Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation. Issue 11 (November 2014)

• Record Type: Journal Article
• Authors: Imagawa, Kei; de Andrés, María C.; Hashimoto, Ko; Itoi, Eiji; Otero, Miguel; Roach, Helmtrud I.; Goldring, Mary B.; Oreffo, Richard O. C.
• Is Part Of: Arthritis & rheumatology. Volume 66:Issue 11(2014)
• Page Start: 3040
• Page End: 3051
• Publication Date: 2014-11
• Subjects: Arthritis -- Periodicals; Rheumatism -- Periodicals; 616.72
• DOI: 10.1002/art.38774
• Languages: English
• ISSNs: 2326-5191

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="art38774-sec-0001" sec-type="section"> <title>Objective</title> <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 <italic>COL2A1</italic> enhancer and <italic>COL9A1</italic> promoter.</p> </sec> <sec id="art38774-sec-0002" sec-type="section"> <title>Methods</title> <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 <italic>COL9A1</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 <italic>COL9A1</italic> promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the <italic>COL9A1</italic> promoter and the influence of DNA methylation.</p> </sec> <sec id="art38774-sec-0003" sec-type="section"> <title>Results</title> <p>Although <italic>COL2A1</italic> messenger RNA (mRNA) levels in OA chondrocytes were 19‐fold higher than those in the controls, all of the CpG sites in the <italic>COL2A1</italic> enhancer were totally demethylated in both samples. The levels of <italic>COL9A1</italic> mRNA in OA chondrocytes were 6, 000‐fold lower than those in controls; 6 CpG sites of the <italic>COL9A1</italic> promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5‐azadeoxycitidine enhanced <italic>COL9A1</italic> gene expression and prevented culture‐induced hypermethylation. In vitro methylation decreased <italic>COL9A1</italic> promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased <italic>COL9A1</italic> promoter activity. Cotransfection with SOX9 enhanced <italic>COL9A1</italic> promoter activity; CpG methylation attenuated SOX9 binding to the <italic>COL9A1</italic> promoter.</p> </sec> <sec id="art38774-sec-0004" sec-type="section"> <title>Conclusion</title> <p>This first demonstration that hypermethylation is associated with down‐regulation of <italic>COL9A1</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.</p> </sec> </abstract>