Fas-associated death domain protein is a Fas-mediated apoptosis modulator in synoviocytes
K. Okamoto1,
T. Kobayashi1,
T. Kobata1,2,
T. Hasunuma1,
T. Kato1,
T. Sumida1 and
K. Nishioka1,
1 Rheumatology, Immunology, and Genetics Program, Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8512, and
2 Department of Immunology, Dokkyo University School of Medicine, Tochigi, Japan
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Abstract
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Objective. To understand the intracellular regulatory mechanisms in Fas-mediated apoptosis of synoviocytes, we examined the involvement of caspases [caspase-1/ICE (interleukin-1ß converting enzyme), caspase-3/CPP32, and caspase-8/FLICE] and Fas-associated death domain protein (FADD) forming a death-inducing signalling complex (DISC) in Fas-mediated apoptosis of synoviocytes.
Methods. Synoviocytes were obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. The number of dead cells was counted after treatment with anti-Fas monoclonal antibody in the presence of caspase-1-, -3-, or -8-specific inhibitors. The involvement of caspases and FADD in Fas-mediated apoptosis of RA synoviocytes was examined by immunoblot and immunoprecipitation analyses.
Results. RA synoviocytes expressed high levels of caspase-3, caspase-8, and FADD compared with OA synoviocytes. Interestingly, Fas ligation activated caspase-8 and caspase-3 with the cleavage of poly(ADP-ribose) polymerase (PARP), corresponding to apoptosis of RA synoviocytes. Furthermore, specific inhibitors for caspase-3 and caspase-8 but not caspase-1 suppressed Fas-induced apoptosis of RA synoviocytes in a dose- and time-dependent manner. Caspase-8-specific inhibitor suppressed the activation of caspase-3 after Fas ligation on RA synoviocytes. Importantly, FADD was selectively recruited to the Fas death domain during Fas-mediated apoptosis of RA synoviocytes, consistent with sensitivity to the Fas-mediated apoptosis.
Conclusion. Our findings suggest that Fas-mediated apoptosis in synoviocytes may be regulated at the level of recruitment of FADD to the DISC, subsequently leading to the activation of the FADD/caspase-8/caspase-3 signalling pathway.
KEY WORDS: Apoptosis, Fas, FADD, Caspase, Rheumatoid arthritis.
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Introduction
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Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by abnormal synovial proliferation with infiltration of various inflammatory cells leading to cartilage and bone destruction [1, 2]. We have previously reported that both synoviocytes and lymphocytes in the rheumatoid synovium expressed functional Fas/APO-1 (CD95) antigen and these cells were sensitive to a Fas-mediated apoptosis 35]. Using animal models of RA, we have also recently demonstrated that active induction of apoptosis in the rheumatoid synovium by anti-Fas monoclonal antibody (mAb) or Fas ligand (FasL) gene transfer improved arthritis due to the elimination of both proliferating synoviocytes and infiltrating lymphocytes in the inflamed synovium [6, 7]. These findings strongly suggest that Fas-mediated apoptosis may be a critical factor in the pathophysiology of RA.
Fas antigen is a well-known mediator of apoptosis and is expressed on a wide variety of cell types [8]. The induction of apoptosis requires oligomerization of the Fas antigen on the cell surface either by FasL or agonist mAbs. Recently, several Fas-interacting signal transducing molecules have been identified using the yeast two-hybrid system or biochemical approaches, including Fas-associated death domain protein (FADD) [911], Fas-associated phosphatase-1 (FAP-1) [12, 13], receptor interacting protein (RIP) [14], and FLICE inhibitory protein (FLIP) [15]. Furthermore, aspartate-specific cystein proteases of the interleukin-1ß (IL-1ß)-converting enzyme (ICE) family, recently renamed the caspases [16], have also been implicated as principal effectors of apoptosis, presumably by their proteolytic action on specific targets, including members of the caspases themselves and poly(ADP-ribose) polymerase (PARP). At present, the caspase family consists of at least 10 homologues [16]. FADD contains a death domain at its C terminus and a death effector domain at its N terminus. After oligomerization of Fas antigen, FADD binds to Fas antigen via interactions between the death domains, at the same time, to caspase-8 (FLICE) via interactions between the death effector domains, and leads to the activation of a cascade of caspases, including caspase-1 (ICE) and caspase-3 (CPP32) [17, 18].
In the present study, we show that in RA synoviocytes, Fas ligation induces sequential activation of the FADD/caspase-8/caspase-3/PARP signalling pathway, corresponding to apoptosis of these cells. Our results strongly suggest that Fas-mediated apoptosis of synoviocytes may be regulated by recruitment of FADD to the Fas death domain.
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Materials and methods
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Cell preparation and culture
RA and osteoarthritis (OA) were diagnosed according to the criteria of the American College of Rheumatology [19]. Following informed consent, synovial tissue was obtained from patients with RA and OA undergoing total joint replacement. Cultured synovial cell lines (six RA and six OA synovial cell lines) were established as described previously [20]. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS) (GIBCO) until examination. Both RA and OA synoviocytes were used from passage 3 to 5 in all experiments.
Antibodies
For cytotoxicity analysis, we used anti-Fas mAb (CH-11) (Medical & Biological Laboratory Co., Nagoya, Japan). For immunoblot analysis, we used mAbs against caspase-3 (Transduction Laboratories, Inc., Lexington, KY, USA), caspase-8 (Medical & Biological Laboratory Co.), FADD (Transduction Laboratories, Inc.), PARP (Pharmingen, San Diego, CA, USA) and
-tublin (Sigma Chemical Co., St. Louis, MO, USA). For immunoprecipitation analysis, we used anti-Fas mAb (UB-2) (Medical & Biological Laboratory Co.).
Cytotoxicity assay
Briefly, synovial cell lines were seeded in 6-well plates at 1 x 105 cells/well. After 24 h, the cells were pre-incubated for 60 min with the indicated concentrations of caspase-1-specific inhibitor (synthetic tetrapeptide; Ac-YVAD-CHO, Peptide Institute Inc., Osaka, Japan), caspase-3-specific inhibitor (synthetic tetrapeptide; Ac-DEVD-CHO, Peptide Institute Inc.), caspase-8-specific inhibitor (synthetic tetrapeptide; Z-IETD-FMK, Medical & Biological Laboratory Co.), or medium alone. The cells were then incubated with 1.0 µg/ml of CH-11 mAb or anti-mouse IgM (Dako, Kyoto, Japan) as an isotype-matched control antibody. The cells were then stained with Trypan blue at 0, 24, 48, 72 h after treatment with CH-11 mAb or isotype-matched control antibody and the number of dead cells was counted under microscopy.
Immunoblot analysis
After incubation of 1 x 106 synoviocytes for the indicated time intervals with or without 1.0 µg/ml of CH-11 mAb, the reaction was terminated by the addition of ice-cold phosphate buffer saline (PBS). After centrifugation at 500 g for 5 min, the pellets were lysed in a lysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1.0% Nonidet P-40 (NP-40), 1.0 mM phenylmethylsulphonyl fluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptine. Insoluble material was removed by centrifugation at 12 000 g for 5 min and detergent soluble proteins (10 µg/lane) were resolved by 10 or 15% polyacrylamide/sodium dodecylsulphate (SDS) gel electrophoresis (SDS-PAGE) before electrophoretic transfer to nitrocellulose membrane. The membrane was blocked with 2.5% bovine serum albumin (BSA) and 2.5% skim milk in PBS for 60 min at room temperature. After washing, the proteins were reacted for 60 min with mAbs for caspase-3, caspase-8, FADD, PARP, or
-tubulin in PBS containing 2.5% BSA, 2.5% skim milk, and 0.1% Tween, respectively. After washing, the membrane was incubated for 60 min with horseradish peroxidase-conjugated second antibodies in PBS containing 2.5% BSA, 2.5% skim milk, and 0.1% Tween, respectively. After washing, the immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Amersham Co., Arlington Heights, IL, USA). To examine the interaction between caspase-8 and caspase-3, synoviocytes were first incubated with 50 µM of caspase-8-specific inhibitor for 60 min, then incubated with 1.0 µg/ml of CH-11 mAb for the indicated time intervals. The reaction was terminated by the addition of ice-cold PBS. Sample preparation, SDS-PAGE, and immunoblotting using anti-caspase-3 mAb were performed as described above.
Immunoprecipitation
Synoviocytes (5 x 106) were incubated with 1.0 µg/ml of CH-11 mAb for the indicated time intervals. The cytosol fraction was prepared as described above using a lysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1.0% NP-40, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM dithiothreitol, 1.0 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptine and then was pre-cleared with protein G Sepharose for 4 h at 4°C. Fas-associated proteins were then immunoprecipitated by incubation with protein G Sepharose after reaction with anti-Fas mAb (UB-2) for 60 min on ice. Following rotation of the Sepharose beads with the cytosol fraction for 24 h, the immunoprecipitates were washed with a lysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1.0 mM ethylenediamine tetraacetic acid (EDTA), 1.0% NP-40, 2.0 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptine. The samples were eluted and immunoblot analysis was performed using anti-FADD mAb.
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Results
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Overexpression of caspase-3, caspase-8, and FADD in RA synoviocytes
RA synoviocytes are sensitive to Fas-mediated apoptosis. In contrast, OA synoviocytes fail to undergo Fas-mediated apoptosis despite the expression of Fas antigen on the surface at levels comparable with those on RA synoviocytes. It is thought that differences in the signal pathway downstream of the Fas antigen may explain the difference in sensitivity to Fas-mediated apoptosis. Thus, to investigate the underlying mechanisms of differences in sensitivities to Fas-mediated apoptosis, we first examined the cytosolic expression of molecules involved in Fas-mediated apoptosis at a protein level. As shown in Fig. 1
, cultured RA synoviocytes expressed relatively abundant caspase-3, caspase-8, and FADD compared with cultured OA synoviocytes. These results suggest that the sensitivity to Fas-mediated apoptosis in synoviocytes may be partly due to the level of expression of caspase-3, caspase-8, and FADD.
Activation of caspase-3 during Fas-mediated apoptosis of RA synoviocytes
To examine the involvement of caspases in Fas-mediated apoptosis of RA synoviocytes, we performed immunoblot analysis using mAbs against anti-caspase-3 and anti-PARP, a specific substrate for caspase-3. Fas ligation on cultured RA synoviocytes resulted in degradation of the inactive form of 32 kDa caspase-3 within 4 h, with subsequent cleavage of 116 kDa PARP to a 85 kDa protein (Fig. 2A
, B
). In contrast, Fas ligation on cultured OA synoviocytes did not activate caspase-3 or the cleavage of PARP, consistent with their insensitivity to Fas-mediated apoptosis (Fig. 2C
, D
). To confirm whether Fas ligation can actually induce the activation of caspase-3 in RA synoviocytes, we evaluated the effect of specific inhibitors for caspase-1 and caspase-3 on Fas-induced apoptosis of cultured RA synoviocytes. As shown in Fig. 3
, caspase-3-specific inhibitor (synthetic tetrapeptide; Ac-DEVD-CHO) clearly inhibited Fas-induced apoptosis of cultured RA synoviocytes in a dose- and time-dependent manner. However, caspase-1-specific inhibitor (synthetic tetrapeptide; Ac-YVAD-CHO) failed to influence Fas-induced apoptosis of cultured RA synoviocytes (Fig. 3
).

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FIG. 2. Activation of the caspase-3/PARP pathway during Fas-induced apoptosis of RA synoviocytes. (A) Immunoblot analysis of caspase-3 activity after Fas ligation on cultured RA synoviocytes. (B) Immunoblot analysis of PARP cleavage after Fas ligation on cultured OA synoviocytes. (C) Immunoblot analysis of caspase-3 activity after Fas ligation on cultured OA synoviocytes. (D) Immunoblot analysis of PARP cleavage after Fas ligation on cultured OA synoviocytes. Cultured RA synoviocytes were treated with anti-Fas mAb for the indicated time intervals and subjected to immunoblot analysis as described in the Materials and methods section. The position of caspase-3 or PARP is shown to the left. Data are representative of six individual experiments.
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FIG. 3. Effect of caspase-1- and caspase-3-specific inhibitors on Fas-induced apoptosis of RA synoviocytes. (A) Dose-dependent effect of caspase-1- or caspase-3-specific inhibitor on Fas-induced apoptosis of cultured RA synoviocytes. Cultured RA synoviocytes were treated for 72 h with anti-Fas or isotype-matched control mAb in the presence or absence of each specific inhibitor for caspase-1 or caspase-3 as described in the Materials and methods section. (B) Time-dependent effects of caspase-1- and caspase-3-specific inhibitor on Fas-induced apoptosis of cultured RA synoviocytes. Cultured RA synoviocytes were treated with anti-Fas or isotype-matched control mAb in the presence or absence of 500 µM of each specific inhibitor for caspase-1 or caspase-3 as described in the Materials and methods section. Data are expressed as mean ± standard error of the mean (S.E.M.). S.E.M. bars were sometimes smaller than the size of the symbol used for the mean data. Data are representative of three individual experiments.
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Activation of the caspase-8/caspase-3/PARP signalling pathway during Fas-mediated apoptosis in RA synoviocytes
We also investigated the involvement of caspase-8 in Fas-mediated apoptosis of cultured RA synoviocytes by immunoblot analysis using anti-caspase-8 mAb. Fas ligation on cultured RA synoviocytes resulted in degradation of the inactive form of 54 and 55 kDa caspase-8 within 4 h (Fig. 4A
). In contrast, Fas ligation on cultured OA synoviocytes did not activate caspase-8, consistent with their insensitivity to Fas-mediated apoptosis (Fig. 4B
). Furthermore, we also examined whether Fas ligation can actually induce the activation of caspase-8 in RA synoviocytes by examining the effect of caspase-8-specific inhibitor (synthetic tetrapeptide; Z-IETD-FMK). As shown in Fig. 5
, caspase-8-specific inhibitor clearly suppressed Fas-induced apoptosis of cultured RA synoviocytes in a dose- and time-dependent manner. Furthermore, we examined whether caspase-8 functions upstream or downstream of caspase-3. Pre-treatment of cultured RA synoviocytes with 50 µM of caspase-8-specific inhibitor clearly suppressed the activation of caspase-3 during the process of Fas-induced apoptosis (Fig. 6
). These results indicate that activation of the caspase-3/PARP pathway requires the activation of caspase-8. In addition, these findings suggest that Fas ligation on cultured RA synoviocytes selectively induces a sequential activation of the caspase-8/caspase-3/PARP pathway, corresponding to the execution of apoptosis.

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FIG. 5. Effect of caspase-8-specific inhibitor on Fas-induced apoptosis of RA synoviocytes. (A) Dose-dependent effect of caspase-8-specific inhibitor on Fas-induced apoptosis of cultured RA synoviocytes. (B) Time-dependent effect of caspase-8-specific inhibitor on Fas-induced apoptosis of cultured RA synoviocytes. Cultured RA synoviocytes were treated with anti-Fas or isotype-matched control mAb in the presence or absence of each specific inhibitor for caspase-1 or caspase-3 as described in the Materials and methods section. Data are expressed as mean ± standard error of the mean (S.E.M.). S.E.M. bars were sometimes smaller than the size of the symbol used for the mean data. Data are representative of three individual experiments.
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FIG. 6. Sequential activation of the caspase-8/caspase-3/PARP pathway during Fas-induced apoptosis of RA synoviocytes. Cultured RA synoviocytes were treated with anti-Fas mAb for the indicated time intervals in the presence or absence of caspase-8-specific inhibitor and subjected to immunoblot analysis using anti-caspase-3 mAb as described in the Materials and methods section. The position of caspase-3 is shown to the left. Data are representative of six individual experiments.
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Recruitment of FADD to Fas death domain after Fas ligation on RA synoviocytes
In the next step, we examined the involvement of functional FADD in Fas-mediated apoptosis of RA synoviocytes. For this purpose, lysates from cultured RA or OA synoviocytes stimulated with anti-Fas mAb (CH-11) for the indicated time intervals were immunoprecipitated with anti-Fas mAb (UB-2) and immunoblotted with anti-FADD mAb. As shown in Fig. 7A
, Fas ligation on cultured RA synoviocytes resulted in recruitment of FADD to the Fas death domain within 1 h. In contrast, Fas ligation did not recruit FADD to the Fas death domain in cultured OA synoviocytes (Fig. 7B
). These results strongly suggest that the sensitivity to Fas-mediated apoptosis in synoviocytes may be regulated by recruitment of FADD to the Fas death domain resulting in the formation of a death-inducing signalling complex (DISC).

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FIG. 7. Recruitment of FADD to Fas death domain after Fas ligation on synoviocytes. (A) Immunoprecipitation analysis of Fas-associated proteins using anti-FADD mAb after Fas ligation on cultured RA synoviocytes. (B) Immunoprecipitation analysis of Fas-associated proteins using anti-FADD mAb after Fas ligation on cultured OA synoviocytes. Both cultured RA and OA synoviocytes were stimulated with anti-Fas mAb (CH-11) for the indicated time intervals. Cytosol was immunoprecipitated with anti-Fas mAb (UB-2) and was then immunoblotted with anti-FADD mAb as described in the Materials and methods section. The position of FADD is shown to the left. Data are representative of six individual experiments.
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Discussion
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We have previously demonstrated that both synoviocytes and lymphocytes in the rheumatoid synovium expressed functional Fas/APO-1 (CD95) antigen 35] and that FasL-positive activated T cells and natural killer (NK) cells are present in the rheumatoid synovium [21]. However, it appears that apoptosis via the Fas/FasL system does not operate perfectly well to remove all Fas-positive proliferating synoviocytes and infiltrating lymphocytes in the rheumatoid synovium, leading to the development of a chronic inflammatory state. Therefore, elucidation of the Fas-mediated apoptosis signalling pathway in RA synoviocytes should provide important insights not only to our understanding of the pathophysiology of RA but also to the development of a novel therapy for RA.
One of the major findings of the present study was that cultured RA synoviocytes expressed high levels of caspase-3, caspase-8, and FADD compared with cultured OA synoviocytes. We have recently reported that tumour necrosis factor-
(TNF
) stimulates non-RA synoviocytes to proliferate as well as sensitize the cells for Fas-mediated apoptosis, at least in part by up-regulation and activation of caspase-8 and caspase-3 [22]. These results suggest that cultured OA synoviocytes may fail to transduce the apoptotic signal into the nuclei irrespective of Fas antigen expression due to insufficient expression of caspase-3, caspase-8, and FADD, which constitute the DISC.
Recent studies have shown that the death effector domain of FADD interacts with caspase-8 leading to proteolytic activation of other caspases during the process of Fas-mediated apoptosis [17, 18]. Furthermore, Fas-mediated apoptosis of cultured RA synoviocytes can be suppressed by a specific inhibitor for caspase-3 but not caspase-1 [23]. In the present study, we confirmed these early findings and further demonstrated that Fas ligation on cultured RA synoviocytes actually induced the activation of caspase-3, with subsequent cleavage of PARP, a specific substrate for caspase-3 [24]. PARP appears to be involved in DNA repair, genome surveillance and integrity [25]. The Ca2+/Mg2+-dependent endonuclease, implicated in internucleosomal DNA cleavage, the hallmark of apoptosis, is negatively regulated by poly(ADP-ribos)ylation [2628]. Therefore, a loss of normal PARP function may highly activate this nuclease in dying cells. Enari et al. have recently reported that the activation of caspase-3 is in turn dependent on the activation of caspase-1, indicating that these caspases are sequentially activated [29]. On the other hand, it is noteworthy that caspase-1-deficient mice do not show any phenotype in apoptosis [30], while mice lacking caspase-3 show hyperplasia and disorganized neuronal cell development in the brain [31]. These findings suggest that the function of caspase-1 in apoptosis is redundant in all cell types, while caspase-3 plays a major role in apoptosis.
Another major finding of the present study was the activation of caspase-8 and inhibition of caspase-3 activity by caspase-8-specific inhibitor during the process of Fas-induced apoptosis of RA synoviocytes. More importantly, we showed that Fas ligation induced the recruitment of FADD to Fas death domain in cultured RA but not OA synoviocytes. These findings strongly suggest that Fas-mediated apoptosis of synoviocytes may be regulated at the level of recruitment of FADD to Fas death domain and that the sequential activation of the FADD/caspase-8/caspase-3/PARP signalling pathway may play a critical role in Fas-induced apoptosis of cultured RA synoviocytes. Phytohaemagglutinin-activated Fas-positive T cells are resistant to Fas-mediated apoptosis at day 1 [32]. After prolonged treatment with interleukin-2, however, these T cells become Fas-mediated apoptosis sensitive at day 6 without changing the level of Fas cell surface expression. It appears that resistance to Fas-mediated apoptosis in T cells can be regulated at the level of recruitment of caspase-8 to the DISC [32]. Moreover, in support of our results, it has been recently reported that dominant-negative FADD inhibited Fas-mediated apoptosis in HeLa cells [33].
We have previously reported that Fas ligation selectively induced a rapid tyrosine phosphorylation of JNK (c-Jun amino-terminal kinase) and formation of AP-1 (activator protein-1) corresponding to apoptosis of RA synoviocytes [34]. In support of our findings, recent studies have shown that in a human T cell line (Jurkat cells) and peripheral blood lymphocytes, Fas ligation strongly activates JNK [3538]. In addition, it has been reported that overexpression of Daxx, a novel Fas death domain binding protein, induced activation of JNK and potentiated Fas-mediated apoptosis [39]. Preliminary results from our laboratory using the reverse transcriptionpolymerase chain reaction (RTPCR) method showed that cultured RA synoviocytes strongly expressed Daxx mRNA (unpublished data). On the other hand, it has been reported that activation of the JNK/AP-1 pathway in Jurkat cells by Fas ligation is probably the result, rather than the cause, of the apoptotic response, and is not required for Fas-mediated apoptosis [40]. Thus, although we found that the JNK/AP-1 pathway was activated during the Fas-mediated signalling process in cultured RA synoviocytes, at present, it is not clear whether activation of the JNK/AP-1 pathway is essential for Fas-mediated apoptosis of RA synoviocytes.
In addition to the death effector function of the caspase family members, the bcl-2 gene family also encodes several proteins that regulate apoptosis in mammalian cells [41]. Bcl-2, Bcl-XL, Bcl-W, and Mcl-1 inhibit apoptosis, whereas others, such as Bax, Bik, Bak, Bad and Bcl-XS, promote apoptosis. RA but not OA synoviocytes also expressed Bcl-2 and Bax abundantly (data not shown). It has been postulated that Bcl-2, Bcl-X, and Bax act upstream the caspase-3 activation step to regulate apoptosis induced by Fas ligation [42]. Therefore, it seems that overexpression of Bcl-2 and Bax may be a homeostatic mechanism to regulate the FADD/caspase-8/caspase-3/PARP pathway in Fas-mediated apoptosis of RA synoviocytes. However, the regulatory effect of Bcl-2 may be insufficient to inhibit Fas-mediated apoptosis of RA synoviocytes since Fas ligation induces apoptosis in RA synoviocytes. Additionally, Fas antigen possesses a unique carboxy-terminal domain important for interaction with FAP-1 (Fas-associated phosphatase type 1), whose expression appears to correlate with the inhibition of Fas-mediated apoptosis [12, 13]. Preliminary results from our laboratory using the RTPCR method showed that both RA and OA synoviocytes strongly express FAP-1 mRNA (unpublished data). The use of a FAP-1-specific inhibitor significantly enhanced apoptosis of RA synoviocytes and induced moderate apoptosis of OA synoviocytes after Fas ligation (unpublished data). These findings suggest that cultured RA synoviocytes may be more sensitive to Fas-mediated apoptosis than OA synoviocytes, since the apoptotic function of FADD may be more advantageous than the inhibitory effects of FAP-1 in RA synoviocytes. Furthermore, FLIP (also called I-FLICE, CASH, CLARP, Casper, FLAME-1, a caspase-8 inhibitory molecule) has recently been identified as a regulator for caspase-8 activation at the DISC [15]. FLIP is involved in the regulation of Fas-mediated apoptosis in RA synoviocytes by interfering with the interaction between FADD and caspase-8 [43].
On the other hand, it has been reported that there are different sub-populations of RA fibroblast-like synoviocytes that are not only involved differently in the disease process but also show different responses to pro-apoptotic stimuli [44, 45]. It seems that the loss of p53 function might be intimately involved with RA synoviocyte proliferation, apoptosis, and invasiveness [44, 45]. However, at present, it is not clear whether the p53 function is involved in the Fas-mediated apoptosis of RA synoviocytes.
We have recently demonstrated beneficial therapeutic effects of intra-articular administration of anti-Fas mAb in the human T cell leukaemia virus type 1 (HTLV-1)-carrying mouse and FasL gene transfer into the human rheumatoid synovium of SCID-RA mouse. Each treatment modality improved the paw swelling and histological features of arthritis by inducing apoptosis of synoviocytes and mononuclear cells in the inflamed synovium [6, 7]. These findings strongly suggest that active induction of apoptosis in proliferating RA synoviocytes may be useful as a new mode of therapy for RA. We believe that a better understanding of the mechanisms of Fas-mediated apoptosis signalling pathways in RA synoviocytes and enhancement of the susceptibility of these cells to apoptosis should be beneficial for the development of effective therapies for RA.
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Acknowledgments
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We thank Miss Ritsuko Sato for her technical assistance. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Health and Welfare of Japan, and Santen Pharmaceutical Company.
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Notes
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Correspondence to: K. Nishioka. 
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Submitted 14 July 1999;
revised version accepted 23 November 1999.