Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

<jats:sec><jats:title>Objective</jats:title><jats:p>To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the <jats:italic>COL2A1</jats:italic> enhancer and <jats:italic>COL9A1</jats:italic> promoter.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Expression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on <jats:italic>COL9A1</jats:italic> promoter activity was determined using a CpG‐free vector; cotransfections with expression vectors encoding SOX9, hypoxia‐inducible factor 1α (HIF‐1α), and HIF‐2α were carried out to analyze <jats:italic>COL9A1</jats:italic> promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the <jats:italic>COL9A1</jats:italic> promoter and the influence of DNA methylation.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Although <jats:italic>COL2A1</jats:italic> messenger RNA (mRNA) levels in OA chondrocytes were 19‐fold higher than those in the controls, all of the CpG sites in the <jats:italic>COL2A1</jats:italic> enhancer were totally demethylated in both samples. The levels of <jats:italic>COL9A1</jats:italic> mRNA in OA chondrocytes were 6,000‐fold lower than those in controls; 6 CpG sites of the <jats:italic>COL9A1</jats:italic> promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5‐azadeoxycitidine enhanced <jats:italic>COL9A1</jats:italic> gene expression and prevented culture‐induced hypermethylation. In vitro methylation decreased <jats:italic>COL9A1</jats:italic> promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased <jats:italic>COL9A1</jats:italic> promoter activity. Cotransfection with SOX9 enhanced <jats:italic>COL9A1</jats:italic> promoter activity; CpG methylation attenuated SOX9 binding to the <jats:italic>COL9A1</jats:italic> promoter.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>This first demonstration that hypermethylation is associated with down‐regulation of <jats:italic>COL9A1</jats:italic> expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.</jats:p></jats:sec>