Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 7;289(6):3308-16.
doi: 10.1074/jbc.M113.515817. Epub 2013 Dec 16.

The transcription factor TEAD1 represses smooth muscle-specific gene expression by abolishing myocardin function

Fang Liu  1 , Xiaobo Wang, Guoqing Hu, Yong Wang, Jiliang Zhou
Affiliations

The transcription factor TEAD1 represses smooth muscle-specific gene expression by abolishing myocardin function

Fang Liu et al. J Biol Chem. .

Abstract

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarily conserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF.

Keywords: Smooth Muscle; Smooth Muscle Phenotypic Modulation; TEAD1; Tissue-specific Transcription Factors; Transcription Factors; Transcriptional regulation; Vascular Biology; Vascular Smooth Muscle Cells.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
TEAD1 expression is up-regulated in injured arteries. A, 7 or 14 days after rat carotid artery balloon injury, contralateral control (−) or injured (+) carotid arteries were harvested for Western blotting to examine protein expression as indicated. At each time point, three rats were subjected to balloon injury as shown. MLCK, myosin light chain kinase; PCNA, proliferating cell nuclear antigen. B, quantification of immunoblot signals of protein expression shown in A. The relative protein expression in uninjured control vessels was set to 1 (n = 3). *, p < 0.05. 14 days post-balloon injury, injured and control rat carotid arteries were sectioned and stained for TEAD1 (C and D, green), SM MHC (C, red), or Ki-67 (D, red) as indicated. Arrowheads point to several representative Ki-67-positive cells. Sections treated with secondary antibody alone served as a negative control. Nuclei were visualized by counterstaining with DAPI (blue). Scale bars = 20 μm. M, media; NI, neointima.
FIGURE 2.
FIGURE 2.
TEAD1 attenuates expression of smooth muscle-specific genes by inhibiting their promoter activity. A, lentivirus expressing shRNA against luciferase (sh-control) or Tead1/3/4 (sh-TEAD1/3/4) was transduced into rat primary aortic SMCs for 48 h. Protein lysates were then harvested for Western blotting using the antibodies indicated. Vinculin served as a loading control. MLCK, myosin light chain kinase. B, cultured rat aortic SMCs were harvested for qRT-PCR to examine Tead family gene expression at the mRNA level using Tead gene-specific primers as indicated. Transcript levels were normalized to the RPLP0 internal loading control and expressed as 2−ΔCt (ΔCt = Ct(TEAD) − Ct(RPLP0)). Silencing Tead1 or scrambled control RNA duplex was transfected into rat aortic SMCs for 48 h and then harvested for Western blotting (C) or qRT-PCR (D). E, rat primary aortic SMCs were transduced with adenovirus encoding TEAD1 and then harvested for Western blotting to evaluate gene expression as indicated. The cells infected with GFP adenovirus served as a control. F, empty vector or TEAD1 expression plasmid was cotransfected with luciferase reporters containing smooth muscle-specific gene promoters as indicated in PAC-1 SMCs, and Dual-Luciferase assay was performed. Reporter activity was normalized to a Renilla luciferase internal control and expressed relative to empty vector transfections (normalized to 1). *, p < 0.05.
FIGURE 3.
FIGURE 3.
TEAD1 abrogates myocardin function. A, luciferase reporter plasmids harboring smooth muscle-specific gene promoters and myocardin expression plasmid were cotransfected into 10T1/2 cells with or without TEAD1 expression plasmid. 36 h post-transfection, Dual-Luciferase assay was performed to measure the promoter activity. Values are presented as relative luciferase activity compared with the basal level of promoter activity (set to 1). *, p < 0.05. B, myocardin-related transcription factor A expression plasmid was transfected with the SM22α promoter-reporter gene into 10T1/2 cells in the absence or presence of TEAD1 expression plasmid, and reporter activity was measured as described for A. *, p < 0.05. C, adenovirus encoding myocardin was transduced into COS-7 cells with or without Myc-tagged TEAD1 adenovirus for 48 h, and cell lysates were harvested for Western blotting to examine the expression of endogenous smooth muscle-specific genes as indicated. The arrowhead points to a non-SM-specific calponin signal. MLCK, myosin light chain kinase.
FIGURE 4.
FIGURE 4.
TEAD1 disrupts myocardin binding to SRF in vitro and in vivo. A, expression plasmids encoding TEAD1 or myocardin were cotransfected into HEK293 cells. Subsequently, nuclear protein was harvested to perform co-immunoprecipitation assay using anti-TEAD1 antibody or control IgG. The immunoprecipitated (IP) protein was detected by Western blotting using the antibodies indicated. B, GST alone or fused to SRF or fragments of myocardin as depicted in C was conjugated to GSH beads and incubated with bacterially expressed TEAD1. After extensive washing, bound protein was examined by Western blotting with anti-TEAD1 antibody (upper panel). The expression of GST or GST-fused proteins by Ponceau staining is shown in the lower panel (marked by asterisks at the top left of each protein). The interaction between TEAD1 and SRF served as a positive control. C, schematic diagram of mouse myocardin domains demonstrating the GST fusion proteins analyzed in B and a summary of myocardin domains mapped to interact with TEAD1. NTD, N-terminal domain; ++, basic domain; Q, polyQ domain; SAP, SAF-A/B, Acinus, and PIAS domains; LZ, leucine zipper domain; TAD, transcriptional activation domain; mut, mutant. D, GST pulldown assay was performed using different combinations of bacterially expressed myocardin (amino acids 1–585) or full-length TEAD1 for incubation with GSH beads conjugated GST-SRF or GST. Subsequently, Western blotting was carried out using anti-myocardin and anti-TEAD1 antibodies as indicated. The expression of GST or SRF-GST bait protein is shown by Ponceau staining (lower panel). E, expression plasmids encoding SRF or myocardin were cotransfected into HEK293 cells with or without TEAD1 expression plasmid for 48 h. Subsequently, nuclear protein was harvested for co-immunoprecipitation assay using anti-SRF antibody or control IgG. The immunoprecipitated protein was analyzed by Western blotting as indicated.
FIGURE 5.
FIGURE 5.
SRF binding to CArG elements within smooth muscle gene promoters is required to mediate TEAD1 function. A, empty vector or TEAD1 expression plasmid was cotransfected with either a wild-type Hic-5 promoter-reporter gene or its CArG mutation reporter (mut) into PAC-1 SMCs, and promoter activity was measured by Dual-Luciferase assay. The basal activity of the wild-type or mutant Hic-5 promoter was set to 1. *, p < 0.05. B, cross-linked chromatin from rat primary aortic SMCs was immunoprecipitated with anti-SRF antibody following transfection with scrambled control or Tead1 siRNA duplexes. Subsequently, the precipitated DNA was amplified by real-time PCR with smooth muscle gene-specific primers spanning the CArG region. The relative SRF binding in control siRNA-transfected cells was set to 1. *, p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Owens G. K., Kumar M. S., Wamhoff B. R. (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84, 767–801 - PubMed
    1. Miano J. M. (2003) Serum response factor: toggling between disparate programs of gene expression. J. Mol. Cell. Cardiol. 35, 577–593 - PubMed
    1. Wang D., Chang P. S., Wang Z., Sutherland L., Richardson J. A., Small E., Krieg P. A., Olson E. N. (2001) Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 105, 851–862 - PubMed
    1. Pipes G. C., Creemers E. E., Olson E. N. (2006) The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev. 20, 1545–1556 - PubMed
    1. Zhou J., Hu G., Herring B. P. (2005) Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1. Mol. Cell Biol. 25, 9874–9885 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources