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.