Abstract
DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-β1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-β1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-β1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-β1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-β1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-β1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-β1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3β, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3β was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-β1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-β1 is known to be crucial, the ability of TGF-β1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-β1 may regulate DNA methylation in these cells.
Highlights
DNA methylation is a key epigenetic process involved in controlling gene expression and cellular phenotype
Treatment with TGF-1 Increased Expression of DNMT1 and DNMT3a in Normal Lung Fibroblasts—To determine whether TGF-1 affects the expression of various DNA methyltransferases (DNMTs) isoforms, adult normal lung fibroblasts (CCL210 cells) were treated with TGF-1 (2 ng/ml) for varying time periods, and expression of DNMT1 and DNMT3a were assayed by immunoblot
The increase in DNMT1 and DNMT3a expression by TGF-1 was supported by immunofluorescence microscopy (Fig. 1, C and D), which demonstrate that TGF-1 treatment increased localization of both DNMT1 and DNMT3a to the cell nucleus
Summary
DNA methylation is a key epigenetic process involved in controlling gene expression and cellular phenotype. Alterations in specific DNMT isoform expression and activity have been identified in cancer [5,6,7], aging [8], autoimmune disease [9, 10], and fibrotic disorders [11,12,13] and have been associated with changes in the global DNA methylation patterns of cells. We previously demonstrated that prostaglandin E2, a lipid mediator that inhibits fibroblast activity and myofibroblast differentiation, induces global DNA methylation changes in fibroblasts that are due, in part, to increased expression of DNMT3a [28].
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