Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Kobayashi
T
T
St Marianna University School of Medicine, Kawasaki, Japan.
Okamoto
K
K
Kobata
T
T
Hasunuma
T
T
Kato
T
T
Hamada
H
H
Nishioka
K
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000096:biosynthesis; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001172:Arthritis, Rheumatoid / Q000473:pathology* / Q000503:physiopathology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000235:genetics; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003602:Cytotoxicity, Immunologic; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D021382:Protein Sorting Signals / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019014:fas Receptor / Q000276:immunology / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D021382:Protein Sorting Signals; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D016222:Fibroblast Growth Factor 2; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases