Steroid receptor coactivator 3 is a coactivator for myocardin, the regulator of smooth muscle transcription and differentiation

Abstract

Abnormal proliferation of vascular smooth muscle cells (VSMCs) constitutes a key event in atherosclerosis, neointimal hyperplasia, and the response to vascular injury. Estrogen receptor alpha (ERalpha) mediates the protective effects of estrogen in injured blood vessels and regulates ligand-dependent gene expression in vascular cells. However, the molecular mechanisms mediating ERalpha-dependent VSMC gene expression and VSMC proliferation after vascular injury are not well defined. Here, we report that the ER coactivator steroid receptor coactivator 3 (SRC3) is also a coactivator for the major VSMC transcription factor myocardin, which is required for VSMC differentiation to the nonproliferative, contractile state. The N terminus of SRC3, which contains a basic helix-loop-helix/Per-ARNT-Sim protein-protein interaction domain, binds the C-terminal activation domain of myocardin and enhances myocardin-mediated transcriptional activation of VSMC-specific, CArG-containing promoters, including the VSMC-specific genes SM22 and myosin heavy chain. Suppression of endogenous SRC3 expression by specific small interfering RNA attenuates myocardin transcriptional activation in cultured cells. The SRC3-myocardin interaction identifies a site of convergence for nuclear hormone receptor-mediated and VSMC-specific gene regulation and suggests a possible mechanism for the vascular protective effects of estrogen on vascular injury.

Fig. 1.

SRC3 transcript and protein are expressed in VSMCs. (A) Expression pattern of SRC3 in human cardiovascular tissues. A human cardiovascular tissue Northern blot (Clontech) was hybridized with a ³²P-SRC3-specific DNA probe encoding a 292-aa fragment (amino acids 400–692). (B) Western blot analysis of SRC3 protein expression in multiple cell lines including human VSMCs. Equal protein loading was confirmed by normalization to GAPDH (data not shown). (C) Immunostaining of SRC3 in aortic VSMC in serum-free media (Upper) or normal DMEM containing 10% FBS (Lower).

Fig. 2.

SRC3 interacts with myocardin family members in vitro. GST pull-down assays with SRF, the N-terminal domain of SRC3 that contains the bHLH and PAS domains [GST-SRC3(1–408)] or GST alone and in vitro-translated ³⁵S- labeled proteins are shown. (A) (Left) GST-SRC3(1–408) interaction with ³⁵S-myocardin. (Center) GST-SRF incubation with ³⁵S-SRC3 (negative control). (Right) GST-SRF incubation with ³⁵S-myocardin (positive control). (B) GST pull-down assays with GST-SRC3(1–408) and myocardin or the related myocardin family members MRTF-A and MRTF-B. The data are representative of at least three individual experiments.

Fig. 3.

Myocardin C-terminal TAD interacts with SRC3. (A) Domain structure of myocardin, including the TAD (amino acids 715–935). RPEL, protein motif containing RPXXXEL sequence involved in actin binding; Q, poly(Q)-rich region; SAP, 35-aa motif conserved among nuclear scaffold attachment proteins SAF-A/B, Acinus, PIAS; LZ, leucine zipper domain. (B and C) ³⁵S-labeled in vitro-translated myocardin domains including the dominant negative mutant [myocd(128–715)] (B Upper), TAD [myocd(715–935)] (B Lower), or SRC3 (C) were incubated with GST-SRC3(1–408) (B) or GST-myocd(581–935) (C) in pull-down assays. Input represents 12.5% of each in vitro-translated protein used in the pull-down assay.

Fig. 4.

Interaction of SRC3 domains with myocardin. (A) Schematic presentation of the SRC3 domain structure. The N-terminal bHLH/PAS domain, receptor interaction domain (RID), and activation domain (AD) are indicated. (B) GST pull-down assay of a series of GST-SRC3 fusion proteins interacting with ³⁵S-myocardin is shown.

Fig. 5.

Coimmunoprecipitiation of myocardin with SRC3 from cells. (A) Myocardin interacts with SRC3 from HEK293 cells transfected with mammalian flag-tagged Myocardin (flag-myocd) and HA-flag-tagged SRC3(1–450) [HA-flag-SRC3(1–450)]. Vector alone (−) was used as negative control. (B) Endogenous SRC3 interacts with myocardin in MCF7 cells. MCF7 cells were transfected with either flag-tagged myocardin (flag-myocd) or vector alone (−). (C) Coimmunoprecipitation of SRC3 with myocardin from vascular smooth muscle (PAC1) cells transfected with mammalian flag-tagged myocardin (flag-myocd) is shown.

Fig. 6.

SRC3 augments myocardin transactivation of vascular smooth muscle-specific promoters. Dose-dependent coactivation of myocardin transcriptional activation by SRC3 is shown. (A and B) Increasing amounts of mammalian expression plasmids pRSV-CBP, pCMX-SRC3(1–450), or pCMX-SRC3 (−, 0 μg; +, 100 ng; +++, 300 ng) were transfected into HEK293 cells with 1 ng of pcDNA-myocardin and either the SM22–445lucifease reporter (A) or smMHC-luciferase reporter construct (B). (C) SRC3 enhances myocardin transactivation of the SM22 reporter in vascular smooth muscle PAC1 cells. Relative fold activation in A–C was determined by comparison with the basal SM22–445 luciferase activity or smMHC-luciferase when transfected with control CMX vector. Data are expressed as the mean ± SD of triplicates from a representative experiment of three independent experiments. Asterisks represent P for comparison of data between SRC3 coactivation and vector only. *, P < 0.02. (D) SRC3 enhances Gal-myocardin TAD activation in Gal4 DBD-luciferase reporter system. Myocardin C-TAD from amino acids 715–935 was expressed as Gal4-DBD fusion protein in the pCMX-Gal vector.

Fig. 7.

Knockdown of SRC3 with siRNA reduces myocardin transactivation of VSMC-specific reporters. HEK293 (A) or PAC1 (B) cells were transfected with 75 nM of SRC3 siRNA (siSRC3) or control scrambled oligo duplex and then cotransfected with 10 ng (A) or 50 ng (B) of pcDNA-myocardin or pcDNA vector and SM22–445luciferase reporter together with 50 ng CMX-β-gal as an internal control. Results shown are mean values of three independent experiments in triplicate. In A, *, P < 0.013; in B, *, P < 0.043. SRC3 protein was assayed by immunoblotting, and expression levels of GAPDH (A) or lamin A (B) were used as protein loading controls.