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 proteasome function mediates apoptosis of synovial cells, and whether cytokines modulate this process.


METHODS
Type B synovial cells (fibroblast-like synovial cells) were cultured with tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta1 (TGFbeta1), and further incubated in the presence of variable concentrations 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 51Cr release assay. The involvement of caspase cascade was examined using enzyme activity assay and blocking experiments by peptide inhibitors. The expression of pro-caspases, Bcl-2-related proteins, and 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-dependent manner by LLL-CHO. Activation of caspase cascade through caspase-8 to caspase-3 was essential during this process. Pretreatment of synovial cells with TNF alpha significantly augmented both the activation of caspases and the proportion of apoptosis in synovial cells induced by LLL-CHO, whereas TGFbeta1 pretreatment markedly suppressed these phenomena. The ratio of the expression of Bcl-2 to Bax or Bcl-xL to Bax, and XIAP expression in synovial cells may not be directly associated with the susceptibility of synovial cells to apoptosis by LLL-CHO.


CONCLUSIONS
Apoptosis of synovial cells was induced by inhibition of proteasome function through the activation of caspase cascade, and this process was 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 therapeutic strategies in rheumatoid arthritis.
Kawakami
A
A
Nagasaki University School of Dentistry, Japan.
Nakashima
T
T
Sakai
H
H
Hida
A
A
Urayama
S
S
Yamasaki
S
S
Nakamura
H
H
Ida
H
H
Ichinose
Y
Y
Aoyagi
T
T
Furuichi
I
I
Nakashima
M
M
Migita
K
K
Kawabe
Y
Y
Eguchi
K
K
D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004347:Drug Interactions; D004789:Enzyme Activation; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000187:drug effects / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2; D013583:Synovial Membrane / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde