Original article Sirt1-deficient mice exhibit an altered cartilage phenotype

tObjective: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here weattempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.Method: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wildtype (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Jointcartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.Results: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibitedlow levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, alreadyshown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KOmice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphataseb) was elevated in the Sirt1 KO mice, in line with previous reports.Conclusion: The findings from this animal model demonstrated that Sirt1 KO mice presented an alteredcartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process

Original article Sirt1-deficient mice exhibit an altered cartilage phenotype

tObjective: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here weattempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.Method: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wildtype (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Jointcartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.Results: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibitedlow levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, alreadyshown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KOmice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphataseb) was elevated in the Sirt1 KO mice, in line with previous reports.Conclusion: The findings from this animal model demonstrated that Sirt1 KO mice presented an alteredcartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process

Kumar et al., 2018 - JCS

Supplemental files _Kumar et al., 2018, JC

Increased apoptotic chondrocytes in articular cartilage from adult heterozygous SirT1 mice.

peer reviewedOBJECTIVE: A growing body of evidence indicates that the protein deacetylase, SirT1, affects chondrocyte biology and survival. This report aims to evaluate in vivo attributes of SirT1 in cartilage biology of 129/J murine strains. METHODS: Heterozygous haploinsufficient (SirT1(+/-)) and wild-type (WT; SirT1(+/+)) 129/J mice aged 1 or 9 months were systematically compared for musculoskeletal features, scored for osteoarthritis (OA) severity, and monitored for chondrocyte apoptosis in articular cartilage. Sections of femorotibial joints were stained for type II collagen and aggrecan. Protein extracts from articular chondrocytes were isolated and immunoblotted for SirT1 and active caspase 3. RESULTS: Phenotypic observations show that, at 1 month of age, SirT1(+/-) mice were smaller than WT and showed a significant decrease in full-length SirT1 (FLSirT1; 110 kDa) protein levels. Levels of FLSirT1 were further decreased in both strains at 9 months. Immunoblot assays for 9-month-old strains revealed the presence of the inactive cleaved SirT1 variant (75 SirT1; 75 kDa) in WT mice, which was undetected in age-matched SirT1(+/-) mice. Nine-month-old SirT1(+/-) mice also showed increased OA and increased levels of apoptosis compared with age-matched WT mice. CONCLUSION: The data suggest that the presence of 75 SirT1 may prolong viability of articular chondrocytes in adult (9-month-old) mice

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC‐chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D‐pellet culture led to significant increases in the expression of ECM genes for type‐II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific –10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N‐acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co‐IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B‐SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC‐chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators

SIRT1 Deficiency, Specifically in Fibroblasts, Decreases Apoptosis Resistance and Is Associated with Resolution of Lung-Fibrosis

In contrast to normal regenerating tissue, resistance to Fas- and FasL-positive T cell-induced apoptosis were detected in myofibroblasts from fibrotic-lungs of humans and mice following bleomycin (BLM) exposure. In this study we show, decreased FLIP expression in lung-tissues with resolution of BLM-induced fibrosis and in isolated-lung fibroblasts, with decreased resistance to apoptosis. Using a FLIP-expression vector or a shFLIP-RNA, we further confirmed the critical need for FLIP to regain/lose susceptibility of fibrotic-lung myofibroblast to Fas-induced apoptosis. Our study further show that FLIP is regulated by SIRT1 (Sirtuin 1) deacetylase. Chimeric mice, with SIRT1-deficiency in deacetylase domain (H355Y-Sirt1y/y), specifically in mesenchymal cells, were not only protected from BLM-induced lung fibrosis but, as assessed following Ku70 immunoprecipitation, had also decreased Ku70-deacetylation, decreasedKu70/FLIP complex, and decreased FLIP levels in their lung myofibroblasts. In addition, myofibroblasts isolated from lungs of BLM-treated miR34a-knockout mice, exposed to a miR34a mimic, which we found here to downregulate SIRT1 in the luciferase assay, had a decreased Ku70-deacetylation indicating decrease in SIRT1 activity. Thus, SIRT1 may mediate, miR34a-regulated, persistent FLIP levels by deacetylation of Ku70 in lung myofibroblasts, promoting resistance to cell-death and lung fibrosis