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 OA) and frorm adjacent macroscopically intact regions (minimal OA). Using small interference RNA we tested if leptin down-regulation would affect MMP-13 activity. We also evaluated the effect of the demethylating agent, 5’-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 down-regulation 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 up-regulated 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 up-regulated after leptin’s epigenetic reactivation, raising the issue of leptin’s therapeutic potential for osteoarthritis.
- DNA methylation
- histone modifications