Session Title: Biology and Pathology of Bone and Joint
Session Type: Abstract Submissions (ACR)
Background/Purpose: Osteoarthritis (OA) reduces mobility and function of the affected joint and is a leading cause of disability among the elderly. Recent evidence points to epigenetic regulation of genes in OA. Interleukin-1β (IL-1β) is the major cytokine involved in cartilage catabolism and induces the expression of pro-inflammatory genes. The aim of this study was to investigate whether IL-1β induces epigenetic changes in human chondrocytes obtained from upper, middle and deep zone cartilage.
Methods: Chondrocytes were derived by enzymatic digestion of upper, middle and deep-zone cartilage obtained from OA patients (n=10). Chondrocytes were stimulated with IL-1β (10ng/ml) in vitro for 24h. Global DNA methylation level was determined using MethylFlashTM Methylated DNA quantification kit (Epigentek). Total RNA was prepared and expression level of DNMT-1, DNMT-3A, DNMT-3B and HDAC-1 was quantified by TaqMan Assays. IL-1β-induced changes in the activity of total DNMT, DNMT-1, DNMT-3A, DNMT-3B, DNA demethylases and Thymine DNA glycosylase (TDG) was determined using ELISA-based assays (Epigentek). Un-stimulated and IL-1β-stimulated chondrocytes obtained from upper and deep zones were used to study the expression of 84 human epigenetic modification enzymes using mRNA array (SA Biosciences). Results were derived using Origin 6.1 software package and p<0.05 was considered significant.
Results: Global DNA methylation estimation showed the differential response of chondrocytes from different zones of the human cartilage to IL-1β. Total DNA methylation was significantly increased in the deep-zone chondrocytes (138%) and in upper-zone chondrocytes (18%) stimulated with IL-1β compared to controls. Expression levels of 84 key genes encoding enzymes known or predicted to modify genomic DNA and histones to regulate chromatin accessibility showed that 30 genes displayed significant differences upon IL-1β-stimulation. Among these, 29 genes were upregulated and 1 gene was downregulated. Interestingly, the mRNA array results showed a significant upregulation of DNMT-1, DNMT-3A and DNMT-3B expression in both upper and deep-zone chondrocytes stimulated with IL-1b and correlated with the increased total DNMT and DNMT-1 activity in the same chondrocytes. Activity of both DNA demethylases and TDG, enzymes essential for active DNA demethylation, was also increased in both deep and upper-zone chondrocytes by IL-1β suggesting a dynamic regulation of DNA methylation and demethylation in these chondrocytes. No significant difference in global DNA methylation, expression and activity of DNMT-1, DNMT-3A, DNMT-3B and demethylases was observed in chondrocytes from middle-zone in response to IL-1β.
Conclusion: We demonstrate a role of IL-1β-mediated epigenetic modifications in chondrocytes of the three histological zones of the human cartilage. Hypermethylation of genomic DNA in OA chondrocytes positively correlated with the expression and activity of DNA methylatranferases. This study also identify for the first time several new candidate genes that may be involved in DNA methylation, demethylation and histone modifications in response to IL-1β in human chondrocytes and thus may play a role in OA pathogenesis.
T. M. Haqqi,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/level-of-il-1a-induced-epigenetic-modifications-differ-in-chondrocytes-from-different-histological-zones-of-human-cartilage/