Glucocorticoids regulate MiR-29c levels in vascular smooth muscle cells through transcriptional and epigenetic mechanisms

Abstract

Aims

The objective of this study was to determine the underlying mechanism by which glucocorticoids (GCs) induce of miR-29c expression in vascular smooth muscle cells.

Main methods

QRT-PCR was used for miR-29c detection. Protein levels were determined by western blotting. Knockdown of SP1, DNMT1 and DNMT3A was achieved through transfection with their specific respective siRNAs. The effect of GCs on SP1 activity was determined by luciferase reporter assay and the methylation status in miR-29c promoter was detected by methylation specific PCR. CHIP assay was used to determine the binding ability of SP1 and glucocorticoid receptor (GR) in miR-29c promoter.

Key findings

Treatment of RASMC with SP1 siRNA or SP1 inhibitor, mithramycin A, as well as DNMT1 and DNMT3A siRNAs and an inhibitor of DNMTs, Decitabine, resulted in increased expression of miR-29c (P < 0.05). Treatment RASMC with dexamethasone (DEX, 0.1 μM) for 24 h reduced the expression of SP1 and phosphorylated SP1 at threonine 453 protein levels, repressed SP1 activity, and inhibited the expression of DNMT1 and DNMT3A proteins (P < 0.05). Treatment with mifepristone, a GR antagonist, blocked the inhibitory effect of DEX on DNMT1 and DNMT3A protein expression. DEX also suppressed SP1 binding ability in miR-29c promoter and inhibited methylation of miR-29c promoter (P < 0.05). Treatment of RASMC with DEX (0.1 μM) significantly (P < 0.05) increased the binding of GR to the miR-29c promoter.

Significance

The stimulatory effect of GCs on miR-29c expression is mediated by these three mechanisms: transcriptionally regulated by SP1, and epigenetically through a methylation-dependent process and GR.

Introduction

Glucocorticoids (GCs) secreted by the adrenal cortex have a broad spectrum of physiological effects including anti-inflammatory, anti-proliferative and immunomodulatory activity [1]. The biological effects of GCs are mediated by binding to the intracellular glucocorticoids receptor (GR) consisting of two splice variants, GRα and GRβ. These receptors are members of the superfamily of ligand-activated nuclear hormone receptor [2]. The activated GRs translocate into nucleus and modulate downstream expression of genes either by binding to glucocorticoid response elements in target gene promoters or through protein-protein interaction with other transcription factors or coactivators [3], [4].

MicroRNAs (miRNAs), a member of short non-coding RNA, with a length of ~ 22 nucleotides have emerged as important post-transcriptional regulators of more than half of protein-coding genes in Homo sapiens [5]. MiRNAs function as evolutionarily conserved post-transcriptional gene regulators through binding to complementary sequences of their target genes to induce degradation of mRNA or inhibit mRNA translation. Several studies have emphasized the importance of miR-29 family (miR-29a, − 29b, − 29c) in the regulation of cell differentiation, proliferation and apoptosis [6], [7], [8]. Although the miR-29 family share a common seed sequence with largely overlapping sets of predicted target genes, their differential expression, subcellular distribution and regulation in different cell and tissue types, suggest their unique functional activities [9]. More specifically, miR-29c expression targets many genes that encode components of extracellular matrix (ECM) and tissue metalloproteinases that influence ECM remodeling [10], [11]. The expression of miR-29c is downregulated in various fibrotic disorders [10], [11], [12], [13], [14]. In our previous study, we demonstrated that maternal undernutrition induces excess GC secretion and decreased expression of vascular miR-29c in the offspring [15]. More recently we reported that GCs directly stimulate the expression of miR-29c in rat primary aortic smooth muscle cells (RASMC) in a dose and time dependent manner, and as a consequence the expression of target genes of miR-29c, including Collagen 3A1 (Col3A1), Collagen 4A5 (Col4A5), elastin (ELN) and matrix metalloproteinase MMP2 protein levels were reduced [10]. Furthermore, co-treatment of these cells with anti-miR-29c partially blocked the effect of GCs on these target proteins [10] suggesting an important role for miR-29c as a mediator of GC effects on vascular smooth muscle composition and function.

Epigenetic mechanisms including molecular modification of DNA (e.g. methylation) or its histone component (e.g. acetylation, phosphorylation and methylation) alters transcription of genes including those that encode for miRNAs [16], [17]. DNA methylation is regulated by three classes of DNA methyltransferases (DNMTs) and is the most common and well-characterized epigenetic process [18], [19]. Increased promoter CpG island methylation results in silencing of specific genes under epigenetic regulation. Two recent studies have provided evidence supporting the regulatory function of promoter DNA methylation as an epigenetic mechanism of miR-29c regulation [20], [21]. Although several transcription factors and enzymes, such as NF-kB, YY1, c-Myc, SP1, Smad3 and HDAC [22], [23], [24], [25], have been shown to regulate miR-29 expression in various tissues, the mechanism by which GCs regulate miR-29c expression in vascular smooth muscle cells (VSMC) remains unknown.