Gene–gene interactions between VDR, OPG and TNFSF11 genes and bone mineral density

Abstract

Epigenetics refers to heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. Epigenetic control of gene expression is achieved by chromatin remodeling as well as DNA methylation. The latter process is catalyzed by DNA (cytosine-5-)-methyltransferases (Dnmts). Bone extra cellular matrix (ECM) consisting mainly of collagen type I is known to play an important role in the regulation of gene expression and differentiation in osteoblasts. Recently, it was found that Dnmt1 is regulated via activator proteincomplex-1 (AP-1), which consists of cFos and cJun. In the present study we demonstrate that collagen regulates Dnmt1 mRNA expression via AP-1 in mouse osteoblast-like cells. Mouse MC3T3-E1 cells were seeded at a density of 2000/cm in alpha-MEM on rat-tail collagen coated dishes and on uncoated dishes as control and cultured for 2 h, 4 h, 12 h, 24 h, 48 h, 4 d and 8 d. To get more information about mechanisms, cells were cultured in presence of resveratrol (40 μM) or cycloheximid (CHX, 10 μg/μl). After the culture time, RNA was isolated and mRNA expression of Dnmt1, c-Fos and c-Jun (components of AP-1)wasmeasured by quantitative RT-PCR. During the first 12 h there was no significant difference in Dnmt1 mRNA expression between collagen coated and control cultures. Then, mRNA expression on collagen increased 5-fold while in controls we found only a 2-fold increase. Moreover, the controls immediately decreased significantly while on collagen the peak lasted for 4 days with a decrease thereafter. We next tested whether protein synthesis is a prerequisite for this regulatory event. Using cycloheximid we inhibited Dnmt1 expression in cells seeded on collagen and in control cultures. Resveratrol treatment, known to inhibit AP-1 mediated gene transcription, completely deleted the collagen stimulation of Dnmt1 transcription. The present results demonstrate that Dnmt1 mRNA expression is regulated by collagen in osteoblasts via AP-1 dependent pathways. From the data we conclude that collagen could influence gene expression via an epigenetic mechanism. This work was supported by FWF project P20646-B11. Conflict of interest: None declared.