Fas activation of a proinflammatory program in rheumatoid synoviocytes and its regulation by FLIP and caspase 8 signaling

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Abstract
Objective The expansion of an aggressive population of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) synovium occurs despite their expression of functional death receptors and exposure to death receptor ligands. FLS can survive Fas challenge because of the constitutive expression of FLIP apoptosis inhibitor. We investigated whether Fas signaling plays a pathogenetic role by activating a nonapoptotic proinflammatory program in RA FLS. Methods Cultured RA FLS were stimulated with an agonistic anti-Fas antibody in the presence or absence of the caspase inhibitor Z-VAD-FMK or after RNA interference with a short hairpin RNA expression plasmid directed against FLIP. NF-κB and activator protein 1 (AP-1) activation was studied by electrophoretic mobility shift assays and p65 immunofluorescence analysis, and expression of messenger RNA (mRNA) for monocyte chemoattractant protein 1, interleukin-8, IκBα, and matrix metalloproteinases (MMPs) 1, 9, and 13 was examined by reverse transcription–polymerase chain reaction. Chemotactic activity of Fas-activated FLS–conditioned media was studied in Transwell migration assays. Results Fas stimulation activated NF-κB and AP-1, and this response required caspase activity, since Z-VAD-FMK inhibitor precluded it. FLIP was processed to p43 protein after Fas stimulation in a caspase-dependent manner, and inhibition of FLIP expression resulted in reduced Fas-triggered NF-κB activation. Fas stimulation increased expression of mRNA for IκBα, MMPs, and chemokines, and Fas-activated RA FLS displayed increased chemotactic activity for monocytic cells. Conclusion Fas triggering may contribute to the proinflammatory features of RA FLS by activating NF-κB and AP-1 and by expression of relevant target genes, such as MMPs and chemokines. Fas proinflammatory signaling is dependent upon caspase activity and FLIP expression. These data implicate FLIP as a potentially important molecular switch that turns the Fas signaling away from apoptosis and toward induction of a proinflammatory phenotype in RA FLS.