Regulation of synovial cell apoptosis by proteasome inhibitor

Objective. Recent studies have shown the importance of proteasome function in the regulation of apoptosis, This study examined whether inhibition of p roteasome function mediates apoptosis of synovial cells, and whether cytoki nes modulate this process. Methods, Type B synovial cells (fibroblast-like synovial cells) were cultur ed with tumor necrosis factor alpha (TNF alpha) or transforming growth fact or beta 1 (TGF beta 1), and further incubated in the presence of variable c oncentrations of Z-Leu-Leu-Leu-aldehyde (LLL-CHO), a proteasome inhibitor. During this process, apoptosis of synovial cells was determined by Hoechst 33258 dye staining and Cr-51 release assay. The involvement of caspase casc ade was examined using enzyme activity assay and blocking experiments by pe ptide inhibitors, The expression of pro-caspases, Bcl-2-related proteins, a nd X chromosome-linked inhibitor of apoptosis (XIAP) in synovial cells was examined by Western blot analysis. Results, Apoptosis of cultured synovial cells was induced in a dose-depende nt manner by LLL-CHO, Activation of caspase cascade through caspase-8 to ca spase-3 was essential during this process, Pretreatment of synovial cells w ith TNF alpha significantly augmented both the activation of caspases and t he proportion of apoptosis in synovial cells induced by LLL-CHO, whereas TG F beta 1 pretreatment markedly suppressed these phenomena. The ratio of the expression of Bcl-2 to Bar or Bcl-xL to Bar, and XIAP expression in synovi al cells may not be directly associated with the susceptibility of synovial cells to apoptosis by LLL-CHO. Conclusion, Apoptosis of synovial cells was induced by inhibition of protea some function through the activation of caspase cascade, and this process w as clearly modulated by cytokines. These data provide new insight into the regulatory mechanisms controlling synovial cells in rheumatoid synovitis by proteasome inhibitors, and might be useful for the design of new therapeut ic strategies in rheumatoid arthritis.