Kobayashi T, Okamoto K, Kobata T, Hasunuma T, Kato T, Hamada H, Nishioka K
St Marianna University School of Medicine, Kawasaki, Japan.
Arthritis Rheum. 2000 May;43(5):1106-14. doi: 10.1002/1529-0131(200005)43:5<1106::AID-ANR21>3.0.CO;2-F.
Fas-mediated apoptosis is associated with the pathophysiology of rheumatoid arthritis (RA). However, the molecular mechanisms of this process remain to be elucidated in rheumatoid synovium. We investigated the behavior of intracellular signaling molecules that regulate Fas-mediated apoptosis in RA synoviocytes.
Anti-Fas monoclonal antibody (mAb) was added to RA synoviocytes after treatment with tumor necrosis factor alpha (TNFalpha) or basic fibroblast growth factor (bFGF) for 5 days. The cytotoxic activity was measured using a lactate dehydrogenase-release assay. The expression of apoptosis-related molecules in RA synoviocytes was examined by immunoblot analysis. The enzymatic activities of caspases 3 and 8 under Fas ligation were examined. Transfer of the FADD (Fas-associated death domain) protein and the FLIP(L) (long form of the FLICE [FADD-like interleukin-1beta-converting enzyme]-inhibitory protein) gene into RA synoviocytes was performed using adenoviral vectors.
Following a 5-day treatment with TNFalpha or bFGF, Fas ligation with its agonistic mAb induced apoptosis of almost all TNFalpha-treated RA synoviocytes but only showed a weak apoptotic activity in bFGF-treated synoviocytes. Although there was no correlation between the induction of Fas-mediated apoptosis and the expression of apoptosis-related molecules among these cells, a high enzymatic activity of caspases 3 and 8 was observed only in the TNFalpha-treated RA synoviocytes after Fas ligation. The bFGF-treated RA synoviocytes were relatively resistant to apoptosis induced by FADD gene transfection, as compared with the TNFalpha-treated synoviocytes. In addition, the expression of FLIP(L), an inhibitory molecule of Fas-mediated apoptosis, was reduced in TNFalpha-treated RA synoviocytes, and the expression of FLIP43 was augmented in bFGF-treated RA synoviocytes. Moreover, Fas-mediated apoptosis in TNFalpha-treated RA synoviocytes was partially inhibited by FLIP(L) gene transfection.
Our results suggest that Fas-mediated apoptosis of RA synoviocytes is differentially regulated by TNFalpha and bFGF. In addition, the regulatory mechanisms of apoptosis involve the formation of the death-inducing signaling complex, especially at the level of caspase 8 activation, and this process may be partly associated with FLIP expression.
Fas介导的细胞凋亡与类风湿关节炎（RA）的病理生理学相关。然而，这一过程在类风湿滑膜中的分子机制仍有待阐明。我们研究了调节RA滑膜细胞中Fas介导的细胞凋亡的细胞内信号分子的行为。
在用肿瘤坏死因子α（TNFα）或碱性成纤维细胞生长因子（bFGF）处理RA滑膜细胞5天后，加入抗Fas单克隆抗体（mAb）。使用乳酸脱氢酶释放试验测量细胞毒性活性。通过免疫印迹分析检测RA滑膜细胞中凋亡相关分子的表达。检测Fas连接下caspase 3和8的酶活性。使用腺病毒载体将FADD（Fas相关死亡结构域）蛋白和FLIP(L)（FLICE [FADD样白细胞介素-1β转化酶]抑制蛋白的长形式）基因导入RA滑膜细胞。
在用TNFα或bFGF处理5天后，用其激动性mAb进行Fas连接可诱导几乎所有TNFα处理的RA滑膜细胞凋亡，但在bFGF处理的滑膜细胞中仅表现出较弱的凋亡活性。虽然在这些细胞中Fas介导的细胞凋亡诱导与凋亡相关分子的表达之间没有相关性，但仅在Fas连接后的TNFα处理的RA滑膜细胞中观察到caspase 3和8的高酶活性。与TNFα处理的滑膜细胞相比，bFGF处理的RA滑膜细胞对FADD基因转染诱导的凋亡相对抗性。此外，Fas介导的细胞凋亡抑制分子FLIP(L)在TNFα处理的RA滑膜细胞中的表达降低，而FLIP43在bFGF处理的RA滑膜细胞中的表达增加。此外，FLIP(L)基因转染部分抑制了TNFα处理的RA滑膜细胞中Fas介导的细胞凋亡。
我们的结果表明，TNFα和bFGF对RA滑膜细胞中Fas介导的细胞凋亡有不同的调节作用。此外，细胞凋亡的调节机制涉及死亡诱导信号复合物的形成，特别是在caspase 8激活水平，并且这一过程可能部分与FLIP表达相关。
