NF-κB-regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α-mediated apoptosis

Objective. Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor a (TNFalpha) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFa. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFalpha-induced NF-kappaB controls the expression of FLIP long (FLIPL) and FLIP short (FLIPS) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFa-induced apoptosis. Methods. RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super-repressor IkappaBalpha (srIkappabalpha). The cells were stimulated with TNFalpha or a control vehicle, and expression of FLIP, and FLIPS was determined by isoform-specific real-time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3. Results. TNFalpha induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIPL was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFa induced the expression of FLIPL and FLIPS mRNA and protein. The TNFalpha-induced, but not the basal, expression of FLIP was regulated by NF-kappaB. When NF-kappaB activation was suppressed by the expression of srIkappaBalpha, TNFalpha-mediated apoptosis was induced. TNFa-induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP, or the caspase 8 inhibitor CrmA. Conclusion. The TNFalpha-induced, but not the basal, expression of FLIP is regulated by NF-kappaB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFa-induced apoptosis is mediated by the NF-kappaB-regulated expression of FLIP. These observations support the role of NF-kappaB and FLIP as attractive therapeutic targets in RA.