75‐kd sirtuin 1 blocks tumor necrosis factor α–mediated apoptosis in human osteoarthritic chondrocytes

Abstract

Objective SirT1 has been previously implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic chondrocytes to TNFα generates a stable and enzymatically inactive 75kDa form of SirT1 (75SirT1) via Cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we assumed it has a distinct role in osteoarthritis (OA) pathology, which we sought out to identify in this study. Methods OA and normal human chondrocytes were analyzed for the presence of Cathepsin B and 75SirT1. Confocal imaging of SirT1 monitored its subcellular trafficking following TNFα stimulation. Co-immunofluorescent staining was carried out for Cathepsin B, mitochondrial Cox IV and Lysosome-associated membrane protein I (LAMP-I) together with SirT1. Human chondrocyte were tested for apoptosis via FACS analysis and immunoblotting for caspase 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1/Cytochrome C association. Results Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes, demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistently, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with Cytochrome C, following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of FLSirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα Cathepsin B and 75SirT1 were elevated in OA vs. normal chondrocytes. Additional analyses shows that human chondrocytes exposed to OA-derived synovial fluid generate the 75SirT1 fragment. Conclusion These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

“…1C, right panel). We previously showed that SIRT1 activity declines due to cleavage of the protein during OA pathogenesis and cartilage aging (Dvir‐Ginzberg et al ., 2011; Gabay et al ., 2012; Oppenheimer et al ., 2012). The cleavage of SIRT1 was also increased in DC compared with IC (Fig.…”

“…Our previous reports provide another facet of regulation for SOX9, dependent on the decreased activity of SIRT1 seen with OA development (Dvir‐Ginzberg et al ., 2011; Gabay et al ., 2012; Oppenheimer et al ., 2012; Dvir‐Ginzberg & Steinmeyer, 2013). With advanced age and Inflammtory insult to the joint, SIRT1, the primary deacetylase of SOX9, becomes cleaved and inactivated, potentially leading to enhanced SOX9 acetylation.…”

LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

“…Because the levels of SIRT1 are reduced in human OA cartilage and chondrocytes (28,38), LEF1 levels are expected to be elevated in the affected tissue, with a corresponding elevation in MMP13. As predicted and shown in Fig.…”

“…Indeed, this report shows that SIRT1 reduced MMP13 gene expression and LEF1 protein levels in human primary chondrocytes. Furthermore, inflammation of the joint may lead to SIRT1 truncation (27), possibly reducing SIRT1 chromatin binding (28), sequentially lifting its inhibitory effects on LEF1 gene expression and promoting downstream MMP13 gene expression.…”

Melanocortin peptides protect chondrocytes from mechanically induced cartilage injury

“…This eventually leads impaired ECM synthesis, cartilage degradation and, ultimately, development of OA [36,37].…”