SOX9 and myocardin counteract each other in regulating vascular smooth muscle cell differentiation

https://doi.org/10.1016/j.bbrc.2012.04.149Get rights and content

Abstract

Transdifferentiation of vascular smooth muscle cells (VSMC) into chondrogenic cells contributes significantly to vascular calcification during the pathogenesis of atherosclerosis. However, the transcriptional mechanisms that control such phenotypic switch remain unclear. This process is characterized by the induction of Sox9 and Col2a1 genes accompanied by the repression of myocardin (Myocd) and SMC differentiation markers such as SM22, SM α-actin and SM-MHC. Here we explore the regulatory role of SOX9, the master regulator for chondrogenesis, in modulating SMC marker gene expression. qRT-PCR and luciferase assays show that over-expression of SOX9 inhibits SMC gene transcription and promoter activities induced by myocardin, the master regulator of smooth muscle differentiation. Such suppression is independent of the CArG box in the SMC promoters but dependent on myocardin. EMSA assay further shows that SOX9 neither participates in SRF (serum response factor) binding to the CArG box nor interacts with SRF, while co-immunoprecipitation demonstrates an association of SOX9 with myocardin. Conversely, myocardin suppresses SOX9-mediated chondrogenic gene Col2a1 expression. These findings provide the first mechanistic insights into the important regulatory role of SOX9 and myocardin in controlling the transcription program during SMC transdifferentiation into chondrocytes.

Highlights

SOX9 inhibits myocardin-induced smooth muscle gene transcription. ► SOX9 does not interfere with SRF binding to the CArG box. ► SOX9 does not interact with SRF but associates with myocardin. ► Myocardin suppresses Sox9-induced Col2a1 promoter activities. ► There is an antagonism between Sox9 and Myocd in controlling gene transcription.

Introduction

Smooth muscle cells (SMCs) are highly plastic and have the capacity to convert from the differentiated contractile phenotype to a variety of synthetic dedifferentiated states exhibiting enhanced proliferation, migration, inflammation, chondrogenesis and osteogenesis during the pathogenesis of vascular diseases such as atherosclerosis [1], [2]. Smooth muscle cell (SMC) differentiation is marked by the expression of a set of contractile proteins including smooth muscle α-actin (Acta2), smooth muscle myosin heavy chain (Myh11), and SM22 (Tagln or SM22α). In response to vascular injury, SMCs undergo phenotypic modulation: this is characterized by the downregulation of SMC contractile genes.

Osteo/chondrocytic conversion of SMCs in calcified atherosclerotic plaques is accompanied by the induction of bone and cartilage differentiation regulators, including Cbfa1 (RUNX2), SOX9 and MSX2 [3], [4]. Previous studies have characterized the role of RUNX2 and MSX2 in promoting osteogenic conversion of VSMCs by repressing SRF/myocardin-mediated SMC differentiation [5], [6], [7], [8]. However, the regulatory roles of SOX9 in SMC phenotypic switching remain not understood.

Serum response factor (SRF), a widely expressed MADS box containing transcription factor, binds to the CArG [CC(A/T)6GG] box in the promoters of SMC genes as well as in the promoters of early growth genes [9]. SRF cooperates with a variety of transcriptional coactivators and corepressors to regulate gene expression in response to different signals [10], [11], [12], [13], [14]. Among them, myocardin is the most potent coactivator of SRF in transactivating cardiac and SMC specific gene transcription [9], [11]. Myocardin is specifically expressed in smooth and cardiac muscle lineages throughout embryonic development and adulthood; myocardin interacts with SRF and/or other transcriptional factors to induce SMC gene transcription in CArG box-dependent and independent manners [11], [15].

It has been demonstrated that SMCs give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries; this process is accompanied by increased expression of SOX9 [3], [7]. However, the functional role of SOX9 in SMC phenotypic modulation during artery calcification has not been determined. We recently showed that carotid injury induces prominent medial chondrogenic differentiation; this is accompanied by the upregulation of Sox9 and Col2a1 transcription and the downregulation of myocardin and SMC marker gene transcription in medial SMCs in SM22−/− mice [16]. Since Sox9 and myocardin are master transcriptional regulators for chondrogenesis and myogenesis respectively, the interplay of SOX9 and myocardin is likely to have a critical role in directing the transcription program switch from myogenesis to chondrogenesis. The goal of the present study is to determine the regulatory role of SOX9 on myocardin-mediated SMC gene transcription, and to explore underlying molecular mechanisms.

Section snippets

Plasmids

The mammalian expression vector plasmids pcDNA3.1-Myc-myocardin, pcDNA3-Flag-SOX9, pCGN-HA-SRF and pCGN-VP16-SRF were described previously [11], [17], [18]. The luciferase reporter plasmids controlled by the promoters of Sm22, Myh11, Aclp, 4× Sm22CArGnear, and 4× FosCArG were described previously [19], [20], [21]. The luciferase reporter controlled by the Col2a1 promoter (pCol2a1-luc) and the 4× SOX9 binding sites promoter (4× SOX9site-luc) were described previously [22], [23].

Cell culture, transfection and luciferase assays

C3H10T1/2 or

SOX9 suppresses SMC marker gene transcription

SOX9, a key transcriptional regulator for chondrocyte differentiation, is highly expressed during SMC osteochondrogenic conversion [3], [7], [25], [26]. We recently showed that carotid injury-induced medial chondrogenic differentiation in SM22−/− mice is accompanied by the upregulation of Sox9 and Col2a1 transcription and the concomitant downregulation of myocardin and SMC marker gene transcription [16]. It is therefore reasonable to propose that SOX9 inhibits SMC differentiation while

Acknowledgments

This work was supported by a grant from the National Heart, Lung, and Blood Institute (HL087014 to L.Li). We are grateful to the Donghong Ju, Hong Jiang for technical assistance and Wei Zhang for graphics. We appreciate the generous support from Dr. Benoit de Crombrugghe Véronique Lefebvre and Mary Goldring and valuable discussion with Drs. Da-zhi Wang, Maozhou Yang, Hui J. Li, Xiaohui Jennifer Liu and Jianpu Zheng.

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