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 (MA) synovial tissues. Tumor necrosis factor α (TNFα) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFα. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFα-induced NF-κB controls the expression of FLIP long (FLIP_L) and FLIP short (FLIP_S) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFα-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 adenoviras vector or one expressing the super-repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIP_L and FLIP_S 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. TNFα induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP_L was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIP_L and FLIP_S mRNA and protein. The TNFα-induced, but not the basal, expression of FLIP was regulated by NF-κB. When NF-κB activation was suppressed by the expression of srIκBα, TNFα-mediated apoptosis was induced. TNFα-induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP_L or the caspase 8 inhibitor CrmA. Conclusion. The TNFα-induced, but not the basal, expression of FLIP is regulated by NF-κB in RA synovial fibroblasts. The resistance of RA synovial fiα broblasts to TNFα-induced apoptosis is mediated by the NF-κB-reguIated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.