Transforming Growth Factor-�1-induced Transcript 1Protein, a Novel Marker for Smooth Muscle ContractilePhenotype, Is Regulated by Serum ResponseFactor/Myocardin Protein*□S

Received for publication, April 13, 2011, and in revised form, August 31, 2011 Published, JBC Papers in Press, October 8, 2011, DOI 10.1074/jbc.M111.250878

Xiaobo Wang‡, Guoqing Hu‡, Courtney Betts§, Erin Yund Harmon‡, Rebecca S. Keller‡, Livingston Van De Water§,and Jiliang Zhou‡1

From the ‡Center for Cardiovascular Sciences and the §Center for Cell Biology and Cancer Research, Albany Medical College,Albany, New York 12208

Background: TGFB1I1 is involved in vascular injury, but the regulation of TGFB1I1 expression is unknown.Results: TGFB1I1 is specifically expressed in smooth muscle cells and regulated by SRF/myocardin.Conclusion: TGFB1I1 is a novel marker for the smooth muscle contractile phenotype.Significance: This study provides the first evidence that TGFB1I1 is an SRF/myocardin-regulated smooth muscle marker andcritical for maintaining the smooth muscle contractile phenotype.

Serum response factor (SRF) plays a central role in regulatingexpression of smooth muscle-specific genes partly by associat-ing with the potent tissue-specific cofactormyocardin. Previousstudies have shown that transforming growth factor-�1-in-duced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-�-responsive gene and is involved in the cellular response to vas-cular injury, but the regulation of TGFB1I1 expression remainselusive. In this report, we demonstrated that TGFB1I1 is a novelmarker for the smoothmuscle contractile phenotype and is reg-ulated by SRF/myocardin. We found that TGFB1I1 is specifi-cally expressed in smooth muscle cells (SMCs) and in smoothmuscle-rich tissues. Furthermore, TGFB1I1 expression is sig-nificantly down-regulated in a variety of models for smoothmuscle phenotypic modulation. The TGFB1I1 promoter con-tains an evolutionarily conserved CArG element, and this ele-ment is indispensible for myocardin-induced transactivationof TGFB1I1 promoter. By oligonucleotide pulldown andchromatin immunoprecipitation assays, we found that SRFbinds to this CArG element in vitro and in vivo. Ectopicexpression of myocardin is sufficient to induce endogenousTGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1expression in SMCs. Furthermore, our data demonstratedthat SRF is essential for TGF-�-mediated induction ofTGFB1I1. Finally, silencing of TGFB1I1 expression signifi-cantly promotes SMC proliferation. Collectively, this studyprovides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical

for maintaining smooth muscle contractile phenotype byinhibiting smooth muscle proliferation.

Smoothmuscle cells (SMCs)2 are themajor contractile com-ponent of most hollow organs, such as esophagus, colon, blad-der, and blood vessels. Unlike striated cardiac and skeletalmus-cles that are terminally differentiated, undermany pathologicalconditions SMCsmaintain the ability tomodulate their pheno-type from contractile to synthetic in response to environmentalcues or growth factors (1).Transforming growth factor-� (TGF-�) is a potent regula-

tory growth factor with diverse functions on vascular SMCs (2).It has been shown to play important roles in vascular develop-ment and in pathogenesis of vascular diseases, including ather-osclerosis and restenosis (2). TGFB1I1 was originally identifiedas a TGF-�-regulated gene in mouse osteoblasts (3, 4).TGFB1I1 is a focal adhesion protein with four terminal LIM(Lin11, lsl, andMec-3) domains that share extensive homologywith paxillin, awell studied focal adhesion protein (5). TGFB1I1can translocate directly from focal adhesions to actin stressfibers uponmechanical stress thereby regulating the contractilecapability of a cell (6). In addition, TGFB1I1 can shuttlebetween the nucleus and cytoplasm through an oxidant-sensi-tive nuclear export sequence (7). Furthermore, TGFB1I1 canfunction as a transcription cofactor in conjunctionwith nuclearreceptors (8, 9) or with a number of other transcription factorsincluding LEF/TCF, Sp1, Smad3, and Smad7 to modulate geneexpression (10–13). During vascular injury TGFB1I1 expres-sion is decreased, and overexpression of exogenous TGFB1I1using adenoviral delivery attenuates injury-induced neointimalexpansion in vivo (14). Consistent with this, neointimal forma-

* This work was supported, in whole or in part, by National Institutes of HealthGrant GM56442 and by American Recovery and Reinvestment Act fundsfrom the NIGMS, National Institutes of Health (to L. V. D. W.). This work wasalso supported by a start-up fund from Albany Medical College and Scien-tist Development Grant from the American Heart Association (to J. Z.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1 and Figs. 1 and 2.

1 To whom correspondence should be addressed: 47 New Scotland Ave., MailCode 8, Albany, NY 12208. Tel.: 518-262-7862; Fax: 518-262-8101; E-mail:zhouj1@mail.amc.edu.

2 The abbreviations used are: SMC, smooth muscle cell; CarG, CC(A/T)6GG; E,embryonic day; HUVEC, human umbilical vein endothelial cell; miR,microRNA; qRT-PCR, quantitative real-time PCR; SRF, serum response fac-tor; TGFB1I1, transforming growth factor-�1-induced transcript 1; MRTF,myocardin-related transcription factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 48, pp. 41589 –41599, December 2, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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tion was enhanced after wire injury of the femoral artery inTGFB1I1-deficient mice (15), suggesting that TGFB1I1 playsan important role in the progression of vascular diseases. Addi-tionally, TGFB1I1 also plays a critical role inmyofibroblast per-sistence during wound healing (16, 17). Because TGFB1I1 playsimportant roles in cell senescence, tumorigenesis, wound heal-ing, vascular injury, and fetal gene expression during cardiachypertrophy (4, 15, 17–19), we sought to determine the mech-anisms by which TGFB1I1 is regulated under physiological andpathological conditions.Serum response factor (SRF) plays a critical role in regulating

expression of many smooth muscle-specific genes. SRF is anevolutionarily conserved MADS (MDM1, agamous, deficiens,SRF) box family transcription factor that binds to a highly con-served cis-regulatory element referred to as the CArG (CC(A/T)6GG) box (20). At least one or more conserved CArG ele-ments can be found in the majority of SMC-restricted genes(20). SRF is a weak activator of gene expression, requiring phys-ical interactions with other cofactors to fully direct smoothmuscle gene transcription. Of the SRF associated proteins,myocardin has been identified as the most potent activator ofCArG-dependent SMC marker genes (21). In this report, weprovide the first evidence that TGFB1I1 is an SRF/myocardin-regulated marker of the smooth muscle contractile phenotype,and the high level of TGFB1I1 expression is critical for main-taining the smooth muscle contractile phenotype.

EXPERIMENTAL PROCEDURES

Cell Culture and PDGF-BB, TGF-� Treatment—Mouse10T1/2 fibroblast cells, green monkey kidney fibroblast COS-7cells, HEK293, rat A10, and A7r5 aortic SMCs were purchasedfrom ATCC and cultured in growth medium (DMEM contain-ing 10%FBS and antibiotics). PAC1SMCswere a gift ofDr. Li Li(Wayne State University). HUVECs, C6 glioma, and MCF-7cells were a gift from Dr. Peter A. Vincent, Dr. Yunfei Huang,and Dr. Chunhong Yan, respectively (AlbanyMedical College).Rat primary aortic SMCs were prepared by enzymatic disper-sion from descending aorta of adult Sprague-Dawley rats asdescribed previously (22). For PDGF-BB treatment experi-ments, rat primary SMCs were grown to 80–90% confluenceand serum-starved for 2 days and then treated with recombi-nant rat PDGF-BB (50 ng/ml; Calbiochem) for 48 h. Cellstreated with vehicle served as control. For TGF-� treatment,10T1/2 cells were grown to 80–90% confluence and serum-starved for 2 days and then treated with 10 ng/ml recombinanthuman TGF-� (BD Biosciences) overnight. For the smoothmuscle calcification model, primary rat SMCs were treatedevery 3 days with differentiation medium (50 ng/ml humanrecombinant BMP-2 (Calbiochem), 10 mM �-glycerol phos-phate (Sigma), and 100 �g/ml ascorbic acid (Sigma)). Cells thatwere cultured in DMEM containing 10% FBS growth mediumserved as control. After 10 days, the cultured cells were rinsedwith PBS, and protein or RNA was harvested for measuringgene expression by Western blotting or real-time PCR.Aortic Tissue Preparation and Aortic Organ Culture—Rat

aorta was dissected to remove adhering tissue and denudedwith a catheter. The aortic tissue was then cut into 2-mm cylin-drical segments and cultured in 10% FBS DMEM for 60 h at

37 °C in a humidified chamber (5% CO2). The fresh isolatedtissues or culture vessels were then harvested with TRIzol fortotal RNA to evaluate gene expression by real-time PCR.Mouse Carotid Artery Ligation Model—All animal studies

were approved by the Institutional Animal Care and Use Com-mittee. Carotid artery ligation was performed on 3-month-oldC57BL/6 mice, and gene expression was analyzed as describedpreviously (23).Western Blotting—Western blot analysis was carried out as

described previously (22–24). Total protein (30 �g) was frac-tionated on 5 or 15% SDS-polyacrylamide gels and transferredelectrophoretically to a nitrocellulose membrane. The mem-brane was then probed with a series of antibodies: �-actin (AC-74, 1:2000; Sigma), calponin (1:5000; Sigma), GAPDH (1:2000;Santa Cruz Biotechnology), TGFB1I1 (1:5000; BDBiosciences),myocardin (M16, 1:2000; Santa Cruz Biotechnology), NMH-CII-B (1:2000; Covance), paxillin (1:5000; BDBiosciences), pro-liferating cell nuclear antigen (1:500; Santa Cruz Biotechnol-ogy), SM �-actin (1:10,000; Sigma), SM22� (1:5000; Sigma),SRF (1:5000; Santa Cruz Biotechnology), and vinculin (1:5000;Sigma).Immunohistochemistry—To detect TGFB1I1 expression, tis-

sues from Sprague-Dawley rats were fixed in 4% paraformalde-hyde and embedded in paraffin for all histological assays. Sec-tions were then stained with TGFB1I1 (1:200; BD Biosciences)or SM �-actin (1:1200; Sigma) antibody. For detection of pri-mary antibodies, we used avidin-biotin method with diamino-benzidine substrate as chromogen (brown; Vector Laborato-ries). Sagittal sections from control wild-type or SRFcardiovasculature-specific knock-out E11.5 embryos were pre-pared as described previously (25) and stainedwith theM.O.M.immunodetection kit (Vector Laboratories). Sections werelightly counterstained using hematoxylin to visualize cell nuclei(blue). Control sections were processed identically as experi-mental samples except no primary antibody was applied. Insome experiments, hematoxylin and eosin staining was per-formed for general morphology by a standard protocol.Immunocytochemistry—Mouse 10T1/2 cells were grown on

coverslips and infected with GFP control or myocardin/GFPadenovirus for 48 h and then fixed, permeabilized, and incu-bated withmonoclonal anti-TGFB1I1 antibody (1:300; BD Bio-sciences), followed by incubation with rhodamine-conjugatedanti-mouse IgG (1:400) secondary antibodies (Jackson Immu-noResearch Laboratories). Cells were counterstained withHoechst (1:5000) to visualize nuclei.Quantitative Real-time RT-PCR (qRT-PCR) Analysis and

MicroRNA (miR) qPCR—Total RNA was isolated with TRIzolreagent, and qRT-PCR was performed with respective gene-specific primers aswe reported previously (22–24) or otherwiselisted in supplemental Table 1. All samples were amplified induplicate, and every experiment was repeated twice indepen-dently. Relative gene expression was converted using the 2��Ct

method against the internal control acidic ribosomal phospho-protein P0 housekeeping gene. For measuringmiR-145 expres-sion, total RNA was isolated with TRIzol, and then small non-coding RNAs were converted into quantifiable cDNAs with aQuantiMir RT kit (System Biosciences). MiR-145 was thenevaluated as described in our recent report (22).

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Adenoviral Construction and Cell Infection—Adenovirusencoding TGFB1I1 was generated as reported previously (16).Adenovirus encoding the full-length cardiac formofmyocardinwas generated, and cell infection was performed as describedpreviously (22).Luciferase Reporter Assays—By using mouse BAC (RP23,

144N15; Invitrogen) DNA as template, a 2.5-kb fragment span-ning TGFB1I1 proximal promoter was amplified by PCR withprimers harboring KpnI and XhoI restriction enzyme sites(primer sequences are listed in supplemental Table 1). The PCRproducts were then cloned into pGL2-Basic (Promega) lucifer-ase reporter vector. Mutation of the CArG box in the TGFB1I1promoter was carried out with QuikChange Site-directedMutagenesis kit (Stratagene). The mouse TGFB1I1 promoter-luciferase reporter included nucleotides �1630 to �930 of themouse TGFB1I1 gene relative to the transcription start site asdetermined by DBTSS. A conserved CArG box within theTGFB1I1 gene promoter was identified by a sequence align-ment among multiple vertebrate species. All plasmids weresequenced to verify the integrity of the insert. Mammalianexpression plasmids for myocardin, MRTF-A and MRTF-B,were described in our previous report (24). Transfectionwas carried out with FuGENE6 transfection reagent (RocheApplied Science) as described previously (24). The level of pro-moter activity was evaluated by measurement of the fireflyluciferase activity relative to the internal control TK-Renillaluciferase activity using the Dual Luciferase Assay System asdescribed by the manufacturer (Promega). A minimum of sixindependent transfections was performed, and all assays werereplicated at least twice. Results are reported as themean� S.E.siRNA Transfection—Control siRNA and siRNA SMART-

pool against rat myocardin and SRF were purchased fromDharmacon. PAC1 cells were transfected either with controlsiRNA or siRNAs against myocardin or SRF using Lipo-fectamine 2000 transfection reagent (Invitrogen) following themanufacturer’s protocol. After 48 h, total RNA or protein washarvested for qRT-PCR or Western blot analysis. For silencingTGFB1I1 in rat primary aortic SMCs, siRNA duplex againstTGFB1I1 purchased from Ambion and transfection was per-formed using the Neon transfection system (Invitrogen) essen-tially following the manufacturer’s protocol.Oligonucleotide PulldownAssays—Bacterially expressed SRF

or myocardin was harvested in binding buffer (20 mM HEPES(pH 7.9), 80 mMKCl, 1 mMMgCl2, 0.2 mM EDTA (pH 8.0), 10%glycerol, 1 mM DTT, 0.1% Triton X-100, 1% protease inhibitormixture) and incubated with biotinylated double-stranded oli-gonucleotides containing the TGFB1I1 CArG box at 4 °C over-night. Then, 50�l of streptavidin-agarose beads were added for1 h at 4 °C to precipitate the oligonucleotide-protein complex.The beads were collected by centrifuging and washed withbinding buffer three times. Subsequently, the precipitated pro-tein was eluted from the beads by adding SDS sample buffer,and SRF or myocardin protein was detected by Western blot-ting. For competition assays, 100� excess nonbiotinylated oli-gonucleotides were used. The wild-type and mutant oligonu-cleotide sequences are shown in supplemental Table 1.Quantitative Chromatin Immunoprecipitation (ChIP) Assays—

PAC1 SMCs were fixed with formaldehyde, and ChIP was per-

formed by using an anti-SRF antibody (G20X; Santa Cruz Bio-technology) or IgG control as described by the manufacturer(Upstate) and in our previous report (24). Primers for quantita-tive evaluation of enrichment of the telokin promoter CArGregion, TGFB1I1 gene CArG region, and exon 10 are listed insupplemental Table 1 or described in our recent report (24).SMC Proliferation Assay—The proliferation of rat primary

aortic SMCs were measured by a cell proliferation WST-1 kit(Roche Applied Science) as described in our previous report(24). For the cell number counting, following transfection of theTGFB1I1-silencing duplex, rat primary aortic SMCs wereserum-starved inDMEM for 48 h and then cultured in 10% FBSmedium. Cell numbers were countedmanually at the indicatedtime points.

RESULTS

TGFB1I1 Expression Is Restricted to SMCs—TGFB1I1 is afocal adhesion protein that is homologous to paxillin and wasoriginally identified as a gene induced by TGF-� and hydrogenperoxide (4). By immunohistochemistry, TGFB1I1 has beenfound to be highly expressed in bothmouse and human smoothmuscle tissues (6, 14, 26). To extend these studies, proteinlysates were harvested from SMC lines (A10 and A7r5 are SMClines that are derived from the rat thoracic aorta (27), PAC1cells are established from the rat pulmonary artery (28)) andnon-SMC lines (HUVEC, COS-7, HEK293, 10T1/2,MCF-7, C6glioma), and Western blotting was carried out to detectTGFB1I1 and paxillin expression (Fig. 1A). Unlike paxillin,TGFB1I1 resembles the expression pattern of other well recog-nized smooth muscle-specific genes, calponin and SM �-actin,in that TGFB1I1 is expressed exclusively in SMC lines (Fig. 1A).We also found that TGFB1I1 was expressed abundantly in allSMC-rich tissues including the aorta, bladder, colon, esopha-gus, lung, spleen, stomach, and trachea of the adult mouse (Fig.1B). To characterize the TGFB1I1 expression pattern further,rat tissues were harvested, and immunohistochemistry stainingwas carried out with a TGFB1I1 antibody. Data from theseexperiments revealed that TGFB1I1 expression resembles SM�-actin tissue distribution that is present exclusively in smoothmuscles but not in cardiac and skeletal muscles (Fig. 1,C andD,and supplemental Fig. 1). The weak TGFB1I1 expressiondetected in heart, kidney, and liver byWestern blotting (Fig. 1B)is due to abundant TGFB1I1 expression in the vasculature ofthese tissues, which was confirmed by immunohistochemistrystaining of TGFB1I1 (Fig. 1C and supplemental Fig. 1).TGFB1I1 Expression Is Down-regulated during Phenotypic

Modulation of Smooth Muscle—Previous studies have sug-gested that TGFB1I1 is down-regulated following vascularinjury (14). To examine the expression of TGFB1I1 duringsmooth muscle phenotypic modulation further, we examinedTGFB1I1 expression in a variety of models in which SMCs areinduced to modulate their phenotype toward a synthetic state.First, we found that TGFB1I1 expression is dramatically atten-uated following passage of primary rat aortic SMCs in culture(P2 and P6), whereas the smooth muscle “synthetic” markerNMHCII-B is induced (29) (Fig. 2A). In contrast, expression ofpaxillin did not change under these conditions (Fig. 2A).PDGF-BB is known as a potent growth factor that inhibits

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smooth muscle marker expression while promoting SMC pro-liferation (30). In response to PDGF-BB treatment, TGFB1I1expression was significantly down-regulated along with othersmooth muscle markers, including calponin, SM22�, and thecardiovascular-specific transcription factor myocardin, at theprotein and/or mRNA level, whereas the marker proliferatingcell nuclear antigen is significantly induced in rat primary aorticSMCs (Fig. 2B). Aortic organ culture has beenwidely used as anex vivo model in which SMCs undergo profound phenotypicmodulation with down-regulation of smooth muscle markersand induction of matrix gene expression (31). In this model,expression of TGFB1I1, SM22�, and myocardin mRNAs weredramatically attenuated, whereas the matrix gene versican wassignificantly increased (6-fold; Fig. 2C). MiR-145, a miR that isconsidered a novel marker for the smooth muscle contractilephenotype (32), was also significantly down-regulated duringorgan culture (Fig. 2C). Smoothmusclemarker gene expressionhas previously been shown to be attenuated when SMCs arecultured in bone differentiation medium to induce SMC calci-fication (33). Similarly, we found that TGFB1I1 expression isalso attenuated at both mRNA and protein levels under theseconditions (Fig. 2D). We further confirmed that TGFB1I1expression is down-regulated along with other smooth musclecontractile genes in vivo in a mouse carotid artery ligationmodel (34). In this arterial injury model,Western blot and real-

time PCR data revealed that expression of TGFB1I1, similar toanother smooth muscle marker SM �-actin, was significantlydown-regulated in injured blood vessels from 1 to 14 days fol-lowing injury (Fig. 2E). Taken together, these data demonstratethat expression of TGFB1I1 is attenuated in tandemwith othersmooth muscle markers following phenotypic modulation ofvascular SMCs.TGFB1I1 Gene Promoter Contains a Functional CArG Ele-

ment Responsive to SRF/Myocardin—Previous studies demon-strated that the complex of SRF/myocardin is a critical deter-minant for smooth muscle-specific gene expression (35). Datadescribed above revealed that TGFB1I1 has a restricted patternof expression in SMCs and TGFB1I1 expression is down-regu-lated in amanner similar to other known smoothmusclemark-ers during smooth muscle phenotypic switch. This raised thepossibility that TGFB1I1 transcription is under the control ofan SRF/myocardin transcriptional complex. To begin exploringthis possibility, we carefully analyzed the TGFB1I1 gene proxi-mal promoter. In agreement with a previous report (36) a puta-tive SRF-binding element (CArG) was identified. This CArGbox is evolutionarily conserved among the species of verte-brates and harbors a C substitution in the A/T-rich core (Fig.3A). To validate experimentally that TGFB1I1 is amyocardin ormyocardin family protein target gene, transient transfectionexperiments were performed using the luciferase reporters

FIGURE 1. TGFB1I1 expression is restricted to SMCs. A, TGFB1I1 expression is restricted to SMC lines. Protein lysates were harvested from multiple cell linesas indicated, and Western blot analyses were performed to detect expression of TGFB1I1, the TGFB1I1 homolog paxillin, as well as smooth muscle-specificgenes calponin and SM �-actin. �-Actin served as loading control. B, TGFB1I1 expression is restricted in SMC-rich tissues in mouse. Mouse tissues wereharvested for Western blotting and expression of TGFB1I1, and smooth muscle-specific genes SM �-actin and SM22� were detected. SK, skeletal. C, TGFB1I1expression is restricted in SMCs in rat. Serial sections were prepared from adult rat heart, and immunohistochemistry staining was used to detect the expressionof TGFB1I1 or SM �-actin as indicated. A section stained with the secondary antibody alone served as a negative control. TGFB1I1, similar to SM �-actin, isexclusively expressed in the arterial smooth muscle layer, as indicated by an arrow, but not in cardiomyocytes. Original magnification, �20. D, immunohisto-chemistry staining was performed in contiguous sections from the rat upper esophagus using antibody against TGFB1I1 or SM �-actin, as indicated. TGFB1I1expression is localized specifically in smooth muscles of perivasculature and muscularis mucosae as indicated by a thin or heavy arrow, respectively. Epithelium(E) and skeletal muscles (M) have no TGFB1I1 expression. L, lumen. Original magnification, �20.

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containing TGFB1I1 promoters with either wild-type ormutated CArG sequences. These assays revealed that luciferaseactivity of the wild-type TGFB1I1 promoter reporter was sig-nificantly activated (7–9-fold) in the presence of myocardin orits family member MRTF-A. In contrast, mutating the CArGbox in the TGFB1I1 promoter completely abolishedmyocardinor MRTF-A-induced transactivation (Fig. 3B). Consistent withprevious studies showing that MRTF-B is a weak transcriptioncoactivator for smoothmuscle genes (37), MRTF-B had amod-

erate effect on the wild-type TGFB1I1 promoter reporter gene(Fig. 3B). To confirm that TGFB1I1 promoter activity is SRF/myocardin-dependent in SMCs, SRF or myocardin expressionwas reduced in PAC1 SMCs using siRNA, and the subsequenteffects on TGFB1I1 promoter activity were determined. Asshown in Fig. 3C, the activity of the wild-type TGFB1I1 pro-moter but not the mutated CArG TGFB1I1 promoter was sig-nificantly reduced (40–50% of control levels) in PAC1 SMCsfollowing knockdown of SRF or myocardin, resembling basal

FIGURE 2. Down-regulation of TGFB1I1 expression during smooth muscle phenotypic modulation. A, TGFB1I1 expression is down-regulated during SMCculture. Aortic arteries were dissected from rat and either harvested directly or cultured to passage 2 or 6 following enzymatic digestion. Subsequently, theprotein extracts of tissue or cultured SMCs were analyzed by Western blotting as indicated. B, PDGF-BB treatment inhibits TGFB1I1 expression in SMCs. Ratprimary aortic SMCs were treated with 50 ng/ml PDGF-BB or vehicle for 48 h. Cells were then harvested, and Western blotting was performed to detect smoothmuscle genes and TGFB1I1 expression as indicated. Vinculin served as loading control. Cells treated with PDGF-BB were also harvested for total RNA, andreal-time PCR was performed to measure TGFB1I1, SM22�, and myocardin mRNA expression (right). The relative expression level of genes in vehicle-treatedcells served as control and was normalized to a value of 1. *, p � 0.05. C, expression of TGFB1I1 is attenuated during arterial organ culture. Rat aortic artery wasdissected and either harvested immediately for total RNA or cultured in 10% FBS DMEM for 3 days and then harvested for total RNA. The changes in gene or miRexpression were measured by qRT-PCR. The relative expression of genes in intact tissue was set to 1. D, down-regulation of TGFB1I1 expression during SMCcalcification is shown. Rat aortic primary SMCs were cultured in growth medium (GM) or differentiation medium (DM) to induce SMC calcification for 10 days.Protein from these cells was then harvested for Western blotting (left), or total RNA was extracted for qRT-PCR (right) to measure TGFB1I1, smooth muscle genescalponin and SM22�, and miR-145 expression. E, left, expression of TGFB1I1 is down-regulated in mouse carotid artery following ligation injury. Carotid arteryligation was performed in 12-week-old C57BL/6 mice. Protein was harvested from uninjured control (con) or injured carotid artery (lig) at postsurgery day 1, 3,7, and 14, and Western blotting was carried out to assess TGFB1I1 expression as indicated. Right, total RNA from 5 or 10 days postoperation injured andcontralateral arterial tissues were harvested for qRT-PCR to measure TGFB1I1 and other smooth muscle gene expression. The relative gene expression inuninjured control vessels was normalized to a value of 1. n � 4.

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CArG mutant levels. Furthermore, CArG mutation in theTGFB1I1 promoter resulted in a 50% reduction in basal activityof TGFB1I1 promoter in PAC1 SMCs, suggesting the intactCArG box in the TGFB1I1 promoter is critical for TGFB1I1promoter activity in SMCs (Fig. 3C).SRF/Myocardin Binds to the TGFB1I1 Gene Promoter in

Vitro and in Vivo—The data described above demonstrate thatthe TGFB1I1 promoter contains a functional CArG boxresponsive to SRF/myocardin. We next sought to determinewhether SRF/myocardin can directly bind to the TGFB1I1 pro-moter CArG element. Oligonucleotide pulldown assaysshowed that SRF binds directly to wild-type but not to themutated TGFB1I1 promoter CArG box. Myocardin alone didnot bind to the probe, but it could be precipitated in the pres-ence of SRF, indicating that myocardin binds indirectly to theCArGbox through SRF (Fig. 4A). The specificity of SRF bindingto the CArG box was demonstrated by a competition assay inwhich excess unlabeled wild-type CArG oligonucleotides butnotmutated oligonucleotides could compete for SRFbinding to

the biotinylated probe (Fig. 4A). To confirm SRF binding to theTGFB1I1 promoter CArG box in vivo further, ChIP assays wereperformed in PAC1 SMCs. In agreement with the oligonucleo-tide pulldown data, ChIP assays unveiled approximately 2-foldenrichment of DNA fragment containing the TGFB1I1 CArGbox region following immunoprecipitation with a SRF anti-body, but not TGFB1I1-coding region exon 10 (Fig. 4B). As apositive control, ChIP assays showed an approximately 4-foldenrichment of DNA containing telokin CArG box region inPAC1 SMCs. Taken together, these data suggest that SRF bindsto the TGFB1I1 promoter CArG box in vitro and in cells.Myocardin Induces TGFB1I1 Expression—Data described

above demonstrate that myocardin can significantly activatetheTGFB1I1 promoter. To testwhethermyocardin is sufficientto induce TGFB1I1 expression, adenovirus encoding myocar-din or GFP control were transduced into rat primary vascularSMCs and non-SMC lines, including HUVECs and COS-7 and10T1/2 cells, and the expression of TGFB1I1 protein andmRNA was analyzed by Western blotting and qRT-PCR,

FIGURE 3. The TGFB1I1 gene promoter contains a functional CArG element responsive to SRF/myocardin. A, schematic diagrams a luciferase reporter ofthe mouse TGFB1I1 promoter (�1630 to �930) containing an evolutionarily conserved CArG box among vertebrate species. The position of CArG element isrelative to the annotated transcription start site (�1). A mutation placed in the CArG box is shown at the bottom. B, mouse myocardin and myocardin familyproteins MRTF-A and B, or pcDNA empty expression plasmid, were co-transfected together with a TGFB1I1 gene promoter luciferase reporter containing eitherwild-type or mutated CArG box, into 10T1/2 cells. The level of promoter activity was determined by measuring the firefly luciferase activity relative to thecontrol Renilla luciferase. Fold activation of promoter activity relative to control vector transfections (normalized to 1) is presented as mean � S.E. (error bars).n � 6. C, PAC1 SMCs were transfected with either 100 pmol of an RNA duplex against myocardin (siMyocardin), SRF (siSRF), or scrambled control RNA duplex(siControl). After 12 h they were then transfected with wild-type or CarG-mutated TGFB1I1 promoter luciferase reporter construct. 24 h following plasmidtransfection cells were harvested, and promoter activity was measured by dual luciferase assay as described in B. Reporter activity is normalized to a Renillaluciferase internal control and expressed relative to siRNA control transfections (normalized to 1). Knocking down endogenous SRF or myocardin in SMCsdecreases wild-type TGFB1I1 promoter activity but not the activity of the CArG mutation reporter.

FIGURE 4. SRF/myocardin binds to TGFB1I1 gene promoter in vitro and in vivo. A, SRF/myocardin binds to the CArG box within the TGFB1I1 promoter invitro. Bacterially expressed SRF or myocardin was incubated with biotinylated double-stranded oligonucleotides harboring TGFB1I1 promoter CArG box, andthen streptavidin-agarose beads were added to precipitate the oligonucleotide-protein complex. Subsequently, the beads were collected, and protein wasdetected by Western blotting as indicated. For competition assays, 100 � excess nonbiotinylated wild-type or mutated CArG oligonucleotides were added.B, SRF specifically binds to the CArG box region of TGFB1I1 promoter in vivo. Cross-linked chromatin from PAC1 SMCs was immunoprecipitated with anti-SRFantibody or control IgG, and the precipitated DNA was amplified by real-time PCR with TGFB1I1 gene-specific primers spanning CArG box region or exon (E) 10.A known CArG region within the smooth muscle-specific gene telokin served as a positive control. The enrichment of SRF binding is indicated relative to IgGcontrol (normalized to 1).

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respectively. Data from these experiments revealed that forcedexpression of myocardin dramatically induces endogenousTGFB1I1 protein and mRNA expression in both non-SMCs(Fig. 5, A–C and E) and SMCs (Fig. 5, D and E). Furthermore,immunofluorescence staining revealed that forced expressionof myocardin dramatically induces endogenous TGFB1I1expression at focal adhesions in 10T1/2 cells (Fig. 5F). Overex-pression of myocardin in HUVECs had no effect on endoge-nous TGFB1I1 family protein paxillin expression (Fig. 5A).Expression of TGFB1I1 Is Dependent on SRF/Myocardin—To

determine the role of endogenous SRF and myocardin in regu-lating TGFB1I1 expression, SRF or myocardin levels weredecreased by siRNA. Knocking down endogenous myocardinor SRF with SMARTpool siRNA duplexes (Dharmacon) inA7r5 and PAC1 SMCs resulted in an approximate 50% reduc-tion of basal TGFB1I1 mRNA and protein expression (Fig. 6,A–G). To determine further whether SRF is required forTGFB1I1 expression in vivo, we detected TGFB1I1 expressionin SRF cardiovasculature-specific knock-out mouse embryos(25) by immunohistochemistry staining. Data from this exper-

iment revealed that TGFB1I1 expression is reduced dramati-cally in the dorsal aorta of SRF knock-out embryo comparedwith control wild-type counterpart (Fig. 6H). Taken together,these results demonstrate that SRF and myocardin are neces-sary for TGFB1I1 expression.SRF Is Required for TGF-�-induced TGFB1I1 Expression—

Previous studies have shown that TGF-� is a potent cytokinethat induces TGFB1I1 expression (4, 17). In response to TGF-�treatment, 10T1/2 fibroblast cells differentiate toward asmoothmuscle- ormyofibroblast-type fatewith induction of anarray of smoothmuscle-specific genes (38). To assess the role ofSRF in TGF-�-mediated induction of TGFB1I1, 10T1/2 cellswere transfected with SRF siRNA overnight and then treatedwithTGF-�. After 48 h, cells were harvested, andWestern blot-tingwas performed to evaluate TGFB1I1 expression. Data fromthis experiment revealed that depletion of SRF significantlydiminished TGF-�-induced TGFB1I1 expression to approxi-mately 50% in a manner similar to other smooth muscle genes,including SM22� and SM �-actin (Fig. 7, A and B). Consistentwith the previous study (39), we also observed that TGF-�treatment increased endogenous SRF expression (Fig. 7A). Fur-thermore, luciferase reporter assays demonstrated that TGF-�significantly increases the WT TGFB1I1 promoter activity2-fold. In contrast, a TGFB1I1 promoter in which the CArGbox was mutated not only exhibited a 50% reduction in basalactivity but also completely lost the ability to be activated byTGF-� (Fig. 7C). These data demonstrated that SRF and anintact CArG box in the TGFB1I1 gene promoter are requiredfor the TGF-�-mediated induction of TGFB1I1.Depletion of TGFB1I1 Expression Promotes SMC

Proliferation—Data described above demonstrated thatTGFB1I1 is a novel marker for smooth muscle contractile phe-notype.We next sought to determine TGFB1I1 function in vas-cular SMCs. To test whether TGFB1I1 is able to affect SMCproliferation, a silencing duplex against TGFB1I1 was trans-fected into rat primary aortic SMCs by electroporation. Thistransfection resulted in a significant decrease of endogenousTGFB1I1 protein expression (Fig. 8A), and SMC proliferationin all culture media examined was significantly promoted com-pared with the control cells transduced with scrambled controlsiRNA (Fig. 8B). Furthermore, silencing endogenous TGFB1I1expression significantly increased rat primary aortic SMCgrowth (Fig. 8C). Taken together, these data suggest thatexpression of TGFB1I1 is critical formaintaining the SMCcon-tractile phenotype by inhibiting SMC proliferation. Overex-pression or silencing TGFB1I1 in rat primary aortic SMCs doesnot promote or attenuate smooth muscle differentiation (sup-plemental Fig. 2).

DISCUSSION

In this study we demonstrated that expression of TGFB1I1 islargely restricted to smooth muscle-enriched tissues (Fig. 1).TGFB1I1 expression is significantly down-regulated duringsmooth muscle phenotypic modulation in a manner parallel toother well documented smooth muscle marker genes (Fig. 2).These findings suggested that TGFB1I1 is a marker for differ-entiated contractile SMCs. Furthermore, consistent with ourprevious finding that TGFB1I1 regulates myofibroblast prolif-

FIGURE 5. Overexpression of myocardin induces endogenous TGFB1I1expression in multiple cell lines. Adenovirus encoding myocardin or con-trol GFP virus was transduced into HUVECs (A), COS-7 cells (B), 10T1/2 cells (C),and rat primary aortic vascular SMCs (D) for 48 h, and subsequently proteinwas harvested for Western blotting to detect TGFB1I1 gene expression atprotein level, or total RNA was extracted for qRT-PCR to detect TGFB1I1expression at mRNA level as indicated (E). C, *, nonspecific signal with anti-myocardin antibody. F, 10T1/2 cells were plated on coverslips and transducedwith the GFP control or GFP/myocardin adenovirus for 48 h and then stainedwith monoclonal anti-TGFB1I1 antibody to detect endogenous TGFB1I1expression (red). Immunofluorescence data are shown with a representativepicture. Overexpression of myocardin in 10T1/2 cells results in a dramaticinduction of TGFB1I1 localized in focal adhesions.

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eration (16), silencing TGFB1I1 in SMCs significantly increasesSMC proliferation (Fig. 8), suggesting a novel role of TGFB1I1in maintaining the smooth muscle contractile phenotype.TGFB1I1 was originally described as a marker for the early

developing heart inmouse (40). At E9.5, TGFB1I1 is detected inthe atria and ventricles of developing heart and in the somites(40). Later, TGFB1I1 expression is diminished in the heart andby E16.5 becomes restricted in smooth muscle layers of multi-

ple organs, including bladder and intestine (40). Consistentwith this finding, we found that the expression of TGFB1I1 isenriched predominantly in the medial smooth muscle layer ofarterial tissues and in other smooth muscle-rich tissues,whereas it is barely detectable in postnatal rat heart and skeletalmuscles (Fig. 1 and supplemental Fig. 1). Interestingly, the spa-tial and temporal regulation of TGFB1I1 expression duringmouse development resembles that of several other SMC-spe-

FIGURE 6. Silencing SRF or myocardin down-regulates endogenous TGFB1I1 expression in SMCs. A–D, A7r5 SMCs were transfected with silencing RNAduplexes against SRF (A and B) or myocardin (C and D) for 48 h, and the cells were harvested for Western blotting (A and C) or qRT-PCR (B and D) to detectTGFB1I1 expression. The cells transfected with scrambled silencing RNA duplex served as control. Blots in A and C are representative of three independentexperiments. Vinculin served as loading control. E and F, PAC1 cells were transfected with silencing myocardin or SRF duplex, and cells were harvested formeasuring TGFB1I1 expression at protein (E) and mRNA level (F) as described in A–D. GAPDH served as loading control. G, densitometric quantification ofTGFB1I1 expression in A, C, and E is shown. TGFB1I1 signals were normalized to vinculin or GAPDH signal to account for variability in loading and expressed asrelative to silencing control samples (normalized to 1). *, p � 0.05. Error bars, S.E. H, sagittal sections from control wild-type (WT) or SRF cardiovasculature-specific knock-out mouse E11.5 embryos (SRF KO) were stained with TGFB1I1 antibody (brown). Staining with secondary antibody alone served as negativecontrol. All sections were lightly counterstained with hematoxylin to visualize cell nuclei (blue). SRF deficiency resulted in a significant reduction of TGFB1I1staining in dorsal aorta (DA) as indicated by an arrow. *, heart.

FIGURE 7. SRF is required for TGF-�-induced TGFB1I1 expression. A, 10T1/2 fibroblast cells were transfected with scrambled control silencing RNA duplexor RNA duplex against SRF for 12 h, and the cells were then treated with 10 ng/ml TGF-� for 48 h. Subsequently, protein lysates were harvested for Westernblotting to detect TGFB1I1 expression, as indicated. B, densitometric quantification of immuno bands of TGFB1I1 and other smooth muscle markers in A isshown. The level of protein expressed was determined by measurement of the protein absorbance units relative to the respective vinculin expression as aninternal control. A value of 1 was arbitrarily assigned to the protein expression level in cells transfected with siRNA control and treated with TGF-�. *, p � 0.05.C, 10T1/2 cells were transfected with wild-type or mutated CArG TGFB1I1 promoter reporter for 12 h and then treated with TGF-� (10 ng/ml) or vehicle controlfor 24 h. Subsequently, cells were harvested for dual luciferase assay to measure TGFB1I1 promoter activity in the presence or absence of TGF-�. The luciferaseactivity of the wild-type TGFB1I1 promoter treated with vehicle served as control (normalized to 1). Data were presented as mean � S.E. (error bars). n � 6.

FIGURE 8. Depletion of TGFB1I1 expression promotes SMC proliferation. A, rat primary aortic SMCs were transfected with control or TGFB1I1 silencing RNAduplex, and protein was then harvested for Western blotting to assess TGFB1I1 expression. B, following transfection with silencing RNA duplex, rat primaryaortic SMCs were plated at equal density either in DMEM with supplement of 50 ng/ml PDGF-BB, 10% FBS medium, or 10% FBS supplemented with 50 ng/mlPDGF-BB, and then the proliferation of SMCs was measured using the cell proliferation WST-1 kit (Roche Applied Science). *, p � 0.05. C, following silencingTGFB1I1 rat aortic primary SMCs were seeded at equal density in 10% FBS medium, and cell numbers were counted at each time point as indicated. *, p � 0.05.Error bars, S.E.

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cific genes including SM �-actin and SM22� that display tran-sient expression and promoter activity in the developing myo-cardium (1). All of these genes contain conserved SRF-bindingCArG elements within their promoter regions or adjacentintrons (20). A previous study suggested that TGFB1I1 is a SRFtarget gene (36), and TGFB1I1 expression was reduced in aSRF-null mouse embryonic heart (41). In this study weextended these findings and identified an evolutionarily con-served CArG box in the TGFB1I1 gene promoter (Fig. 3). Inagreement with these studies, TGFB1I1 expression is signifi-cantly attenuated in the dorsal aorta of SRF cardiovascularknock-out mouse embryos (Fig. 6H). We showed that thisCArG box in TGFB1I1 gene promoter is vital for myocardin/SRF-dependent transactivation (Fig. 3) and for activation of thepromoter by TGF-� (Fig. 7). We also confirmed that SRF bindsto the TGFB1I1 promoter CArG sequence in vitro and in vivo(Fig. 4). Interestingly, this CArG box we identified within theTGFB1I1 gene promoter harbors a conserved C substitution inthe central A/T-rich region (Fig. 3). Previous studies haveshown that this type of CArG degeneracy diminishes bindingaffinity for ubiquitously expressed SRF (42). In agreement withthis, quantitative ChIP assays demonstrated relatively weakbinding of SRF to the TGFB1I1 CArG box compared with thecanonical CArG box in the telokin promoter in SMCs (Fig. 4B).Previous studies demonstrated that this reduced SRF bindingaffinity is critical for controlling gene expression in response topathophysiological stimuli such as vascular injury (42).Although the TGFB1I1 promoter reporter gene we cloned canbe readily activated by myocardin and TGF-� treatment, fur-ther work is needed to test directly whether this fragment of theTGFB1I1 promoter can recapitulate the expression patterns ofthe endogenous gene in transgenic mice and whether thedegenerated CArG box is required for changes in promoteractivity following vascular injury.The effects of TGF-� on smoothmusclemarker gene expres-

sion are thought to be mediated by activation of the SMADfamily of proteins (43–45). Previous studies have shown thatTGF-� activates the SM22� promoter via either a CArG box-dependent manner by direct association of Smad3 with SRF orby a CArG box-independent pathway involving direct interac-tion of Smad-binding element-bound Smad3 and myocardin(45, 46). Although we found that TGF-� stimulationof TGFB1I1 requires SRF, and the intact CArG box in theTGFB1I1 promoter is similar to the CArG-dependent activa-tion of the SM22� promoter, we cannot rule out the possibilitythat an alternative CArG-independent mechanism is involvedin TGF-�-inducedTGFB1I1 expression. In future studies it willbe interesting to examine whether the TGFB1I1 promoter con-tains any putative Smad-binding element sites that are impor-tant for TGFB1I1 expression.Unlike myocardin whose expression is restricted to cardiac

and smooth muscle and localizes exclusively within nucleus,MRTF-A is broadly expressed, and the function of MRTF-Acan be regulated by interactions with the actin cytoskeleton,resulting in subcellular distribution either in cytoplasm ornucleus (47). In response to Rho/ROCK signaling that can alsobe activated by TGF-� treatment, MRTF-A translocates intothe nucleus where it binds to SRF, thereby activating a subset of

CArGbox-containing genes, including SM�-actin (48). Similarto SM �-actin, TGFB1I1 is also highly expressed in myofibro-blasts and induced by TGF-� during wound healing (16, 17).Previous studies demonstrated that up-regulation of TGFB1I1expression following TGF-�-induced epithelial-mesenchymaltransition is bothRhoA- andROCK-dependent (49). In the cur-rent study we demonstrated that MRTF-A strongly activated aTGFB1I1 promoter reporter gene to an extent similar to myo-cardin. Consistently, in our accompanying study3we found thatforced expression of MRTF-A can dramatically induce endog-enous TGFB1I1 expression in human fibroblast cells. However,myocardin is almost undetectable in myofibroblasts comparedwith abundant expression of MRTF-A. Consistent with thisfinding, silencing MRTF-A attenuated TGF-�-inducedTGFB1I1 expression in fibroblasts. Taken together, these stud-ies demonstrated that myocardin family proteins play impor-tant roles for TGFB1I1 expression through various mecha-nisms whereby using the distinct availability of myocardinfamily proteins in SMCs versusmyofibroblasts.In summary, this study identified TGFB1I1 as a novelmarker

for the contractile phenotype of vascular SMCs that is regulatedby SRF/myocardin and the expression of TGFB1I1 is critical formaintaining smooth muscle contractile phenotype.

Acknowledgments—We thank Dr. Paul Herring for a critical readingof the manuscript, DebbieMoran for help with the preparation of thismanuscript, Dr. Joseph Miano for sharing SRF cardiovasculature-specific knock-out mouse embryo sections, and Christina Rotondi inthe Albany Medical College Histology Core for excellent technicalsupport with immunohistochemistry staining.

REFERENCES1. Owens, G. K., Kumar, M. S., and Wamhoff, B. R. (2004) Physiol. Rev. 84,

767–8012. Bobik, A. (2006) Arterioscler. Thromb. Vasc. Biol. 26, 1712–17203. Shibanuma,M.,Mashimo, J.,Mita, A., Kuroki, T., andNose, K. (1993) Eur.

J. Biochem. 217, 13–194. Shibanuma,M.,Mashimo, J., Kuroki, T., andNose, K. (1994) J. Biol. Chem.

269, 26767–267745. Thomas, S. M., Hagel, M., and Turner, C. E. (1999) J. Cell Sci. 112,

181–1906. Kim-Kaneyama, J. R., Suzuki, W., Ichikawa, K., Ohki, T., Kohno, Y., Sata,

M., Nose, K., and Shibanuma, M. (2005) J. Cell Sci. 118, 937–9497. Shibanuma,M., Kim-Kaneyama, J. R., Ishino, K., Sakamoto,N., Hishiki, T.,

Yamaguchi, K., Mori, K., Mashimo, J., and Nose, K. (2003)Mol. Biol. Cell14, 1158–1171

8. Aghajanova, L., Velarde, M. C., and Giudice, L. C. (2009) Endocrinology150, 3863–3870

9. Heitzer, M. D., and DeFranco, D. B. (2006)Mol. Endocrinol. 20, 56–6410. Wang, H., Song, K., Krebs, T. L., Yang, J., and Danielpour, D. (2008) On-

cogene 27, 6791–680511. Ghogomu, S.M., vanVenrooy, S., Ritthaler,M.,Wedlich, D., andGradl, D.

(2006) J. Biol. Chem. 281, 1755–176412. Wang, H., Song, K., Sponseller, T. L., and Danielpour, D. (2005) J. Biol.

Chem. 280, 5154–516213. Shibanuma, M., Kim-Kaneyama, J. R., Sato, S., and Nose, K. (2004) J. Cell.

Biochem. 91, 633–64514. Kim-Kaneyama, J. R., Wachi, N., Sata, M., Enomoto, S., Fukabori, K., Koh,

K., Sh, M., and Nose, K. (2008) Biochem. Biophys. Res. Commun. 376,

3 C. B. Betts, S. D. Varney, X. Wang, J. Zhou, and L. Van De Water, submitted.

Transcriptional Regulation of TGFB1I1 Expression

41598 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 48 • DECEMBER 2, 2011

by guest on Decem

ber 8, 2020http://w

ww

.jbc.org/D

ownloaded from

682–68715. Kim-Kaneyama, J. R., Takeda, N., Sasai, A., Miyazaki, A., Sata, M., Hi-

rabayashi, T., Shibanuma,M., Yamada, G., andNose, K. (2011) J.Mol. Cell.Cardiol. 50, 77–86

16. Dabiri, G., Tumbarello, D. A., Turner, C. E., and Van DeWater, L. (2008)J. Invest Dermatol. 128, 280–291

17. Dabiri, G., Tumbarello, D. A., Turner, C. E., and Van DeWater, L. (2008)J. Invest Dermatol. 128, 2518–2525

18. Yund, E. E., Hill, J. A., and Keller, R. S. (2009) J. Mol. Cell. Cardiol. 47,520–527

19. Li, X., Martinez-Ferrer, M., Botta, V., Uwamariya, C., Banerjee, J., andBhowmick, N. A. (2011) Oncogene 30, 167–177

20. Miano, J. M. (2003) J. Mol. Cell. Cardiol. 35, 577–59321. Pipes, G. C., Creemers, E. E., and Olson, E. N. (2006) Genes Dev. 20,

1545–155622. Wang, X., Hu, G., and Zhou, J. (2010) J. Biol. Chem. 285, 23241–2325023. Hu, G., Wang, X., Saunders, D. N., Henderson, M., Russell, A. J., Herring,

B. P., and Zhou, J. (2010) J. Biol. Chem. 285, 11800–1180924. Zhou, J., Hu, G., and Wang, X. (2010) J. Biol. Chem. 285, 23177–2318525. Miano, J. M., Ramanan, N., Georger, M. A., de Mesy Bentley, K. L., Emer-

son, R. L., Balza, R.O., Jr., Xiao,Q.,Weiler,H., Ginty,D.D., andMisra, R. P.(2004) Proc. Natl. Acad. Sci. U.S.A. 101, 17132–17137

26. Yuminamochi, T., Yatomi, Y., Osada, M., Ohmori, T., Ishii, Y., Nakazawa,K., Hosogaya, S., and Ozaki, Y. (2003) J. Histochem. Cytochem. 51,513–521

27. Kimes, B. W., and Brandt, B. L. (1976) Exp. Cell Res. 98, 349–36628. Rothman, A., Kulik, T. J., Taubman, M. B., Berk, B. C., Smith, C. W., and

Nadal-Ginard, B. (1992) Circulation 86, 1977–198629. Kuro-o, M., Nagai, R., Nakahara, K., Katoh, H., Tsai, R. C., Tsuchimochi,

H., Yazaki, Y., Ohkubo, A., and Takaku, F. (1991) J. Biol. Chem. 266,3768–3773

30. Holycross, B. J., Blank, R. S., Thompson, M. M., Peach, M. J., and Owens,G. K. (1992) Circ. Res. 71, 1525–1532

31. Zheng, J. P., Ju, D., Shen, J., Yang,M., and Li, L. (2010)Exp.Mol. Pathol. 88,52–57

32. Cordes, K. R., Sheehy, N. T., White, M. P., Berry, E. C., Morton, S. U.,Muth, A. N., Lee, T. H., Miano, J. M., Ivey, K. N., and Srivastava, D. (2009)Nature 460, 705–710

33. Tanaka, T., Sato, H., Doi, H., Yoshida, C. A., Shimizu, T., Matsui, H.,Yamazaki, M., Akiyama, H., Kawai-Kowase, K., Iso, T., Komori, T., Arai,M., and Kurabayashi, M. (2008)Mol. Cell. Biol. 28, 1147–1160

34. Kumar, A., and Lindner, V. (1997) Arterioscler. Thromb. Vasc. Biol. 17,2238–2244

35. Parmacek, M. S. (2007) Circ. Res. 100, 633–64436. Sun, Q., Chen, G., Streb, J. W., Long, X., Yang, Y., Stoeckert, C. J., Jr., and

Miano, J. M. (2006) Genome Res. 16, 197–20737. Wang, D. Z., Li, S., Hockemeyer, D., Sutherland, L., Wang, Z., Schratt, G.,

Richardson, J. A., Nordheim, A., and Olson, E. N. (2002) Proc. Natl. Acad.Sci. U.S.A. 99, 14855–14860

38. Hirschi, K. K., Rohovsky, S. A., and D’Amore, P. A. (1998) J. Cell Biol. 141,805–814

39. Sandbo, N., Kregel, S., Taurin, S., Bhorade, S., and Dulin, N. O. (2009)Am. J. Respir. Cell Mol. Biol. 41, 332–338

40. Brunskill, E. W., Witte, D. P., Yutzey, K. E., and Potter, S. S. (2001) Dev.Biol. 235, 507–520

41. Niu, Z., Iyer, D., Conway, S. J., Martin, J. F., Ivey, K., Srivastava, D., Nord-heim, A., and Schwartz, R. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105,17824–17829

42. Hendrix, J. A., Wamhoff, B. R., McDonald, O. G., Sinha, S., Yoshida, T.,and Owens, G. K. (2005) J. Clin. Invest. 115, 418–427

43. Sinha, S., Hoofnagle, M. H., Kingston, P. A., McCanna, M. E., and Owens,G. K. (2004) Am. J. Physiol. Cell Physiol. 287, C1560–1568

44. Shi, Y., and Massagué, J. (2003) Cell 113, 685–70045. Qiu, P., Feng, X. H., and Li, L. (2003) J. Mol. Cell. Cardiol. 35, 1407–142046. Qiu, P., Ritchie, R. P., Fu, Z., Cao, D., Cumming, J., Miano, J. M., Wang,

D. Z., Li, H. J., and Li, L. (2005) Circ. Res. 97, 983–99147. Miralles, F., Posern, G., Zaromytidou, A. I., and Treisman, R. (2003) Cell

113, 329–34248. Morita, T.,Mayanagi, T., and Sobue, K. (2007) J. Cell Biol. 179, 1027–104249. Tumbarello, D. A., and Turner, C. E. (2007) J. Cell. Physiol. 211, 736–747

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Livingston Van De Water and Jiliang ZhouXiaobo Wang, Guoqing Hu, Courtney Betts, Erin Yund Harmon, Rebecca S. Keller,

Factor/Myocardin Proteinfor Smooth Muscle Contractile Phenotype, Is Regulated by Serum Response

1-induced Transcript 1 Protein, a Novel MarkerβTransforming Growth Factor-

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