Itoh K, Hase H, Kojima H, Saotome K, Nishioka K, Kobata T
Division of Immunology, Institute for Medical Science, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan.
Rheumatology (Oxford). 2004 Mar;43(3):277-85. doi: 10.1093/rheumatology/keh039. Epub 2003 Nov 17.
Fas-mediated apoptosis is preferentially observed in synoviocytes of patients with rheumatoid arthritis (RA) and is associated with the pathophysiological process of RA. To clarify the molecular mechanisms of Fas-mediated apoptosis of RA synoviocytes, we investigated the role of the mitochondrial pathway and tumour suppressor p53 in this process.
Cultured synovial fibroblasts were prepared from RA patients. After treatment of RA synovial fibroblasts with anti-Fas monoclonal antibody, the expression levels of activated caspase-9 and -3, Bid cleavage, cytochrome c release and phosphorylation of p53 at Ser15 were assessed using immunoblot analysis. The mitochondrial membrane potential (DeltaPsim) was evaluated with a fluorescence-based detection assay. Apoptotic cells were determined by a DNA fragmentation assay in the presence or absence of caspase inhibitors. Expression of p53-regulated apoptosis-inducing protein 1 (p53AIP1) was measured by real-time PCR. RA synovial fibroblasts stably transfected with a dominant-negative (DN) p53 were prepared in order to investigate the role of p53 during Fas-induced apoptosis.
Fas ligation induced Bid cleavage, loss of DeltaPsim, cytochrome c release to the cytosol and activation of caspase-9 and -3 in RA synovial fibroblasts. Treatment with a caspase-9-specific inhibitor almost completely inhibited Fas-mediated apoptosis. Moreover, p53 activation after Fas ligation was evidenced by its phosphorylation at Ser15 and up-regulation of the p53 target gene p53AIP1. Fas-mediated apoptosis was significantly suppressed by anti-sense p53 oligonucleotides and by p53DN.
Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.
在类风湿关节炎（RA）患者的滑膜细胞中优先观察到Fas介导的细胞凋亡，且其与RA的病理生理过程相关。为阐明RA滑膜细胞中Fas介导的细胞凋亡的分子机制，我们研究了线粒体途径和肿瘤抑制因子p53在此过程中的作用。
从RA患者制备培养的滑膜成纤维细胞。用抗Fas单克隆抗体处理RA滑膜成纤维细胞后，采用免疫印迹分析评估活化的caspase-9和-3的表达水平、Bid裂解、细胞色素c释放以及p53在Ser15位点的磷酸化。用基于荧光的检测方法评估线粒体膜电位（ΔΨm）。在存在或不存在caspase抑制剂的情况下，通过DNA片段化测定法确定凋亡细胞。通过实时PCR测量p53调节的凋亡诱导蛋白1（p53AIP1）的表达。制备稳定转染显性阴性（DN）p53的RA滑膜成纤维细胞，以研究p53在Fas诱导的细胞凋亡过程中的作用。
Fas连接诱导RA滑膜成纤维细胞中的Bid裂解、ΔΨm丧失、细胞色素c释放到细胞质中以及caspase-9和-3的活化。用caspase-9特异性抑制剂处理几乎完全抑制了Fas介导的细胞凋亡。此外，Fas连接后p53的活化通过其在Ser15位点的磷酸化和p53靶基因p53AIP1的上调得到证实。反义p53寡核苷酸和p53DN显著抑制了Fas介导的细胞凋亡。
我们的研究结果强烈表明线粒体和p53参与了RA滑膜成纤维细胞中Fas介导的细胞凋亡。
