The Transcription Factor TEAD1 Represses Smooth Muscle-specific Gene Expression by Abolishing Myocardin Function*♦

Background: The function of TEAD1 in the expression of smooth muscle-specific genes is unknown. Results: TEAD1 is induced after arterial injury and suppresses the expression of smooth muscle-specific genes by abolishing myocardin function. Conclusion: TEAD1 is a novel repressor for smooth muscle contractile gene expression. Significance: This study provides novel evidence that TEAD1 is critical for promoting phenotypic switching in smooth muscle cells. 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.

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 ␣-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 ␣-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.
As a major component of blood vessels, vascular smooth muscle cells (SMCs) 3 not only provide structural support to the vasculature but also play a critical role in the maintenance of vascular homeostasis. In response to arterial injury and other environmental stimuli, vascular SMCs are able to modulate their phenotype from a contractile to a synthetic state that is associated with down-regulation of smooth muscle contractile protein expression (1). The mechanisms underlying down-regulation of contractile proteins during the phenotypic modulation of smooth muscle are incompletely understood.
Serum response factor (SRF), a member of the MADS (MCM1/Agamous/Deficiens/SRF) box transcription factor family, plays a central role in smooth muscle phenotypic modulation (2). SRF regulates expression of nearly all smooth muscle-specific genes by binding to highly conserved CArG (CC(A/T) 6 GG) elements within these genes. SRF is widely expressed and is generally a weak transcriptional activator; thus, it requires physical interaction with other tissue-specific coactivators to regulate gene expression. Among these SRF coactivators, myocardin, which is expressed only in cardiac and smooth muscle cells is a very powerful and specific activator of CArG-dependent cardiac-and smooth muscle-specific genes. Myocardin binds to the SRF MADS domain through its basic and polyQ domain (3,4). Although a number of negative and positive regulators of myocardin activity have been identified (5)(6)(7)(8), the mechanisms modulating myocardin-SRF function in SMCs are incompletely understood.
The TEAD (transcriptional enhancer activator domain) family of proteins consist of four members, TEAD1 (TEF-1, NTEF-1), TEAD2 (ETF, TEF-4), TEAD3 (DTEF-1, TEF-5, ETFR-1), and TEAD4 (TEF-3, RTEF-1) (9). In mammals, TEAD genes are expressed widely in embryonic and adult tissues and display distinct but overlapping expression patterns (10,11). The TEAD proteins share an evolutionarily conserved 72-amino acid DNA-binding TEA domain, which forms a three-helix bundle with a homeodomain fold to bind to a consensus DNA sequence (5Ј-CATTCC-3Ј) named the MCAT (muscle CAT) element (12). The MCAT element has been identified in a number of cardiac-specific (13,14), smooth muscle-specific (15), and skeletal muscle-specific (16) genes. Ablation of the Tead1 gene in mice results in embryonic lethality between embryonic days 11 and 12 from cardiac defects (17). Previous studies have shown that MCAT elements within the smooth muscle (SM) ␣-actin promoter, which bind TEAD proteins, are required for the initial activation of SM ␣-actin gene transcription in embry-onic SMCs and myofibroblasts but are not required for the promoter activity in adult differentiated SMCs (15,18). However, it remains undetermined whether the function of TEAD proteins in vascular SMCs is unique to the SM ␣-actin gene or whether TEAD proteins also regulate expression of other smooth muscle-specific genes. In this study, we found that expression of TEAD1 is significantly induced during SMC phenotypic modulation and that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including SM ␣-actin, via disrupting myocardin binding to SRF.

EXPERIMENTAL PROCEDURES
Rat Carotid Balloon Angioplasty Injury Model-Rat balloon angioplasty was carried out as described previously (19). The use of experimental animals for arterial injury procedures was approved by the Institutional Animal Care and Use Committee of Georgia Regents University.
Quantitative Real-time RT-PCR (qRT-PCR) Analysis-Total RNA from tissue or SMCs was isolated with TRIzol reagent, and qRT-PCR was performed with the respective gene-specific primers as reported previously (6,7,19,20,22). All samples were amplified in duplicate, and every experiment was repeated independently two times. Relative gene expression was converted using the 2 Ϫ⌬⌬Ct method against the internal control housekeeping gene Rplp0 (ribosomal phosphoprotein, large, P0). The sequences of the primer set for rat TEAD1 are 5Ј-TTTGTG-CAGCAGGCCTACCCCATC-3Ј and 5Ј-GGCGAAGCTTGGT-TGTGCCAATGGA-3Ј.
Gene Silencing-TEAD1/3/4 shRNA was designed for a region identical in rat TEAD1/3/4 and cloned into the pLKO.1 lentiviral vector as described previously (23). Scrambled control and silencing siRNA duplexes targeting Tead1 were designed and purchased from Dharmacon. The sequence of the siRNA targeting mouse and rat Tead1 is 5Ј-AGACGGAGTAT-GCGAGGTT-3Ј. Rat SMCs were transfected with siRNA duplexes using the Neon transfection system (Invitrogen) as described in our recent report (19).
Luciferase Reporter Assays-Transient transfection and reporter assays in PAC-1 SMCs or 10T1/2 fibroblasts were carried out with FUGENE 6 transfection reagent (Roche Applied Science) as described previously (19,21,24). The level of promoter activity was evaluated by measurement of firefly luciferase activity relative to the internal control thymidine kinase-Renilla luciferase activity using the Dual-Luciferase assay system (Promega) as described by the manufacturer. A minimum of six independent transfections were performed, and all assays were replicated at least twice. Results are reported as the mean Ϯ S.E.
Adenoviral Construction and Cell Infection-The TEAD1 expression plasmid was kindly provided by Dr. Kun-Liang Guan (University of California, San Diego) (23), and TEAD1 cDNA was subcloned into the AdTrack adenoviral vector with a Myc tag. Adenoviral packaging and transduction into cultured rat aortic SMCs were performed as described previously (6,7,22). Myocardin adenovirus was described in our previous report (22). Empty adenovirus served as a negative control.
Co-immunoprecipitation-HEK293 cells were cotransfected with expression plasmids encoding TEAD1, myocardin, and SRF. 36 h after transduction, nuclear protein was harvested, and co-immunoprecipitation assays were performed using a nuclear complex co-immunoprecipitation kit (Active Motif) as described in our previous reports (6,25). The co-immunoprecipitated protein was detected by Western blotting using the antibodies indicated in the figures.
GST Pulldown Assays-TEAD1 cDNA was cloned into the pET28 vector (Novagen), and GST pulldown assays were performed as described in our previous reports (24,25).
Quantitative ChIP Assays-Rat primary aortic SMCs were transfected with control or TEAD1 silencing RNA duplex by Neon electroporation for 36 h. ChIP was performed essentially following the protocol of Active Motif. After immunoprecipitation with anti-SRF antibody (G-20), the recovered DNA was subjected to qPCR using primers encompassing the CArG box element within SMC markers as described previously (6,24).
Statistical Analysis-Data are expressed as means Ϯ S.E., and statistical analysis using one-way analysis of variance was done with GraphPad Prism software. Differences with p values of Ͻ0.05 were considered significant.

TEAD1 Expression Is Significantly Induced following Arterial
Injury-To explore a potential role of Tead family genes in vascular SMCs, we first examined TEAD1 expression during smooth muscle phenotypic modulation. The rat carotid artery balloon injury model that resembles the angioplasty procedure in humans (26) is a well accepted procedure to induce smooth muscle phenotypic modulation from a contractile phenotype to a proliferative phenotype after endothelial denudation. In this arterial injury model, we found that TEAD1 protein was significantly elevated by 1.5-2-fold at 7 and 14 days following surgery (Fig. 1, A and B). Furthermore, the increased TEAD1 expression coincided with the reduced expression of smooth muscle contractile protein genes, including SM MHC, myosin light chain kinase, Hic-5, SM ␣-actin, calponin, and SM22␣, whereas it positively correlated with increased expression of the proliferative marker proliferating cell nuclear antigen (Fig. 1, A and  B). Immunohistochemical staining demonstrated that TEAD1 was highly induced in the nuclei of synthetic neointima SMCs where the expression of SM MHC was reduced (Fig. 1C). Furthermore, the Ki-67-positive cells in the neointima were mainly positive for TEAD1 staining (Fig. 1D). Taken together, these data demonstrate that TEAD1 is induced after arterial injury and positively correlates with the "proliferative" smooth muscle phenotype.
TEAD1 Is a Potent Repressor of Smooth Muscle-specific Gene Expression-As TEAD1 expression was significantly induced during smooth muscle phenotypic modulation and negatively correlated with the expression of smooth muscle-specific genes, we next sought to determine whether the induction of TEAD1 is sufficient to attenuate smooth muscle-specific gene expression. A previous study reported that a lentiviral construct with shRNAs designed in a region identical in Tead1/3/4 can simultaneously knock down expression of all three genes in cells (23). To test the function of TEAD family proteins in vascular SMCs, control or shRNA lentivirus against TEAD1/3/4 was transduced into rat primary aortic SMCs, and Western blotting was performed to examine smooth muscle-specific gene expression. The data from this experiment revealed that simultaneous knockdown of Tead1/3/4 markedly induced all of the smooth muscle-specific gene expression examined ( Fig.  2A). We next examined the expression of Tead family genes in SMCs by qRT-PCR. We found that Tead1 had the highest expression level, followed by Tead3 and Tead4, whereas Tead2 was almost undetectable in rat primary aortic SMCs (Fig. 2B). Given the dominant expression of TEAD1 relative to other Tead family genes in vascular SMCs, we next sought to specifically test TEAD1 function by specific depletion of TEAD1 in SMCs. Compared with scrambled silencing control samples, specific knockdown of Tead1 alone was sufficient to up-regulate smooth muscle-specific proteins to comparable levels as seen following knockdown of Tead1/3/4 together, suggesting that TEAD1 plays a dominant role in repressing the expression of smooth muscle-specific proteins (Fig. 2C). Furthermore, knockdown of Tead1 slightly, albeit significantly, induced myocardin mRNA expression in addition to increasing expression of smooth muscle-specific gene mRNA (Fig. 2D). To test whether overexpression of TEAD1 is sufficient to suppress smooth muscle-specific gene expression, adenovirus encoding GFP or TEAD1 was transduced into rat primary aortic SMCs, and the expression of smooth muscle-specific genes was assessed by Western blotting . The data from this experiment showed that overexpressed TEAD1 markedly down-regulated smooth muscle-specific protein expression (Fig. 2E). Furthermore, Dual-Luciferase reporter assays revealed that overexpressed TEAD1 significantly abrogated the promoter activities of smooth muscle-specific genes such as SM ␣-actin, SM22␣, and Hic-5 (Fig. 2F). These data suggest that the inhibitory effects of TEAD1 on smooth muscle markers are at the transcriptional level. Taken together, these data from loss/gain-offunction assays suggest that TEAD1 is a potent repressor of the transcription of smooth muscle-specific genes.
TEAD1 Represses the Myocardin-induced Activation of Smooth Muscle-specific Genes-Previous studies suggested that TEAD1 regulates SM ␣-actin gene expression by binding to the MCAT element within the SM ␣-actin gene promoter during development of embryonic SMCs and myofibroblasts but that the MCAT elements are not required for SM ␣-actin gene expression in differentiated SMCs (18). The data described above demonstrated that silencing TEAD1 in differentiated rat primary aortic SMCs up-regulated the expression of not only SM ␣-actin but also other CArG-dependent smooth musclespecific genes that do not contain any obvious MCAT elements (Fig. 2). These data suggest that TEAD1 inhibits smooth muscle gene expression through a common mechanism shared by all CArG-dependent smooth muscle-specific genes, rather than via TEAD binding to MCAT elements. As myocardin and its binding partner SRF have been shown to be "master" regulators in activating all CArG-dependent smooth muscle-specific genes, we hypothesized that TEAD1 may suppress myocardin function, thereby abrogating smooth muscle-specific gene expression. To test this hypothesis, myocardin-induced activation of smooth muscle-specific gene promoters was assessed in the absence or presence of TEAD1 by Dual-Luciferase reporter assays. The data from this experiment revealed that TEAD1 significantly abrogated the ability of myocardin to activate the promoters of smooth muscle-specific genes (Fig. 3A). Furthermore, in the presence of TEAD1, the transactivation of the SM22␣ promoter induced by myocardin-related transcription factor A was significantly reduced (Fig. 3B). To further evaluate the effects of TEAD1 on myocardin-induced endogenous smooth muscle gene expression, adenovirus encoding myocardin was transduced into COS-7 cells with or without co-infection with adenovirus encoding TEAD1, and the expression of endogenous smooth muscle-specific proteins was assessed by Western blotting. The data from this experiment demonstrated that TEAD1 significantly abolished myocardin-induced expression of smooth muscle-specific proteins, including SM MHC, myosin light chain kinase, Hic-5, SM ␣-actin, calponin, and SM22␣ (Fig. 3C). Taken together, these data demonstrate that TEAD1 is a potent repressor of myocardin function, leading to suppression of smooth muscle-specific gene expression.
TEAD1 Disrupts Myocardin-SRF Binding in Vitro and in Vivo-As TEAD1 is a potent repressor of myocardin function, we next sought to explore the underlying mechanism by which TEAD1 abolishes myocardin function. First, we tested whether TEAD1 can form a complex with myocardin and SRF in vivo by co-immunoprecipitation assay. Expression plasmids encoding TEAD1 or myocardin were cotransfected into HEK293 cells. Subsequently, nuclear protein was harvested to perform coimmunoprecipitation assay using anti-TEAD1 antibody, and Western blotting was carried out to determine the immunoprecipitated components. The data from these experiments revealed that TEAD1 formed a complex together with myocardin and SRF in vivo (Fig. 4A). To further test whether TEAD1 directly binds to myocardin, a series of GST proteins fused with truncation mutants of myocardin were generated, and GST pulldown assays were performed after incubation with bacterially expressed TEAD1. The data from these experiments demonstrated that TEAD1 bound to the N-terminal region of myocardin, especially within the basic and polyQ domain (amino acids 221-350), where SRF also binds (Fig. 4, B and C). We also confirmed that TEAD1 can directly interact with SRF as reported previously (27). As both TEAD1 and SRF bind to an overlapping region of myocardin at the basic and polyQ region, we next examined whether TEAD1 competes with SRF for binding to myocardin, thereby suppressing myocardin function. In vitro competitive GST pulldown assays revealed that myocardin binding to SRF was decreased in the presence of TEAD1 (Fig. 4D, compare myocardin signal in lanes 3 and 4). Conversely, TEAD1 binding to SRF was diminished by increased input of myocardin (Fig. 4D, compare TEAD1 signal in lane 6 with lanes 4 and 5). Moreover, data from co-immunoprecipitation assays further showed that myocardin binding to SRF was significantly decreased in the presence of TEAD1 in cells (Fig. 4E, compare myocardin signal in lanes 1 and 3).
Together, these data suggest a novel mechanism by which TEAD1 represses expression of smooth muscle-specific genes through disrupting myocardin-SRF complex formation.
The CArG Element Is Critical in Mediating the Inhibitory Effects of TEAD1 on Smooth Muscle-specific Genes-As myocardin-SRF binding to CArG elements within smooth musclespecific genes is critical for myocardin-mediated smooth muscle gene activation, we next sought to examine the role of CArG boxes in TEAD1-induced smooth muscle gene down-regulation. Luciferase reporter assays using the smooth muscle-specific Hic-5 promoter revealed that TEAD1 significantly attenuated the activity of this promoter by 80%, whereas a CArG box mutant promoter was completely refractory to TEAD1 inhibition (Fig. 5A). Quantitative ChIP assays revealed that silencing endogenous TEAD1 in rat primary aortic SMCs resulted in a significant increase in SRF binding to CArG box regions of smooth muscle-specific genes (Fig. 5B).

DISCUSSION
In this study, we discovered a novel role of TEAD1 in vascular SMCs whereby TEAD1 competes with SRF for binding to myocardin and thereby represses smooth muscle-specific gene expression. TEAD1 have been reported to function as either an activator or a repressor of muscle gene expression. For instance, TEAD1 binding to the MCAT elements in the ␣-tropomyosin 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 Ϫ⌬Ct (⌬C t ϭ C t(TEAD) Ϫ C t(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 Ͻ 0.05. gene is required for maximal promoter activity in all three muscle types. Furthermore, overexpression of TEAD1 mRNA in Xenopus embryonic cells leads to activation of both the endogenous ␣-tropomyosin gene and the exogenous ␣-tropomyosin promoter, suggesting that TEAD1 is an activator for the ␣-tropomyosin gene (28). However, TEAD1 has been reported to act as a mild activator at low concentration and as a repressor at higher concentrations on the cardiac ␣-MHC gene (29). Of the smooth muscle-specific genes examined thus far, only the SM ␣-actin gene has been identified to contain two MCAT elements that bind TEAD proteins (15,18,30). However, these elements are not required for SM ␣-actin-specific expression in SMCs, and it is surprising that silencing Tead1/3/4 in SMCs has been reported to increase endogenous SM ␣-actin mRNA expression, in agreement with our findings (18). Our data presented in this study demonstrated that silencing Tead1 in rat primary aortic SMCs up-regulated expression of not only SM ␣-actin but also other smooth muscle-specific genes that do not contain any obvious MCAT elements (Fig. 2). These data suggest that TEAD1 can inhibit smooth muscle gene expression dependent on or independent of TEAD binding to MCAT elements. The functions of TEAD family proteins in regulating gene expression appear complex and to be cell-context dependent, operating through both MCAT-independent and MCATdependent mechanisms.
Mechanistically, we found TEAD1 competes with myocardin for binding to SRF, resulting in disruption of myocardin-SRF complexes and thereby attenuating the expression of smooth muscle-specific genes (Figs. 4 and 5). Previous studies demonstrated that the DNA-binding domain of TEAD1 directly interacts with the MADS domain of SRF in vivo and in vitro (12,27). We confirmed this physical interaction by GST pulldown assays (Fig. 4B). In this study, we further found that TEAD1 directly interacts with the basic and polyQ domain of myocardin, which is also the binding site of SRF (Fig. 4B). Therefore, it is possible that the inhibitory effects of TEAD1 on SRF-myocardin binding occur through its binding to both the SRF MADS domain and the myocardin basic and polyQ domain. As myocardin binding to SRF has been shown to increase the binding of SRF to the promoters of smooth muscle-specific genes (31), disrupting myocardin-SRF interactions thus decreases SRF binding to CArG elements, thereby resulting in down-regulation of smooth muscle-specific gene expression.
A previous study identified a TEAD-binding element in the myocardin promoter, and TEAD2 can significantly activate a myocardin promoter-reporter gene (32). However, consistent with a previous report (18), we found that TEAD1 is the most abundant TEAD family member in cultured SMCs, with TEAD2 being undetectable ( Fig. 2A). The lack of expression of TEAD2 in cultured SMCs is unlikely due to the loss of its expression during cell culture, as we found no detectable TEAD2 expression in rat aortic tissues either (data not shown). Our results are consistent with previous reports demonstrating that TEAD2 was expressed only in a subset of mouse embryonic tissues but not in adult tissues, including smooth muscles (11,18). In contrast to the myocardin promoter, in which TEAD2 is a transcriptional activator as demonstrated by reporter assays and that a MCAT element mediates myocardin expression specifically in branchial arch arteries and aortas in mice (32), we found that TEAD1 is a transcriptional repressor that inhibits the promoters of smooth muscle-specific genes. Furthermore, silencing Tead1 in rat vascular SMCs resulted in moderate induction of myocardin expression at the mRNA level, suggesting that TEAD1 is also a weak repressor of myocardin expression in these cells (Fig. 2D). Given the small degree of induction of myocardin after silencing Tead1 in SMCs (Fig. 2D), it is unlikely that the up-regulation of smooth muscle-specific genes by depletion of Tead1 is through increasing myocardin expression, rather than increasing its effective activity. On the basis of the notion that TEAD1 can function as either an activator or a repressor of other muscle-specific genes as shown previously, we propose that TEAD factors can activate myocardin during smooth muscle development but can function as a repressor of myocardin function to abrogate smooth muscle-specific gene expression during arterial injury. . 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 Ͻ 0.05. B, myocardin-related transcription factor A expression plasmid was transfected with the SM22␣ 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 Ͻ 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.
Previous studies have shown that TEAD1 mediates YAP (Yes-associated protein)-dependent changes in gene expression (23). YAP is a major effector in the Hippo signaling pathway (33). We have recently reported that expression of YAP is significantly induced in arterial injury models and that knock-down of YAP expression in vascular SMCs in vitro significantly up-regulates endogenous smooth muscle-specific gene expression and inhibits SMC proliferation (19). We also found that smooth muscle-specific genes are significantly down-regulated by wild-type YAP but not YAP S94A, a mutant that is deficient 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; ϩϩ, 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.
in its ability to interact with TEAD1 (data not shown). In the present study, we found that similar to YAP, TEAD1 is significantly induced following arterial injury (Fig. 1) and that overexpression of TEAD1 alone in rat aortic primary SMCs is sufficient to repress, whereas knockdown of Tead1 markedly up-regulates smooth muscle-specific gene expression (Fig. 2), mimicking the inhibitory effects of YAP on smooth muscle gene expression in SMCs. These data collectively suggest that there is a coordinated activation of YAP and up-regulation of its cofactor TEAD1 in response to the stimuli that promote smooth muscle phenotypic modulation. Further studies are needed to confirm the functional role of TEAD1 in smooth muscle-specific gene expression and proliferation in vivo and to verify its role as a cofactor in the Hippo-YAP pathway-mediated phenotypic modulation of SMCs. Indeed, previous reports have shown that TEAD1 mediates YAP function to promote cell proliferation by directly activating some cell cycle genes, including cyclin D 1 (23,34). In summary, this study has provided the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with the binding of SRF and myocardin.