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.

Objective A growing body of evidence indicates that the protein deacetylase SirT1 affects chondrocyte biology and survival. This report aims to evaluate in-vivo effects of SirT1 on cartilage biology in 9 month-old mice 129/J murine strains. Methods Heterozygous (SirT1+/−) and wild type (WT; SirT1+/+) mice were systematically compared for musculoskeletal features, scored for OA severity and chondrocyte viability in articular cartilage at 1 month and 9 months post-natally. Sections of femoro-tibial joints were stained for type II collagen, aggrecan and active caspase 3. Articular murine chondrocytes were isolated and immunoblotted for SirT1 and active caspase 3. Results Phenotypic observations show that at the age of 1 month SirT1+/− mice were smaller than the wild type and presented a significant reduction in full-length SirT1 (FLSirT1; 110kDa) protein levels. Nine month-old SirT1+/− and WT mice did not express FLSirT1, however immunoblot assays of 9M articular cartilage-derived protein extracts revealed the inactive cleaved variant of SirT1 (75SirT1; 75kDa) was decreased in the SirT1+/− compared to WT mice. Nine month-old SirT1+/− mice possessed enhanced OA and enhanced levels of active caspase 3, compared to age-matched WT mice. Conclusion The data suggest that the presence of 75SirT1 may prolong viability of articular chondrocytes in adult 9 month-old mice.

Type II collagen is a key cartilaginous extracellular protein required for normal endochondral development and cartilage homeostasis. COL2A1 gene expression is positively regulated by the NAD-dependent protein deacetylase Sirtuin 1 (SirT1), through its ability to bind chromatin regions of the COL2A1 promoter and enhancer. Although SirT1/Sox9 binding on the enhancer site of COL2A1 was previously demonstrated, little is known about its functional role on the gene promoter site. Here, we examined the mechanism by which promoter-associated SirT1 governs COL2A1 expression. Human chondrocytes were encapsulated in three-dimensional (3D) alginate beads where they exhibited upregulated COL2A1 mRNA expression and increased levels of SirT1 occupancy on the promoter and enhancer regions, when compared to monolayer controls. Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA-binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site. ChIP reChIP assays revealed that SirT1 and Set7/9 form a protein complex on the COL2A1 promoter region of 3D-cultured chondrocytes, which also demonstrated elevated trimethylated lysine 4 on histone 3 (3MeH3K4), a hallmark of Set7/9 methyltransferase activity. Advanced passaging of chondrocytes yielded a decrease in 3MeH3K4 and Set7/9 levels on the COL2A1 promoter and reduced COL2A1 expression, suggesting that the SirT1/Set7/9 complex is preferentially formed on the COL2A1 promoter and required for gene activation. Interestingly, despite SirT1 occupancy, its deacetylation targets (ie, H3K9/14 and H4K16) were found acetylated on the COL2A1 promoter of 3D-cultured chondrocytes. A possible explanation for this phenotype is the enrichment of the histone acetyltransferases P300 and GCN5 on the COL2A1 promoter of3 D-cultured chondrocytes. Our study indicates that Set7/9 prevents the histone deacetylase activity of SirT1, potentiating euchromatin formation on the promoter site of COL2A1 and resulting in morphology-dependent COL2A1 gene transactivation.

BackgroundIt has been shown that the proportion of natural killer T cells is markedly elevated during liver regeneration and their activation under different conditions can modulate this process. As natural killer T cells and liver injury are central in liver regeneration, elucidating their role is important.MethodsThe aim of the current study is to explore the role of natural killer T cells in impaired liver regeneration. Concanvalin A was injected 4 days before partial hepatectomy to natural killer T cells- deficient mice or to anti CD1d1-treated mice. Ki-67 and proliferating cell nuclear antigen were used to measure hepatocytes proliferation. Expression of hepatic cyclin B1 and proliferating cell nuclear antigen were evaluated by Western Blot and liver injury was assessed by ALT and histology.ResultsNatural killer T cells- deficient or mice injected with anti CD1d antibodies exhibited reduced liver regeneration. These mice were considerably resistant to ConA-induced liver injury. In the absence of NKT cells hepatic proliferating cell nuclear antigen and cyclin B1 decreased in mice injected with Concanvalin A before partial hepatectomy. This was accompanied with reduced serum interleukin-6 levels.ConclusionsNatural killer T cells play an important role in liver regeneration, which is associated with cyclin B1 and interleukin-6.

Objective: Collagen type II (Col-II) plays a significant role in skeletal development and cartilage homeostasis and its loss with age is associated with degenerative joint diseases such as osteoarthritis (OA). Previously findings establish that the protein deacetylase SirT1 is required for enhanced expression of Col-IIa1, a major cartilage structural component of Col-II. Here we profile the mechanism by which SirT1 activates promoter-driven Col-IIa1 expression. Methods: Chondrocytes derived from human osteoarthritic knee joints were encapsulated in three-dimensional alginate hydrogel microbeads (3D) and compared to paired monolayer (2D, passage 2) cultures derived from the same donor tissue. 3D-cultured cells displayed augmented expression of Col-IIa1 and were subject to serial chromatin immunoprecipitation (ChIP) and ChIP reChIP analyses to characterize epigenetic variations as compared to 2D cultures. Results: ChIP analyses exhibited enrichment for SirT1 and the histone methyl transferase Set7/9, which formed a complex in 3D cultures wherein Col-IIa1 was augmented in expression. Consistent with enriched Set7/9 levels on the Col-IIa1 promoter, ChIP data revealed enriched levels of trimethylation on histone 3 lysine 4 (3MeH3K4). Acetylated H3K9/14 and H4K16 on Col-IIa1 promoters of 3D-cultured chondrocytes were also elevated indicating that SirT1 is enzymatically repressed by Set7/9 on the transactivated Col-IIa1 promoter. Elevated marks of the Histone acetyl transferases GCN5 and P300 also supported the local enrichment of their acetylated histone marks (i.e. H3K9/ 14,H4K5 and H4K16). Conclusions: The data support that Set7/9 is capable of locally repressing SirT1 enzymatic activity on the promoter of Col-IIa1, resulting in its augmented expression dependent on cell morphology.