NF-κB–regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α–mediated apoptosis
Shaochun Bai
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorHongtao Liu
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorKun-Hung Chen
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorPolikseni Eksarko
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorHarris Perlman
Saint Louis University School of Medicine, St. Louis, Missouri
Search for more papers by this authorTerry L. Moore
Saint Louis University School of Medicine, St. Louis, Missouri
Search for more papers by this authorCorresponding Author
Richard M. Pope
Saint Louis University School of Medicine, St. Louis, Missouri
Northwestern University Feinberg School of Medicine, Department of Medicine, Division of Rheumatology, McGaw Pavilion, Suite 2300, 240 East Huron Street, Chicago, IL 60611Search for more papers by this authorShaochun Bai
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorHongtao Liu
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorKun-Hung Chen
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorPolikseni Eksarko
Northwestern University Feinberg School of Medicine, and Chicago Veterans Administration Medical Center, Chicago, Illinois
Search for more papers by this authorHarris Perlman
Saint Louis University School of Medicine, St. Louis, Missouri
Search for more papers by this authorTerry L. Moore
Saint Louis University School of Medicine, St. Louis, Missouri
Search for more papers by this authorCorresponding Author
Richard M. Pope
Saint Louis University School of Medicine, St. Louis, Missouri
Northwestern University Feinberg School of Medicine, Department of Medicine, Division of Rheumatology, McGaw Pavilion, Suite 2300, 240 East Huron Street, Chicago, IL 60611Search for more papers by this authorAbstract
Objective
Little apoptosis has been observed in rheumatoid arthritis (RA) 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 (FLIPL) and FLIP short (FLIPS) 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 adenovirus vector or one expressing the super-repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIPL 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
TNFα 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, TNFα induced the expression of FLIPL and FLIPS 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 FLIPL 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 fibroblasts to TNFα-induced apoptosis is mediated by the NF-κB–regulated expression of FLIP. These observations support the role of NF-κB and FLIP as attractive therapeutic targets in RA.
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