Background Osteoarthritis (OA) is a chronic degenerative joint disorder of high prevalence that remains the leading cause of disability in aged people. A balance between anabolic and catabolic mechanisms maintains extracellular matrix homeostasis in articular cartilage. Among mechanisms to control gene expression, genomic DNA methylation, modification of nucleosome histone tails, and chromosome remodeling are essential contributors to the mechanisms of epigenetic control.
Objectives We tested the hypothesis that methylation status of SOX-9 gene promoter region and gene expression is differential in osteoarthritic cartilage compared with normal cartilage.
Methods Articular cartilage was obtained from normal human femoral heads from femoral neck fractures without OA and from OA patients undergoing total hip arthroplasty. For Methylation Specific Polymerase Chain Reaction (MS-PCR), CpG rich regions within upstream sequences 5 kb from the transcription start site were analyzed. Putative CpG-rich islands and respective primers for MS-PCR were derived from the CpG Island Searcher and the MethPrimer program, respectively. In 5-aza-2’-deoxycytidine treatment, Chondrocytes were treated with 5μM 5-aza-2’-deoxycytidine dissolved in medium for 8 days; medium was changed daily.
Results We tested that DNA methylation of SOX-9 promoter is differential in OA cartilage compared with normal cartilage via MSP and bisulfite sequencing analysis. We found that methylation of SOX-9 promoter region increased in OA cartilage compared to normal cartilage. Methylated CpG sites significantly increased in all the examined regions and total methylated CpG sites increased about eight-fold in OA cartilage (14.04%) than in normal (1.66%). We also found that chondrogenesis- or degradation-related gene expression level in OA cartilage compared with normal cartilage and 5-aza-2’-deoxycytidine treatment results in changes in gene expression. We confirmed that While it had decreased SOX-9 expression by 52%, it had increased chondrogenic degradation-related genes (MMP 3, 9, 13, ADAMTS4, IL-1β and IL-6) by 1.12 ∼60 fold in OA cartilages compare with normal cartilages. In 5-aza-2’-deoxycytidine treatment, increased chondrogenesis- and degradation- related gene expression compare with untreated samples.
Conclusions In present study, we tested methylation status of chondrogenesis- or degradation-related gene promoter region in OA cartilage compared with normal cartilage. We found DNA methylation of SOX-9 promoter region via MSP analysis. Increased methylation of SOX-9 promoter regions in OA cartilages appeared to be consistent via MSP and bisulfite sequencing analysis. In summary, we found that methylation in SOX-9 promoter increased in OA cartilages. We also found that 5-aza-2’-deoxycytidine treatment results in changes in gene expression of several in osteoarthritic and normal chondrocyte. Our study suggests that the increased methylation status in the SOX-9 promoter region may have a close relation to the progression of OA.
Acknowledgements This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea. (A100010)
Ezura, Y. et al. (2009). Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium-derived mesenchymal stem cells. Arthritis Rheum 60: 1416-1426.
Disclosure of Interest None Declared