Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention

Abstract

Objective: To investigate whether epigenetic mechanisms can regulate leptin's expression and affect its downstream targets as metalloproteinases 3,9,13 in osteoarthritic chondrocytes. Methods: DNA methylation in leptin promoter was measured by DNA bisulfite sequencing, and mRNA expression levels were measured by real-time quantitative PCR in osteoarthritic as well as in normal cartilage. Osteoarthritic articular cartilage samples were obtained from two distinct locations of the knee ( n = 15); from the main defective area of maximum load ( advanced osteoarthritis ( OA)) and from adjacent macroscopically intact regions ( minimal OA). Using small interference RNA, we tested if leptin downregulation would affect matrix metalloproteinase ( MMP)-13 activity. We also evaluated the effect of the demethylating agent, 59-Aza-2-deoxycytidine ( AZA) and of the histone deacetylase inhibitor trichostatin A ( TSA) on leptin expression in chondrocyte cultures. Furthermore, we performed chromatin immunoprecipitation in leptin's promoter area. Results: We found a correlation between leptin expression and DNA methylation and also that leptin controls MMP-13 activity in chondrocytes. Leptin's downregulation with small interference RNA inhibited MMP-13 expression dramatically. After 5-AZA application in normal chondrocytes, leptin's methylation was decreased, while its expression was upregulated, and MMP-13 was activated. Furthermore, TSA application in normal chondrocyte cultures increased leptin's expression. Also, chromatin immunoprecipitation in leptin's promoter after TSA treatment revealed that histone H3 lysines 9 and 14 were acetylated. Conclusion: We found that epigenetic mechanisms regulate leptin's expression in chondrocytes affecting its downstream target MMP-13. Small interference RNA against leptin deactivated directly MMP-13, which was upregulated after leptin's epigenetic reactivation, raising the issue of leptin's therapeutic potential for osteoarthritis.