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Départements de
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Maladies Infectieuses and
Génétique Développement et Pathologies Moléculaires, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U567, Centre National de la Recherche Scientifique UMR8104, Université Paris 5, Paris, France;
Laboratoire de Physiologie Cellulaire, Institut National de la Santé et de la Recherche Médicale EMI0228, Université Lille 1, Villeneuve d’Ascq, France; and
Institut National de la Santé et de la Recherche Médicale U526, Faculté de Médecine Pasteur, Nice, France
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Abstract |
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Introduction |
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Death receptors, such as TNFR or Fas, are members of a superfamily of receptors that play pivotal roles in transducing extrinsic cell death or survival signals (6, 7). The TNFR superfamily consists of more than 20 structurally related type I transmembrane proteins that can be divided into two subclasses depending on whether they contain a death domain (DD)4 within their cytoplasmic region. Ligation of death receptors by their ligands provokes the recruitment of a death-inducing signaling complex through homotypic interactions between the death cytoplasmic domain of the receptor and the adaptor molecules also containing a DD. Formation of the death-inducing signaling complex then triggers a signaling cascade that leads to the final execution of cell death (8, 9, 10). Unlike death receptors, members of the TNFR superfamily that do not contain a DD within their cytoplasmic tail primarily transduce signals for cell growth and survival. However, some receptor members of the family can be involved, under different environmental circumstances, in either cell proliferation or apoptosis (7).
CD27 is such a receptor of the TNFR superfamily expressed on B and T cells, which can equally provide stimulatory signals for cell growth as well as apoptosis (11, 12, 13). The binding to CD27 of its ligand CD70, a member of the TNF family also expressed on discrete subpopulations of lymphocytes, results in T and B cell proliferation and B cell Ig production (14, 15, 16, 17). The costimulatory function of CD27 in the generation and maintenance of T cell immunity was confirmed in vivo in CD27-deficient mice (18). However, it was documented that ligation of CD27 could also induce an apoptotic process, despite its cytoplasmic region lacking a DD (13). This CD27-mediated apoptosis has been related to the identification of a new intracellular ligand of CD27, called Siva, which is highly expressed in lymphoid cells and exhibits proapoptotic activity (13, 19). The full-length predominant form of Siva, referred to as Siva-1, is a 175 aa protein containing a DD homology region (DDHR) in its central part (13), but a minor form, Siva-2, which is generated by alternative splicing and lacks most of the DDHR coding sequence, is also expressed (20). Interestingly, Siva can also bind to the cytoplasmic domain of glucocorticoid-induced TNFR family-related gene (GITR) (21), another member of the TNFR family that is mainly expressed on T lymphocytes and involved in the control of T cell activation and programmed cell death (22). The cytoplasmic tails of CD27 and GITR display a striking homology and associate with the cysteine-rich region found at the C-terminal end of both Siva forms (13, 20).
Several studies show that Siva-1 is overexpressed in various pathological circumstances, such as acute ischemic injury (23) and Coxsackievirus infection (24). Moreover, up-regulation of Siva was also detected in hepatocarcinoma cells treated with the cisplatin anticancer drug (25), whereas the metastasis suppressor TIP30, which inhibits metastasis of the small cell lung carcinoma by predisposing cells to apoptosis, also induces Siva overexpression (26). Although Siva-1 participates in multiple apoptotic processes in lymphoid cells in which both CD27 and GITR are primarily expressed (13, 19, 22, 24, 27), little is known about the signaling pathway underlying the Siva-induced apoptosis in T lymphocytes. We have thus explored the mechanism of the proapoptotic activity of both Siva-1 and Siva-2 in T cells. Siva-overexpressing cells display stereotypical features of apoptosis, including caspase activation, mitochondrial events, and morphological changes, which are modulated by overexpression of Bcl-2 or Bcl-xL antiapoptotic proteins. In contrast with previous reports, we show that the DDHR of Siva-1 is dispensable for induction of apoptosis, whereas the main determinants required for this activity are located in both the N- and C-terminal regions shared by both Siva-1 and Siva-2 proteins. These results indicate that both Siva proteins are able to mediate apoptosis in lymphoid cells through activation of a caspase-dependent mitochondrial signaling pathway.
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Materials and Methods |
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Vectors for expression of the human wild-type Siva-1, Siva-2, and deletion mutants of Siva-1 (see Fig. 7A) fused to the green fluorescent protein (GFP) were constructed by PCR with specific primers, and the amplified fragments were inserted between the EcoRI and BamHI sites of pEGFPC1 (Clontech Laboratories, Palo Alto, CA). Vector for expression of Siva-1 fused to the flag M2 epitope at its N terminus was also constructed by PCR with specific primers in the pSG-flag vector (28). Vector for expression of the CD27 receptor (pcDNA3-CD27) was kindly provided by K. V. Prasad (University of Illinois, Chicago, IL) (13).
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CD4/CD8 double-positive human peripheral blood-acute lymphocytic leukemia (HPB-ALL) T cells were kindly provided by G. Bismuth (Institut Cochin, Paris, France), and were maintained in RPMI 1640 medium with Glutamax-1 (Invitrogen, San Diego, CA) supplemented with 10% FCS and 100 U/ml penicillin/streptomycin at 37°C under 5% CO2. CD4 single-positive Jurkat T cells stably expressing Bcl-2 (29) (kindly provided by N. Israel, Institut Pasteur, Paris, France) or Bcl-xL (30) (kindly provided by K. Schulze-Osthoff, University of Muenster, Muenster, Germany) as well as the control Jurkat cells stably transfected with the neomycin-resistant vector were cultured as HPB-ALL cells, but in the presence of 500 µg/ml G418 (Invitrogen). SKW6.4 B cells stably overexpressing Bcl-2 and the control SKW6.4 cells stably transfected with the neomycin-resistant vector (kindly provided by B. Fadeel, Institute of Environmental Medicine, Stockholm, Sweden) were cultured as already described (31). Transient transfections of HPB-ALL, Jurkat, and SKW6.4 cells were performed by electroporation. A total of 10 x 106 cells were resuspended in 200 µl of RPMI 1640 medium with Glutamax-1 and supplemented with 10% FCS and 10 mM HEPES buffer. Electroporations were performed with 10 µg of plasmid DNA using 4-mm gap cuvettes (EquiBio, Needham Heights, MA) in a Bio-Rad Gene Pulser II (260 V, 950 microfarads). Cells were then washed once in culture medium supplemented with 10 mM HEPES buffer and cultured subsequently for the indicated period of time. Where indicated, HPB-ALL transfected cells were cultured for 48 h in the presence of the caspase inhibitors, Boc-Asp-fluoromethylketone (boc-D-fmk; 40 µM) or N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk; 20 µM) purchased from Calbiochem (La Jolla, CA), or were stimulated 24 h after transfection by incubation with an anti-CD27 mAb (1 µg/ml) purchased from Immunotech (clone 1A4-CD27; Marseilles, France). The hepatocarcinoma cell line Hep3B (kindly provided by C. Desbois-Mouthon, Hopital Saint-Antoine, Paris, France) were maintained in MEM (Invitrogen) supplemented with 10% FCS and 100 U/ml penicillin/streptomycin at 37°C under 5% CO2. Where indicated, Hep3B cells were treated with cisplatin (16 µg/ml) for 16 h.
Flow cytometry assays
The flow cytometry analyses were performed on GFP-positive cells with a FACSCalibur cytofluorimeter (BD Biosciences, San Jose, CA) while gating the forward and the side scatters on viable cells. A minimum of 1 x 104 events were acquired in list mode and analyzed with the CellQuest software (BD Biosciences). Cell shrinkage, cell viability, and phosphatidyl-serine (PS) exposure at the cell surface were analyzed as previously described (32). Briefly, cell shrinkage was evaluated by analysis of the light scattering properties, whereas viability and PS exposure were assessed by analysis of the cell permeability to propidium iodide (1 µg/ml) and by cell surface staining with Alexa 633-conjugated annexin V (Molecular Probes, Eugene, OR), respectively. Caspase-3 activity was determined using the caspase-3 Intracellular Activity Assay kit II purchased from Calbiochem, whereas caspase-8 and caspase-9 activities were determined using the Caspase-8 Detection kit (Red-IETD-fmk) and Caspase-9 Detection kit (Red-LEHD-fmk), purchased from Oncogene Research Products (San Diego, CA). The determination of the caspase activities was done following the manufacturer’s instructions.
Electron microscopy
After 48 h transfection, 7 x 105 HPB-ALL GFP-positive cells were sorted by flow cytometry using an Epics-ELITE EST cytometer and then processed for electron microscopy analysis. The cell pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, postfixed in 1% osmium tetroxide in the same buffer and subsequently processed for embedding in Epon resin. The contrasted sections were observed on a Hitachi H600 transmission electron microscope.
Western blot analysis
A total of 7 x 105 HPB-ALL GFP-positive cells were sorted 24 or 48 h after transfection as previously described and then lysed in a buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, and an antiprotease mixture (Sigma-Aldrich, St. Louis, MO). The protein concentration of the cell lysates was measured (Bio-Rad, Hercules, CA), and proteins (50 µg) were then separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ). The membrane was saturated with 5% nonfat dry milk in TBS containing 0.5% Tween 20 and then incubated with primary Ab in the saturation solution for 1 h at room temperature. The following Abs were used to detect actin, pro-caspase-3, pro-caspase-8, and Bid, respectively: a goat polyclonal anti-actin (I-19; Santa Cruz Biotechnology), a rabbit polyclonal anti-caspase-3 (CCP32; BD PharMingen, San Diego, CA), a goat polyclonal anti-caspase-8 (T-16; Santa Cruz Biotechnology, Santa Cruz, CA) and a goat polyclonal anti-Bid (AF860; R&D Systems, Minneapolis, MN). The membrane was then incubated with an appropriate HRP-conjugated secondary Ab (DAKO, Carpinteria, CA) in TBST for 30 min at room temperature. The proteins were visualized with the ECL kit (Amersham Biosciences), and the signals were quantified using NIH Image software. The expression of the endogenous Siva protein, either in HPB-ALL cells or in Hep3B cells treated with cisplatin, was determined by Western blot analysis on the cell lysate (25 µg of total protein) using anti-Siva (C-20; Santa Cruz Biotechnology). As a control, 
-tubulin was detected using anti--tubulin (TU-02; Santa Cruz Biotechnology). The expression of wild-type Siva-1, Siva-2, and the Siva-1 deletion mutants was monitored by Western blotting of total lysates of 1 x 106 HPB-ALL transfected cells using a mixture of anti-GFP mAbs (clones 7.1 and 13.1; Boerhinger Mannheim, Mannheim, Germany).
Immunofluorescence staining
HPB-ALL cells (2 x 106) were concentrated in 300 µl of culture medium without FCS and put on cover glass pretreated with poly-L-lysine (Sigma-Aldrich). After adhesion, cells were washed in PBS, fixed with 4% paraformaldehyde, and treated with 0.1 M glycine. Cells were then permeabilized for 30 min at room temperature with 0.05% saponin (Sigma-Aldrich) in PBS containing 0.2% BSA for anti-cytochrome c staining. For anti-GFP staining, cells were permeabilized either with 0.1% Triton X-100 for 10 min or with 0.05% digitonin (Sigma-Aldrich) in a buffer containing Tris-HCl (pH 7.5) 10 mM, NaCl 150 mM, EDTA 1 mM for 5 min. Cytochrome c was detected with the 6H2.B4 mAb (BD PharMingen) and GFP fusions with the mixture of anti-GFP mAbs, followed by incubation with a Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing 4',6'-diamidino-2-phenylindole (DAPI) for staining of the nuclear DNA. Images were acquired with a Zeiss Axiophot microscope 80x (Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, NY) equipped with a CCD camera controlled by Metaview software (Universal Imaging, Downingtown, PA).
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Results |
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The capacity of the full-length Siva-1 protein (see Fig. 7A) to trigger an apoptotic process in lymphoid cells was investigated by transient overexpression in T cell lines. A vector for expression of Siva-1 fused at its N terminus to the GFP was generated and used to transfect HPB-ALL (Fig. 1) or Jurkat T cells (data not shown). We first explored whether Siva overexpression induces a cell death process associated with some features of apoptosis. HPB-ALL cells were transfected with the GFP-Siva-1 expression vector, and 48 h later, cell shrinkage, cell viability, and PS exposure at the outer face of the plasma membrane were assessed by flow cytometry on GFP-positive cells. As shown in Fig. 1A, 67.5% of cells overexpressing GFP-Siva-1, vs only 16.3% of control cells expressing GFP, had a reduced forward scatter and a slightly increased side scatter, corresponding to a smaller size and a higher cell granularity reminiscent of cell shrinkage. Additionally, a higher percentage of cells expressing GFP-Siva-1 (51% vs <10% of GFP expressing cells) showed PS exposure as detected by the cell surface annexin V staining (Fig. 1B). This significant PS externalization was observed among GFP-Siva-1 expressing cells as early as 24 h after transfection, with progressive exposure exhibited in following days (Fig. 1C). The PS exposure was accompanied by a decrease of the mitochondrial membrane potential in a significant fraction of GFP-Siva-1-expressing cells (data not shown), suggesting that mitochondria contribute to the Siva-induced cell death. A similar cell death induction was observed by overexpression of the well-characterized DD-containing proapoptotic Fas-associated DD protein in HPB-ALL cells (data not shown). Altogether, these results indicate that overexpression of Siva-1 in T lymphocytes leads to an apoptotic cell death process.
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The ultrastructural features of HPB-ALL cells overexpressing Siva-1 were analyzed by transmission electron microscopy to further define the morphological changes induced by Siva-1 in T lymphocytes. HPB-ALL cells expressing either GFP-Siva-1 or GFP were sorted 48 h after transient transfection by flow cytometry, fixed, and then processed for electron microscopy (Fig. 2). Although control cells overexpressing the GFP protein appeared with a normal phenotype (Fig. 2, right panels), dying cells expressing GFP-Siva-1 displayed many characteristic changes of apoptosis (Fig. 2, left panels). The vacuolar system as well as the endoplasmic reticulum were dramatically expanded, and the chromatin was condensed at the nuclear periphery and detached from the nuclear membrane. Compared with control cells, mitochondria from GFP-Siva-1 expressing cells were electron translucent and swollen, and often appeared either with a bulging swollen matrix or with a partly disrupted outer membrane (Fig. 2, lower panels), suggesting that intermembrane space proteins were released into the cytosol during cell death. All these morphological changes demonstrate that Siva-1 overexpression results in the induction of a stereotypical apoptotic cell death, including mitochondrial damage events.
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To analyze the role of Siva in apoptotic signaling pathways, we first checked that the level of Siva overexpression induced in our transient experimental system was comparable to the up-regulation of Siva expression observed in some physiopathological processes, such as cell treatment with the cisplatin anticancer drug. In agreement with the results previously reported (25), Hep3B hepatocarcinoma cells treated with cisplatin led to a 6.2-fold overexpression of the Siva protein (Fig. 3A). This up-regulation was similar to what we estimated from the Western blot analysis of HPB-ALL transfected cells using anti-Siva Abs to detect both GFP-Siva and the endogenous Siva protein (Fig. 3B). Because the transfection efficiency of the GFP-Siva expression vector in HPB-ALL cells was 
20% as evaluated by FACS analysis (Fig. 3B), we calculated that a 5-fold overexpression of Siva is induced in our transient expression experimental system.
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Siva-induced apoptosis is related to a caspase-dependent pathway
Caspase proteases are key factors involved in both the initiation and execution phases of most apoptotic processes (8). To characterize the signaling pathway triggered by Siva-1 overexpression, we first investigated whether caspase inhibitors could block the Siva-induced apoptosis. After transfection with the GFP-Siva-1 expression vector, HPB-ALL cells were cultured for 2 days in the presence of the broad-spectrum caspase inhibitors, boc-D-fmk and z-VAD-fmk. GFP-positive cells were then assessed for PS exposure and cell viability by flow cytometry analysis. As shown in Fig. 4A, both caspase inhibitors largely decreased the level of GFP-Siva-1-expressing cells displaying PS exposure and also preserved the cell viability (data not shown), indicating that Siva-1 induces apoptosis in a caspase-dependent manner. Direct activation of caspases by Siva-1 overexpression was further explored by analysis of activation of caspase-8 and caspase-3, which are the major initiator and effector caspases, respectively (8). HPB-ALL cells expressing GFP-Siva-1 were sorted 24 and 48 h after transfection, and caspase-3- and caspase-8-activation was first analyzed by Western blot detection of the proteolytic cleavage of the pro-caspase precursors (Fig. 4B). Compared with untransfected cells or cells expressing the GFP control, a significant decrease of the pro-caspase-8 was already observed in cell lysate from GFP-Siva-1-expressing cells as soon as 24 h after transfection, whereas the decrease of the pro-caspase-3 could be detected only 48 h after transfection, even though a similar amount of protein was loaded, as evidenced by probing the same blots with an anti-actin Ab. This suggests that activation of the initiator caspase-8 precedes activation of the effector caspase-3 in the Siva apoptotic pathway. In addition, the Siva-induced apoptosis was significantly reduced by expression of the viral CrmA inhibitor (data not shown), which inhibits caspase-8 without affecting caspase-3 activity (33).
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The Siva-1 apoptotic pathway triggers mitochondrial events
The mitochondria play a pivotal role in multiple caspase-dependent apoptotic pathways (9, 10). Because the main molecular link connecting caspase and mitochondrial pathways is the caspase-8-mediated activation of Bid, a proapoptotic member of the Bcl-2 protein family (34), we initially explored whether overexpression of Siva-1 leads to the activating proteolytic cleavage of Bid. HPB-ALL cells were transfected with either GFP-Siva-1 or GFP expression vector and then sorted 24 and 48 h later by flow cytometry. Processing of Bid was evaluated on GFP-positive cells by Western blot using an anti-Bid Ab (Fig. 5A). Compared with untransfected and GFP-expressing cells, lysate from GFP-Siva-1-expressing cells showed a marked disappearance of Bid as soon as 24 h after transfection, whereas probing the same blot with anti-actin evidenced that a similar amount of cellular proteins was loaded. This indicates that Siva-1 overexpression induces the activation of Bid, which usually results in the permeabilization of mitochondrial membranes and the release of mitochondrial apoptogenic components, such as cytochrome c (10). We thus checked that the Siva-mediated apoptosis led to cytochrome c release from mitochondria by indirect immunofluorescence staining. As shown in Fig. 5B, Siva-1 expression induced a change in the cytochrome c staining pattern. A typical punctuate mitochondrial profile was observed in a large majority of both untransfected and GFP-expressing cells, whereas a significant proportion of GFP-Siva-1-expressing cells displayed a diffuse cytosolic distribution of cytochrome c as soon as 24 h posttransfection. Quantitative analysis from immunofluorescence images showed that around 30 to 40% of GFP-Siva-1-expressing cells display a mitochondrial release of cytochrome c 2 days after transfection, whereas such a phenotype was observed in fewer than 5% of cells expressing the GFP control. As previously mentioned, overexpression of Siva-1 also provoked a marked drop in the membrane mitochondrial potential (data not shown), thus confirming that the Siva-1 apoptotic pathway includes a mitochondrial step in T lymphocytes.
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Siva-1 accumulates in nuclear dot-like structures
To analyze the subcellular distribution of Siva-1 in T lymphocytes, HPB-ALL cells were transfected with the GFP-Siva-1 expression vector and the fusion was then visualized by direct fluorescence at various time points after transfection. GFP-Siva-1 was detected by Western blot analysis as soon as 3 h after transfection (data not shown), and accumulated in the nucleus in a diffuse staining pattern (Fig. 6A). This nucleoplasmic localization was still observable 6 and 9 h after transfection, but a fraction of the fusion appeared to form dot-like structures. At 24 h after transfection, GFP-Siva-1 was almost exclusively localized into dot-like structures, whereas the GFP control showed a diffuse nucleo-cytoplasmic staining distribution. The GFP-Siva-1-positive dots were often found at the periphery of the nucleus, and their number varied between 5 and 20. In addition, identical dotted structures were detected by indirect immunofluorescence on HPB-ALL cells expressing a flag-tagged Siva-1 form (Fig. 6A), indicating that this typical localization is independent of the epitope tag added to the Siva-1 sequence.
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Deleted forms of Siva-1 corresponding to the three distinct regions of the 175 aa long protein (see Fig. 7A) were then generated to determine which regions were responsible for the nuclear-dotted localization of the protein. Although Siva-1 is the predominant form expressed in T cell populations, the minor Siva-2 form, generated by alternative splicing and lacking most of the DDHR, is also produced (19, 20). HPB-ALL cells expressing the wild-type or deleted GFP-Siva forms were examined 24 h after transfection by direct fluorescence microscopy (Fig. 7). Whereas both GFP-SivaDDHR and GFP-SivaC were equally distributed between the nucleus and the cytoplasm, the GFP-SivaN mutant still retained the ability of GFP-Siva-1 to stain nuclear dot-like structures. A similar dotted staining was observed in cells expressing the GFP-Siva-2 form. These findings indicate that the determinants required to target Siva-1 and Siva-2 into the nuclear dot-like structures are contained within the N-terminal part of the proteins.
The N- and C-terminal regions of Siva-1 and Siva-2 proteins contain the proapoptotic determinants
Because the Siva-2 form lacks most of the DDHR (see Fig. 7A), we explored whether Siva-2 was also able to induce apoptosis in T lymphocytes to characterize the role of the DDHR in the activity of the protein. As evidenced by caspase-3 activation, GFP-Siva-2 overexpression led to an apoptosis induction as efficient as the full-length Siva-1 form in HPB-ALL cells (Fig. 8A). Similar results were obtained by analyzing PS exposure on GFP-Siva-2-expressing cells (data not shown). These results show that both Siva forms display proapoptotic activity, and also indicate that the DDHR of Siva-1 is dispensable for this activity.
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Discussion |
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Both the N- and C-terminal domains of the Siva proteins equally display the ability to induce a cell death process in T lymphocytes. These two regions could act in synergy in the same apoptotic pathway or participate in various pathways because the two domains show a distinct pattern of intracellular distribution when they are separately expressed. The C-terminal domain equally distributes between the cytoplasm and the nucleus, whereas the N-terminal region mediates the accumulation of both Siva forms into nuclear dot-like structures. Therefore, Siva-1 and Siva-2 are nuclear proteins that initially show a diffuse nucleoplasmic localization and subsequently translocate in dot-like structures at steady state. Interestingly, many recent studies reported that some DD-containing proteins, including DAXX, DEDAF, DEDD, or TRADD are involved in the regulation of various apoptotic pathways by playing specific roles in either the cytoplasmic or nuclear compartments (34, 37, 38, 39, 40, 41, 42). Similarly, Siva-1 and Siva-2 could be nucleo-cytoplasmic shuttling proteins that lead to the activation of distinct cytoplasmic and nuclear apoptotic pathways through the two N- and C-terminal modular domains, respectively.
Although the C terminus of the Siva proteins is required for direct association with the cytoplasmic domain of the CD27 and GITR receptors (13, 21), the N-terminal region likely forms the effector domain of the proteins in the nucleus. In agreement with this modular organization, it was reported that Siva-1 directly interacts with the antiapoptotic Bcl-xL protein, but not with Bcl-2, through a putative amphipathic helical structure encompassing the N-terminal region of the protein (19). This binding may specifically inhibit Bcl-xL activity, leading to an increased susceptibility of Siva-1-expressing cells to UV radiation-induced apoptosis. However, these observations do not fit with our data showing that both Bcl-2 and Bcl-xL are equally able to prevent the apoptotic process provoked by Siva-1 overexpression. Because the previous analyses were done in nonlymphoid cells (13, 19, 27), our results suggest that Siva-1 uses distinct mechanisms to induce apoptosis in T lymphocytes. Siva-1 is indeed a ubiquitous protein (13) that could participate in various apoptotic pathways in a cell type-dependent manner.
The N-terminal domain of Siva-1 and Siva-2 does not show any homology with known proteins, but the C-terminal region contains 13 cysteine residues that potentially form a Ring-finger and also a Zn-finger motif (13). This cysteine-rich region of Siva proteins is involved in the association with CD27 and GITR, and thus plays a pivotal role for the Siva-transducing apoptotic signal provided by ligation of these receptors (13, 21). Interestingly, some TNFR-associated factor (TRAF) proteins, which like Siva contain both Ring- and Zn-finger motifs, also directly interact with the CD27 and GITR cytoplasmic tails (43, 44). Because it is proposed that TRAF proteins function as important mediators of antiapoptotic signals (45), we can hypothesize that Siva and TRAF molecules are respectively involved in either the cell death or survival signals mediated by CD27 or GITR in T lymphocytes.
In contrast to the N- and C-terminal regions, the central part of the long Siva-1 form containing the DDHR is not necessary for the proapoptotic activity of the protein, at least in T lymphocytes. Although in some DD-containing proteins (46), such as TRADD, the DD is sufficient by itself to trigger apoptosis (42, 47, 48), the Siva-1 DDHR is, like the DD of the Fas-associated DD protein, devoid of intrinsic apoptotic activity (49). Overexpression of a deletion mutant covering this region is not able to induce apoptosis, whereas the Siva-2 form, lacking most of this region, still retains an apoptotic activity. These data are in contradiction with the previous reports (20, 27) indicating that both human and murine Siva-2 proteins lacked the proapoptotic activity of Siva-1 in nonlymphoid cells. Again, these discrepancies may reflect differences in the mechanism used by Siva proteins to induce apoptosis in T lymphocytes.
The signaling pathway triggered by Siva-1 in T cell lines results in both activation of caspases and induction of mitochondrial events. Although our analysis suggests that Siva-1 is involved in various apoptotic pathways, it likely participates in a prototypical death receptor-initiated pathway in lymphoid cells. Siva-1 interacts through its C-terminal region with the cytoplasmic tails of CD27 and GITR (13, 20, 22), two members of the TNFR family that, in contrast to Fas or TNFR, lack a DD in their cytoplasmic domain (7). Association of Siva-1 with CD27 or GITR may thus mediate subsequent activation of the initiator caspase-8. However, we failed to detect a direct binding of Siva-1 to caspase-8 in two-hybrid assay (data not shown), and Siva probably requires a downstream adaptor protein not yet identified. Because DDs usually form an interface for homotypic interactions (46), the DDHR of Siva-1 could similarly form a module required for homodimerization of the protein (B. Py and S. Benichou, unpublished observations) and for interactions with some other DD-containing proteins. Caspase-8 then leads to activation of effector caspases either directly or through a Bid-mediated mitochondrial pathway (35). Siva-induced apoptosis resulted in proteolytic activation of Bid and release of cytochrome c from mitochondria, and was efficiently blocked by overexpression of Bcl-2 or Bcl-xL proteins, at least in type II Jurkat cells. Cytosolic cytochrome c then associates with Apaf-1 in a complex with caspase-9 (the apoptosome) allowing activation of effector caspases, such as caspase-3, and subsequently the final execution of the apoptotic process (9, 10). In agreement with such a pathway, we observed caspase-9 activity preceding caspase-3 activation in Siva-induced cell death. However, the results obtained in SKW6.4 type I cells suggest that the Siva-induced activation of caspase-8 can also bypass the mitochondria to directly activate the caspase-3.
In summary, our results show that both Siva-1 and Siva-2 display proapoptotic activity in T lymphocytes. Because Siva proteins participate in CD27- and GITR-mediated apoptosis pathways (this study and Ref. 22), they likely contribute to lymphocyte function and homeostasis regarding the essential roles of CD27 and GITR in the T cell functions in vivo (18, 44). Because several recent studies indicate that the expression of Siva-1 is modulated in multiple pathological circumstances, further investigations will aim to pinpoint the physiological roles of Siva proteins to understand their contribution in these pathological processes.
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Acknowledgments |
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Footnotes |
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2 P.X.P. and S.B. contributed equally to this work as co-senior authors.
3 Address correspondence and reprint requests to Dr. Serge Benichou, Département de Maladies Infectieuses, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U567, Batiment Gustave Roussy, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France. E-mail address: benichou{at}cochin.inserm.fr
4 Abbreviations used in this paper: DD, death domain; DDHR, DD homology region; GITR, glucocorticoid-induced TNFR family-related gene; GFP, green fluorescent protein; DAPI, 4',6'-diamidino-2-phenylindole; PS, phosphatidyl-serine; TRAF, TNFR-associated factor.
Received for publication March 14, 2003. Accepted for publication January 20, 2004.
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References |
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B and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-B-inducing kinase. J. Biol. Chem. 273:13353.B activation. Cell 81:495.[Medline]
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) and the cysteine-rich region (
) are shown. Numbering of the N- and C-terminal ends of the deletion mutants refers to the Siva-1 form. B, Expression of GFP-Siva-1, GFP-Siva-2, and GFP-Siva deletion mutants in HPB-ALL cells is shown. Lysates from cells transfected with the vector for expression of the wild type or deleted forms of Siva-1 and Siva-2 were analyzed by Western blotting with anti-GFP Abs. C, Cellular distribution of GFP-Siva deletion mutants. Twenty-four hours after transfection, HPB-ALL cells expressing GFP-Siva-1, GFP-Siva-2, or GFP-Siva deletion mutants were fixed and directly examined by fluorescence microscopy. Nuclear DNA was stained with DAPI (middle row). Scale bar represents 10 µm.
) or GFP (
