DNA methylation of the RUNX2 P1 promoter mediates MMP13 transcription in chondrocytes

Atsushi Takahashi,Ko Hashimoto,Miguel Otero,Richard O C Oreffo,María C De Andrés,Eiji Itoi,Mary B Goldring

doi:10.1038/s41598-017-08418-8

Abstract

The Runt-related transcription factor 2 (RUNX2) is critical for bone formation as well as chondrocyte maturation. Matrix metalloproteinase (MMP)-13 is a major contributor to cartilage degradation in osteoarthritis (OA). We and others have shown that the abnormal MMP13 gene expression in OA chondrocytes is controlled by changes in the DNA methylation status of specific CpG sites of the proximal promoter, as well as by the actions of different transactivators, including RUNX2. The present study aimed to determine the influence of the methylation status of specific CpG sites in the RUNX2 promoter on RUNX2-driven MMP13 gene expression in OA chondrocytes. We observed a significant correlation between MMP13 mRNA levels and RUNX2 gene expression in human OA chondrocytes. RUNX2 overexpression enhanced MMP13 promoter activity, independent of the MMP13 promoter methylation status. A significant negative correlation was observed between RUNX2 mRNA levels in OA chondrocytes and the percentage methylation of the CpG sites in the RUNX2 P1 promoter. Accordingly, the activity of the wild type RUNX2 promoter was decreased upon methylation treatment in vitro. We conclude that RUNX2 gene transcription is regulated by the methylation status of specific CpG sites in the promoter and may determine RUNX2 availability in OA cartilage for transactivation of genes such as MMP13.

Highlights

The Runt-related transcription factor 2 (RUNX2) is critical for osteoblast differentiation and bone formation[1,2,3]
We hypothesized that the altered methylation status of specific CpG sites in the P1 promoter of RUNX2 determines the availability of the expressed gene product, which in turn influences the levels of MMP13 gene expression in human OA chondrocytes
High MMP13 gene expression in OA chondrocytes is associated with demethylation of specific CpG sites in the MMP13 proximal promoter

Summary

Introduction

The Runt-related transcription factor 2 (RUNX2) is critical for osteoblast differentiation and bone formation[1,2,3]. Matrix metalloproteinase (MMP)-13 gene expression is decreased in Runx2-null mutant mice[12,13,14], and Runx[2] interacts with Osterix in regulating MMP13 gene transcription in growth plate chondrocytes[15] Both Runx2-deficient mice and chondrocyte-specific Runx2–transgenic mice display abnormal cartilage development[8, 9], and Runx2-haploinsufficient mice show reduced type X collagen and MMP13 protein and mRNA levels accompanied by decreased cartilage degradation in osteoarthritis (OA) models[16]. We and others have shown that the abnormal MMP13 gene expression in OA chondrocytes is controlled by changes in the DNA methylation status of specific CpG sites of the proximal promoter[28, 42], as well as by the actions of different transactivators[28, 43, 44], including RUNX236, 45, 46. We hypothesized that the altered methylation status of specific CpG sites in the P1 promoter of RUNX2 determines the availability of the expressed gene product, which in turn influences the levels of MMP13 gene expression in human OA chondrocytes

Methods

Results

Conclusion