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(Circulation Research. 1996;78:589-595.)
© 1996 American Heart Association, Inc.

Articles

Regulated Expression of the Ets-1 Transcription Factor in Vascular Smooth Muscle Cells In Vivo and In Vitro

Anna Hultgårdh-Nilsson, Bojan Cercek, Jian-Wei Wang, Shinji Naito, Cecilia Lövdahl, Behrooz Sharifi, James S. Forrester, James A. Fagin

From the Division of Cardiology (A.H.-N., B.C., J.-W.W., S.N., B.S., J.S.F., J.A.F.), Cedars-Sinai Medical Center, UCLA Medical School, Los Angeles, Calif, and the Department of Cell and Molecular Biology (A.H.-N., C.L.), Medical Nobel Institute, Laboratory of Medical Cell Biology, Karolinska Institutet, Stockholm, Sweden.

Correspondence to Dr James A. Fagin, Division of Endocrinology and Metabolism, University of Cincinnati, College of Medicine, PO Box 670547, Cincinnati, OH 45267.

	Abstract

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Abstract Ets-1 regulates the transcription of several genes encoding extracellular matrix proteins (ie, osteopontin and tenascin) as well as enzymes involved in degradation and remodeling of the extracellular matrix (ie, stromelysin and urokinase plasminogen activator). In the present study, we investigated the regulation of c-ets-1 in cultured rat vascular smooth muscle cells as well as in the arterial wall after balloon injury in vivo. Serum-starved smooth muscle cells exposed to serum for various time points express a major c-ets-1 mRNA transcript of 5.3 kb and minor bands of 4.0 and 2.5 kb with a peak at 2 hours after stimulation. These effects were concentration dependent. Western blotting revealed an increase in 55- and 40-kD immunoreactive ets-1 proteins in cells treated with serum for 2 hours, and binding to an oligonucleotide containing the ets-1 consensus cis-acting motif was demonstrated by electrophoretic mobility shift assay. Ets-1 mRNA abundance was induced with a peak at 2 hours after stimulation with platelet-derived growth factor-BB and with angiotensin II. There was a distinct increase of ets-1 immunoreactivity in the inner layer of the media 2 hours after balloon catheter injury of rat arteries, which declined after 6 hours and returned to the basal level 1 day after vessel wall damage. Arterial c-ets-1 mRNA content was induced with an identical time course. These findings suggest that c-ets-1 may be of importance in the mitogenic signaling pathway of smooth muscle cells grown in culture. In addition, ets-1 may play a role in the activation of smooth muscle cells in vivo after mechanical injury of the vessel wall. Because the ets-1 transcription factor activates the gene expression of a number of mRNA species involved in matrix deposition and degradation, these data are compatible with a role for ets-1 in vascular remodeling and/or cell migration.

Key Words: ets-1 • smooth muscle cells • matrix metalloproteinases

	Introduction

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Vascular SMCs play a central role in the development of atherosclerotic lesions¹ and in the formation of the hyperplastic response that follows arterial injury, including interventions for occlusive vascular disease.² The role of SMCs has been carefully examined in animal models of intimal hyperplasia. Balloon injury of the arterial wall initiates activation of SMC DNA synthesis. These events are followed by migration of cells into the intima, where they undergo further cell division and secrete extracellular matrix, leading to the obstruction of blood flow.³ Production of enzymes that allow degradation of extracellular matrix is one important mechanism in the process of cellular migration. MMPs degrade matrix components at neutral pH.^{4 5} As an initial step, collagenase (MMP-1) degrades interstitial collagens (types I, II, and III),⁶ followed by stromelysin (MMP-3), which has a broad activity against denatured collagen as well as laminin, fibronectin, and proteoglycans. Collagenase and stromelysin may both be activated by plasmin generated from plasminogen by either urokinase or tissue-type plasminogen activator.⁷ A positive regulation of the expression of metalloproteinases has been reported from a variety of agents, including hormones, growth factors, cytokines, and oncogene products,⁵ whereas repression is mediated by transforming growth factor-ß and retinoic acid. In atherosclerotic coronary arteries, in situ hybridization demonstrates stromelysin mRNA in SMCs.⁸ Evidence has also been presented that collagenase is upregulated in serum-stimulated arterial SMCs grown in culture.⁹ Furthermore, Kenagy et al¹⁰ have shown that phorbol 12-myristate 13-acetate, a phorbol ester, increases the levels of mRNA of collagenase, 92-kD gelatinase, and stromelysin and that this induction could be blocked by heparin.

v-Ets was originally identified as part of the avian leukemia virus E26. The cellular ets-1 gene is the proto-oncogene of the viral gene and has been shown to be oncogenic when expressed inappropriately.¹¹ The ets family of proteins includes >20 DNA-binding proteins that have been grouped on the basis of a highly conserved 85-residue region, referred to as the ETS domain.¹² The ETS domain mediates the binding of ets proteins as monomers to a 20-bp DNA site. Homologues of many of the ets genes have been found in organisms ranging from Drosophila to humans. Ets proteins have been implicated in a number of cellular processes, such as thymocyte/lymphocyte development and differentiation,¹³ as a component of the serum response ternary complex¹⁴ and in angiogenesis.¹⁵ Ets-1 transcription is transiently observed in groups of mesodermal cells engaged in morphogenetic processes such as organ formation and tissue modeling. Throughout development, c-ets-1 is highly expressed in endothelial cells at the onset of the formation of the blood vessel.¹⁶ Recent articles demonstrate that several ets proteins, including ets-1, act as transcription factors. Transcription of the stromelysin ¹⁷ and urokinase plasminogen activator¹⁸ genes, among others, has been shown to be efficiently activated by ets-1. Furthermore, PEA3, a member of the ets family of transcription factors, activates the collagenase promoter.¹⁹ Ets-1 may also participate in the regulation of tumor invasion, presumably by controlling the transcriptional activation of matrix-degrading protease genes in stromal fibroblasts.²⁰ TNF affects growth and differentiation of several different cell types, including SMCs, and is a potent mediator of inflammation, and it has been suggested that ets-1 plays an essential role in the activation of TNF gene transcription.²¹ Furthermore, an ets-1 binding site has been demonstrated in the promoter of the osteopontin gene.²² In the present study, we demonstrate serum-induced expression of ets-1 in SMCs in vitro and in medial SMCs after balloon damage in rat arteries. We postulate that ets-1 is an interesting candidate as an early mediator of vascular remodeling after arterial injury.

	Materials and Methods

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Cell Culture
SMCs were isolated from the aortas of 7- to 9-month-old Sprague-Dawley rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind) by digestion in 0.2% collagenase (type I, Sigma Chemical Co), as described previously.²³ The cells were then seeded in 75-cm² plastic flasks (3-5x10⁶ cells per flask, Costar Corp) and grown in Ham's F-12 medium supplemented with 50 mg/mL L-ascorbic acid, 50 mg/mL gentamicin, and 10% NCS at 37°C in 5% CO₂ in air. The cells were passaged by treatment with 0.25% trypsin/0.02% EDTA in PBS. Cells used in the experiments were in their second or third passage. All cell culture media and sera were purchased from GIBCO.

RNA Extraction
RNA was extracted by the method of Chirgwin et al.²⁴ Briefly, cells were washed twice at 4°C with PBS. A lysis solution containing 4 mol/L guanidine isothiocyanate, 3 mol/L sodium acetate (pH 6.0), 0.5% sodium N-lauroylsarcosine, and 0.83% ß-mercaptoethanol was added to each dish, and the cells were scraped with a rubber policeman. The suspensions were homogenized before being layered onto 6 mL of 5.7 mol/L cesium chloride and centrifuged for 18 to 20 hours at 170 000g at 20°C. The supernatant was carefully removed, and the RNA pellet was resuspended in 0.3 mol/L sodium acetate. The solubilized RNA was precipitated overnight with 2.5 vol ethanol at -20°C. The RNA was then pelleted, washed with 80% ethanol, dried, and resuspended in sterile water. UV spectrophotometry at 260 and 280 nm was used for quantification and to determine purity of the total RNA.

Northern Blot Analysis
Gel electrophoresis of 20 µg total RNA was performed on 1.1% agarose gels containing 2.2 mol/L formaldehyde as previously described.²⁵ The filters were hybridized in a buffer containing 50% formamide, 5x SSC (43.8 g/L NaCl and 22 g/L sodium citrate), 5x Denhardt's solution (1 g/L polyvinylpyrrolidone, 1 g/L BSA, and 1 g/L Ficoll 400), 0.1% SDS, 100 µg/mL salmon sperm DNA, and 10% dextran sulfate for 20 hours at 42°C with ³²P-labeled DNA probes. Probes were labeled with [-³²P]dCTP using the random-primer technique²⁶ following the manufacturer's protocol (Stratagene, Inc). The following probes were used: ets-1 (pmEts100a, HindIII fragment, American Type Culture Collection) and cyclophilin (pCD15.8.1, BamHI fragment).²⁷

Western Blot Analysis
SMCs were seeded in 100-mm dishes in serum containing Ham's F-12 medium for 24 hours. After serum starvation for 48 hours, cells were exposed to 10% NCS for 2 and 6 hours. Cells were rinsed and scraped in cold PBS and subsequently lysed in 10 mmol/L CHAPS in PBS containing 5 mmol/L EDTA, 1 mmol/L PMSF, and 5 mmol/L benzamidine. The cell lysates were concentrated 10 times by ultrafiltration through a Centricon-10 microconcentrator (Amicon). For quantification of total protein, a small aliquot was measured from each retentate by the method of Bradford according to the manufacturer's protocol (Pierce). Total protein (100 µg) from each sample was mixed with sample buffer (ratio, 4:1; 62.5 mmol/L Tris, 10% glycerol, 2.3% SDS, and 10 mmol/L DTT, pH 6.8) and 2 mL of mercaptoethanol before heating to 100°C for 5 minutes. This mixture was electrophoresed through a 10% discontinuous SDS-polyacrylamide gel in nondenaturing conditions at a constant current of 15 mA for 3 to 4 hours until 15 minutes after the dye front reached the bottom of the gel. Proteins were transferred in electroblotting buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol) onto a prewetted nitrocellulose membrane (Hybond ECL, Amersham) in a transfer cell at 200 mA for 40 minutes. Nonspecific binding sites were blocked by immersing the membrane in PBS-T supplemented with 5% nonfat dry milk for 16 hours. The membrane was rinsed three times in PBS-T and subsequently incubated for 1 hour at room temperature with a rabbit polyclonal ets-1 antibody (Santa Cruz Biotechnology, Inc) diluted 1:1000 in PBS-T. After rinsing in PBS-T, the membrane was incubated with a biotinylated anti-rabbit antibody (Amersham) diluted 1:5000 in PBS-T. To minimize background, the membrane was washed once for 15 minutes and four times for 5 minutes each. Immunoreactive protein bands were visualized by enhanced chemiluminescence (Amersham).

Preparation of Nuclear Extracts
Subconfluent serum-starved SMCs were washed twice in ice-cold PBS, and cells were scraped off their dishes with a rubber policeman. Nuclear extracts were prepared essentially as described by Alksnis et al.²⁸ Briefly, cells were washed with 1 mL PBS and resuspended in 100 µL hypotonic buffer (10 mmol/L HEPES [pH 7.3], 10 mmol/L KCl, 1.5 mmol/L MgCl₂, 1 mmol/L DTT, 1 mmol/L PMSF, 0.7 µg/mL leupeptin, and 16.7 µg/mL aprotinin). After centrifugation, cells were lysed by resuspension in 300 µL of lysis buffer (10 mmol/L HEPES [pH 7.3], 10 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.4% Nonidet P-40, 1 mmol/L DTT, 1 mmol/L PMSF, 0.7 µg/mL leupeptin, and 16.7 µg/mL aprotinin). The isolated nuclei were resuspended in 15 µL of 20 mmol/L KCl buffer, and 60 µL of 0.6 mol/L KCl buffer (20 mmol/L HEPES [pH 7.3], 22% glycerol, 0.6 mol/L KCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 0.7 µg/mL leupeptin, and 16.7 µg/mL aprotinin) was added. Nuclear proteins were extracted by incubation on ice for 30 minutes. After centrifugation for 15 minutes at 8000g, the supernatant containing nuclear proteins was transferred to precooled microcentrifuge tubes, and an aliquot of the extract was diluted 40 times with 484 mmol/L KCl buffer for protein assay. Protein concentration was determined spectrophotometrically according to the following equation: Protein Concentration (µg/mL)=184xA (230 nm)-81.7xA (260 nm).

EMSA
EMSA was performed as described by Schütze et al.²⁹ Equal amounts of protein from nuclear extracts (2 µg) were incubated on ice with 3 µg poly(dI-dC) (Pharmacia) in binding buffer (giving the final concentrations stated below) for 10 minutes. The oligonucleotide probe (7500 cpm in 3 µL) was added, and the reaction mixture (25 µL) was incubated for 30 minutes at room temperature. Final concentrations in the binding reactions were as follows: 50% glycerol, 100 mmol/L Tris (pH 7.5), 500 mmol/L NaCl, 1 mmol/L DTT, and 1 mmol/L PMSF. DNA-protein complexes were separated from unbound DNA probe on native 4% polyacrylamide gels in low ionic strength buffer (4.45 mmol/L Tris, 4.45 mmol/L borate, and 0.1 mmol/L EDTA, pH 8.0). The sequences of the double-stranded oligonucleotide probes labeled with T4 kinase and [-³²P]dATP were as follows: ets-1 consensus, 5'-GTC AGT TAA GCA GGA AGT GAC TAA C-3' (the underlined nine bases are required for ets-1 binding)¹⁷ ; ets-1 mutant, 5'-GTC AGT TAA GCA GGC AGT GAC TAA C-3' (Scandinavian Gene Synthesis).

Balloon Arterial Injury of Rats
Adult male Sprague-Dawley rats (400 to 500 g) were anesthetized with sodium pentobarbital (20 mg/kg IP). The left iliac artery was exposed, and a 2F embolectomy catheter was advanced to the aortic arch. The balloon was inflated with 0.7 mL saline and withdrawn into the abdominal aorta at least three times. The iliac artery was subsequently ligated. For the immunohistochemistry experiments, the carotid arteries were catheterized and balloon-injured. Rats were killed at 2 hours, 6 hours, 1 day, and 2 days after injury, and their arteries were removed and either snap-frozen and stored at -70°C for RNA extraction or immersed in 4% paraformaldehyde for a minimum of 16 hours and embedded in OTC medium for cryosectioning.³⁰

RNase Protection Assay for c-Ets-1 mRNA Expression In Vivo
A rat ets-1 riboprobe complementary to a fragment of exon 8 and 9 of the ets-1 cDNA (a region not subject to alternative splicing) was constructed. A 323-bp fragment of pc-ets-1³¹ was amplified by polymerase chain reaction with the following primers: 5'-ACACAGGAAGTGGGCCGATC-3' and 5'-TTCACATCGTATAGGGCATG-3'. The polymerase chain reaction product was then inserted into pBluescript by T/A cloning. The resulting plasmid pETS-8-9 was linearized with Xho I. A [-³²P]UTP–labeled riboprobe was generated by T3 RNA polymerase, predicted to protect a c-ets-1 mRNA fragment of 293 bases. After in vitro transcription, the reaction product was electrophoresed through 5% Sequagel, and the full-length riboprobe was excised. RNase protection was performed by incubating 20 µg of total aortic tissue RNA according to the manufacturer's instructions (Ambion, Inc), and the protected c-ets-1 mRNA fragments after RNase digestion were resolved by electrophoresis through 5% Sequagel. The resulting gel was exposed to x-ray film at -80°C with intensifying screens.

Immunohistochemistry
The 6-mm-thick sections were mounted on Superfrost slides, rinsed in PBS, and incubated with blocking serum for 1 hour. Before permeabilization in PBS/0.2% Triton X-100, the sections were repeatedly washed in PBS. They were then incubated for 16 hours in a humidified chamber with a rabbit polyclonal ets-1–specific antibody (Santa Cruz Biotechnology, Inc) diluted 1:50 in PBS/0.1% Triton X-100. After rinsing in PBS, sections were incubated for 30 minutes with a biotinylated anti-rabbit IgG (Pierce). After rinsing in PBS, slides were exposed to an avidin-biotinylated alkaline phosphatase complex (Pierce) for 30 minutes. A preadsorbtion of the primary antibody with excess recombinant ets-1 peptide (Santa Cruz Biotechnology, Inc) was used as a negative control. By following the manufacturer's protocol (Pierce), the antigen was detected by a red precipitate color.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Serum-Induced c-Ets-1 mRNA Expression in SMCs In Vitro
Northern blotting demonstrates low but detectable levels of 5.3- and 4.0-kb c-ets-1 mRNA transcripts in SMC cultures serum-starved for 48 hours and subsequently treated with serum for 0.5 and 1 hour (Fig 1). A marked transient increase in the abundance of c-ets-1 transcripts, particularly the 5.3-kb band, was observed, with a maximum level achieved 2 hours after the addition of serum (Fig 1). c-Ets-1 mRNA content was shown to be concentration dependent, with a maximal effect in cells exposed to 10% NCS (Fig 2). The effects on ets-1 mRNA abundance of PDGF-BB and angiotensin II, two specific factors present in serum which are known to be involved in the regulation of SMC growth and matrix remodeling, are shown in Fig 3. Ets-1 mRNA levels were induced by PDGF-BB and angiotensin II with a time course identical to that for serum (Fig 3, panels A and B, respectively).

View larger version (73K): [in this window] [in a new window]   Figure 1. Induction of c-ets-1 mRNA transcripts by 10% NCS. A, Northern blot demonstrating c-ets-1 mRNA levels in SMCs serum-starved for 48 hours and subsequently treated with 10% NCS for 0.5, 1, 2, 4, and 6 hours before RNA extraction. Arrows indicate ets-1 mRNA transcripts of 5.3 and 4.0 kb. B, Blot hybridized with cyclophilin cDNA. Control represents mRNA expression in untreated serum-starved SMCs.

View larger version (100K): [in this window] [in a new window]   Figure 2. Dose-response relationship of serum-induced c-ets-1 mRNA expression. Serum-starved SMCs were treated with 0.1%, 1%, 10%, or 20% NCS for 2 hours and harvested for RNA analysis by Northern blotting. Arrows indicate ets-1 mRNA transcripts of 5.3 and 4.0 kb. The blot was sequentially hybridized with c-ets-1 cDNA (A) and cyclophilin cDNA (B). Control represents untreated serum-starved SMCs.

View larger version (91K): [in this window] [in a new window]   Figure 3. Time course of induction of c-ets-1 mRNA abundance by PDGF-BB or angiotensin II (AII). Serum-starved SMCs were incubated with the indicated concentration of the agents (nanograms per milliliter) and harvested for RNA extraction at the indicated times. A, Northern blot hybridized with c-ets-1 cDNA. Arrows indicate ets-1 mRNA transcripts of 5.3 and 4.0 kb. B, Ethidium bromide staining of ribosomal RNA bands.

Serum-Induced Immunoreactive Ets-1 in SMCs In Vitro
Stimulation of quiescent SMCs with 10% NCS was also associated with an increase in immunoreactive ets-1, as determined by Western blotting with a polyclonal rabbit antibody to ets-1 (Fig 4). The abundance of the 55- and 40-kD proteins was maximal at 2 hours and decreased 6 hours after the addition of serum. The smaller immunoreactive ets-1 band was also regulated coordinately, which is in agreement with earlier findings.³²

View larger version (52K): [in this window] [in a new window]   Figure 4. Expression of immunoreactive ets-1 in SMCs. SMCs were serum-starved for 48 hours and subsequently treated with 10% NCS for 2 or 6 hours. Ets-1 protein expression was determined by Western blotting. Control represents immunoreactive ets-1 in untreated serum-starved SMCs. Arrows indicate bands of 55 (top arrow) and 40 (bottom arrow) kD.

Detection of Activated Ets-1 in SMCs
SMCs contained an ets-1–like protein that binds specifically to an ets DNA cis-acting motif (Fig 5, lane 2) but not to an oligomer containing a single base substitution (Fig 5, lane 1). Gel-shift experiments revealed one distinct band and one weaker band, both specific for ets-1. Addition of excess unlabeled mutant ets-1 oligonucleotide (with one point mutation in the binding site) did not compete for binding (Fig 5, lane 4), whereas competition with unlabeled wild-type probe completely abolished ets-1 DNA binding activity (Fig 5, lane 3). Furthermore, the addition of ets-1 peptide antibodies (directed against the N-terminal domain of the ets-1 protein; Santa Cruz Biotechnology, Inc) to the reaction resulted in the disappearance of specific binding, whereas no effect could be detected with rabbit preimmune serum or an antibody specific for the Rb p110–related protein, p107 (Fig 5, lanes 5 and 6).

View larger version (103K): [in this window] [in a new window]   Figure 5. Binding activity of ets-1 protein in serum-starved SMCs. EMSA blot is shown of nuclear extracts from cells treated with NCS for 2 hours and incubated with either mutated c-ets-1 oligonucleotide (lane 1) or the wild-type ets-1 binding consensus motif (lane 2), the wild-type probe incubated with either unlabeled wild-type antibody (x100) (lane 3), mutant antibody (x100) (lane 4), ets-1 antibody (lane 5), preimmune serum (lane 6), or a p107 antibody (lane 7).

c-Ets-1 mRNA Expression in Aortic Tissue
A 2F embolectomy catheter was used to balloon-distend the aorta of adult male rats. c-Ets-1 mRNA abundance in aortic tissue was induced by about fourfold after balloon injury, with an early peak after 2 hours (Fig 6). c-Ets-1 mRNA content remained higher than in uninjured aortas through 3 days, before declining on days 7 and 14. Three replicate experiments confirmed both the magnitude and kinetics of the injury-mediated stimulation of c-ets-1 expression.

View larger version (38K): [in this window] [in a new window]   Figure 6. Time course of arterial c-ets-1 mRNA expression after balloon aortic injury. A rat-specific c-ets-1 riboprobe complementary to a region bracketing part of exons 8 and 9 was incubated with 20 µg RNA from aortas harvested before (control [C]) or at the indicated time points after balloon injury. The mixture was exposed to RNase to degrade single-stranded RNA. Lane M shows molecular weight markers. Yeast RNA (Y. RNA) was incubated to the riboprobe as a negative control. An aliquot of free riboprobe is displayed in the outer right lane. Aortic samples exhibit a protected c-ets-1 mRNA fragment of 293 bases.

Ets-1–Like Protein Expression in SMCs In Vivo
In sections from aortas of rats killed 2 hours after injury, a strong ets-1 immunoreactivity was detected. The staining is localized to the inner layers of the media and declines as soon as 6 hours after injury, returning to near-basal levels on day 1 after injury (Fig 7). Sections from the control arteries as well as sections incubated with a preadsorbed ets-1 antibody did not reveal any immunoreactivity (Fig 7, control and right panels).

View larger version (122K): [in this window] [in a new window]   Figure 7. Ets-1–like protein expression in vivo. Immunohistochemistry demonstrates ets-1–like protein immunostaining in rat arteries 2 hours, 6 hours, and 1 day after injury. Control represents uninjured artery. Right panels demonstrate adjacent sections incubated with the ets-1 antibody preadsorbed with excess ets-1 peptide.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present findings demonstrate that the c-ets-1 gene is expressed in vascular SMCs and that an activated DNA-binding ets-1 protein is present in the cell nucleus of these cells. Exposure of SMCs to serum results in a dose-dependent increase in c-ets-1 mRNA levels and ets-1 protein, with a peak value at 2 hours. The cells were found to contain two transcripts (5.3 and 4.0 kb) as well as two ets-1 immunoreactive proteins (55 and 40 kD). Indeed, earlier studies have documented the presence of two ets-1 proteins in mesodermal cells.³³ The two different transcripts may be due to alternative splicing or differential polyadenylation, events that previously have been shown to occur within the c-ets-1 locus in T cells from different species.^{31 34} The expression of the c-ets-1 gene in SMCs appears to be regulated by serum factors. The observation that serum, PDGF-BB, and angotensin II increase c-ets-1 transcript levels indicates that the expression of this gene is controlled by serum factors regulating cell migration and growth. Analysis of nuclear extracts isolated after 2 hours of incubation with 10% NCS demonstrated the presence of a protein capable of binding the ets-1–specific consensus sequence GCA GGA AGT. This protein was identified as ets-1 by a change in mobility after the addition of an ets-1–specific antibody. The function of ets-1 in SMCs remains to be characterized.

Ets-1 has recently been shown to be present in mesodermal cells involved in tissue remodeling.¹⁶ Furthermore, Wernert et al²⁰ have detected c-ets-1 transcripts in stromal cells surrounding human carcinomas. The expression of c-ets-1 was often increased in fibroblasts adjacent to neoplastic cells, whereas fibroblasts of corresponding noninvasive lesions and of normal tissues were negative. These results suggest a role for ets-1 in the regulation of tumor invasion in vivo, possibly by activating the transcription of genes encoding enzymes that degrade the tissue surrounding the tumor. The enzymes necessary for degradation and remodeling of the extracellular matrix include the matrix-degrading metalloproteinases and the plasminogen activators.^{5 6 7} Ets-1 is known to regulate transcription of matrix-degrading metalloproteinases and the plasminogen activator.^{17 18} The sequence used in our EMSA experiments is identical to the ets-1 binding site in the promoter of the stromelysin gene.¹⁷ Thus, the function of the ets-1 transcription factor during these morphogenetic processes may be to regulate transcription of genes involved in matrix degradation and remodeling. Activation of SMC migration and proliferation plays a key role in the development of atherosclerotic plaque and restenosis following arterial angioplasty. Both these processes involve degradation and remodeling of the vascular extracellular environment. The fact that ets-1 has been shown to regulate genes coding matrix-degrading enzymes in other cell types suggests that it also may be of importance in the control of vascular SMC migration and growth. This concept is supported by the observation of Southgate et al³⁵ that synthetic inhibitors of MMPs inhibit explant outgrowth and proliferation of rabbit SMCs. Moreover, osteopontin, another ets-1–regulated gene,²² demonstrates increased expression in proliferating SMCs. Osteopontin is an extracellular matrix protein believed to play an important role in the adhesion of SMCs to the surrounding matrix and in their migration.³⁶

Besides matrix proteins and the enzymes that degrade them, ets-1 is known to participate in the transcriptional control of other gene products expressed in the vessel wall. An ets-1 binding motif has been reported in the human TNF promoter, the site-specific mutation of which leads to a complete loss of responsiveness to the transcription factor, suggesting an essential role of ets-1 for the activation of TNF gene transcription.²¹ TNF affects the growth and differentiation of a multitude of cell types, including SMCs, and is also produced by cultured SMCs. Using in situ hybridization, it has been demonstrated that SMCs in intimal lesions express TNF- mRNA, whereas medial SMCs do not.³⁷ We have recently reported that balloon injury of the rat aorta is associated with increased expression of PTHrP.³⁸ PTHrP is believed to function as an autocrine/paracrine vasodilator and to play a role in SMC compliance to mechanical stimuli. Interestingly, transcription of PTHrP is also under the control of ets-1.³⁹

Balloon injury of the rat carotid artery has been used to study the activation and control of SMC migration and proliferation in vivo. This insult results in a removal of the endothelium and death of SMCs in the luminal part of the media. Within a few days, medial SMCs migrate into the intima and start to proliferate. Studies performed on cultured SMCs have shown that mechanical injury of SMCs is associated with enhanced expression of MMP genes. Furthermore, as SMCs enter the intima of injured rat carotid arteries, they begin to produce osteopontin. The present observation that balloon injury of the rat aorta is associated with increased expression of both c-ets-1 mRNA and ets-1 protein within 2 hours is in accordance with the notion that this transcription factor regulates genes encoding matrix-degrading enzymes and growth-stimulating matrix proteins, which would enable SMC migration. Induction of stromelysin gene expression activated by mechanical injury has been detected in the vascular smooth muscle–derived cell line Rb-1.⁴⁰ The mechanisms involved in the upregulation of c-ets-1 remain to be elucidated.

The present studies also show that serum stimulates the expression of c-ets-1 mRNA in a dose-dependent manner. In the human T-cell lines CEM and HSB2, serum activation of c-ets-1 transcription was linked to induction of the c-fos/c-jun AP-1 dimer and activation of an AP-1 binding site in the c-ets-1 promoter.⁴¹ A similar mechanism may also be responsible for serum-induced activation of the c-ets-1 gene in SMCs. This notion is also supported by the finding of an increased expression of c-fos/c-jun in rat SMCs treated with serum for 30 minutes.⁴² Majesky et al⁴³ have previously shown that balloon injury of the rat aorta results in an increased accumulation of c-fos mRNA within 1 hour, suggesting that a similar mode of activation could be involved in c-ets-1 expression in the vascular wall.

In conclusion, the present findings demonstrate a serum-inducible production of the ets-1 transcription factor in cultured rat SMCs. The observation of an increased expression of ets-1 following balloon injury of the rat carotid artery, taken together with previous studies demonstrating that ets-1 regulates genes involved in matrix structure and degradation, suggests that ets-1 may play a key role in the regulation of vascular SMC migration and vessel remodeling.

	Selected Abbreviations and Acronyms

 

DTT = dithiothreitol EMSA = electrophoretic mobility shift assay MMP = matrix metalloproteinase NCS = newborn calf serum PBS-T = PBS-Tween PDGF = platelet-derived growth factor PMSF = phenylmethylsulfonyl fluoride PTHrP = parathyroid hormone–related peptide SMC = smooth muscle cell TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL-43802, the Swedish Heart and Lung Foundation, the Tore Nilson Fund, the King V 80th Birthday Fund, and the Funds of Karolinska Institutet. Dr Fagin is a recipient of an Established Investigator Award of the American Heart Association and Bristol-Myers Squibb.

Received July 31, 1995; accepted December 29, 1995.

	References

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