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Am J Physiol Cell Physiol 275: C608-C618, 1998;
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Vol. 275, Issue 2, C608-C618, August 1998

Two MCAT elements of the SM alpha -actin promoter function differentially in SM vs. non-SM cells

Ellen A. Swartz, A. Daniel Johnson, and Gary K. Owens

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22906

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transcriptional activity of the smooth muscle (SM) alpha -actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM alpha -actin promoter are two MCAT motifs, binding sites for transcription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM alpha -actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significance. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resolution gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts from the different cell types, although no single band was unique to SM. Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple reactive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-related protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCAT elements may contribute to cell type-specific regulation of the SM alpha -actin gene. However, it remains to be determined whether the differential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins or to unique (cell type specific) combinatorial interactions of the MCAT elements with other cis-elements and trans-factors.

vascular smooth muscle cells; transcription enhancer factor 1; transcriptional regulation

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SMOOTH MUSCLE CELLS (SMC) in both atherosclerotic and myointimal lesions exhibit alterations in their differentiated state (52, 57). These changes include decreased expression of contractile proteins characteristic of SMC as well as altered growth responsiveness, changes in lipid metabolism, and increased production of extracellular matrix molecules. It is believed that such alterations in the differentiated state of the vascular SMC play a critical role in the progression of vascular disease. To understand the cellular and molecular regulation of differentiation, it is important to identify mechanisms that regulate the expression of genes that distinguish one cell type from another and are required for their differentiated function.

The process of differentiation requires the coordinate regulation of many sets of genes that enable the mature cell to perform its specialized functions (51, 69). Genes that are co-regulated often share cis-elements that are targets for common transcription factors. For example, studies of skeletal muscle development have led to the characterization of several important transcriptional regulatory factors, such as the MyoD family of helix-loop-helix factors and the myocyte-specific enhancer-binding factor-2 (MEF-2) family (for review, see Refs. 51, 69). It is likely that SMC differentiation is governed by an analogous system of transcriptional regulation by specific families of factors. However, as yet no smooth muscle (SM)-specific differentiation control factors have been identified.

Genes encoding the contractile proteins are candidates for studies of SM-specific transcriptional regulation (reviewed in Ref. 52). In particular, the SM myosin heavy chain, SM-22alpha , h1-calponin, and SM alpha -actin genes are appropriate for studies of SM-specific transcriptional regulation, since they are products of single genes and are required for contractile function of SMC. SM alpha -actin is the first known marker of differentiated SMC to be expressed in the developing vasculature (27), and it is the most abundant of the contractile proteins in mature vascular SMC. Although it is transiently expressed in developing skeletal and cardiac muscle (55, 70) and in myofibroblasts within tumors (8) and healing wounds (12), it is exclusively expressed in SMC in the normal adult animal (21, 70). Interestingly, SM alpha -actin expression is reduced in SMC within human atherosclerotic lesions (20, 23, 66), although the mechanisms that mediate reduced expression are presently unknown, including whether changes occur at the transcriptional or posttranscriptional level.

Earlier studies of the chicken, human, mouse, and rat SM alpha -actin gene promoters have demonstrated that regulation is cell specific and ultimately determined by a complicated orchestration of both positive and negative signals from interactions of cis-elements and trans-factors (5, 6, 47, 50, 61). The SM alpha -actin promoter contains a number of highly conserved cis-elements. For example, we (61) and others (38, 63, 68) have demonstrated that two highly conserved CArG boxes within the 5'-flanking region of the SM alpha -actin gene are required for tissue-specific transcription and that serum response factor (SRF) or an SRF-like protein binds to these elements (61). The SM alpha -actin promoter also contains two highly conserved MCAT elements at -184 (designated MCAT-1) and at -320 (designated MCAT-2), located in a region of the promoter, which, based on deletion analysis, contains elements that have negative regulatory activity within a SMC context (61). MCAT elements bind the transcription enhancer factor 1 (TEF-1) family of transcription factors (16, 62, 71) and have been implicated in the transcriptional activation of cardiac and skeletal troponin T (29, 43-45), skeletal alpha -actin (37, 42), beta -myosin heavy chain (18, 35, 36, 59, 60, 65), alpha -myosin heavy chain (24, 48), and the beta -acetylcholine receptor (2). Several nonmuscle promoters have also been shown to be regulated by MCAT elements, such as the viral SV40 enhancer (13, 28, 71), HPV-16 E6 and E7 oncogenes (30), and the human chorionic somatomammotropin (hCS) enhancer (31, 33, 67). MCAT-dependent regulation of gene expression has been shown to be extremely complex, involving binding of multiple different TEF-1 and TEF-1-related binding proteins (17). For example, there are four TEF-1 genes, including N-TEF-1, which encodes at least eight different isoforms (17, 62; P. Simpson, personal communication). In addition, MCAT-dependent regulation has been shown to involve multiple TEF-1 cofactors as well as interaction with other cis-regulatory elements, their binding factors, and basal transcriptional regulatory machinery (17, 62).

Nothing is known regarding the contributions of MCAT elements to transcriptional regulation in SMC. Strauch and colleagues (9, 63, 64) provided evidence of functionality of the MCAT-1-containing region of the murine SM alpha -actin gene promoter in AKR-2B fibroblasts. Based on promoter deletion studies, transcriptional activity in AKR-2B cells was shown to be limited to a construct containing the first 191 bp of the 5'-flanking sequence. Their interpretation was that transcriptional activity in AKR-2B fibroblasts was dependent on a positive element between -150 and -191 (this region includes MCAT-1) and removal of a negative element between -191 and -224 that suppressed promoter activity in AKR-2B and BC3H1 myoblasts (19, 63). Mutation of the MCAT-1 element in the context of the 191-bp construct decreased transcriptional activity in the AKR-2B cell line but had no effect on transcriptional activity in pre- and postconfluent myoblasts (9). Although these results demonstrated a transcriptional regulatory role for the MCAT-1 element of the 191-bp promoter construct in fibroblasts and myoblasts, studies did not address the function of either MCAT-1 or MCAT-2 in SMC or the function of MCAT-2 in non-SMC. The aims of the present study were 1) to determine whether the two MCAT elements of the rat SM alpha -actin promoter are important in cell type-specific transcriptional regulation and 2) to perform initial characterization of the interactions of nuclear proteins with these cis-elements.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of mutant promoter-CAT expression plasmids. Site-directed mutagenesis was performed using the Altered Sites II in vitro mutagenesis system (Promega) as recommended by the manufacturer. The oligonucleotides that were used to mutate the alpha -actin promoter sequence were as follows (underlined bases show mutations): mutated MCAT-1 oligo, 5'-P-GGTCTCTTCCACTG<UNL>GG</UNL>T<UNL>A</UNL>CCTCTGCTCTGCTC-3'; mutated MCAT-2 oligo, 5'-P-AGGCATGGTTTGCA<UNL>GG</UNL>T<UNL>A</UNL>CCTCAGAAGATGCC-3'. The mutants were subcloned into p371CAT, the pCAT-Basic plasmid (Promega) containing the first 371 bp of the rat SM alpha -actin promoter. The mutant clones were sequence verified by the Sanger dideoxy sequencing procedure (56) using a Sequenase kit (US Biochemical).

All promoter-CAT plasmid DNAs used for transient transfections were prepared by an alkaline lysis procedure (4), followed by banding on two successive ethidium bromide-cesium chloride gradients. The integrity of each plasmid preparation was examined by electrophoresis on 1% agarose gels, and preparations were judged acceptable if >50% of the DNA was supercoiled and the relative amount of supercoiled to nicked plasmid DNA was roughly the same for all constructs used in the same set of transfections.

Cell culture. SMC from rat thoracic aorta were isolated and cultured as previously described (22). Rat aortic SMC used in the present studies were between passages 13 and 30 and cultured under conditions that we have previously shown to maximize expression of a variety of SM-specific proteins including SM alpha -actin (53), SM myosin heavy chain (54), SM myosin light chain (49), and SM alpha -tropomyosin (26). Rat L6 skeletal myoblasts, originally isolated by Yaffe (72), were obtained from the American Type Culture Collection and cultured as recommended. L6 myoblasts were maintained in 10% FBS-containing medium. Myoblast differentiation into myotubes was induced by reducing the FBS concentration to 1% when cells reached confluence and by maintaining cells for a minimum of 3 days. More than 70% of myoblasts were induced to differentiate into myotubes by this procedure. Bovine aortic endothelial cells (EC) were isolated (41) and cultured (5) as previously described. AKR-2B mouse embryonic fibroblasts were the gift of Dr. Harold Moses (Vanderbilt University, Nashville, TN) and were cultured as previously described in McCoy's 5A medium (63).

DNA transfections. Cells for transient transfection assays were plated into six-well plates (Corning Glass, Corning NY) at a density of 2 × 104 cells/cm2 for SMC and for AKR-2B, 3 × 104 cells/cm2 for EC, and 4 × 104 cells/cm2 for L6 myoblasts and myotubes. These densities were chosen so that the cells would be at 60-80% confluence at the time of transfection at 20-22 h postplating. Transient transfection experiments consisted of transfecting each promoter-CAT plasmid in triplicate using DOTAP reagent according to the manufacturer's recommendations (Boehringer Mannheim) or Transfectam (Promega) for EC as previously described (61): specifically, 4 µg DNA plus 30 µl DOTAP/well for SMC, myoblasts, and myotubes; 3 µg DNA plus 15 µl DOTAP for AKR-2B; and 2.5 µg DNA plus 7.5 µl Transfectam for EC. After 48 h from the time of transfection, the cells were washed twice in 4 ml of ice-cold PBS, harvested by scraping in harvesting buffer of 40 mM Tris (pH 7.5), 1 mM EDTA, and 150 mM NaCl, pelleted, resuspended in 100 µl 25 mM Tris (pH 7.5), and stored at -70°C.

Reporter gene assays. Extracts from transfected cells were prepared by three freeze-thaw cycles, followed by centrifugation to remove cellular debris. The supernatant was heat inactivated at 65°C for 15 min and then pelleted, and a 55-µl aliquot was assayed for CAT activity by enzymatic butyrylation of tritiated chloramphenicol (DuPont-NEN) as previously described (58). All CAT activity values were normalized to the protein concentration of each cell lysate as measured by the Bio-Rad microtiter plate assay. Early transfection studies were performed by cotransfection with a beta -galactosidase (beta -Gal) vector to correct for changes in transfection efficiency. Because the beta -Gal measurements did not result in qualitative changes in the data and viral-driven beta -Gal genes could potentially compete for limited trans-factors that regulate SM alpha -actin transcription (15), the cotransfections were discontinued. In each experiment, a promoterless CAT construct, pCAT-Basic, was transfected to serve as the baseline indicator of CAT activity; the activity of the other constructs was expressed relative to pCAT-Basic set equal to one, unless otherwise stated. In addition, an SV40 enhancer/promoter-CAT construct, pCAT-Control, served as a positive control of transfection and CAT activity. All CAT activity values represent at least three independent transfection assays with each construct tested in triplicate per experiment and are expressed as means ± SE. Data from replicate independent experiments for each cell type were analyzed using a two-factor ANOVA with "experimental group" differences as the main effect and the "experimental day" as the interacting factor. The latter was necessary due to experiment-to-experiment differences in the absolute levels of reporter activity that are common in transient transfection experiments and would result in a nonparametric distribution if experimental data were simply pooled and analyzed by one-way ANOVA. Where appropriate, post hoc comparisons between experimental groups for a given cell type were made using a Student-Newman-Keuls multicomparison test. Values of P < 0. 05 were considered statistically significant.

Preparation of nuclear extracts and electrophoretic mobility shift assays. Crude nuclear extracts (NE) were purified by the method of Dignam et al. (14) with the addition of 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma) to all solutions. The protein concentration of each NE was measured by the microtiter plate assay of Bio-Rad. The HeLa cell NE were purchased commercially (Santa Cruz Biotechnologies, Santa Cruz, CA).

Oligonucleotides were synthesized and HPLC purified by Operon Technologies (Alameda, CA). The 17-mer MCAT oligonucleotides consisted of the 7-bp motif plus the additional 5 bp immediately 5' and 3' to the motif. Complementary strands were end labeled separately with T4 polynucleotide kinase (Promega) and [gamma -32P]ATP (6,000 Ci/mmol, DuPont-NEN) and annealed. Unincorporated nucleotides were removed by Nuc-Trap push columns (Stratagene), and the oligonucleotides were gel purified. The appropriate bands were excised, eluted from the acrylamide overnight at 37°C, precipitated, and resuspended in Tris-EDTA.

Electrophoretic mobility shift assays (EMSA) were performed in a 20-µl binding reaction that contained ~50 pg probe, 8 µg NE, 10 mM Tris (pH 7.5), 5 mM HEPES, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2 µg poly(dIdC), 10% glycerol, and competitor oligonucleotides when indicated. Reactions were incubated 20 min at room temperature, loaded onto a 5% acrylamide gel (30:1 acrylamide/bis-acrylamide), which had been prerun at 170 V for 30 min, and electrophoresed at 170 V in 0.5× TBE (45 mM Tris borate-1 mM EDTA) for 1.5 h. Gels were dried onto filter paper and exposed to film at -70°C. For supershift assays, 2 µg of NE were preincubated for 30 min with 1.5 µl of an IgY antibody (6 µg/µl) to a GAL-4/TEF-1-related protein recombinant fusion protein (antibody provided by N. Shimizu, University of South Carolina, Columbia, SC; Ref. 73) or control chicken IgY (Charles River). After addition of radiolabeled probe, the samples were incubated another 15 min at room temperature before electrophoresis. High-resolution EMSA were similarly performed (17) with the following modifications: binding reactions consisted of 3 ng probe, 15 µg NE, and 3 µg poly(dIdC). Reactions were run on a 6% acrylamide gel (44:1 acrylamide/bis-acrylamide) for 4 h.

Western analysis. NE (15 µg) were boiled for 2 min, centrifuged 3 min to pellet debris, and separated by SDS-PAGE on an 11% gel. After transfer to nitrocellulose (Amersham), membranes were probed with polyclonal antisera to TEF-1 kindly provided by I. Farrance (Veterans Affairs Medical Center, San Fransisco, CA; Ref. 62), followed by a secondary antibody direct conjugate donkey alpha -rabbit-peroxidase (Amersham) and detected using enhanced chemiluminescence (Amersham) as described previously (17).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SM alpha -actin MCAT elements exhibited differential activity in SMC vs. non-SMC. The SM alpha -actin promoter contains two MCAT elements, designated MCAT-1 (-184 to -178) and MCAT-2 (-320 to -314), that are highly conserved across mammalian species (61). To evaluate the functional significance of the MCAT motifs in the transcriptional regulation of the rat SM alpha -actin gene, mutations of either or both elements were made in the context of the first 371 bp of the promoter linked to a CAT reporter gene (Fig. 1). The mutations tested were based on the work of Mar and Ordahl (44), who showed that these mutations abolished activity of the cTNT promoter in transfection assays and disrupted binding in footprinting assays. To assess the transcriptional activity of the constructs, wild-type and mutant promoter-CAT plasmids were transiently transfected into cultured rat aortic SMC, which express high levels of the endogenous SM alpha -actin gene, and several non-SMC lines. The latter included 1) AKR-2B mouse embryonic fibroblasts, which do not normally express the endogenous SM alpha -actin gene but have been shown to express a promoter/reporter construct containing 191 bp of the 5'-region of the SM alpha -actin promoter in response to serum stimulation (63), and 2) L6 skeletal myoblasts, which express SM alpha -actin at very low levels but show marked induction of expression when induced to differentiate into myotubes (1), and bovine aortic EC, which do not express the endogenous gene but do express the p371CAT reporter construct at modest levels (61). Results of transient transfections demonstrated that mutations of the MCAT elements had different effects in different cell lines (Fig. 2). In SMC, the p371CAT wild-type construct had approximately eightfold greater activity than a promoterless control construct. Mutation of the MCAT-1 element resulted in a twofold increase in activity compared with the wild-type construct, whereas mutation of MCAT-2 led to a threefold increase (Fig. 2). Simultaneous mutation of both the MCAT-1 and -2 elements resulted in activity equivalent to that of the MCAT-1 mutation, rather than having an additive effect. This suggests that at least part of the increase in activity seen with the MCAT-2 mutation was dependent on having an intact MCAT-1 element or that mutation of the MCAT-1 element resulted in a state of promoter upregulation which was stable and could not be altered by additional mutation of MCAT-2. Taken together, data demonstrate that, in the context of the first 371 bp of the promoter, the two MCAT elements function as repressors of transcription in SMC in that mutation of either or both resulted in a two- to threefold increase in activity compared with the wild-type promoter.


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  		Fig. 1.   A: schematic illustration of MCAT mutations of rat smooth muscle (SM) alpha -actin promoter tested in present studies. Site-directed mutations were introduced using an Altered Sites in vitro mutagenesis system (Promega) into a construct containing first 371 bp of rat SM alpha -actin promoter linked to a CAT reporter gene. B: sense strand sequences of MCAT oligonucleotide probes used in electrophoretic mobility shift assays (EMSA).


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  		Fig. 2.   Effects of MCAT mutations in SM alpha -actin p371CAT constructs transfected into SM cells (SMC) and non-SMC (EC, endothelial cells; AKR-2B, AKR-2B fibroblasts; tubes, myotubes; blasts, myoblasts). Subconfluent cultures were transiently transfected with wild-type p371CAT constructs or with constructs containing mutations of MCAT-1, MCAT-2, or both elements. CAT activities were expressed relative to that of promoterless control (pCAT-Basic) set to one. An SV40 enhancer/promoter-CAT construct served as a positive control for transfection and CAT activity (not shown). Data represent means ± SE of 5 independent experiments for SMC, 4 for myoblasts, and 3 for other non-SMC lines, each performed with triplicate samples/plasmid construct. Statistical analyses were performed by 2-way ANOVA, followed by a Student-Newman-Keuls multicomparison test as described in MATERIALS AND METHODS, with P < 0.05 considered significant. * Significantly different from wild-type p371CAT; + significantly greater than pM1 or pM1M2; ddager  significantly greater than pM1M2; # significantly greater than pM2 or pM1M2.

In contrast to observations in SMC, mutations of the MCAT elements had either no effect or decreased transcriptional activity in L6 myotubes, and EC (Fig. 2). In L6 myotubes, mutation of the MCAT-1 motif, either alone or in combination with the mutation of MCAT-2, resulted in significant decreases in transcriptional activity, whereas mutation of the MCAT-2 element alone had no effect. In EC, mutation of the MCAT-2 element alone or in combination with MCAT-1 resulted in a marked decrease in activity, whereas mutation of the MCAT-1 element alone had no effect. In AKR-2B fibroblasts and L6 myoblasts, mutation of MCAT-1 or both MCAT-1 and -2 had no effect, whereas mutation of the MCAT-2 element alone resulted in a statistically significant increase in reporter activity. However, the biological significance of the latter observation is uncertain given the extremely low activity of all constructs in these cell types.

In summary, the preceding results provide clear evidence of differential function of the SM alpha -actin MCAT elements in the various cell types tested. The elements functioned as potent repressors in SMC, whereas they acted as activators in L6 myotubes and EC. Moreover, we found evidence of differential function of MCAT-1 vs. MCAT-2 within a given cell type as indicated by observations of differential effects of mutation of MCAT-1 vs. MCAT-2 in SMC, EC, and L6 myotubes. Finally, we found evidence for interaction of the two MCAT elements in SMC, in that maximal effects of MCAT-2 mutations were dependent on the presence of an intact MCAT-1.

MCAT elements bound nuclear factors from SMC and non-SMC. To study the interaction of proteins with the MCAT elements of the SM alpha -actin promoter, EMSA were performed with NE from both SMC and non-SMC. Double-stranded 17-mer oligonucleotide probes, containing the 7-bp MCAT motif plus 5 bp of the 5'- and 3'-flanking sequence, were end labeled with 32P. Wild-type MCAT probes formed several shift bands with SMC NE (Fig. 3A, lanes 2 and 9) that were abolished with addition of excess cold wild-type oligonucleotides (lanes 3-5 and 10-12). However, mutant oligonucleotide competitor DNA at a 250-fold molar excess failed to compete for binding (lanes 6-7 and 13-14).


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  		Fig. 3.   Binding of SMC nuclear factors to wild-type MCAT-1 and -2 oligonucleotide probes (A) or binding of non-SMC factors to MCAT-1 (B) or to MCAT-2 (C) oligonucleotide probes. MCAT oligonucleotide probes (17 bp) were end labeled with [gamma -32P]ATP and T4 polynucleotide kinase and annealed to form double-stranded duplexes. After gel purification, each radiolabeled probe was incubated with 8 µg nuclear extract (NE). Competitions were performed with 17-bp double-stranded oligonucleotides of wild-type (wt) or mutated (mut) MCAT elements, added as labeled for A at a 50- to 250-fold molar excess relative to labeled DNA probe and for B and C at a 100-fold molar excess for wt and at a 250-fold excess for mut.

EMSA were also performed using both MCAT-1 and -2 probes and NE from the non-SMC lines (Fig. 3, B and C). Shift complexes were seen with extracts from each cell type that were competed for by wild-type but not mutant oligonucleotides. Results demonstrated that nuclear factors from each of the cell lines specifically bound the MCAT-1 and -2 oligonucleotide probes.

Although the MCAT elements possessed different functional activities in different cells (Fig.2), under the conditions of our initial EMSA analysis, shift complexes of roughly equivalent mobility were seen with extracts from each of the cell lines. To better distinguish the mobilities of the shift complexes, high-resolution gel shifts were performed using the methods of Farrance and Ordahl (17). With this technique, the broad bands were resolved into two, three, or four bands, dependent on the origin of the NE (Fig. 4, A and B). As a positive control, shift analyses included use of HeLa cell NE, which are known to contain multiple TEF-1 or TEF-1-related proteins that bind MCAT elements (13, 71). Because the MCAT elements functioned as negative regulatory elements in SMC but either had no effect or functioned as positive elements in the non-SMC lines, the focus was on determining whether there were differences in shift complexes between SMC and non-SMC. The mobilities of complexes formed with the MCAT-1 and -2 oligonucleotide probes appeared to be the same within each cell type with the exception of the highest mobility shift band seen with the MCAT-1 probe with HeLa cell NE that was absent with the MCAT-2 probe (Fig. 4, cf. A and B). However, there were both similarities and differences in binding between the different cell lines. For example, the doublet bands in the SMC lane comigrated with the shift complexes in the AKR-2B, myoblast, and myotube lanes (lanes 1-4). In addition, although both bands were present in SMC, AKR-2B, myoblasts, and myotubes, the relative intensities of the bands varied (Fig. 4, A and B, lanes 1-4). NE from EC formed three shift complexes, whereas HeLa cell NE formed four shift complexes (Fig. 4, A and B, lanes 5-6). The uppermost shift complex in the HeLa cell lane was unique to that cell line. The lower three bands from the HeLa cell binding reactions comigrated with those in EC but not in the other cell types. Taken together, results of EMSA showed clear differences in MCAT binding complexes between the various cell types tested that could contribute to the functional differences in activity of MCAT elements observed (Fig. 2). However, there was no single shift band that was unique to SM, thus suggesting that the mechanisms that control the differential activity of these elements are likely to be complex and involve interaction of multiple cis-elements and trans-factors.


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  		Fig. 4.   Binding of SMC and non-SMC nuclear factors to MCAT-1 (A) and MCAT-2 (B) oligonucleotide probes. High-resolution EMSA were performed as described in MATERIALS AND METHODS.

NE from SMC and non-SMC lines contained multiple proteins in 50- to 60-kDa size range that cross-reacted with antibodies to TEF-1. Because there were multiple and different complexes formed in the high-resolution EMSA, we hypothesized that there might be multiple TEF-1 or TEF-1-related proteins responsible for the formation of these different complexes. To ascertain the presence of TEF-1 family members in SMC and non-SMC, NE were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with TEF-1 antiserum (generous gift of I. Farrance). The antiserum was raised against pooled peptide sequences derived from conserved regions of the TEF-1 gene family so that the antiserum would have a broad specificity for diverse TEF-1 proteins (17). Results of immunoblot analyses with this TEF-1 antibody showed that NE from each of the cell lines tested contained proteins that were antigenically related to TEF-1 and that there were differences in the mobilities of the immunoreactive bands (Fig. 5). Of particular interest, a single distinct reactive band was seen with SMC NE that was not seen with NE from any of the other cell types tested with the possible exception of HeLa cells. These observations are provocative and provide a potential basis to explain the differential functional activity of the SM alpha -actin MCAT elements in SMC vs. non-SMC (Fig. 2). However, whereas results of these Western analyses suggest that different complements of TEF-1-related proteins exist in SMC vs. non-SMC extracts, they do not provide evidence that such proteins actually bind to the MCAT elements. It is also possible that at least some of the differences in mobility of TEF-1 immunoreactive proteins reflect species differences rather than unique TEF-1-related proteins.


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  		Fig. 5.   Anti-transcription enhancer factor 1 (TEF-1) Western blot analysis of SMC and non-SMC NE. Extracts were subjected to SDS-PAGE, transferred to nitrocellulose, immunoblotted with rabbit polyclonal TEF-1 antisera (gift of I. Farrance), incubated with donkey anti-rabbit secondary antibody (Amersham), and detected using enhanced chemiluminescence. Positions of molecular mass standards are indicated. In each cell line, TEF-1 immunoreactive bands were observed in 50- to 60-kDa range. No bands were observed when TEF-1 antisera were excluded from blotting protocol (data not shown).

SM alpha -actin MCAT elements bound TEF-1 or TEF-1-related proteins present in SMC and non-SMC NE. To determine whether TEF-1 or TEF-1-related proteins were present in the MCAT shift complexes, we attempted supershift analyses with the same antibody employed in the Western analyses shown in Fig. 5. Despite repeated attempts under many gel shift conditions and using various antibody and NE concentrations, no effect on MCAT-1 or -2 shift complexes was observed. However, this antibody is known to be of relatively low affinity and does not work well in supershift analyses unless the site affinity for TEF-1 is extremely high (I. Farrance and P. Simpson, personal communication). As such, we repeated supershift assays employing a polyclonal chicken IgY antibody raised against a mouse GAL-4/TEF-1-related protein fusion protein (a generous gift of N. Shimizu). This antibody has previously been shown to react specifically with recombinant TEF-1 proteins under the EMSA conditions employed in the present studies (73). Addition of this antibody to gel shifts resulted in formation of supershift complexes with each of the cell extracts, and reduced but did not abolish the shift complexes. Binding reactions lacking NE but including antisera did not contain any shifted complexes (data not shown). Control chicken IgY antibodies did not affect the shift complexes (Fig. 6, A and B). These results suggest that at least some of the MCAT binding factors are TEF-1 or TEF-1-related proteins, thus implicating a possible role for this family of transcription factors in control of cell type-specific expression of SM alpha -actin. However, results do not identify which, if any, of the known TEF-1 or TEF-1-related protein isoforms are involved. In addition, our observations that MCAT shift complexes were not abolished by inclusion of high concentrations of antibody in the EMSA reaction leave open the possibility that non-TEF-1 proteins and/or TEF-1 family members that are not recognized by the GAL-4/TEF-1-related protein antibody may be involved as well.


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  		Fig. 6.   Characterization of nuclear factor binding to MCAT-1 (A) or MCAT-2 (B) elements. NE (2 µg) from SMC and non-SMC were incubated for 30 min at room temperature with a chicken polyclonal antibody raised against a fusion protein consisting of GAL-4/TEF-1-related protein (T; gift of N. Shimizu) or a control chicken IgY antibody (C). After addition of MCAT oligonucleotide probes, reactions were incubated for another 15 min before electrophoresis. Supershifted bands are indicated by arrows.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is well established that TEF-1 protein interactions with MCAT elements are involved in and are sometimes required for the cell-specific transcriptional activation of many cardiac and skeletal muscle-specific genes in vitro (2, 18, 24, 29, 35, 37, 42-45, 48, 59, 60, 65). However, our report is the first to demonstrate a transcriptional regulatory role for MCAT elements in SMC. Results of the present studies demonstrated that the MCAT motifs were important for the transcriptional activity of the SM alpha -actin promoter/reporter constructs and that these elements had surprisingly different effects in SMC vs. non-SMC. In SMC, the MCAT elements functioned as repressors, whereas, in L6 myotubes and EC, the MCAT elements acted as activators (Fig. 2). To our knowledge, this is the first time that an MCAT element has been shown to serve as a negative regulatory element in the context of a muscle-specific promoter, although there are several reports of MCAT elements exerting repressor effects in nonmuscle promoters. For example, Berger et al. (3) presented evidence indicating that TEF-1 functioned as a repressor of the SV40 late promoter. Jiang and Eberhardt described repression by TEF-1/MCAT interactions of the hCS gene enhancer (32) and heterologous RSV and TK promoters in BeWo choriocarcinoma cells (33). However, in none of the preceding cases have the molecular mechanisms responsible for MCAT repressor activity been elucidated.

The mechanisms by which MCAT elements mediate the repression of SM alpha -actin transcription in SMC are also not known at this time. Moreover, the importance of the MCAT elements in repressing SM alpha -actin transcription under physiological or pathological circumstances is unclear. Unfortunately, studies in this area are hampered by the lack of a model system in which the SM alpha -actin promoter is repressed at the transcriptional level. There is extensive evidence showing that SM alpha -actin expression can be repressed in a highly selective manner by stimulation with platelet-derived growth factor BB in cultured SMC (10, 11). However, decreases in expression of the protein under these circumstances are mediated at the posttranscriptional level through mRNA and protein destabilization, not a change in transcription rate as measured by run-on analyses. Likewise, whereas it is well established that expression of SM alpha -actin, as well as other SMC differentiation markers, is reduced within intimal SMC in vivo in response to vascular injury or in atherosclerotic lesions, it is unclear if these changes are mediated at the transcriptional level (52). As such, there is no system currently available with which to test for the repressor function of the MCAT elements of the SM alpha -actin promoter.

Results of our EMSA analyses demonstrated that multiple and different shift complexes were formed between MCAT oligonucleotide probes and NE from SMC and non-SMC (Figs. 3 and 4). However, consistent with extensive studies of the role of MCAT elements in multiple skeletal- and cardiac-specific genes (17), our results provided no simple or obvious explanation for the differential effects of MCAT-1 and -2 mutations in different cell types. For example, similar shift bands were seen between SMC and L6 myotubes (Fig. 4, A and B, lanes 1 and 4), despite the fact that MCAT mutations had opposite effects in these two cell types, i.e., a two- to threefold increase in activity in SMC but a 60% reduction in activity in L6 myotubes (Fig. 2). There are several plausible mechanisms, which are not mutually exclusive, to explain these observations. First, differences in functional activity may be the result of binding of different TEF-1 family members or their splice variants that are not resolvable in mobility shift assays. This is certainly possible given the known complexity of the TEF-1 gene family (17, 62) and results of Western analyses in the present studies suggesting that SMC may express a unique TEF-1-related protein (Fig. 5). There is also direct precedence for this mode of MCAT-dependent regulation. For example, Stewart et al. (62) described the isolation of two splice variants of chicken RTEF-1 and demonstrated that chimeric proteins containing the activation domains of these isoforms possessed differing abilities to transactivate GAL-4-dependent reporter constructs. A second possibility is that the transcriptional activity of TEF-1 proteins may be differentially regulated in different cell types by posttranslational modifications. Farrance and Ordahl (17) found that three species of TEF-1 proteins in chicken primary muscle cultures were differentially phosphorylated and suggested that these modifications may modulate the interaction of TEF-1 proteins with MCAT elements or with cofactors to provide cell-specific transcriptional activity. A third possiblity is that the observed differences in binding properties and functional activities of the SM alpha -actin MCAT elements may be due to 1) binding of TEF-1 cofactors, 2) binding of factors other than TEF-1 proteins, and/or 3) combinatorial interactions with other regulatory cis-elements within the promoter and their binding factors. A recent report by Larkin et al. (40) demonstrated that the sequences flanking the MCAT motifs of the cardiac troponin T promoter directed the binding of TEF-1 cofactors that conferred tissue-specific activity to promoter/reporter constructs in cultured cells. Indeed, cofactors that are required for TEF-1 activity have been partially purified from HeLa cells (7, 48). Jiang and Eberhardt (32) reported that COS and BeWo cells contain a protein distinguishable from TEF-1, designated CSEF-1, that binds to the MCAT elements of the hCS gene enhancer. Importantly, activation of the skeletal alpha -actin promoter in response to alpha 1-adrenergic stimulation required not only the MCAT element, but also the CArG and Sp1 elements (37). Furthermore, studies published by the Robbins laboratory (39) demonstrated that in vivo, combinatorial interactions between the MCAT, C-rich, and beta e3 elements were required for high level tissue-specific activity of the beta -myosin heavy chain gene promoter. Of interest, our laboratory has previously demonstrated that transcriptional activity of the SM alpha -actin gene in SMC is dependent on multiple cis-elements. This includes two highly conserved CArG elements at -62 and -112 (61), a novel transforming growth factor-beta 1 control element at -42 (25), and at least one of two E-box elements at -214 and -252 respectively (34) (see Fig. 7). Although direct evidence is lacking, it is thus possible that the differential function of MCAT elements in SMC is dependent on interaction with one or more of these cis-elements in a manner analogous to the MCAT elements found in a number of skeletal- and cardiac-specific gene promoters (37, 39). However, in contrast to these genes, a unique feature of the SM alpha -actin MCAT elements is that they act as cell type-specific repressor elements rather than activators. As such, the repressor activity of the MCAT elements within a SMC context may be due to interaction with one or more of the known positive regulatory elements of the promoter, an as yet unidentified positive regulatory element, or with the basal transcriptional machinery (or some combination thereof). Thus elucidation of the mechanisms whereby the SM alpha -actin MCAT elements act as repressors is likely to be difficult and will require clear identification of the specific TEF-1 proteins and cofactors that bind to these control elements and how these factors influence other control elements within the promoter.


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  		Fig. 7.   Regulatory elements of SM alpha -actin gene. Schematic summary of various cis-elements that are highly conserved across species and have been shown to influence expression of SM alpha -actin gene in cultured SMC and/or non-SMC. Differential functional activity of SM alpha -actin MCAT elements in SMC vs. non-SMC may reflect 1) differences in expression of TEF-1 or TEF-1-related proteins that bind MCAT elements and/or 2) unique combinatorial interactions of MCAT elements with other cis-elements and trans-factors that are cell type specific (i.e., identical TEF-1 proteins may exhibit opposite activities via MCAT elements in different cell contexts). 5'-Region CArG-like elements have been shown to exhibit cell specificity in that they are required for transcriptional activity in SMC and L6 myotubes but not in non-SMC, including EC (19, 61). The intronic CArG element has been shown to be contained within a larger conserved region that has enhancer activity (50), although specific boundaries of this enhancer and whether it exhibits SMC specificity have not yet been determined. The TGF-beta 1 control element (TCE) has been shown to be required for both basal and TGF-beta 1-induced expression of SM alpha -actin in cultured SM cells (25). However, its role in regulation of SM alpha -actin expression in non-SMC has not been reported. E-box elements (designated E1 and E2) have been shown to be required for expression in L6 myotubes, and also exhibit some positive activity in SMC (34). The TGTTT element has been shown to exhibit positive transcriptional activity based on mutational analysis of chicken SM alpha -actin promoter in rat SMC (46) but actually exhibits negative activity within a homologous transfection system (rat SM alpha -actin promoter in rat SMC; F. Jung, M. Hautmann, B. Wei, A. D. Johnson, G. K. Owens, and C. McNamara, unpublished observation). Nucleotide sequence of each of these elements in rat, mouse, human, and chicken can be found in Shimizu et al. (61).

In conclusion, we have demonstrated that the MCAT elements of the rat SM alpha -actin promoter bind TEF-1 or TEF-like proteins and contribute to the differential regulation of transcription of this gene in different cell types (61). The elements acted as repressors in SMC, whereas they had either no effect or functioned as activators in non-SMC. Although the precise mechanisms that govern this cell-specific regulation remain to be elucidated, this report is the first to demonstrate functional activity of the SM alpha -actin MCAT motifs within SMC. Moreover, it is the first report, to our knowledge, to show a repressor activity of MCAT elements in a muscle-specific promoter context. These studies underscore the utility of the SM alpha -actin promoter in dissecting the variable and very complex interactions between MCAT elements and the large family of TEF-1 and TEF-1-related proteins, although it remains to be determined whether and by what mechanisms the MCAT elements mediate transcriptional repression of this gene in vivo under conditions in which expression of the SM alpha -actin gene is decreased at the transcriptional level.

    ACKNOWLEDGEMENTS

We thank Dr. Iain Farrance for the gift of the rabbit TEF-1 antiserum and his very helpful comments and suggestions; Dr. Noriko Shimizu for sharing the chicken GAL-4/TEF-related protein antibodies; Drs. Alexandre Stewart and Paul Simpson for helpful discussions; Dr. Harold Moses for the AKR-2B cell line; and Andrea Tanner, Diane Raines, and Jennifer Clatterbuck for outstanding technical advice and assistance.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants 5T32-HL-07824-19 (E. A. Swartz), F32-HL09648-01 (to A. D. Johnson), RO1-HL-38854 (to G. K. Owens), and PO1-HL-19242 (to G. K. Owens).

Address for reprint requests: G. K. Owens, Dept. of Molecular Physiology and Biological Physics, PO Box 10011, Charlottesville, VA 22906-0011.

Received 22 October 1997; accepted in final form 18 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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