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(Circulation Research. 1997;81:600-610.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II–Induced Stimulation of Smooth Muscle -Actin Expression by Serum Response Factor and the Homeodomain Transcription Factor MHox

Martina B. Hautmann, Maria M. Thompson, Ellen A. Swartz, Eric N. Olson, , Gary K. Owens

From the Department of Molecular Physiology and Biological Physics (M.B.H., M.M.T., E.A.S., G.K.O.), University of Virginia Health Sciences Center, Charlottesville, and the Hamon Center for Basic Cancer Research (E.N.O.), The University of Texas Southwestern Medical Center, Dallas.

Correspondence to Dr Gary K. Owens, Department of Molecular Physiology and Biological Physics, Box 449, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail gko{at}virginia.edu

	Abstract

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Abstract The objective of the present study was to examine the molecular mechanisms whereby angiotensin II (Ang II) stimulates smooth muscle (SM) -actin expression in rat aortic smooth muscle cells (SMCs). Nuclear run-on analysis and transfection studies indicated that the effects of Ang II on SM -actin were mediated at least in part at the transcriptional level. Transfection of various rat SM -actin promoter/chloramphenicol acetyltransferase (CAT) constructs into SMCs demonstrated that the first 155 bp of the SM -actin promoter was sufficient to confer maximal Ang II responsiveness, conferring an 4-fold increase in reporter activities in these SMCs compared with vehicle-treated SMCs. Mutation of either of two highly conserved CArG elements, designated A (-62) and B (-112), completely abolished Ang II–induced increases in reporter activity, whereas mutation of a homeodomain-like binding sequence at -145 (ATTA) reduced reporter activity by half. Results of EMSAs showed that nuclear extracts from Ang II–treated SMCs exhibited enhanced binding activity of serum response factor (SRF) to the CArG elements and of a homeo-domain factor, MHox, to the ATTA element. Northern analyses showed that Ang II also stimulated marked increases in MHox mRNA levels. Western analyses demonstrated that Ang II–induced increases in SRF binding were not due to increased SRF protein expression. Recombinant MHox markedly enhanced binding activity of SRF in EMSAs. Finally, MHox overexpression transactivated a SM -actin promoter/CAT reporter construct by 3.5-fold in transient cotransfection studies. These results provide evidence for involvement of a homeodomain transcription factor, MHox, in Ang II–mediated stimulation of SM -actin via a CArG/SRF-dependent mechanism.

Key Words: smooth muscle -actin promoter • vascular smooth muscle cell • angiotensin II • MHox

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arteries from hypertensive patients and animals have a greater SMC mass than those from their normotensive counterparts.^{1 2 3 4} The arterial thickening is believed to represent an important adaptive response to normalize the elevated wall stress that occurs secondary to increased blood pressure.³ Previous studies in this and other laboratories^{4 5} have demonstrated that the growth response of SMCs in hypertension varies as a function of the blood vessel examined and the specific model of hypertension studied. For example, medial hypertrophy in the aorta of spontaneously hypertensive rats⁶ or two-kidney, one clip Gold-blatt hypertensive rats⁷ was due almost exclusively to hypertrophy of preexisting SMCs, with little to no change in SMC number. In contrast, medial hypertrophy in intermediate-sized resistance vessels in the spontaneously hypertensive rat was the consequence of increased cell number.^{8 9} Results of drug intervention studies have implicated a role for Ang II in the mediation of SMC hypertrophy in large vessels during chronic hypertension.¹⁰ Angiotensin-converting enzyme inhibitors and Ang II receptor antagonists have been shown to inhibit development of SMC medial hypertrophy in a variety of hypertensive animal models.^{6 11 12} Of particular interest, effects of angiotensin-converting enzyme inhibitors were not simply due to blood pressure lowering, since other antihypertensive drugs were not as efficacious in blocking hypertrophy despite inducing similar reductions in blood pressure.⁶ Consistent with in vivo studies, we¹³ and others¹⁴ have shown that Ang II stimulated a dose-dependent increase in protein synthesis and cellular hypertrophy in cultured SMCs.

There has been considerable interest in identifying the molecular mechanisms whereby Ang II stimulates SMC hypertrophy. Previous studies from our laboratory have demonstrated that Ang II–induced hypertrophy of SMC is associated with generalized increases in protein synthesis and content, as well as increased rRNA content,^{13 15} suggesting that part of the regulation is through alterations in translational control. Hershey et al¹⁶ demonstrated that Ang II markedly increased 18S rRNA transcription and also stimulated increased phosphorylation and nuclear localization of upstream binding factor, which is believed to be an important control step in regulation of rRNA transcription in response to serum or growth factor stimulation.^{17 18 19} Ang II has also been shown to stimulate phosphorylation of 4E-binding protein 1²⁰ and translation initiation factor 4E,²¹ factors involved in control of translation initiation, indicating that Ang II modulates protein synthesis at multiple levels. In addition to these effects on overall protein synthesis, Ang II has also been shown to stimulate selective increases in the expression of a number of contractile proteins, including SM -actin, SM -tropomyosin, and SM MHC, that far exceed overall increases in cellular protein.²² For example, Ang II–induced stimulation of SM -actin expression was accompanied by a 5- to 8-fold increase in SM -actin mRNA. Previous studies, however, have not identified the mechanisms whereby Ang II upregulates SM -actin mRNA levels or determined whether increases are mediated at the transcriptional or posttranscriptional level. These issues are of particular interest, since hypertrophy of SMCs in hypertensive animal models is associated with marked increases in expression of SM -actin and other SMC contractile proteins.²³

The SM -actin promoter contains a number of highly conserved cis elements, including CArG elements.²⁴ CArG elements are also found in the promoters of skeletal and cardiac -actin genes as well as many other muscle-specific genes and have been shown to direct developmental and tissue-specific expression.^{25 26 27 28 29 30} Previous studies have demonstrated that two CArG elements (designated A and B) located within the first 125 bp of the rat SM -actin promoter were required for basal, cell-specific, and TGF-ß–stimulated SM -actin expression in cultured vascular SMCs.^{24 31} It is thus reasonable to hypothesize that CArG elements may play a role in mediating Ang II effects on SM -actin expression.

The aims of the present study were to address the following questions: (1) Is Ang II–induced stimulation of SM -actin expression mediated at least in part at the transcriptional level? If so, (2) what regions of the SM -actin promoter are required for Ang II responsiveness, and what trans-acting factor(s) mediates the effects of Ang II?

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of Promoter/CAT Expression Plasmids
The generation of various truncated SM -actin promoter/CAT reporter constructs, including the CArG A and B mutants, has been previously reported.²⁴ Site-directed mutagenesis of the ATTA motif at -145 was performed by polymerase chain reaction using the following primer: 5' CACCCAG CAGTGAGAGTTTTGTG 3'. The mutated fragments were PCR-amplified and subcloned into pCAT-Basic (Promega). In experiments designed to determine the minimal SM -actin promoter sequence required to confer Ang II responsiveness, it was necessary to subclone the 125- and 155-bp promoter constructs into a pBLCAT 6 vector, since the pCAT-Basic vector contains a putative homeodomain (ie, ATTA) binding element in the multicloning site. The sequence was verified by the Sanger dideoxy-sequencing procedure³² using a Sequenase kit (U.S. Biochemical Corp).

All promoter/CAT plasmid DNAs used for transfections were prepared using an alkaline lysis procedure,³³ followed by banding on two successive ethidium bromide/cesium chloride gradients. Multiple independent plasmid preparations were tested for each construct.

Cell Culture, Transient and Stable Transfections, and Reporter Gene Assays
SMCs were isolated from thoracic aortas and cultured as previously described.¹³ Cells were plated at a density of 3x10³/cm², grown to confluence in 10% serum–containing medium, and then growth-arrested for 4 days in SFM²² before stimulation with Ang II (10^-6 mol/L, Peninsula Laboratories) or SFM. Cells used for the experiments described were between the 8th and the 12th passage. SMCs that are growth-arrested in this fashion express multiple SMC differentiation marker proteins, including SM -actin, SM MHC, h-caldesmon, h₁-calponin, SM tropomyosin, and SM MLC₂₀ (References 34³⁴ to 36 and M.M. Thompson and G.K. Owens, unpublished data, 1994).

Confluent growth-arrested SMCs were transiently transfected (in triplicate in six-well plates) with 5 µg DNA using the transfection reagent DOTAP (Boehringer-Mannheim) according to the manufacturer's recommendations. After an incubation period of 12 to 14 hours, the medium was replaced with fresh SFM, and Ang II (10^-6 mol/L) or vehicle was added. Cells were harvested 72 hours later by scraping into ice-cold buffer A (mmol/L: Tris 15 [pH 8.0], KCl 60, NaCl 15, EDTA 2, spermine tetrahydrochloride 0.15, and dithiothreitol 1).³⁷ Cell lysates were prepared by four freeze/thaw cycles, followed by 10 minutes of heat inactivation at 65°C; 95 µL aliquots of each cell extract were assayed for CAT activity by enzymatic butyrylation of tritiated chloramphenicol (Du Pont NEN).³⁸ CAT activities were normalized as described previously.²⁴ Experiments were repeated two to six times, and relative CAT activity data were expressed as the mean±SD unless otherwise noted.

Nuclear Run-on Analysis
Nuclear run-on reactions were performed as described previously, with minor modifications.³⁹ Briefly, confluent growth-arrested SMCs were grown on 150-cm² flasks and treated either with Ang II or SFM for the times indicated. The nuclei were harvested, resuspended in storage buffer, and frozen in liquid nitrogen.

An equal number of nuclei were added to a reaction mixture containing 0.625 mmol/L ATP, 0.312 mmol/L GTP, 320 µCi of [³²P]UTP (>3000 Ci/mmol), 40 mmol/L Tris-HCl (pH 8.3), 150 mmol/L NH₄Cl, 7.5 mmol/L MgCl₂, and 200 U/mL RNasin (Promega). The reaction mix was incubated for 35 minutes at 30°C, and then RQ1 DNase (30 U, Promega) and CaCl₂ (final, 1.25 mmol/L) were added and incubated for 30 minutes. Extraction buffer (100 µL) was added, and the reaction was incubated for 2 hours at 42°C. The reaction was phenol/chloroform-extracted and ethanol-precipitated, and the resulting pellet was resuspended in Tris-EDTA buffer. Unincorporated counts were removed by Sephadex G-50 column chromatography (Pharmacia). The eluates were hybridized to a single-stranded cRNA probe consisting of the 3' untranslated region of the rat SM -actin cDNA and an EcoRI fragment of the rat fibronectin cDNA⁴⁰ denatured in 0.3 mol/L NaOH at 65°C for 1 hour. Both probes were immobilized to a nylon membrane. Hybridization was carried out at 65°C for 40 to 45 hours in 5x SSPE, 10x Denhardt's solution, 1% SDS, 0.5 mg/mL herring sperm DNA, and 0.05% sodium pyrophosphate. The cDNA blot was washed at high stringency according to the Church and Gilbert method,⁴¹ and the cRNA blots as described previously.³⁵ Densitometric analysis of autoradiographs was performed using a Visage 2000 (BioImage).

Northern Blot Analysis
Total RNA was isolated using TRI REAGENT (Molecular Research Center) 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 cDNA probe used for hybridization was an EcoRI fragment encoding amino acids 9 to 217 of MHox. The probe was labeled with [-³²P]dCTP (Du Pont NEN) by random primer extension (Prime it, Stratagene). For Northern analysis, 10 µg of total RNA was diluted in loading buffer consisting of 0.2 mol/L MOPS, 0.05 mol/L sodium acetate, 0.01 mol/L EDTA, 4% formaldehyde, and 65% formamide, denatured by treating for 10 minutes 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) was carried out overnight in 10x SSPE buffer (2 mol/L EDTA, 20 mol/L NaH₂PO₄ · H₂O, and 298 mol/L NaCl). Blots were air-dried, exposed to an UV transilluminator for 1.5 minutes, and baked for 2 hours under vacuum at 80°C.

Hybridization to cDNA probes and subsequent washes were carried out at 65°C as previously described by Church and Gilbert.⁴¹ Blots were then dried and subsequently exposed to Kodak X-OMAT K film at -70°C. A 5.8-kb EcoRI human cDNA fragment for 18S RNA was released from pBR322 and used in Northern analysis for standardization of RNA loading and transfer.⁴²

Western Blot Analysis
Cell lysates were prepared from confluent growth-arrested SMC cultures stimulated with Ang II or vehicle for 4 hours. Briefly, cells were rinsed with PBS, scraped into 0.6 mL ice-cold RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) plus protease inhibitors (10 mg/mL phenylmethylsulfonyl fluoride, 30 µg/mL aprotinin, and 100 mmol/L sodium orthovanadate), and passed through a 21-gauge needle several times. Cell lysates were then incubated on ice for 30 minutes and microfuged for 20 minutes at 4°C (protocol provided by Santa Cruz). Sample loading was normalized to DNA content determined with a DNA fluorometer (Hoefer Scientific). DNA was loaded (600 ng per well) on a 7.5% SDS-PAGE Mini-Protean gel (Bio-Rad). The proteins were transferred onto a PVDF membrane at 100 V for 1.5 hours. Blocking of the membrane and probing with appropriate antibodies was performed according to the enhanced chemiluminescence Western blotting protocol from Amersham, 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 EMSAs
Crude nuclear extracts were prepared by the method of Dignam et al⁴³ using confluent growth-arrested SMCs stimulated with Ang II or SFM for 4 hours. Protein concentrations were measured by the Bradford assay (BioRad). Probes for EMSA were obtained by end-labeling 20 µmol/L of single-stranded oligonucleotides with 150 µCi of [-³²P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase. Labeled single-stranded oligonucleotides were annealed, and unincorporated nucleotides were removed using Nuc Trap Push columns (Stratagene) as recommended by the manufacturer. The 95-bp promoter segment (-137 to -43) was generated by polymerase chain reaction amplification in the presence of 33.3 pmol of [-³²P]dCTP (3000 Ci/mmol, NEN) using a 207-bp SM -actin promoter/CAT construct as a template. The following nucleotides were used either as a probe or as cold competitors (only the sense strand is shown): MHox, 5' CACCCAGATTA GAGAGTTTTGTG 3'; MHox mutant, 5' CACCCAGCAGT GAGAGTTTTGTG 3'.

EMSAs were performed with 20 µL binding reactions that contained 50 pg of ³²P-labeled annealed oligonucleotides or 0.1 to 0.5 ng of the 95-bp probe, 5 µg nuclear extracts (in Dignam buffer D), human recombinant SRF, 150 mmol/L KCl, 5 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 1.125 mmol/L dithiothreitol, 10% glycerol, and 0.125 to 1 µg poly (dI-dC) as a nonspecific competitor. Approximately 10 to 30 ng bacterially expressed GST-MHox was added to some binding reactions. Specific competitor oligonucleotides and specific antibodies against SRF (Santa Cruz) or hyperimmune serum from rabbits immunized against MHox was included when indicated. The binding reaction was incubated for 20 minutes at room temperature before radiolabeled probe was added, followed by another 20-minute room temperature incubation unless otherwise noted. All binding reactions were loaded on a 4.5% polyacrylamide gel and electrophoresed at 170 V in 0.5x TBE. The gels were 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) with the human SRF cDNA clone p T7 ATG as a template.⁴⁴

Preparation of GST-MHox Fusion Protein
The expression vector encoding the GST-MHox fusion protein used in the present study was constructed as described previously⁴⁵ and contains amino acids 9 to 217 (the carboxyl terminus). Fusion protein was purified on glutathione agarose beads (Pharmacia) according to the method of Chakraborty et al.⁴⁶ Concentration and purity of the fusion protein was estimated on Coomassie blue staining after separation by SDS-PAGE and comparison with BSA standards.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Upregulated SM -Actin Transcription
Previous studies by our laboratory demonstrated that Ang II increased SM -actin mRNA expression 5- to 8- fold.²² To determine whether increased transcription of the SM -actin gene contributed to this increase, we performed nuclear run-on analyses. Nuclei were harvested from growth-arrested rat SMCs stimulated with Ang II (10^-6 mol/L) or SFM for the times indicated (Fig 1). Results demonstrated that compared with SFM, Ang II transiently enhanced SM -actin transcription by 2.8±0.47-fold at 6 hours after treatment (three independent experiments). The transient increase of SM -actin transcription at 6 hours, but not at 24 hours, after Ang II treatment is consistent with previous studies from our laboratory²² demonstrating transient increases in SM -actin mRNA expression at 4 and 6 hours, but not 24 hours, as measured by Northern analysis. In contrast to SM -actin, Ang II treatment did not increase fibronectin transcription under the same conditions (Fig 1, middle panel) and other constitutively expressed genes, including cathepsin D and glyceraldehyde-3-phosphate dehydrogenase (data not shown), indicating that Ang II effects on SM -actin transcription were selective. No hybridization signal was obtained when nonspecific control DNA (pGEM plasmid DNA) was used as a probe (Fig 1, bottom panel), and the addition of -amanitin at a concentration that selectively inhibits RNA polymerase II and III (80 µg/mL) to the in vitro reaction resulted in loss of the hybridization signal (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1. Nuclear run-on analysis demonstrating the effects of Ang II (A-II) on SM -actin transcription. Nuclei were isolated from cultured rat aortic SMCs that were treated with A-II (10 -6 mol/L) or SFM (vehicle) for the times indicated. Resulting RNA transcripts were hybridized to a single-stranded cRNA probe consisting of the 3' untranslated region of the rat SM -actin cDNA (top panel) or a rat fibronectin cDNA probe (middle panel). No hybridization signal was obtained when nonspecific control DNA (pGEM plasmid DNA) was used as a probe (bottom panel). Because of density and feed effects that are part of our experimental design, basal SM -actin expression increased at 24 hours. Thus, comparison between groups must be restricted to individual time points. The experiments were repeated three times.

The First 155 bp of the SM -Actin Promoter Was Sufficient to Confer Ang II Responsiveness
To determine the minimal promoter sequence required to confer Ang II inducibility of the SM -actin gene, we tested a construct containing 2.8 kb of the 5' flanking sequence of the SM -actin gene linked to a promoterless CAT reporter gene (designated pProm/CAT) and a series of deletion mutants of this construct in transient transfection assays. Transfection data indicated that the first 155 bp was sufficient to confer full Ang II responsiveness (Fig 2). Ang II induced an 4-fold increase in reporter activity in all SM -actin promoter constructs tested. Inclusion of upstream sequences between -155 bp and -2.8 kb, previously shown to contain negative regulatory elements required for cell-specific expression of the SM -actin gene,²⁴ did not alter Ang II effects, suggesting that these additional sequences are required for cell-specific expression of the SM -actin gene but not for Ang II inducibility. Ang II did not increase CAT activity in SMCs transfected with an SV40-driven viral promoter/CAT construct (pCAT control, Promega), suggesting that Ang II effects were promoter specific and not due to general changes in CAT expression via posttranscriptional mechanisms.

View larger version (25K): [in this window] [in a new window]   Figure 2. Expression of pProm CAT, various deletion mutants, and an SV40-driven viral promoter/CAT construct (pCAT control, Promega) in cultured rat aortic SMCs in response to Ang II (AII). SMC cultures were grown to confluence and growth-arrested in SFM for 4 to 5 days. Cells were then transiently transfected with the constructs indicated and stimulated with AII (10-6 mol/L) or SFM for 72 hours. CAT activities of AII-treated groups were expressed relative to vehicle-treated groups (set to 1). Data represent mean±SE. AII significantly increased reporter activity of all SM -actin promoter constructs, but no statistically significant differences (ANOVA, P.05) were observed for the AII-treated groups with the different constructs.

Two Highly Conserved CArG Elements, A and B, Contained Within the First 155 bp of the SM -Actin Promoter Were Required for Ang II–Induced Stimulation of SM -Actin Gene Expression
CArG elements found in the promoters of a number of -actin genes have been shown to be important for regulation of transcriptional activity.^{24 28 47} Consistent with this, recent studies from our laboratory demonstrated that both CArG elements in the SM -actin promoter were required for basal, cell-specific, and TGF-ß–inducible expression of SM -actin.^{24 48} Thus, we tested whether Ang II responsiveness of the SM -actin gene was also dependent on CArG elements. Mutation of either CArG A or B alone completely abolished Ang II–induced increases in CAT activity in transient transfection assays (Fig 3). Similar results were obtained when CArG mutations (either alone or in combination) were tested within the context of the 2.8-kb construct, pProm/CAT (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of CArG mutations on Ang II (AII)–induced stimulation of reporter activity. Wild-type SM -actin pBL 155 CAT and constructs containing mutations of either CArG box A or B (subcloned into a promoterless pBL CAT vector) were transiently transfected into growth-arrested rat SMCs as described in the legend to Fig 2. CAT activities of AII (10-6 mol/L)–treated or SFM-treated groups were expressed relative to the baseline CAT activity of a promoterless CAT construct. Data represent the mean±SD of triplicate samples.

Ang II Enhanced Binding of SRF to CArG A and B but Did Not Increase SRF Expression
The functional importance of the CArG elements for Ang II responsiveness of the SM -actin gene prompted us to test whether Ang II affected binding to the SM -actin CArGs. Previous studies from this laboratory have demonstrated that the SM -actin CArG boxes bind SRF or an SRF-like factor.²⁴ EMSAs were performed with labeled 20-bp CArG A or B probes and nuclear extracts from either Ang II–treated or SFM-treated SMCs. Ang II treatment markedly enhanced binding to both CArG elements (Fig 4A, lanes 2 and 5) compared with no treatment (lanes 1 and 4). Consistent with our previous studies,²⁴ the binding complex could be supershifted with multiple SRF antibodies (data not shown). The enhanced SRF binding in response to Ang II was not associated with changes in mobility of the SRF-containing complex compared with the complex formed with recombinant SRF alone (lanes 3 and 6), suggesting that Ang II did not induce formation of a stable higher order complex, at least under the assay conditions used in these studies. Ang II also enhanced SRF binding activity to a larger probe (*Wt 95; Fig 4B, lane 1), which contained both CArG boxes. This longer probe gave rise to two SRF-containing bands (designated bands 1 and 2), which we previously demonstrated are specific for SMCs.²⁴ As observed with the 20-bp CArG-containing oligonucleotides, Ang II–induced stimulation of SRF binding to the 95-bp probe was not associated with formation of a higher order complex compared with SFM control (lane 2). These results are important in that they suggest that CArG-dependent stimulation of SM -actin does not involve formation of a ternary complex, eg, a complex between SRF and the ets domain binding SRF accessory proteins, such as SAP-1 or ELK-1, as observed with the c-fos SRE.^{49 50} Consistent with this, the SM -actin promoter CArGs lack a 5' ets binding site, and we have been unable to detect a known ets domain containing SRF accessory proteins (eg, SAP-1 and ELK-1) in the SM -actin CArG-binding complexes (C.P. Mack and G.K. Owens, unpublished data, 1996).

View larger version (46K): [in this window] [in a new window]   Figure 4. Gel-shift analysis of the effects of Ang II (AII) on binding activities 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 to 3) or CArG A (lanes 4 to 6) double-stranded oligonucleotide was incubated with nuclear extracts (5 µg) from rat SMCs treated with AII (10-6 mol/L), SFM, or in vitro–translated SRF (rSRF) for 20 minutes at room temperature. Although not visible on this autoradiogram, longer exposure times revealed a shift band (lane 6) of the same mobility as the shift band formed with rSRF and a CArG B probe in lane 3. B, Gel-shift analysis with a 95-bp radiolabeled probe containing both CArG A and B (-137 to -43) is shown. The probe was incubated with 5 µg nuclear extracts from rat SMCs treated with AII or SFM for 20 minutes at room temperature. Gel-shift assays confirming specificity of SRF binding were performed as previously described.24

To test whether Ang II–induced increases in SRF protein expression contributed to enhanced SRF binding activity, we performed Western analysis of cell lysates obtained from Ang II–treated or SFM-treated SMCs. Results showed that Ang II did not significantly increase SRF protein levels compared with SFM control (Fig 5). In contrast, treatment of SMCs with 10% serum markedly stimulated SRF expression. These results suggest that Ang II–induced increases in SRF binding were not due to increased SRF expression.

View larger version (50K): [in this window] [in a new window]   Figure 5. Western blot analysis of SRF expression in cell lysates from SMCs stimulated with Ang II (AII), SFM vehicle, or 10% FBS. Western blot analyses were performed on cell lysates obtained from growth-arrested SMC cultures treated with AII (10-6 mol/L) or SFM for 4 hours. Cell lysates were analyzed by SDS-PAGE with loadings normalized to DNA content (600 ng). Resolved proteins were then transferred to a PVDF membrane and immunoblotted with a rabbit polyclonal SRF antibody (Santa Cruz). Immunoreactive bands with a size of 67 kD were detected. No reactivity was observed when the membrane was immunoblotted with preimmune rabbit serum (data not shown). Whereas serum induced an increase in all three experiments, no consistent change was observed with AII treatment.

Recombinant MHox Increased SRF Binding to CArG B
To further elucidate the mechanisms whereby Ang II enhanced SRF binding, we tested whether interaction of SRF with homeodomain transcription factors might play a role. We⁵¹ and others⁴⁵ have demonstrated that SMCs express MHox, the murine homologue of Phox 1, which has been shown to increase SRF binding to the c-fos SRE.⁵² Thus, we tested whether recombinant MHox, expressed as a GST fusion protein, affected SRF binding. A 20-bp–labeled CArG B oligonucleotide was used as a probe and incubated with a constant amount (2 µL) of recombinant SRF produced in a reticulolysate system (Fig 6, lanes 2 to 4). Addition of MHox markedly enhanced SRF binding to CArG B. However, as observed with Ang II–treated SMC nuclear extracts, enhanced SRF binding to CArG B did not result in formation of a higher order complex (lanes 3 and 4). No shift bands were formed at the position of the CArG-containing complex when GST-MHox or unprogrammed lysate was incubated with the CArG B probe. In addition, GST-MHox did not form any retarded complexes when incubated with the CArG B probe (data not shown). Addition of GST alone to the binding reaction at approximately the same concentration as GST-MHox did not affect SRF binding (data not shown). Likewise, addition of BSA to binding reactions did not affect SRF binding (data not shown), indicating that nonspecific protein-protein interactions did not contribute to enhanced SRF binding. We also tested whether MHox increased SRF binding to CArG A. Consistent with previous studies⁵³ demonstrating that Phox 1 did not effectively increase SRF binding to weak SRF binding CArGs, MHox failed to significantly increase SRF binding to CArG A.

View larger version (40K): [in this window] [in a new window]   Figure 6. Effects of GST-MHox on binding activity of recombinant SRF to CArG B. Gel-shift assays were performed with a 20-bp radiolabeled CArG B probe and in vitro–translated SRF (rSRF) (lanes 1 and 2). Various amounts of bacterially expressed GST-MHox fusion protein were added to binding reactions, which contained constant amounts of rSRF (2 µL) (lanes 3 and 4). Reactions were incubated for 10 minutes at room temperature in the presence of labeled probe before loading on the gel. As a control, unprogrammed (unprog.) reticulocyte lysate (lane 5) and GST-MHox alone (lane 6) were incubated with the CArG B probe.

Ang II Increased MHox mRNA Expression
To further explore the potential role of MHox in mediating Ang II effects on SM -actin transcription, we investigated whether Ang II had an effect on MHox expression in SMCs. Northern analyses were performed on total RNA isolated from SMCs stimulated with Ang II (10^-6 mol/L) or SFM for either 1, 4, 8, or 24 hours (Fig 7). Consistent with previous reports, the MHox cDNA probe hybridized to two major transcripts of 3.6 and 4 kb in size.⁴⁵ Ang II markedly enhanced the expression of the smaller transcript at all time points examined. Expression of the larger transcript, however, was less affected by Ang II treatment. The significance of this finding is uncertain, since the mechanisms of MHox mRNA processing and how they might be altered by Ang II are poorly understood.⁴⁵

View larger version (83K): [in this window] [in a new window]   Figure 7. Northern analysis of Ang II (AII) on MHox mRNA expression. Growth-arrested cultures of SMCs were treated with AII or SFM and harvested at the times indicated. Total RNA was loaded (10 µg per lane). Northern hybridization was performed with a MHox cDNA probe encoding amino acids 9 to 217 (top blot). The blots were rehybridized with an 18S rRNA probe to verify similar RNA loadings for each lane (bottom blot).

SMC Nuclear Extracts From Ang II–Treated SMCs Exhibited Enhanced MHox Binding Activity to an ATTA-Containing Element Located at -145 of the SM -Actin Promoter
Previous studies have demonstrated that MHox and other Antennapedia-type homeodomain proteins recognize A/T-rich sequences with an ATTA core motif.^{45 52} Such a motif is found within a highly conserved region of the SM -actin promoter at -145. Thus, we performed gel-shift assays to determine whether MHox bound to this element and whether Ang II treatment was associated with alterations in MHox binding activity within SMC nuclear extracts. Incubation of a 23-bp–labeled oligonucleotide containing the ATTA motif (designated as "MHox probe") with nuclear extracts from SFM-treated or Ang II–treated SMCs led to formation of two DNA-protein complexes (Fig 8, lanes 1 and 2). This binding pattern is consistent with previous reports⁴⁵ demonstrating that MHox proteins of different molecular sizes derived from two different in-frame translation initiation sites give rise to two DNA-protein complexes with slightly different mobilities. Ang II treatment markedly enhanced formation of the slower migrating band, consistent with the observation that the higher-molecular-weight MHox variant is the predominant form in vivo.⁴⁵ Addition of cold competitor MHox oligonucleotides (lanes 3 and 4) completely abolished formation of the MHox-containing complexes, whereas mutated MHox oligonucleotides failed to do so (lanes 5 and 6). No MHox binding was observed when a mutated MHox oligonucleotide was used as a probe (data not shown). To further confirm the specificity of MHox binding to the SM -actin ATTA, we added hyperimmune serum derived from rabbits immunized with GST-MHox (lane 7) to the binding reaction. Addition of hyperimmune serum disrupted binding of MHox proteins to the MHox probe. In contrast, addition of preimmune serum (lane 8) had no effect on MHox binding. The observation that Ang II increased MHox mRNA levels and binding suggests that increased MHox binding might be due to enhanced MHox protein expression. However, it cannot be ruled out that Ang II–mediated posttranscriptional modifications also contribute to increased MHox binding. Further studies are required to resolve these issues.

View larger version (47K): [in this window] [in a new window]   Figure 8. EMSA analysis of the effects of Ang II (AII) on MHox binding. Gel-shift assays were performed with a 23-bp radiolabeled oligonucleotide spanning from -156 to -131 (*MHox-oligo) containing an ATTA motif and 5 µg of nuclear extracts from SMCs treated with AII (10-6 mol/L) (lanes 2 to 8) or SFM (lane 1). Competition reactions were performed with oligonucleotide duplexes containing the wild-type (lanes 3 and 4) or mutated ATTA sequence (lanes 5 and 6). Three microliters of preimmune rabbit serum (lane 8) or hyperimmune serum from rabbits immunized against MHox (lane 7) were added to the binding reaction. Incubation times were as described in "Material and Methods."

The MHox Binding Site at -145 Within the SM -Actin Promoter Was Required for Full Ang II Responsiveness of the SM -Actin Gene
To test the functional importance of MHox binding for Ang II responsiveness of the SM -actin gene, we mutated the MHox binding site within a pBL 155 CAT construct and transiently transfected it along with a wild-type pBL 155 CAT construct into SMCs. As shown in Fig 9, mutation of the ATTA site reduced Ang II–induced stimulation of reporter activity by 50% compared with the wild-type construct. Consistent with this finding, Ang II stimulated only a 2-fold increase in reporter activity of a pBL 125 CAT construct that lacks the MHox binding site and some additional sequences 5' to CArG B. These results indicate that the ATTA MHox binding site is required for maximal Ang II responsiveness of the SM -actin gene but that some Ang II–induced activation occurs independent of this site.

View larger version (22K): [in this window] [in a new window]   Figure 9. Effects of MHox binding site on Ang II (AII)–induced stimulation of SM -actin expression. Growth-arrested rat SMCs were transiently transfected with the following SM -actin promoter CAT constructs: pBL 125 CAT, pBL 155 CAT, and a pBL 155 CAT Mut containing a mutation of the ATTA motif (ATTACAGT). SMC cultures were stimulated with AII (10-6 mol/L) or SFM. The promoter activity of each construct was expressed relative to the baseline activity of a promoterless CAT construct (set to 1). Data represent the mean±SD of triplicate samples.

Overexpression of MHox Transactivated SM -Actin Gene Transcription
To determine whether MHox alone was able to stimulate SM -actin expression, we cotransfected an MHox expression vector and a p155 CAT construct into SMCs (Fig 10). Control SMCs were cotransfected with the empty expression vector. Overexpression of MHox transactivated SM -actin transcription 3.5-fold. When a p155 CAT construct containing a mutation of the MHox binding site was cotransfected along with the MHox expression vector, MHox-mediated transactivation was substantially reduced, suggesting that MHox binding was important for maximal transactivation.

View larger version (18K): [in this window] [in a new window]   Figure 10. Effects of MHox overexpression on SM -actin expression. Subconfluent SMCs, growing in 10% FBS, were cotransfected with a wild-type p155 CAT construct or the p155 CAT construct containing a mutation of the MHox binding site as described in Fig 9 (0.75 µg each) and an MHox expression vector (4 µg). Control SMCs were cotransfected with the empty p Zeo SV vector (4 µg). Cells were harvested 48 hours after transfection. CAT activities were expressed relative to the baseline CAT activities of a promoterless-CAT construct set to one.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of the present study was to investigate the molecular mechanisms whereby Ang II stimulates SM -actin expression. Our results demonstrated that (1) Ang II responsiveness of the SM -actin gene was, at least in part, transcriptionally mediated; (2) the first 155 bp of the rat SM -actin promoter was sufficient to confer Ang II–induced activation; and (3) Ang II responsiveness was completely dependent on two highly conserved CArG elements, at -62 (CArG A) and -122 (CArG B), and partially dependent on an MHox binding site at -145.

Consistent with our findings, a number of studies have provided evidence indicating that growth factor–induced stimulation of cell-specific genes is mediated through CArG-dependent mechanisms.^{48 54 55 56} For example, CArG elements were also found to be essential for TGF-ß inducibility of the SM -actin gene,⁴⁸ as well as the ₁-adrenergic and TGF-ß-mediated induction of skeletal -actin.^{54 55} Of particular interest, a study by Van Putten et al⁵⁶ demonstrated that AVP, which induces a hypertrophic response similar to that of Ang II,²² also stimulated increased transcription of SM -actin. AVP responsiveness of the SM -actin gene was contained within the first 152 bp of the promoter but was lost by deletion of the region between -152 and -102 that contains CArG B and the ATTA element. These results suggest that AVP and Ang II responsiveness might be conferred by similar cis elements. Our results are in contrast to a study of Andrawis et al,⁵⁷ who showed that Ang II–induced increases in SM -actin expression in rat SMCs were dependent on a region of the human SM -actin promoter from -258 to -896. There are several possible explanations for the observed differences. First, these authors determined the activity of the human SM -actin promoter in rat SMCs. As previously shown in our laboratory, a different pattern of promoter activity can result from testing within a heterologous system.⁵⁸ However, this seems unlikely, given the high degree of conservation of the -155-bp region of the SM -actin promoter in humans and rats. A second possibility is that contrasting results reflect differences in experimental design and/or methods of cell culture. For example, the studies by Andrawis et al were carried out in SMCs that were not growth-arrested before Ang II stimulation. We¹³ and others⁵⁹ have previously shown that growth arrest is a condition required for demonstrating hypertrophic effects of Ang II. As such, it is unclear whether Ang II stimulated hypertrophy or hyperplasia in the experiments of Andrawis et al and whether there was altered expression of the endogenous SM -actin gene under the conditions of their experiments.

Our results demonstrate that Ang II inducibility of SM -actin expression was associated with markedly enhanced SRF binding to the CArG boxes. These observations are consistent with an extensive body of evidence showing that enhanced SRF binding is associated with increased transcriptional activity of a variety of genes, ranging from immediate-early response genes (such as c-fos,^{44 60} which is expressed in all cells) to -actin genes^{61 62} (which are expressed in a tissue-specific fashion). The precise mechanisms involved in mediating CArG-dependent transcription of these diverse genes have not been completely elucidated but have been shown to include (1) an increase in SRF expression^{47 48 61 62} and (2) alteration in SRF binding and/or activity through interaction with SRF accessory proteins or posttranslational modifications of SRF.^{53 60 63} Whereas results of the present study have indicated that increased SRF expression does not contribute to Ang II–induced increases in SRF binding (Fig 5) and SM -actin transcription, the following observations provide strong evidence for involvement of the homeodomain factor MHox in the mediation of Ang II effects: (1) Ang II treatment was associated with marked increases in MHox mRNA levels (Fig 7) as well as MHox binding activity in nuclear extracts derived from Ang II–treated SMCs (Fig 8). (2) MHox enhanced SRF binding to CArG B (Fig 6). (3) Overexpression of MHox transactivated SM -actin reporter constructs (Fig 10), with the magnitude of stimulation being almost identical to that seen after Ang II stimulation of SMCs, as measured by run-on analysis (Fig 1) or by transfection studies with -actin promoter/CAT constructs (Fig 2). (4) Ang II stimulation of SM -actin (Fig 9) and MHox-induced transactivation of the SM -actin reporter (Fig 10) were greatly diminished by mutation of an MHox binding site within the SM -actin promoter. Taken together, these studies provide strong evidence for an important role for MHox in mediation of Ang II–induced increases in SM -actin transcription, although direct proof of this will require development of means to specifically inhibit MHox function using dominant negatives, neutralizing antibodies, etc.

Of interest, the effects of MHox in enhancing SRF binding were not associated with formation of a stable higher order complex with SRF. This observation is consistent with previous studies demonstrating that Nkx-2.5, a cardiac-specific homeodomain factor, enhanced SRF binding to the cardiac -actin SREs without formation of a higher order complex with SRF.⁶⁴ Likewise, studies by Grueneberg et al⁵² showed that the human counterpart of MHox, Phox 1, which differs from MHox by only a single amino acid substitution outside the active region, enhanced SRF binding to the c-fos SRE without formation of a higher order complex in gel-shift assays. Furthermore, these investigators showed that Phox 1–enhanced binding of SRF was due to an increased rate of exchange of SRF with its binding site and that it was not dependent on binding of Phox 1 to DNA. They postulated that this effect occurred through protein-protein interactions between SRF and Phox 1 that enhanced SRF binding to the c-fos SRE but that Phox 1 then dissociated from the complex once SRF had bound to DNA. Consistent with these results, the present study showed that MHox increased SRF binding to a CArG B oligonucleotide that lacked any detectable MHox binding activity. However, although MHox/Phox 1 binding to DNA does not appear to be necessary for its ability to enhance SRF binding to the CArG element in vitro, binding may be important for effects in vivo. Consistent with this, Grueneberg et al⁵³ demonstrated that a mutation within the homeodomain of Phox 1 that abolished its ability to bind to DNA resulted in loss of ability to transactivate c-fos transcription. Thus, activation in vivo may require higher order complex formation between MHox/Phox 1 and SRF, but this may have not been detectable in gel-shift assays because (1) protein-protein interactions were either transient or unstable under the gel-shift conditions used, and/or (2) stable higher complex formation might require posttranslational modifications of SRF, and MHox/Phox 1 not present in the recombinant proteins tested. Clearly, further studies will be required to resolve these issues.

An interesting question is whether MHox might be involved in coordinate regulation of other SM differentiation marker genes through a CArG-dependent mechanism. Virtually all SM-specific contractile protein genes characterized to date have been shown to be regulated by CArG elements, including the SM MHC,⁶⁵ SM 22,⁶⁶ telokin,⁶⁷ and caldesmon⁶⁸ genes. Since a number of homeobox genes have been shown to be retinoic acid inducible,⁶⁹ it is of interest that a previous study from our laboratory⁵¹ demonstrated that retinoic acid treatment of undifferentiated P19 embryonal carcinoma cells led to the coexpression of MHox and several SM-specific contractile proteins, including SM -actin and MHC. The observation that MHox is coexpressed along with SM differentiation marker genes raises the possibility that MHox is involved in control of SMC differentiation. However, the fact that MHox is also expressed by other mesodermally derived cell types that do not normally express SM -actin⁴⁵ suggests that cell type–specific gene expression is probably not mediated by MHox itself but through recruitment of a cell type–specific protein (or proteins) interacting with MHox and SRF and/or some unique combinatorial interaction between CArG binding factors and other cis elements and their trans factors in a manner analogous to that of many skeletal and cardiac specific genes.^{25 70} Consistent with the first possibility, we previously presented evidence for formation of two unique SRF-containing complexes with SMC nuclear extracts and a 95-bp SM -actin promoter probe containing both CArG elements (bands 1 and 2 in Fig 4B).²⁴ These complexes were not observed using nuclear extracts from a variety of non-SMCs, suggesting involvement of an SMC-specific SRF accessory protein in control of SM -actin gene expression.²⁴ MHox itself does not appear to be responsible for formation of this higher order complex observed with the 95-bp probe, since addition of recombinant MHox to SRF in the presence of the 95-bp probe did not result in formation of a higher order complex, suggesting that it does not stably interact with SRF in vitro, at least under the gel-shift conditions used (M.B. Hautmann and G.K. Owens, unpublished data, 1996). However, it is tempting to speculate that MHox might play a role in recruitment of this as-yet-unidentified SMC factor in vivo.

Our observations that mutation of an MHox binding site reduced Ang II inducibility but did not abolish it suggest that residual Ang II responsiveness is mediated by MHox interacting with SRF through protein-protein interaction and/or that MHox independent mechanisms are involved. Consistent with the latter, we have shown that Ang II enhanced SRF binding to both CArG boxes but that recombinant MHox enhanced SRF binding only to CArG B in vitro, suggesting that alternative mechanisms might be involved in mediating Ang II–induced increases to CArG A. One possibility is that enhanced SRF binding to CArG A could depend on proteins other than MHox (Fig 11). Alternatively, effects of MHox on SRF binding to CArG A might require posttranscriptional modifications of SRF, MHox, or as yet unidentified proteins not present in our in vitro gel-shift reconstitution experiments using recombinant proteins. It is also of interest that Hill et al⁷¹ demonstrated that lysophosphatidic acid stimulation of a modified c-fos SRE that lacks an ets binding site was dependent on the activation of Rho A. Of interest, Rho A activation was shown to be involved in signaling pathways of agonists that signal via seven transmembrane G protein–coupled receptor families,^{71 72} of which the Ang II AT₁ receptor is a member. Given that the SM -actin promoter also lacks an ets binding site, it is interesting to speculate that Ang II may also increase SM -actin transcription via a Rho-dependent mechanism. In preliminary studies (M.B. Hautmann and G.K. Owens, unpublished data, 1996), we demonstrated that Rho A overexpression could transactivate SM -actin transcription. In addition, SM -actin transcription was blocked by C3 transferase, a specific inhibitor of Rho A. Taken together, the preceding results clearly indicate that further studies will be required to determine the role of Rho A and other potential downstream targets in Ang II–induced stimulation of SM -actin.

View larger version (11K): [in this window] [in a new window]   Figure 11. A simplified model of possible mechanisms whereby Ang II (A-II) might increase SM -actin expression. Rx indicates receptor.

An additional possibility is that autocrine-produced growth factors might contribute to Ang II responsiveness of the SM -actin gene, since Ang II has been shown to increase PDGF-AA and biological active TGF-ß.^{15 73} In addition, we⁴⁸ and others^{74 75 76} have provided clear evidence that TGF-ß can stimulate SM -actin expression in a variety of cell types. However, several lines of evidence suggest that the effects of Ang II on SM -actin expression are not mediated through autocrine production of TGF-ß and PDGF-AA: (1) Ang II–induced stimulation of SM -actin mRNA expression was not affected by neutralizing TGF-ß antibodies (M.B. Hautmann and G.K. Owens, unpublished data, 1994). (2) Ang II–induced stimulation of SM -actin expression was not accompanied by increased SRF expression, whereas TGF-ß inducibility of SM -actin involved increased SRF expression.⁴⁸ (3) We have shown that PDGF-AA did not contribute to Ang II–induced hypertrophy in SMCs.⁷⁷ Moreover, the Sprague-Dawley rat–derived SMC cultures used in the present study lack any detectable PDGF -receptors.

In summary, the present study provides evidence for involvement of a homeodomain transcription factor, MHox, in Ang II–induced stimulation of SM -actin expression through a CArG-dependent mechanism. Further studies are required to determine whether MHox also plays a role in stimulation of other SM differentiation marker proteins and whether MHox is involved in regulation of these genes in vivo under normal and pathological conditions.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AVP = arginine vasopressin CAT = chloramphenicol acetyltransferase EMSA = electrophoretic mobility shift assay GST = glutathione S-transferase MHC, MLC = myosin heavy chain, myosin light chain PDGF = platelet-derived growth factor PVDF = polyvinylidene difluoride SFM = serum-free medium SM = smooth muscle SMC = smooth muscle cell SRE = serum response element SRF = serum response factor SV = simian virus TGF-ß = transforming growth factor-ß1

	Acknowledgments

 
This study was supported by grants RO1 HL-38854 and PO1 HL-19242 from the National Institutes of Health (to Dr Owens) and Fellowship Grant VA-94-F-14 (to Dr Hautmann) from the American Heart Association, Virginia Affiliate, Inc. We gratefully acknowledge the expert technical assistance of Diane Raines and Andrea Tanner.

Received January 28, 1997; accepted July 9, 1997.

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