Okamoto K, Kobayashi T, Kobata T, Hasunuma T, Kato T, Sumida T, Nishioka K
Rheumatology, Immunology, and Genetics Program, Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8512, Japan.
Rheumatology (Oxford). 2000 May;39(5):471-80. doi: 10.1093/rheumatology/39.5.471.
To understand the intracellular regulatory mechanisms in Fas-mediated apoptosis of synoviocytes, we examined the involvement of caspases [caspase-1/ICE (interleukin-1beta 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.
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.
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.
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.
为了解滑膜细胞Fas介导的凋亡中的细胞内调节机制，我们检测了半胱天冬酶（半胱天冬酶-1/ICE（白细胞介素-1β转换酶）、半胱天冬酶-3/CPP32和半胱天冬酶-8/FLICE）以及形成死亡诱导信号复合物（DISC）的Fas相关死亡结构域蛋白（FADD）在滑膜细胞Fas介导的凋亡中的作用。
从类风湿关节炎（RA）和骨关节炎（OA）患者中获取滑膜细胞。在用半胱天冬酶-1、-3或-8特异性抑制剂存在的情况下，用抗Fas单克隆抗体处理后，对死细胞数量进行计数。通过免疫印迹和免疫沉淀分析检测半胱天冬酶和FADD在RA滑膜细胞Fas介导的凋亡中的作用。
与OA滑膜细胞相比，RA滑膜细胞高水平表达半胱天冬酶-3、半胱天冬酶-8和FADD。有趣的是，Fas连接通过聚（ADP-核糖）聚合酶（PARP）的裂解激活了半胱天冬酶-8和半胱天冬酶-3，这与RA滑膜细胞的凋亡相对应。此外，半胱天冬酶-3和半胱天冬酶-8而非半胱天冬酶-1的特异性抑制剂以剂量和时间依赖性方式抑制了Fas诱导的RA滑膜细胞凋亡。半胱天冬酶-8特异性抑制剂抑制了Fas连接后RA滑膜细胞上半胱天冬酶-3的激活。重要的是，在RA滑膜细胞Fas介导的凋亡过程中，FADD被选择性募集到Fas死亡结构域，这与对Fas介导凋亡的敏感性一致。
我们的研究结果表明，滑膜细胞中Fas介导的凋亡可能在FADD募集到DISC的水平上受到调节，随后导致FADD/半胱天冬酶-8/半胱天冬酶-3信号通路的激活。
