Fas-associated death domain protein is a Fas-mediated apoptosis modulator in synoviocytes.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Okamoto
K
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.
Kobayashi
T
T
Kobata
T
T
Hasunuma
T
T
Kato
T
T
Sumida
T
T
Nishioka
K
K
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000473:pathology*; D002352:Carrier Proteins / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction; D013583:Synovial Membrane / Q000473:pathology*; D019014:fas Receptor / Q000276:immunology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011506:Proteins; D019014:fas Receptor; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases