Rheumatology Advance Access originally published online on November 17, 2003
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Rheumatology 2004; 43: 277-285
Rheumatology Vol. 43 No. 3 (c) British Society for Rheumatology 2003; all rights reserved
Basic Science |
Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts
1Division of Immunology, Institute for Medical Science and 2Department of Orthopedic Surgery, Dokkyo University School of Medicine, Tochigi and 3Institute of Medical Science, St Marianna University School of Medicine, Kawasaki, Japan.
Correspondence to: T. Kobata, Division of Immunology, Institute for Medical Science, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan. E-mail: tkobata{at}dokkyomed.ac.jp
Objective. 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.
Methods. 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 (
m) 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.
Results. Fas ligation induced Bid cleavage, loss of m, 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.
Conclusion. Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.
KEY WORDS: Rheumatoid arthritis, Synoviocyte, Apoptosis, Fas, Mitochondria, Caspase-3, Caspase-9, Bid, p53, p53AIP1.
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