Searching potential transcription factors regulating MMP13 overexpression via promoter CpG methylation status in human osteoarthritic chondrocytes: A microarray screening

Abstract

Purpose: Matrix metalloproteinase 13 (MMP-13) is known to play important roles in the progress of osteoarthritis (OA) in human articular cartilage. Our previous studies have demonstrated that the elevated MMP13 gene expression in human chondrocytes is regulated by the CpG methylation status in its proximal promoter. Also, hypoxia-inducible factor (HIF) -2α, a transcription factor (TF) mainly induced under the hypoxic condition, was found to regulate MMP13 expression via methylation status of a specific CpG site in the proximal promoter. In this study, we screened TFs that have a potential to regulate MMP13 expression via its promoter CpG methylation status by a microarray analysis comparing healthy and OA human chondrocytes. Methods: Four healthy and five OA cartilage pieces were harvested from the human femoral head collected from the hemiarthroplasty for fracture of neck of femur (#NOF) and total hip arthroplasty for hip OA, respectively. Then mRNA was extracted (QIAGEN, RNeasy Lipid Kit®), from those cartilage pieces. The mRNA samples with RNA integrity number (RIN) >6.5 by Bioanalyzer® (Agilent Technologies) were proceeded to a microarray analysis (TORAY, 3D-Gene®). The expression level of MMP13 and various TF genes were compared between healthy and OA human chondrocytes. The TF gene expression with 2-time difference between healthy and OA chondrocytes were selected for further analysis. The potential binding sites of the selected TFs, and the presence of CpG sites in those binding sites, were detected in up to −5000bp of MMP13 promoter using TF binding site databases. Results: The relative expression levels of MMP13 were 32 ± 43 and 538 ± 337 in healthy and OA chondrocytes, respectively (P=0.022). In the microarray analysis, 41 TFs were more expressed, and 40 TFs were less expressed in OA compared to healthy chondrocytes, respectively. Of them, 13 TFs were found to be potentially bindable to up to −5000-bp of MMP13 promoter. Moreover, transcription factors FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 and E2F2 were found to have their consensus sequences close to CpG sites in the MMP13 promoter sequence. Conclusions: In addition to HIF-2α, the function of FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 or E2F2 can be also regulated by the methylation status of CpG sites in MMP13 promoter. Further investigation is needed to prove the detailed function of those transcription factors, especially for a relationship with CpG methylation status of the MMP13 promoter.