In human chondrocytes DNMT-1 plays a key role in the expression of several genes associated with the pathogenesis of osteoarthritis (OA)

Abstract

Introduction: Recent evidence points to chromatin remodeling as a key event in the expression of several genes associated with OA pathogenesis. IL-1β is the principal cytokine involved in cartilage catabolism in OA. The aim of this study was to investigate (a) whether IL-1β modulates DNA methylation content in human chondrocytes; and if so (b) how the DNA methylation status affect the expression of selected downstream genes; and (c) whether the effect on DNA methylation is the same or different in chondrocytes from the 3 defined histological zones of the human articular cartilage. Methods: Femoral head cartilage samples (n=12) obtained at the time of hip arthroplasty were stained with India ink and only the unaffected, smooth cartilage was used. A portion of the cartilage was fixed, decalcified and analyzed histologically. Chondrocytes were derived by enzymatic digestion of superficial zone (SZ), transition zone (TZ) and deep zone (DZ) of the cartilage and cultured in monolayer. Chondrocytes stimulated with IL-1β (10ng/ml) in vitro for 24h were used in these studies. Gene expression of 84 human epigenetic modification enzymes was studied using Human Epigenetic Chromatin Modification Enzymes RT2 Profiler™ PCR Array. Expression levels of DNMT-1, -3A,-3B, Thymine DNA glycosylase (TDG) and of selected genes was quantified by TaqMan Assays. Expression of Global DNA methylation level was determined using MethylFlashTM Methylated DNA quantification kit. IL-1β-induced changes in the activity of DNMTs and DNA demethylases was studied using commercially available kits. Effect of DNA demethylation by a pharmacological agent 5-Aza-2 deoxycytidine (5-Aza) on the expression of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), cyclooxygenase-2 (COX-2) and SOX-9 was also studied. SiRNA-mediated knockdown of DNMT-1 expression was used to study its role in the regulation of selected genes. Results were derived using Origin 6.1 software package and p<0.05 was considered significant. Results: In IL-1β-stimulated chondrocytes, global DNA methylation showed significant increase in the DZ chondrocytes (128%) and in SZ chondrocytes (44%), but not in TZ chondrocytes. Expression analysis of 84 genes whose products are known or predicted to modify genomic DNA and histones to regulate gene expression, identified 30 genes that displayed significant differences in expression upon IL-1β-stimulation. Results of both the mRNA array and TaqMan assays showed a significant up-regulation of DNMT-1, -3A and -3B gene expression upon IL-1β-stimulation which correlated with the increased total DNMT and DNMT-1 enzyme activity in both the SZ and DZ chondrocytes but not in TZ chondrocytes. Interestingly, expression and activity of DNA demethylases and TDG enzymes was also increased in SZ chondrocytes by IL-1β-stimulation. Pharmacological DNA demethylation and knockdown of DNMT-1 resulted in significant increase in the expression of MMP-3, MMP-9, MMP-13, TIMP-2, and COX-2 and transcription factor SOX-9 in SZ chondrocytes. Conclusions: We identify for the first time several new candidate genes that show epigenetic regulation in response to IL-1β in human chondrocytes. We also show that IL-1β induce hypermethylation of genomic DNA in SZ and DZ chondrocytes, but not in TZ chondrocytes, which positively correlated with the expression and activity of DNA methylatranferases. IL-1β stimulation also increased the expression and activity of DNA demethylases suggesting a dynamic regulation of DNA methylation and demethylation in SZ chondrocytes. Our results also suggest a role of DNMT1 activity in mediating epigenetic modifications that regulate the expression of MMP-3, -9, -13, TIMP-2, SOX-9 and COX-2 in human chondrocytes.