Wild-type Tax, its mutants M22 and d3, and the dominant negative NF-B (DNp50: the truncated mutant of NF-B p105) were expressed using the vector pCG (Shimizu et al. 2003). The dominant negative mutant of IB (IB DN), in which 32Ser and 36Ser were replaced by alanine, was kindly provided by J. Inoue (the University of Tokyo, Tokyo, Japan). The expression vector for green GFP was from K. Umesono (Kyoto University, Kyoto, Japan). The expression vectors for dominant negative mutants of AP-1, A-Fos (Olive et al. 1997) and TAM67 (Brown et al. 1993) were kindly provided by C. Vinson and M. J. Birrer, respectively. cDNAs of v-FLIP E8 and v-FLIP MC159 were prepared as previously described (Nakagiri et al. 2005).
Antibodies
Anti-human Fas mAb CH-11 was previously described (Yonehara et al. 1989). Humanized anti-Fas monoclonal antibody HFE7A, used for immunoprecipitation of Fas, was provided by K. Ichikawa (Sankyo Co. Ltd, Tokyo, Japan) (Ichikawa et al. 2000; Haruyama et al. 2002). MAbs against Tax (Lt-4 and WATM-1) (Tanaka et al. 1992) were generous gifts from Y. Tanaka (Ryukyu University, Okinawa, Japan). The other Abs for Western blotting were purchased from the following sources: anti-Caspase-8 mAb (5F7), MBL; anti-FLIP mAb (Dave-2) and anti-Fas mAb (3D5), ALEXIS Biochemicals; anti-caspase-3 and anti-active caspase-3 Abs, Cell Signaling; anti-caspase-7, anti-Bcl-XL, anti-Bcl-2 and anti-ß-tubulin mAbs, BD Transduction Laboratories; anti-myc-tag mAb, Upstate group Inc.; anti-Actin mAb, CHEMICON International Inc; and anti-cIAP2 Ab, R & D Systems Inc.
Luciferase assay
The luciferase assay was carried out with the Dual-luciferase reporter assay system (Promega) as previously described (Nakagiri et al. 2005). WT-Luc and B-Luc indicate firefly luciferase expression plasmids regulated by the HTLV-I-LTR 21 bp-enhancer element for the detection of Tax-induced CREB/ATF1 activity and the B element for the detection of NF-B activity, respectively (Mori et al. 1999).
Western blotting and DISC analysis
For detection of caspase cleavage, cells were lyzed in lysis buffer (20 mM Tris pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM PMSF and protease inhibitor cocktail (SIGMA)) after stimulation with anti-Fas mAb CH-11 for given periods, and Western blotting was performed as previously described (Lee et al. 2001). For analyzing the expression of c-FLIP in CEM-C7 cells transiently expressing Tax, M22 and d3 together with GFP, cells expressing GFP were enriched by a cell sorter (EPICS ELITE) after 36 h transfection by electroporation. After GFP-positive cells were cultured overnight at 37 °C, cell lysates were analyzed by Western blotting.
To analyze DISC formation, cells (1 x 108/5 mL) were treated with or without 0.5 µg/mL anti-Fas mAb CH-11 for 15 min at 37 °C, washed twice with phosphate-buffered saline (PBS) on ice and then lyzed in the lysis buffer. Cell lysate was incubated with 5 µg anti-Fas mAb HFE7A for 16 h at 4 °C and then Fas was immunoprecipitated with protein G-Sepharose and protein A-Sepharose beads (Amersham Biosciences) for detecting caspase-8 and c-FLIP, respectively.
Assay of Fas-mediated apoptosis
In CEM-C7 cells, transfection was performed by electroporation at 300 V with a capacitance of 960 µF using a Gene Pulser (Bio-Rad). At 48 h after the co-transfection of expression vectors of various genes and that of GFP, cells were stimulated with 100 ng/mL of CH-11 for 16 h, washed twice with PBS and lyzed in the lysis buffer. The relative fluorescence intensities of GFP in the lysates were measured with a plate reader. Cell viability was represented as the percentage of the emission intensity of anti-Fas-treated cells against that of untreated cells, which was confirmed by analysis of nuclear morphology by staining with Hoechst 33342 (Sigma) and observing under a fluorescence microscope. For analysis of apoptosis-specific DNA fragmentation, the TUNEL assay was performed with MEBSTAIN Apoptosis KitII (MBL) according to the manufacturer's instructions by flow cytometer (EPICS EXPO32, Beckman Coulter). In lentiviral vector-infected MT-2 and HUT-102 cells, apoptosis was quantified by counting subdiploid (sub-G1) populations by DNA content analysis as previously described (Watanabe et al. 2004). In CEM-C7 cells expressing MerCreMer, apoptosis was analyzed by flow cytometer using phycoerythrin (PE)-conjugated anti-active caspase-3 mAb (BD Bioscience) in accordance with the manufacturer's instructions, and cell viability was assessed by CellTiter-GloTM Luminescent Cell viability Assay (Promega).
Preparation of lentiviral vectors
For the preparation of lentiviral vectors, we used the packaging vector pCAG-HIVgp encoding HIV-1 viral proteins (Gag and Pol), the vesicular stomatitis virus G glycoprotein- and Rev-expressing vector pCMV-VSV-G-RSV-Rev and the self-inactivating (SIN) vector, all of which were generous gifts from H. Miyoshi (RIKEN, Tsukuba, Japan) and H. Nakauchi (the University of Tokyo, Japan) (Tahara-Hanaoka et al. 2002; Gyobu et al. 2004). For gene silencing with the RNAi system, we used lentivirus-based vectors, CSII-U6-MCS-EGFP and CSII-U6-MCS-puro, that express shRNA under the control of the U6 promoter and were engineered to co-express EGFP as a reporter gene (kindly provided by Y. Satoh and M. Matsuoka, Kyoto University, Kyoto, Japan) and puror gene, respectively. The oligonucleotides of shRNA for silencing endogenous c-FLIP (shFLIP) were generated from nt 139-157 of c-FLIP cDNA. The oligonucleotides of shRNA for silencing GFP (shGFP) were generated from nt 240-258 of GFP cDNA. For expression of v-FLIP or mouse c-FLIP, each cDNA fragment was inserted into the SIN-vector (CSII-EF-MCS-IRES-EGFP).
To produce recombinant lentiviruses, the transfer vector was co-transfected with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev into 293T cells using the calcium phosphate method. For infection with lentivirus, culture supernatants containing viruses were collected 48 h after transfection, and then cells (1 x 105) were cultured with 1 mL of viral solution for 24 h in the presence of 8 µg/mL polybrene (Sigma). After infection, cells were cultured for 2 days, and then used for various analyses.
Development of a conditional Cre-loxP-mediated induction system
The lentiviral vector-based Cre-loxP-inducible expression vector (Lex-Puro/Tax) was constructed as follows. The puror cDNA and the fragment containing human G-CSF poly(A) signal were isolated from pIRES2-puro (BD Biosciences), and pEF-BOS (Mizushima & Nagata 1990), respectively, and subcloned in the BamHI/HindIII site of a pBS246 (Invitrogen) (pBS246-puro). An EF1 promoter fragment was deleted by AgeI digestion from a SIN-vector CSII-EF-MCS (CSII-MCS). Because internal poly(A) signals can abrogate the transcription of full-length lentiviral RNA, the floxed puror-poly(A) fragment from pBS246-puro, a fragment containing human growth hormone poly(A) signal isolated from plck-hGH (gifted from R. M. Perlmutter), Tax cDNA and the EF1 promoter fragment were inversely inserted in multicloning site of CSII-MCS as described in Fig. 5A, resulting in generating high-titer infectious lentiviruses. CEM-C7 cells were transfected with pCAGGS-MerCreMer (kindly provided by Y. Shinkai, Kyoto University, Kyoto, Japan) by electroporation using a Gene Pulser and stably MerCreMer-expressing CEM-C7 cells were cloned based on the appropriate expression of MerCreMer analyzed by Western blotting. The clones were infected with lentiviruses expressing Lex-Puro/Tax. After 2 days infection, cells were incubated with 3 µg/mL Puromycin (nacalai tesque) and puromycin-resistant cells were amplified. MerCreMer recombinase, which is insensitive to the natural ligand 17ß-estradiol, was activated by the synthetic ligand 4-OHT. In the absence of 4-OHT, enzymatic activity of MerCreMer remains inactive, resulting in expression of the puror gene. In the presence of 4-OHT, however, Cre-loxP-mediated recombination leads to deletion of the puror-poly(A) sequence, thereby allowing expression of Tax gene.
EMSA
EMSA was carried out as previously described (Murakami et al. 2004) by using the 32P-labeled CD28RE probe derived from the human IL-2 gene (Good et al. 1996). For super-shift assay, the following Abs were used; anti-RelA, anti-cRel, anti-RelB, anti-p50 and anti-p52 Abs from Santa Cruz Biotechnology, and anti-NFAT1 Ab from Upstate group Inc.
Northern blotting
Procedures for RNA isolation and Northern blotting were previously described (Murakami et al. 2004). The BamHI fragment (nt1-585) (FLIP(N)) and HindIII fragment (nt 696-1443) (FLIP(C)) of FLIPL cDNA, and MluI-BamHI fragment (nt 184-1062) of Tax cDNA were used for hybridization as probes. The probe for hEF1 cDNA was used as a standard.
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Acknowledgements |
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This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government.
K. Okamoto was supported by the 21st century program of the MEXT of the Japanese Government to Graduate School of Biostudies, Kyoto University.
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Footnotes |
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* Correspondence: E-mail: yonehara{at}lif.kyoto-u.ac.jp
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Received: 15 August 2005
Accepted: 15 November 2005
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