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Tumor necrosis factor ?-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes.

ABSTRACT: OBJECTIVE:The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor ? (TNF?) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNF?, resulting in altered gene expression. METHODS:Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the ?2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNF?. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNF? were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNF?-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?. RESULTS:TNF?-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?-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNF? exposure blocked the generation of the 75-kd SirT1 fragment. CONCLUSION:These data indicate that TNF?, 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?-treated cells.

SUBMITTER: Dvir-Ginzberg M

PROVIDER: S-EPMC3174010 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Tumor necrosis factor α-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes.

Dvir-Ginzberg Mona M Gagarina Viktoria V Lee Eun Jin EJ Booth Richard R Gabay Odile O Hall David J DJ

Arthritis and rheumatism 20110801 8

<h4>Objective</h4>The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression.<h4>Methods</h4>Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were ...[more]

PMID: 21305533

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Dataset Information

Tumor necrosis factor ?-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes.

Publications

Tumor necrosis factor α-mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes.

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