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Tumor necrosis factor [alpha]-mediated cleavage and inactivation of sirT1 in human osteoarthritic chondrocytes.
Dvir-Ginzberg, Mona 1; Gagarina, Viktoria 1; Lee, Eun Jin 1; Booth, Richard 1; Gabay, Odile 1; Hall, David J. 1
[Miscellaneous Article] Arthritis & Rheumatism. 63(8):2363-2373, August 2011.

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Objective. The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor [alpha] (TNF[alpha]) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNF[alpha], resulting in altered gene expression.

Methods. Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the [alpha]2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNF[alpha]. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNF[alpha] were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNF[alpha]-treated extracts was identified by mass spectroscopy, and its amino-terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNF[alpha].

Results. TNF[alpha]-treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full-length 110-kd protein and a smaller 75-kd fragment. The 75-kd SirT1 fragment was found to lack the carboxy-terminus. Cathepsin B was identified as the TNF[alpha]-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNF[alpha] exposure blocked the generation of the 75-kd SirT1 fragment.

Conclusion. These data indicate that TNF[alpha], a cytokine that mediates joint inflammation in arthritis, induces cathepsin B-mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage-specific gene expression evident in these TNF[alpha]-treated cells.