Regulation of tumor necrosis factor alpha-mediated apoptosis of rheumatoidarthritis synovial fibroblasts by the protein kinase Akt

Objective. To determine if tumor necrosis factor alpha (TNF alpha)-driven p roliferation of rheumatoid arthritis synovial fibroblasts (RASF) is associa ted with upregulation of the activity of serine/threonine kinase B/Akt and with survival of RASF. Methods. Staining of phosphorylated Akt was done using anti-phosphorylated Thr(308) Akt antibody. Levels of phosphorylated Akt were analyzed by Wester n blot and Akt activity was analyzed using a kinase assay. TUNEL staining w as used to analyze the cytotoxicity of TNF alpha treatment or TNF alpha com bined with either the Akt activity inhibitor wortmannin, an adenovirus expr essing dominant-negative mutant (AdAkt-DN), or an adenovirus expressing pho sphatase and tensin homolog deleted on chromosome 10 (AdPTEN). Results. The levels of phosphorylated Akt were higher in RASF than in osteo arthritis synovial fibroblasts (OASF), as demonstrated by immunohistochemic al staining, immunoblot analysis, and an Akt kinase assay. The levels of ph osphorylated Akt and Akt kinase activity were increased by stimulation of p rimary RASF with TNF alpha (10 ng/ml). Treatment of RASF with the phosphati dylinositol 3-kinase inhibitor wortmannin (50 nM) plus TNF alpha resulted i n apoptosis of 60 +/- 8% (mean +/- SEM) of RASF within 24 hours. This pro-a poptosis effect was specific for Akt, since equivalent levels of apoptosis were observed upon TNFa treatment of RASF transfected with AdAkt-DN and wit h AdPTEN, which opposes the action of Akt. Conclusion. These results indicate that phosphorylated Akt acts as a surviv al signal in RASF and contributes to the stimulatory effect of TNF alpha on these cells by inhibiting the apoptosis response. This effect was not obse rved in OASF and may reflect the pathophysiologic changes associated with t he proliferating synovium in rheumatoid arthritis.