A Transforming Growth Factor beta  (TGFbeta ) Control Element Drives TGFbeta -induced Stimulation of Smooth Muscle alpha -Actin Gene Expression in Concert with Two CArG Elements*

(Received for publication, August 6, 1996, and in revised form, December 13, 1996)

Martina B. Hautmann , Cort S. Madsen and Gary K. Owens Dagger

From the Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta  (TGFbeta ) increases smooth muscle (SM) alpha -actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha -actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha -actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta -induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta -treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha -actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.


INTRODUCTION

Restenosis after balloon angioplasty results from abnormal proliferation of phenotypically modulated vascular smooth muscle cells (SMC)1 that synthesize large amounts of extracellular matrix (1). A variety of growth factors have been shown to play a role in the development of restenotic lesions including transforming growth factor beta 1 (TGFbeta ) (2, 3). The expression of TGFbeta mRNA and protein in the arterial wall is increased following balloon injury in rats (3) and overexpression of TGFbeta by gene transfer into normal arteries results in substantial extracellular matrix production accompanied by intimal and medial hyperplasia (4). Additionally, it has been shown that intravenous administration of neutralizing anti-TGFbeta antibodies significantly reduced intimal lesion size following carotid injury in the rat (5). The precise role of TGFbeta in lesion development, however, is not known, and is confounded by observations that TGFbeta can have very diverse functions in cultured SMC (6, 7). In particular, TGFbeta can either stimulate or inhibit cell proliferation depending on the cell line and culture conditions employed (8, 9). In addition to its effects on cell growth, TGFbeta has also been implicated in differentiation of SMC and other mesenchymal-derived cells during development (10). For example, TGFbeta treatment of human SMC (11), granulation tissue myofibroblasts (12), and pericytes (13), has been shown to increase expression of SM alpha -actin, a marker of differentiated SMC (14). Whereas SM alpha -actin is also expressed in cardiac and skeletal muscle during development (15), and in fibroblasts during wound repair (16), its expression is highly specific for SMC under normal circumstances in adult animals (14). It is the single most abundant protein in SMC, and is required for the principle function of mature SMC, contraction (14). Of particular interest, a recent study by Shah et al. (17), demonstrated that TGFbeta stimulated differentiation of neural crest cells into SMC or SMC-like cells based on morphological criteria and induction of SM alpha -actin and calponin. However, the molecular mechanisms whereby TGFbeta up-regulates SM alpha -actin expression are not known, nor is it known whether TGFbeta alters expression of other SMC differentiation marker proteins.

SM alpha -actin expression has been shown to be governed by a complex interplay of both positive and negative acting cis elements (18-21) depending on the cell context studied. The SM alpha -actin promoter contains several highly conserved cis elements including CArG elements. CArG elements are also found in the promoters of skeletal and cardiac alpha -actin genes as well as many other muscle-specific genes and have been shown to direct developmental and tissue-specific expression of these genes (22-28). Studies in our laboratory demonstrated that two CArG elements designated A and B, that are located within the first 125 bp of the rat SM alpha -actin promoter were required for basal and cell-specific expression (29). These latter results are of particular interest since MacLellan et al. (30) previously demonstrated that CArG elements were also important for TGFbeta inducibility of the skeletal alpha -actin gene in cardiac myocytes, thus suggesting a possible mechanism whereby TGFbeta might alter expression of the SM alpha -actin gene in vascular SMC.

The aims of the present study were to address the following questions: 1) what sequences impart TGFbeta responsiveness to the SM alpha -actin promoter? 2) Do the identified sites confer both basal and TGFbeta inducible expression, or are these sequences unique to TGFbeta induction? 3) What trans-acting factors interact with these sites? 4) Is TGFbeta inducibility restricted to the SM alpha -actin gene or does TGFbeta also stimulate expression of other SM differentiation marker genes including SM MHC and h1 calponin?


MATERIALS AND METHODS

Construction of Promoter-CAT Expression Plasmids

The generation of various truncated SM alpha -actin promoter/CAT reporter constructs, including the CArG A and B mutants have been previously reported (29). Site-directed mutagenesis of the TGFbeta control element (TCE)-like sequence spanning from -48 to -57 (TCE wild type: 5'-ATGAGG-3') was performed using the Altered Sites in vitro mutagenesis system (Promega) according to the manufacturer's recommendations. Three separate mutants of this putative TGFbeta response element were made: TCE mut 1: 5'-ATGGGAGG-3'; TCE mut 2: 5'-GAGTGAGG-3'; TCE mut 3: 5'-AGAGG-3'. The mutated fragments were polymerase chain reaction amplified and subcloned into pCAT-Basic (Promega). The sequence was verified by the Sanger dideoxy-sequencing procedure (31) using a Sequenase kit (U. S. Biochemical Corp.).

All promoter-CAT plasmid DNAs used for transfections were prepared using an alkaline lysis procedure (32) followed by banding on two successive ethidium bromide-cesium chloride gradients. Multiple independent plasmid preparations were tested for each construct.

Cell Culture, Transient Transfections, and Reporter Gene Assays

Rat SMC were isolated from thoracic aorta and cultured as described previously (33). Cells were plated at a density of 3 × 103/cm2, grown to confluency in 10% serum containing medium, and then growth-arrested for 4 days in serum-free medium (34) prior to stimulation with TGFbeta (human TGFbeta 1 from R&D Systems, 2.5 ng/ml) diluted with vehicle (4 mM HCl, bovine serum albumin, 1 mg/ml). Control cultures were treated with vehicle only. Cells used for the experiments described were between the 8th and the 22nd passage. SMC that are growth-arrested in this fashion express multiple SMC differentiation marker proteins including SM alpha -actin, SM MHC, h-caldesmon, h1 calponin, SM alpha -tropomyosin, and SM myosin light chain (MLC20) (35-37).2

Confluent, growth-arrested SMC in 6-well plates were transiently transfected (in triplicates) with 5 µg of DNA using the transfection reagent DOTAP (Boehringer Mannheim) according to the manufacturer's recommendations. After an incubation period of 12-14 h, the medium was replaced with fresh serum-free medium and TGFbeta (2.5 ng/ml) or vehicle were added. Cells were harvested 72 h later by scraping into ice-cold buffer A (15 mM Tris, pH 8.0, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.15 mM spermine tetrahydrochloride, 1 mM dithiothreitol) (38). Cell lysates were prepared by four freeze-thaws, followed by 10 min heat inactivation at 65 °C; 95-µl aliquots of each cell extract were assayed for CAT activity by enzymatic butyrylation of tritiated chloramphenicol (DuPont NEN) (39). CAT activities were normalized as described previously (29). Experiments were repeated two to six times and relative CAT activity data were expressed as the mean ± S.D. unless otherwise noted.

RNA Isolation, Probe Synthesis, and Northern Blot Analysis

Total RNA was isolated using TRI REAGENTTM (Molecular Research Center, Cincinnati, OH) according to the manufacturer's recommendations. Extracted RNA was dissolved in sterile water and stored at -70 °C until use. RNA concentration was measured spectrophoretically. The probe used for SM alpha -actin detection consisted of a 512-bp EcoRI fragment that encoded for amino acids 202 to 374 of the human skeletal alpha -actin cDNA (a gift kindly provided by Drs. Gunning and Kedes, Veterans' Administration Medical Center, Palo Alto, CA). For detection of SM MHC transcripts, a 373-bp cDNA probe corresponding to amino acids 1659 to 1929 of the SM2 isoform of SM MHC (kindly provided by Drs. P. Babij and M. Periasamy) was used. This cDNA probe recognizes both the SM1 and SM2 isoforms of SM MHC but not nonmuscle MHC (40, 41). H1 calponin transcripts were identified by a cDNA probe corresponding to the coding region spanning nucleotides 144 to 715 (kindly provided by M. Parmacek, University of Chicago). All cDNA probes were isolated from plasmid vector DNA by appropriate restriction enzyme digestions and gel purified (Bio-Rad, Prep-A-Gene). Probes were labeled with [alpha -32P]dCTP (DuPont NEN) by random primer extension (Prime-It, Stratagene). For Northern analysis, 10 or 15 µg of total RNA was diluted in loading buffer consisting of 0.2 M MOPS, 0.05 M sodium acetate, 0.01 M EDTA, 4% formaldehyde, and 65% formamide, denatured by treating for 10 min at 65 °C, and subsequently resolved on a 1.2% agarose gel containing 6.1% formaldehyde and 10% loading buffer. Capillary transfer of RNA to a nylon membrane (Micron Separating, Westboro, MA) was carried out overnight in 10 × saline/sodium/phosphate/EDTA buffer (2 M EDTA, 20 M NaH2PO4 H2O, 298 M NaCl). Blots were air dried, exposed to an UV transilluminator for 1.5 min, and baked for 2 h under vacuum at 80 °C.

Hybridization to cDNA probes and subsequent washes were carried out at 65 °C as described previously by Church and Gilbert (42). Blots were then dried, quantified using an Image Quant PhosphorImager, and subsequently exposed to Kodak X-Omat K film at -70 °C. A 5.8-kb EcoRI human cDNA fragment for 18 S rRNA was released from pBR322 and used in Northern analysis for standardization of RNA loading and transfer (43).

Western Blot Analysis

Cell lysates were prepared from confluent, growth-arrested SMC cultures stimulated with TGFbeta (2.5 ng/ml) or vehicle for 4 h. Briefly, cells were rinsed with phosphate-buffered saline, scraped into 0.6 ml of ice-cold RIPA buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibitors (10 mg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, 100 mM sodium orthovanadate), and passed through a 21-gauge needle for several times. Cell lysates were then incubated on ice for 30 min and microfuged for 20 min at 4 °C (protocol provided by Santa Cruz). Sample loading was normalized to DNA content determined with a DNA fluorometer (Hoefer Scientific, San Francisco). 600 ng of DNA was loaded per well on a 7.5% SDS-PAGE Mini-Protean gel (Bio-Rad). The proteins were transferred onto a polyvinylidene difluoride membrane at 100 V for 1.5 h. Blocking of the membrane and probing with appropriate antibodies were performed according to the ECL Western blotting protocol from Amersham and Life Science. Affinity-purified rabbit polyclonal SRF antibodies (Santa Cruz), raised against a peptide corresponding to SRF amino acids 486 to 505, were used as primary antibodies at a concentration of 1 µg/ml.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)

Crude nuclear extracts were prepared by the method of Dignam et al. (44) using confluent, growth-arrested SMC stimulated with TGFbeta or vehicle for 4 h. Protein concentrations were measured by the Bradford assay (Bio-Rad). Probes for EMSA were obtained by end labeling 20 µM single-stranded oligonucleotides with 150 µCi of [gamma -32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase. Labeled single-stranded oligonucleotides were annealed and unincorporated nucleotides were removed using NucTrap Push columns (Stratagene) as recommended by the manufacturer. The following nucleotides were used either as a probe or as cold competitors (only sense strand shown): TCE, 5'-GAAGCGAGTGGGAGGGGAT-3'; TCE mut 2, 5'-GAAGCGAGTGTTAGGGGAT-3'; Sp1, 5'-GATCGATCGGGGCGGGGCGATC-3'; AP-1, 5'-CTAGTGATGAGTCAGCCGGATC-3'. The 20-µl binding reaction contained ~20,000 cpm labeled probe, 5 µg of nuclear extracts in Dignam buffer D, 20 mM Hepes, pH 7.9, 50 mM KCl, 4 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 15% glycerol, 2.5% Nonidet P-40, 0.5 µg of poly(dA-dT) (Sigma), and cold competitor oligonucleotides where indicated. Recombinant human Sp1 was added at a concentration of 9 fpU per reaction (1 fpU defined as the amount of protein required to give full protection against DNase I of Sp1 sites within SV 40 early promoter (Promega)). All binding reactions were incubated for 20 min at room temperature. For Sp1 or Sp3 supershift assays, antibodies (2 µg/reaction) were added to the binding reaction and incubated for 20 min at room temperature. The radiolabeled DNA was subsequently added to the binding reaction and incubated for additional 20 min at room temperature. Protein-DNA complexes were resolved on a 4.5% polyacrylamide gel (30:1 acrylamide/bis-acrylamide (Bio-Rad)) and electrophoresed at 170 V in 0.5 × TBE (45 mM Tris borate, 1 mM EDTA). The gels were then dried and subjected to autoradiography at -70 °C.

In Vitro Synthesis of SRF

In vitro synthesis of SRF was performed using a TNT coupled reticulocyte lysate translation system (Promega) and using the human SRF cDNA clone pT7Delta ATG (45) as a template.


RESULTS

TGFbeta Up-regulated SM alpha -Actin mRNA Expression

To examine the effects of TGFbeta on SM alpha -actin mRNA expression, rat aortic SMC were grown to confluency, growth-arrested in SFM, and treated with 2.5 ng/ml TGFbeta or vehicle. Total RNA was extracted from these cultures at the times indicated and subjected to Northern blot analysis for SM alpha -actin (Fig. 1). SM alpha -actin mRNA levels peaked at 3 h (9-fold) and then decreased over time (4.5-fold at 8 h and 2.5-fold at 32 h, data not shown). Addition of the protein synthesis inhibitor cycloheximide (20 µg/ml) completely abolished TGFbeta -induced increases in SM alpha -actin mRNA levels, suggesting that effects were dependent on new proteins of ongoing synthesis of proteins with relatively short half-lives. TGFbeta also slightly induced nonmuscle (NM) beta -actin mRNA expression.


Fig. 1. Northern analysis of the effects of TGFbeta on SM alpha -actin. Quiescent cultures of SMC in a defined serum-free medium were treated with TGFbeta (2.5 ng) or vehicle (-) either alone or in combination with cycloheximide (20 µg/ml) and harvested at the times indicated. 10 µg of total RNA were loaded per lane. Northern analysis was performed with a radiolabeled 512-bp fragment of the coding region of a human skeletal alpha -actin cDNA, which hybridizes to both a 1.7-kb SM alpha -actin transcript and a 2.1-kb nonmuscle (NM) beta -actin transcript (upper panel). The blots were rehybridized with a 18 S rRNA probe to verify similar RNA loadings for each lane (lower panel).
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The First 125 bp of the SM alpha -Actin Promoter Were Sufficient to Confer TGFbeta Responsiveness

To determine whether increases in SM alpha -actin gene transcription contributed to TGFbeta -induced increases in SM alpha -actin mRNA expression, transient transfection studies were performed in cultured rat aortic SMC using a construct containing 2.8 kb of the 5'-flanking sequence of the SM alpha -actin gene linked to a promoterless CAT reporter gene (pProm/CAT) (29). Results demonstrated that TGFbeta induced an ~7-fold increase in activity of pProm/CAT above vehicle-treated controls (Fig. 2) suggesting that TGF-induced increases in SM alpha -actin mRNA were due, at least in part, to increased SM alpha -actin gene transcription. To identify the minimal sequences within the pProm/CAT construct that were required for TGFbeta responsiveness, we tested a series of deletion mutant constructs. Transfection data indicated that the first 125 bp of the SM alpha -actin promoter were sufficient to confer full TGFbeta responsiveness. Previous analysis of the promoter regions between -125 bp and -2.8 kb demonstrated that it contained negative regulatory elements required for cell-specific expression of the SM alpha -actin gene (29). Inclusion of these upstream sequences, however, did not alter TGFbeta -induced stimulation of SM alpha -actin gene expression in SMC.


Fig. 2. Transfection analysis of a 2.8-kb fragment of the SM alpha -actin promoter linked to a CAT-reporter gene (pProm CAT) and various deletion mutants in cultured rat aortic SMC. Growth-arrested SMC cultures were transiently transfected with the constructs indicated and stimulated with TGFbeta (2.5 ng/ml) or vehicle for 72 h. CAT activities of TGFbeta -treated groups were expressed relative to vehicle treated groups set to one. Data represent means ± S.E. TGFbeta treatment significantly increased reporter activity for all constructs tested but no statistically significant differences (ANOVA, p >=  0.05) were observed for TGFbeta -treated groups between the different constructs. black-square, vehicle; , TGF.
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Identification of a TGFbeta Responsive Element within the First 125 bp of the SM alpha -Actin Promoter

To identify potential TGFbeta response elements within the first 125 bp, we first performed a sequence analysis to determine whether the initial 125 bp of the SM alpha -actin promoter contained any known TGFbeta response elements (46-50). A 10-bp sequence spanning from -48 to -57 shared sequence similarities with a previously reported TGFbeta response element designated as TGFbeta inhibitory element (TIE) paradoxically found in promoters of several genes whose expression were inhibited by TGFbeta (51, 52; Table I). We analyzed this region by site-directed mutagenesis to test its importance for TGFbeta responsiveness (Fig. 3A). The design of mutants was based on those shown by Pietenpol et al. (51) to abolish TGFbeta -mediated inhibition of the c-myc gene. Results of transient transfection studies demonstrated that TGFbeta -induced stimulation of the SM alpha -actin gene was nearly completely abolished in all three mutant constructs (Fig. 3B). Mutations of this TGFbeta responsive control element (henceforth designated "TCE") also reduced basal transcriptional activity of p125 CAT by approximately half, even in the absence of exogenous TGFbeta . We (53) and others (54) have previously shown that cultured rat SMC produce active TGFbeta . Thus, it is possible that the effect of mutation of the TCE on "basal" transcription was also TGFbeta -dependent. To test this possibility, we incubated SMC with a TGFbeta neutralizing antibody (raised in chicken, R&D) during the entire period following transfection. Control cells were treated with chicken IgG. In an attempt to maximize inhibition of autocrine produced TGFbeta , high concentrations of TGFbeta antibodies (2 µg/ml) were administered. Results demonstrated that addition of TGFbeta antibodies reduced the basal transcriptional activity of wild-type p125 CAT by ~50% as compared with controls (Fig. 4). In contrast, TGFbeta neutralizing antibodies had no effect on the activity of TCE mutants, although in the presence of neutralizing TGFbeta antibodies, basal transcriptional activity of the TCE mutants was about 50% lower than wild-type p125 CAT. Results of these studies indicate that endogenously produced TGFbeta contributes to basal transcriptional activity of p125 CAT. It cannot be ascertained from these data, however, whether there is also a TGFbeta independent mechanism that contributes to the reduced basal transcription seen with the TCE mutants (Fig. 4), since it cannot be determined if complete neutralization of TGFbeta was achieved over the entire time course of the experiment.

Table I.

Comparison of a consensus TIE/TCE sequence first described in the promoters of genes inhibited by TGFbeta (51, 60) with TCE-like elements found in the promoter regions of several SM differentiation genes: SM alpha -actin (rat, mouse) (29), SM MHC (rat, rabbit) (57, 58), SM-22alpha (mouse) (71), and h1 calponin (mouse) (59)

SM-22alpha is a putative calcium-binding protein and structurally related to calponin. In the adult animals, it is highly expressed in smooth muscle-containing tissues (74).


Consensus TIE/TCE G a/c G T t/g G G t/g G A

Rat SM alpha -actin  -57 G A G T G G G A G G
Mouse SM alpha -actin  -57 G A G T G G G A G G
Rat SM MHC  -1074 G A G T G G G A G G
Rab SM MHC  -1994 G T G T G G G A G G
Mouse h1 calponin  -320 G A T T G G G T G G
Mouse SM 22alpha  -106 G A G T G G G G C G


Fig. 3. A, cartoon illustrating site-directed mutants of the TCE element tested in transient transfection studies in B. Mutants were generated using an Altered Sites in vitro mutagenesis system (Promega). B, effect of TCE mutations on TGFbeta -induced up-regulation of CAT activity. SM alpha -actin p125 CAT constructs containing the three different TCE mutations (TCE m1 to 3) and the wild-type p125 CAT were transiently transfected into growth-arrested rat SMC. CAT activities of TGFbeta (2.5 ng/ml) treated versus vehicle-treated controls were expressed relative to the base-line CAT activity of a promoterless CAT construct set to one. Data represent the mean ± S.D. of triplicate samples. Results shown are for one representative experiment out of a total of four experiments that were done. black-square, vehicle; , TGF.
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Fig. 4. Analysis of the contribution of autocrine TGFbeta to reporter activity of wild-type p125 CAT and TCE mutants. The p125 CAT wild-type construct or p125 CAT constructs containing the TCE mutants (TCE m1 to 3) were transiently transfected into growth-arrested rat SMC cultures. After transfection, cultures were treated with TGFbeta antibodies (R&D) (2 µg/ml) or chicken IgG daily for three subsequent days and then harvested for CAT assays. CAT activities of TGFbeta antibody treated groups versus control groups were expressed relative to the base-line CAT activity of a promoterless-CAT construct set to one. Data represent the mean ± S.D. of triplicate samples. Similar results were seen in two independent repeats. black-square, TGF-AB; , control AB.
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Two Highly Conserved CArG Elements (A and B) Contained Within the First 125 bp of the SM alpha -Actin Promoter Were Also Required for TGFbeta -induced Up-regulation of SM alpha -Actin Gene Expression

CArG elements have been shown to be important for basal SM alpha -actin transcription in SMC (29) as well as for basal and TGFbeta -induced stimulation of the skeletal alpha -actin gene in cardiac muscle cells (30). Thus, we tested whether the CArG elements in the SM alpha -actin promoter were required for TGFbeta responsiveness. Mutation of either CArG A or CArG B, alone or in combination, completely abolished TGFbeta -induced increases in CAT activity in transient transfection assays (Fig. 5). Mutation of the CArG elements also completely abolished TGFbeta responsiveness within the context of the 2.8-kb promoter construct, pProm/CAT (data not shown).


Fig. 5. Effect of CArG mutations on TGFbeta -induced stimulation of reporter activity. Wild type SM alpha -actin p125 CAT or constructs containing mutations of either CArG box A or CArG B alone or both CArGs were transiently transfected into growth-arrested rat SMC. CAT activities of TGFbeta () (2.5 ng/ml) or vehicle treated (black-square) groups were expressed relative to the base-line CAT activity of a promoterless-CAT construct. Data represent the mean ± S.D. of triplicate samples. Results shown are representative of three independent experiments.
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TGFbeta Markedly Enhanced Binding of Nuclear Factor(s) to the TCE

To characterize protein interactions with the TCE, EMSA were performed with a 19-bp probe containing the TCE (henceforth designated as "TCE probe") and nuclear extracts from TGFbeta - or vehicle-treated SMC. Results of EMSA with the TCE used as a probe showed a single shift band with nuclear extracts from TGFbeta -treated SMC (henceforth designated as shift band 1, Fig. 6, lane 2) that was barely detectable in extracts from vehicle-treated cells (Fig. 6, lane 1). Addition of cold double-stranded wild-type TCE competitor oligonucleotides inhibited formation of shift band 1 (lanes 3 and 4), indicating that shift band 1 represented a sequence-specific protein-DNA complex. Competition with mutant TCEm2 cold double-stranded competitor oligonucleotides did not affect formation of shift band 1 (lanes and 6). No shift band was formed when the mutant TCEm2 oligonucleotide was used as labeled probe (lane 13). These results are consistent with data obtained from transfection studies showing that mutations of the TCE element resulted in loss of TGFbeta responsiveness. Previous studies have shown binding of the transcription factor AP-1 to the TIE in the transin/stromelysin promoter (52). To determine if AP-1 binding contributed to formation of shift band 1, cold oligonucleotides containing a canonical AP-1 consensus sequence were used in competition assays. AP-1 oligonucleotides did not affect formation of shift band 1 (lanes 7 and 8). Further evidence that AP-1 is not likely to be responsible for formation of shift band 1 is provided by a study by Angel et al. (55) which demonstrated that a T at position 1 of the AP-1 consensus sequence (TGAGTCAG) is critical for AP-1 binding. Moreover, the TCE in the SM alpha -actin promoter shares only 4 bp (GAGT) with the AP-1 consensus sequence and the T at position 1 is replaced by a C. Taken together, these results suggest that AP-1 was not part of shift band 1 (lanes 7 and 8).


Fig. 6. Gel shift analysis of nuclear factor(s) binding to the TCE. Nuclear extracts were obtained from rat SMCs either stimulated with TGFbeta (2.5 ng/ml) or vehicle. 5 µg of nuclear extracts were incubated with a radiolabeled 19-bp wild-type (lanes 1-11) or mutated TCE TCE m2 probe (lane 13). In lane 12, a 22-bp Sp1 consensus oligonucleotide was used as a probe. Competition reactions were performed with oligonucleotide duplexes of the wild-type and mutated TCE element and oligonucleotide duplexes containing the sequences for the transcription factors AP-1 and Sp1. Competitor oligonucleotides were added at a 100-750 fold molar excess relative to the radiolabeled DNA.
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The Nuclear Factor That Bound to the TCE Exhibited Binding Properties That Distinguished It from Sp1 or Sp3

Since part of the TCE sequence (GGGAGGG) shares similarities with a transcription factor Sp1 recognition site, and in addition Sp1 has been implicated in TGFbeta -mediated gene expression (47, 49, 50), additional experiments were performed to determine whether Sp1 or an Sp1-like factor bound to the TCE. Formation of shift band 1 was decreased by the addition of high excesses of cold oligonucleotides containing a canonical Sp1-binding site indicating that the nuclear protein responsible for formation of shift band 1 could bind to a Sp1-binding site, albeit weakly (Fig. 6, lanes 9-11). Several lines of evidence, however, suggested that the factor binding to the TCE sequence was not Sp1. First, incubation of labeled TCE probe with nuclear extracts from TGFbeta -treated SMC or with large amounts of recombinant Sp1 resulted in formation of shift bands (Fig. 7, compare lanes 6 and 9) with different mobilities. Second, the shift complex formed by labeled TCE and recombinant Sp1 co-migrated with those formed by labeled Sp1 probe and nuclear extracts from TGFbeta -treated SMC (Fig. 7, lane 2). Both of these complexes were supershifted by Sp1 antibodies (Fig. 7, lanes 3 and 10) indicating that the Sp1 antibodies specifically recognized human recombinant Sp1 and Sp1 present in nuclear extracts from rat aortic SMC. However, Sp1 antibodies did not affect shift band 1 formation, when labeled TCE probe was incubated with nuclear extracts from TGFbeta -treated cells (Fig. 7, lane 7). These results strongly suggest that Sp1 was not the TGFbeta -induced nuclear factor binding to the TCE probe.


Fig. 7. Characterization of nuclear factor binding to the TCE. 5 µg of nuclear extract from rat SMC treated with TGFbeta (2.5 ng/ml) or vehicle were incubated with radiolabeled TCE (lanes 5-10) or Sp1 oligonucleotide duplexes (1-4). Polyclonal antibodies (Santa Cruz) raised against Sp1 (lanes 3, 7, and 10) (2 µg/reaction) or Sp3 (lanes 4 and 8) (2 µg/reaction) were added to the binding reaction in the absence of radiolabeled probe and incubated for 20 min at room temperature. Recombinant human Sp1 (rh Sp1) was added at a concentration of 3 fpu units (lanes 9 and 10).
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Recently, other members of the Sp1 transcription factor family have been cloned and sequenced (56). One of these family members, Sp3, has also been partially characterized and shown to bind to similar sequences as Sp1 (56). We therefore tested whether Sp3 might be responsible for formation of shift band 1. Sp3 antibodies did not affect formation of shift band 1, indicating that the TCE binding factor was not Sp3 (Fig. 7, lane 8). The ability of the Sp3 antibody to form supershifted complexes was verified using an Sp3 containing nuclear extract (data not shown).

TGFbeta -enhanced Binding of SRF to CArG A and Carg B Was at Least Partly Due to Increased SRF Protein Expression

Since transfection data provided evidence for functional importance of the CArG elements for TGFbeta inducibility of the SM alpha -actin gene, we tested whether TGFbeta treatment affected binding to the SM alpha -actin CArGs. EMSA were performed with labeled 20-bp CArG A or B oligos and nuclear extracts from either TGFbeta - or vehicle-treated SMC. TGFbeta treatment markedly enhanced binding to both CArG elements (Fig. 8, lanes 2 and 5) when compared with vehicle-treated controls. Consistent with our previous studies (29), two shift bands were identified that supershifted with addition of SRF antibodies (data not shown). These results demonstrate that SRF is present in the protein-DNA binding complex formed with nuclear extracts from TGFbeta -treated SMC. TGFbeta -enhanced SRF binding was not associated with changes in mobility as compared with the shift bands formed with recombinant SRF, suggesting that TGFbeta did not induce formation of a stable higher order complex, at least under the gel-shift assay conditions employed in these studies (Fig. 8A, lanes 3 and 6). TGFbeta treatment also markedly enhanced binding activity to the Wt 95 probe (Fig. 8B, lane 2) compared with vehicle controls (lane 1). Addition of SRF antibodies (Fig. 8B, lane 3) led to formation of supershifted complexes and the disappearance of bands 1 and 2, indicating that these two bands contained SRF. The SRF antibodies employed also appeared to inhibit SRF binding since bands 1 and 2 are not quantitatively supershifted. To test whether TGFbeta -induced increases in SRF protein levels might have contributed to the enhanced binding activity noted for the SM alpha -actin CArGs, we performed Western analysis of cell lysates obtained from TGFbeta -treated SMC. Results showed increased immunoreactive SRF in TGFbeta -treated cells as compared with vehicle-treated cells (Fig. 9), suggesting that enhanced CArG binding following TGFbeta treatment was due, at least in part, to increased SRF expression.


Fig. 8. Gel shift analysis of the effects of TGFbeta treatment of SMC on binding to CArG A and B oligonucleotides (A) or a 95-bp probe containing both CArG elements and flanking sequences (WT 95) (B). A, a radiolabeled 20-bp CArG B (lanes 1-3) or CArG A (lanes 4-6) double-stranded oligonucleotide was incubated with nuclear extract (5 µg) from rat SMC treated with TGFbeta (2.5 ng/ml), vehicle or in vitro translated human SRF (rSRF). B, a 95-bp radiolabeled, double-stranded probe was incubated with 5 µg of nuclear extracts from rat SMC treated with TGFbeta or vehicle. Binding reactions were carried out as previously described (29). Polyclonal SRF antibody (Santa Cruz) raised against the COOH terminus of human SRF was added at a concentration of 2 µg/reaction.
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Fig. 9. Western blot analysis of cell lysates from SMC stimulated with TGFbeta . Western blot analysis was performed on cell lysates obtained from growth-arrested SMC cultures treated with TGFbeta (2.5 ng/ml) or vehicle for 4 h. Cell lysates, normalized to DNA content (600 ng), were analyzed by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted with a polyclonal SRF antibody (Santa Cruz). Immunoreactive bands with a size of ~67 kDa were detected. However, it cannot be ruled out that TGFbeta may have induced post-translational modifications in SRF that might have affected reactivity with the antibody employed. No reactivity was observed when the membrane was immunoblotted with control rabbit serum (data not shown). The experiment was repeated three times.
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TGFbeta Treatment of SMC Also Enhanced Expression of SM MHC and Calponin

The promoters of the SM MHC (rat, rabbit) and h1 calponin (mouse) have recently been cloned and partially sequenced (57-59). A computer-assisted search revealed that both promoters contain TCE-like sequence motifs (Table I). Thus, we tested whether TGFbeta treatment of SMC also stimulated expression of these genes. A comparative Northern analysis of control versus TGFbeta -treated SMC showed that TGFbeta markedly increased both SM MHC (Fig. 10A) and h1 calponin (Fig. 10B) mRNA levels. These results suggest that TGFbeta might act as a positive SMC differentiation factor by coordinately up-regulating expression of several SM differentiation marker genes.


Fig. 10. Northern analysis of the effects of TGFbeta on SM MHC (A) and h1 calponin mRNA expression (B). Growth-arrested cultures of SMC were treated with TGFbeta (2.5 ng/ml) or vehicle, and harvested at the times indicated. Northern hybridization was performed with a 373-bp fragment of SM2 which specifically hybridized to SM specific MHC isoforms (41) (A, upper panel) or a 571-bp fragment corresponding to bp 144 to 715 of the murine h1 calponin cDNA (B, upper panel). The blots were rehybridized with a 18 S rRNA probe to verify similar RNA loadings for each lane (lower panels).
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DISCUSSION

The goal of the present study was to investigate the molecular mechanisms whereby TGFbeta up-regulates SM alpha -actin expression. Through both deletion and mutational analysis, we identified the promoter sequences required for TGFbeta activated SM alpha -actin transcription. Results showed that the first 125 bp of the SM alpha -actin promoter were sufficient to confer TGFbeta -induced activation and that three positive-acting cis-elements contained within this region were essential for this response, CArG box A at -62 and B at -112 and a TCE at -42. The fact that mutation of any one of these elements completely abolished transcriptional activity suggests that these elements operate interactively rather than independently to confer TGFbeta responsiveness.

Mutation of the TCE resulted in both loss of TGFbeta induction of SM alpha -actin expression, and loss of binding of an as yet unidentified TGFbeta -inducible factor to the TCE, suggests that the TCE functions as a positive-acting cis-element. Paradoxically, this SM alpha -actin TCE shares sequence similarities with a previously described TIE found in a number of genes inhibited by TGFbeta (60), including transin/stromelysin (52) and c-myc (51). Of interest, however, Pietenpol et al. (51) presented evidence suggesting that TGFbeta -induced inhibition of c-myc transcription in keratinocytes was mediated through down-regulation of a positive trans-acting factor (51). As such, the TCE in the c-myc promoter might also function as a positive-acting cis-element as we observed for the SM alpha -actin TCE. However, whether the TCE binding factors in SMC and keratinocytes are related, and whether they are regulated in a cell-type specific manner, will require further studies.

TGFbeta responsiveness of the SM alpha -actin gene was also dependent on two highly conserved CArG elements, A and B, that bind SRF (29). Evidence for functional importance of CArG elements in TGFbeta -mediated gene transcription has been shown previously in studies by MacLellan et al. (30) who showed that the most proximal CArG box in the skeletal alpha -actin promoter was essential for TGFbeta responsiveness in cardiac myocytes whereas mutation of the more distal CArG elements only partially inhibited TGFbeta responsiveness. Several lines of evidence indicate that these genes are regulated differently by TGFbeta in cardiac myocytes versus SMC. First, TGFbeta -mediated activation of the skeletal alpha -actin gene was found to be dependent on an M-CAT site which binds members of the TEF-1 family (61) in that mutation of this site abolished basal and TGFbeta -inducible expression. The M-CAT sites in the SM alpha -actin promoter, however, are located upstream (-178 and -314) of the minimal sequence required for full TGFbeta responsiveness. Second, the region on the skeletal alpha -actin promoter shown to be TGFbeta -responsive does not contain a TCE or TCE-like element. Third, there is evidence that CArG-dependent regulation of these two genes is different with respect to TGFbeta activation. For example, our results demonstrated that TGFbeta markedly enhanced SRF binding to the CArG boxes, and increased SRF protein expression. In contrast, MacLellan et al. (30) did not observe TGFbeta -induced changes in SRF binding to the skeletal alpha -actin SRE by gel shift analysis. It should be noted, however, that the proximal SRE of the skeletal alpha -actin promoter contains overlapping sites for SRE and YY1 and thus, SRF binding to the proximal SRE could have been influenced by YY1 competing with SRF binding.

Our observation that TGFbeta increased SRF in SMC suggests that TGFbeta -mediated activation of SM alpha -actin might be due in part to increased SRF expression and binding. Consistent with this, there is extensive evidence demonstrating that increased SRF expression and binding to CArG elements is associated with increased transcriptional activity of several genes including skeletal alpha -actin (62, 63), SM alpha -actin (64), and c-fos (65). The fact that TGFbeta -induced stimulation of SM alpha -actin mRNA expression was dependent on ongoing protein synthesis (Fig. 1) is consistent with a role for increased synthesis of SRF in mediation of TGFbeta inducibility of the SM alpha -actin gene. Alternative mechanisms, however, must also be considered whereby TGFbeta might mediate increased SRF binding. These include post-translational modifications of SRF (66), and interaction with homeodomain containing transcription factors that have been shown to modify binding of SRF to CArG elements (67). Of interest, we have recently demonstrated that MHox, the murine homologue of the homeodomain containing protein Phox 1 can potentiate binding of SRF to CArG B (68). It remains to be determined, however, whether TGFbeta influences MHox expression and/or activity, and whether MHox is involved in mediating TGFbeta effects on SM alpha -actin expression.

Our observations that TGFbeta also stimulated increased expression of SM MHC and h1 calponin raise the interesting possibility that it may function as a positive differentiation factor for SMC. Moreover, the fact that the promoters of each of these three genes contain paired CArG elements and at least one TCE-like element (Table I) suggest a potential common mechanism that might contribute to coordinate regulation of expression of these genes during SMC differentiation. Consistent with this, we have recently demonstrated functionality of the two SM MHC CArGs at -1103 and -1221 by site-directed mutagenesis (69). Moreover, with the possible exception of h1 calponin (70) CArG elements are also required for maximal expression of a number of other SMC differentiation marker genes including the SM-22alpha and h-caldesmon gene in cultured SMC (71, 72). The effects of mutations of the TCE-like elements on TGFbeta inducibility of the SM MHC, h1 calponin, and other SMC differentiation marker genes will need to be tested in further studies. Further evidence that TGFbeta might act as a positive differentiation factor in SMC is provided by studies of Shah et al. (17), who demonstrated that TGFbeta treatment stimulated differentiation of pluripotent neural crest stem cells into smooth muscle cells or SMC-like cells based on morphology and induction of expression of SM alpha -actin and calponin. Moreover, Grainger et al. (73) demonstrated that TGFbeta treatment inhibited down-regulation of SM MHC isoforms in primary cultures of SMC suggesting that TGFbeta might be involved in maintaining of SMC in a more differentiated state. However, in contrast to our results, TGFbeta failed to induce re-expression of SM MHC in subcultured SMC. The reason for these differences is not clear but may relate to culture methodologies (14). In particular, the studies reported by Grainger et al. (73) were done in the presence of serum, whereas ours involved initial growth arrest in a defined serum-free medium prior to TGFbeta stimulation.

A number of caveats need to be considered before concluding that TGFbeta might be a positive differentiation factor for SMC. First, our observation that TGFbeta also stimulated small increases in NM beta -actin expression would appear to be inconsistent with TGFbeta being a positive differentiation factor for SMC. However, in separate studies, we have shown that TGFbeta specifically increased SM alpha -actin mRNA expression when administered at lower concentrations than employed in the present studies (i.e. 0.1 ng/ml versus 2.5 ng/ml).3 Second, one must consider the possibility that the TGFbeta effects on SM differentiation marker expression may simply be secondary to growth effects. However, this seems to be highly unlikely, since TGFbeta had no effect on cell growth under the experimental conditions used in the present studies which involved initial growth arrest of SMC in a defined serum-free medium.

In summary, the present study showed that the two CArG elements A and B and a novel TCE were required for TGFbeta responsiveness of the SM alpha -actin gene. Regulation appears to involve SRF binding to the CArGs as well as an yet unidentified factor or factors that bind to the TCE in a TGFbeta -dependent manner. Identification of this factor may help to elucidate mechanisms that control SMC differentiation during vasculogenesis and in diseases such as atherosclerosis and injury-induced restenosis which are characterized by altered expression of multiple SMC differentiation proteins as well as TGFbeta 1.


FOOTNOTES

*   This work was supported by Grants RO1 HL 38854 and PO1 HL 19242 from the National Institutes of Health (to G. K. O.) and Fellowship Grants VA-94-F-14 (to M. H.) and VA-95-F-18 (to C. M.) from the Virginia Affiliate of the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, Box 10011, University of Virginia Health Sciences Center, Charlottesville, VA 22906-0011. Tel.: 804-924-2652; Fax: 804-982-0055.
1   The abbreviations used are: SMC, smooth muscle cell(s); SM MHC, smooth muscle myosin heavy chain; bp, base pair(s); CArG element, CC(A/T-rich)6GG; SRF, serum response factor; SRE, serum response element; CAT, chloramphenicol acetyltransferase; TGFbeta , transforming growth factor beta  1; TCE, TGFbeta control element; kb, kilobase pair(s); MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate.
2   M. M. Thompson and G. K. Owens, unpublished observations.
3   M. B. Hautmann and G. K. Owens, unpublished observations.

ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Diane Raines and Andrea Tanner.


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