MiR-217 Regulates SIRT1 Expression and Promotes Inflammatory and Apoptotic Responses in Osteoarthritis.

Previous studies have reported miR-217 uregulation in age-related pathologies. We investigated the impact of miR-217-5p on sirtuin 1 (SIRT1) regulation in human osteoarthritic (OA) chondrocytes. MiR-217 target enrichment analyses were performed using three public databases, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. MiR-217-5p expression levels were quantified in normal and OA chondrocytes. SIRT1 expression levels, nuclear factor kappa-B p65 subunit (NF-κBp65) and p53 acetylation levels, and expression levels of OA-related pro-inflammatory markers [tumor necrosis factor α (TNFα), interleukin 1ß (IL-1ß), IL-6], pro-apoptotic markers [Bax, pro-caspase 3, cleaved caspase 3] and matrix regulators [matrix metalloproteinase (MMP)-1, MMP-13, MMP-9, Collagen 2 (COL2A1), Aggrecan (ACAN)] were evaluated in miR-217 mimic-treated and/or miR-217 inhibitor-treated OA chondrocytes, with/without subsequent treatment with siRNA against SIRT1 (siSIRT1). MiR-217-5p was upregulated in OA chondrocytes, while target prediction/enrichment analyses revealed SIRT1 as miR-217 target-gene. Deacetylation of NF-κBp65 and p53 in miR-217 inhibitor-treated OA chondrocytes was reversed by siSIRT1 treatment. MiR-217 inhibitor-treated OA chondrocytes showed increased COL2A1, ACAN and decreased IL-1ß, IL-6, TNFα, Bax, cleaved caspase 3 and MMPs expression levels, which were reversed following miR-217 inhibitor/siSIRT1 treatment. Our findings highlight the impact of miR-217-5p on SIRT1 downregulation contributing to OA pathogenesis.

DNA methylation regulates Sirtuin 1 expression in osteoarthritic chondrocytes.

PURPOSE: Sirtuin 1 (SIRT1) comprises a major anti-aging longevity factor with multiple protective effects on chondrocyte homeostasis. Previous studies have reported that downregulation of SIRT1 is linked to osteoarthritis (OA) progression. In this study, we aimed to investigate the role of DNA methylation on SIRT1 expression regulation and deacetylase activity in human OA chondrocytes. MATERIALS AND METHODS: Methylation status of SIRT1 promoter was analyzed in normal and OA chondrocytes using bisulfite sequencing analysis. CCAAT/enhancer binding protein alpha (C/EBPα) binding to SIRT1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Subsequently, C/EBPα's interaction with SIRT1 promoter and SIRT1 expression levels were evaluated after treatment of OA chondrocytes with 5-Aza-2'-Deoxycytidine (5-AzadC). Acetylation and nuclear levels of nuclear factor kappa-B p65 subunit (NF-κΒp65) and expression levels of selected OA-related inflammatory mediators, interleukin 1ß (IL-1ß) and IL-6 and catabolic genes (metalloproteinase-1 (MMP-1) and MMP-9) were evaluated in 5-AzadC-treated OA chondrocytes with or without subsequent transfection with siRNA against SIRT1. RESULTS: Hypermethylation of specific CpG dinucleotides on SIRT1 promoter was associated with downregulation of SIRT1 expression in OA chondrocytes. Moreover, we found decreased binding affinity of C/EBPα on the hypermethylated SIRT1 promoter. 5-AzadC treatment restored C/EBPα's transcriptional activity inducing SIRT1 upregulation in OA chondrocytes. Deacetylation of NF-κΒp65 in 5-AzadC-treated OA chondrocytes was prevented by siSIRT1 transfection. Similarly, 5-AzadC-treated OA chondrocytes exhibited decreased expression of IL-1ß, IL-6, MMP-1 and MMP-9 which was reversed following 5-AzadC/siSIRT1 treatment. CONCLUSIONS: Our results suggest the impact of DNA methylation on SIRT1 suppression in OA chondrocytes contributing to OA pathogenesis.

Leptin-depended NLRP3 inflammasome activation in osteoarthritic chondrocytes is mediated by ROS.

Leptin and ROS are implicated in the regulation of inflammatory pathways including NLRP3-inflammasome. We investigated the functional link between leptin, ROS and NLRP3-inflammasome formation/activation in osteoarthritis (OA), an age-related disease. We found that inflammasome components' (NLRP3, ASC, Caspase-1 and cleaved Caspase-1) protein expression were increased in OA cartilage biopsies and chondrocytes compared to healthy cartilage and chondrocytes. Immunofluorescence showed increased co-localization of NLRP3/ASC and NLRP3/Caspase-1, ASC-specks formation and ROS levels in OA compared to normal chondrocytes. NOX4 mRNA expression and IL-1ß/IL-18 secretion levels were also elevated in OA chondrocytes. Furthermore, NLRP3-siRNA in OA chondrocytes revealed significant MMP-9/MMP-13 downregulation. To elucidate leptin/ROS/NLRP3-inflammasome interactions, OA chondrocytes were treated with ROS-inhibitor NAC, NOXs-inhibitor DPI, NOX4-inhibitor GLX351322 and leptin-siRNA, while normal chondrocytes were incubated with leptin with or without DPI or GLX351322. We observed attenuated ROS levels and NLRP3-inflammasome formation/activation in NAC-, DPI- or GLX351322-treated OA chondrocytes, while the same effect was shown after transfection with leptin-siRNA. Furthermore, incubation of normal chondrocytes with leptin enhanced ROS production and inflammasome formation/activation, while pretreatment with DPI or GLX351322 abolished leptin's stimulatory effects confirming leptin-NOX4-ROS-inflammasome regulatory axis. Overall, our findings provide novel evidence indicating that leptin-induced NLRP3-inflammasome formation/activation in OA chondrocytes is mediated by NOX4-dependent ROS production.

Dual Role of SIRT1 in Autophagy and Lipid Metabolism Regulation in Osteoarthritic Chondrocytes.

Background and Objectives: Osteoarthritis (OA) is one of the most common and highly prevalent types of arthritis, also considered a multiphenotypic disease with a strong metabolic component. Ageing is the primary risk factor for OA, while the age-related decline in autophagic activity affects cell function and chondrocyte homeostasis. The aim of this study was to investigate the role of sirtuin 1 (SIRT1) in autophagy dysregulation and lipid metabolism in human OA chondrocytes. Materials and Methods: OA chondrocytes were treated with Resveratrol, Hydroxycloroquine (HCQ) or 3-Methyladenine (3-MA) and HCQ or 3-MA followed by siRNA against SIRT1 (siSIRT1). Then, SIRT1, AcNF-κBp65, LOX-1 and autophagy-related proteins ATG5, ATG13, PI3K class III, Beclin-1, LC3 and ULK protein levels were evaluated using Western blot. Normal articular chondrocytes were treated under serum starvation and/or siSIRT1, and the protein expression levels of the above autophagy-related proteins were evaluated. The staining patterns of LC3/p62 and LOX-1 were analyzed microscopically by immunofluorescence. SIRT1/LC3 complex formation was analyzed by immunoprecipitation. Results: SIRT1 and LOX-1 protein expression were negatively correlated in OA chondrocytes. SIRT1 regulated LOX-1 expression via NF-κΒ deacetylation, while treatment with Resveratrol enhanced SIRT1 enzymatic activity, resulting in LOX-1 downregulation and autophagy induction. In OA chondrocytes, SIRT1 was recognized as an autophagy substrate, formed a complex with LC3 and was consequently subjected to cytoplasmic autophagosome-lysosome degradation. Moreover, siSIRT1-treated normal chondrocytes showed decreased autophagic activity, while double-treated (siSIRT1 and serum starvation) cells showed no induction of autophagy. Conclusions: Our results suggest that SIRT1 regulates lipid homeostasis through LOX-1 expression regulation. Additionally, we indicate that the necessity of SIRT1 for autophagy induction in normal chondrocytes, together with its selective autophagic degradation in OA chondrocytes, could contribute to autophagy dysregulation in OA. We, therefore, suggest a novel regulatory scheme that functionally connects lipid metabolism and autophagy in late-stage OA.