INTRODUCTION

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THE PROTOONCOGENE c-ets is the cellular progenitor of v-ets and codes for a transcription factor that activates the oncogene responsive unit of the polyoma virus enhancer and interacts with the promoters of several eukaryotic genes (28). The v-ets was originally identified as a second transforming-specific sequence fused to the v-myb oncogene in the avian leukemia retrovirus E26. The c-ets-1, ets-2, erg, elk-1, and elk-2 genes belong to a family of transcription factors that play an important role in the regulation of cell proliferation and differentiation (4). The c-ets-1 gene product cooperates with the c-fos/c-jun complex [activator protein 1 (AP-1)] to activate expression of certain promoters (28). Furthermore, the ets-1 binding site, containing a central GGAA/T core motif (PEA3), has been identified in the promoter of some matrix-degrading metalloproteinase genes such as stromelysin, collagenase, and urokinase plasminogen activator, suggesting possible roles for ets-1 in the regulation of matrix degradation and tissue remodeling (10, 15).

After balloon intra-arterial injury, a program is initiated that results in vascular smooth muscle cell (VSMC) migration, proliferation, and a change in the composition and structure of the vascular extracellular matrix (20). The signaling process that orchestrates the injury response is multifactorial and involves a number of mesenchymal cell growth factors, some of which are released from platelets adhering at the injury site, whereas others are produced within the vessel wall itself. In this vascular remodeling process, matrix-degrading metalloproteinases are believed to be necessary for smooth muscle cell (SMC) proliferation and migration into the neointima (3). Ets-1 is expressed by periluminal arterial SMC, with a first peak occurring 2 h after balloon injury (12).

Serum induces ets-1 expression in quiescent VSMC with a time course comparable to that seen after arterial injury in vivo (12). The objective of this study was to determine which factor, among the growth and chemotactic factors believed to play a role in the early stages of the arterial response to balloon denudation, stimulates the expression of this transcription factor and to explore the signal transduction pathways involved.

We report that platelet-derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) are potent activators of ets-1 gene expression in VSMC. Furthermore, protein kinase C (PKC) activation and release of Ca²⁺ stored in the endoplasmic reticulum (ER) play an essential role in ets-1 gene expression induced by PDGF-BB and ET-1, which is not abrogated by inhibition of protein synthesis. Induction of ets-1-mediated transcriptional activity may be a common mechanism by which certain growth and/or chemotactic factors activate a program of extracellular matrix remodeling in vascular smooth muscle.

	EXPERIMENTAL PROCEDURES

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Cell culture. VSMC were isolated from the aorta of 6-mo-old Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) by digestion in 0.2% type I collagenase (Sigma Chemical, St. Louis, MO) as described by Chamley-Campbell et al. (5), with minor modifications. The cells were then seeded in 75-cm² plastic flasks (Costar, Cambridge, MA; 3- 5 × 10⁶ cells/flask) and grown in Dulbecco's modified Eagle's medium-F-12 supplemented with 2.438 g/l sodium bicarbonate, 50 µl/ml streptomycin and penicillin, and 10% newborn calf serum at 37°C in 5% CO₂ in air. Cells were passaged by treatment with 0.25% trypsin-0.02% EDTA in phosphate-buffered saline (PBS). The cells used in the experiments were in their second or third passage. All cell culture media and sera were purchased from GIBCO (Grand Island, NY).

Reagents and cDNA probes. PDGF-BB, insulin-like growth factor I (IGF-I), transforming growth factor- (TGF-), and basic fibroblast growth factor (bFGF) were purchased from Austral Biologicals (San Ramon, CA). ET-1 was from Bachem Bioscience (King of Prussia, PA). Herbimycin A, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), phorbol 12-myristate 13-acetate (PMA), forskolin, thapsigargin, and ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) were purchased from Sigma Chemical. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) was from Molecular Probes (Eugene, OR). The following cDNA probes were used: the rat ets-1 probe was a 1.4-kilobase (kb) BamH I fragment of ets-1 cDNA cloned in pLXSN plasmid vector; the human collagenase probe was a 2.2-kb Xho I fragment of p35-1 (American Type Culture Collection 79062). For loading controls, blots were rehybridized with a 1.4-kb Pst I fragment of rat glyceraldehyde-3-phosphate dehydrogenase cDNA. Probes were gel purified and ³²P-labeled by random priming as described previously (7).

Northern blot hybridization. Total RNA was extracted from cultured rat VSMC with the TRIzol reagent (GIBCO), according to the manufacturer's instructions. Twenty micrograms of total RNA were size separated by electrophoresis through 1% agarose-formaldehyde gels. RNA samples were transferred to nylon membranes (Micron Separation, Westborough, MA) and hybridized to the indicated random prime-labeled cDNA probe. Hybridization reactions were carried out for 16-24 h at 65°C in 0.25 M Na₂HPO₄ (pH 7.2), 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS), and 30% formamide. Membranes were washed in 20 mM Na₂HPO₄ (pH 7.2), 1 mM EDTA (pH 8), and 1% SDS. All experiments were performed at least three times. Blots were exposed to Kodak X-Omat AR film at 80°C for 12-72 h and/or quantified with a PhosphorImager (Molecular Dynamics).

Inositol phosphate formation. Total inositol phosphate formation was measured essentially as described previously (1, 2). Briefly, VSMC were prelabeled with 2.4 µCi/ml [³H]inositol (Amersham, Arlington Heights, IL) for 24 h in inositol-free medium containing 10% dialyzed fetal calf serum. VSMC were then washed and incubated in PBS containing 20 mM Li²⁺ with the indicated agents at 37°C for 5 min. Metabolic activity was stopped by addition of 100 µl of 10% perchloric acid and 10 µl phytic acid (20 mg/ml) on ice for 20 min followed by centrifugation. The supernatants were neutralized with 2 M KOH and 1 mM EDTA and subjected to anion exchange chromatography. The final eluant was dissolved in scintillation fluid and counted.

Measurement of intracellular Ca²⁺ concentration. Intracellular free Ca²⁺ was assessed using the Ca²⁺-sensitive fluorescent dye fura 2 (Sigma) according to the method of Grynkiewicz et al. (9). Cells were seeded in 35-mm-bottom glass dishes. After they were plated, cells were incubated in 2 ml of complete culture medium containing 5 µM fura 2-AM and 0.05% cremophor for 60 min at 37°C under 5% CO₂-95% air. The cells were washed three times in a 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered physiological saline solution with a composition of (in mmol/l) 140 NaCl, 4.7 KCl, 1.2 NaH₂PO₄, 20 MOPS, 0.02 EDTA, 1.2 MgSO₄, 2.5 CaCl₂, and 11 glucose (pH 7.4) and allowed to equilibrate for 15 min at room temperature with or without the indicated reagents. The fluorescence images of the cells were recorded with a video image analysis system (InCa system, Intracellular Imaging). Fluorescence intensities were not corrected for background, as in control experiments autofluorescence of these cells was negligible. The cells were excited sequentially at 340 and 380 nm, and emissions at 510 nm were measured. The 340- and 380-nm images were ratioed on a pixel-by-pixel basis, and the ratios were converted to Ca²⁺ concentrations using a previously generated standard curve. Standard solutions of free Ca²⁺ (0-10 mM Ca²⁺-EGTA, 100 mM KCl, and 10 mM MOPS, pH 7.2; calcium calibration buffer kit II, Molecular Probes) containing 10 µM fura 2 were used to make the standard curve. All Ca²⁺ measurements were carried out at room temperature.

	RESULTS

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Effect of growth and chemotactic factors on ets-1 mRNA levels. As a first approach to examine the mechanisms controlling ets-1 gene expression in VSMC, serum-deprived cells were treated with selected growth or chemotactic factors believed to play a role in VSMC proliferation and/or migration. Figure 1 shows that serum induced a concentration-dependent induction of ets-1 mRNA. In addition, PDGF-BB, ET-1, and PMA induced expression of a major ets-1 mRNA transcript of 5.3 kb and of minor bands of 4.0 and 2.5 kb at 2 h after exposure (by ~7-fold). In contrast, IGF-I, TGF-, and bFGF had only a modest effect (1.8-, 2.2-, and 2.2-fold respectively) through a range of concentrations (1-100 ng/ml, not shown). Notably, all factors increasing ets-1 mRNA abundance are known to activate PKC. In contrast, IGF-I and TGF- signal through alternative pathways, whereas the signaling intermediates for bFGF in SMC are unclear. Activation of adenylyl cyclase activity by treatment with forskolin appeared to decrease basal levels of ets-1 mRNA (Fig. 1). The effects of ET-1 and PDGF-BB on ets-1 mRNA were maximal at 2 h (Figs. 2 and 5) and were concentration dependent (Fig. 3).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effect of growth and chemotactic factors on ets-1 mRNA abundance. Vascular smooth muscle cells (VSMC) were serum starved for 48 h and then treated with or without 5% serum, 10% serum, platelet-derived growth factor-BB (PDGF-BB; 25 ng/ml), insulin-like growth factor I (IGF-I; 50 ng/ml), transforming growth factor- (TGF-; 5 ng/ml), basic fibroblast growth factor (bFGF; 50 ng/ml), endothelin-1 (ET-1; 100 ng/ml), phorbol 12-myristate 13-acetate (PMA; 100 ng/ml), or forskolin (100 µM) for 2 h before RNA extraction. After electrophoresis of 20 µg RNA per sample and transfer to a nylon membrane, the blot was sequentially hybridized with 32P-labeled ets-1 cDNA (top) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (bottom). Arrows indicate ets-1 mRNA transcripts of ~5.3, 4, and 2.5 kilobases (kb). Size of GAPDH is ~1.4 kb.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Time course of ET-1 induction of ets-1 mRNA. VSMC were serum starved for 48 h and then treated with either vehicle (control) or ET-1 (100 ng/ml) for indicated times. Representative Northern blot of 20 µg RNA was sequentially hybridized to either ets-1 (top) or GAPDH cDNA (bottom) probes. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Dose response of PDGF-BB and ET-1 induction of ets-1 mRNA. Serum-starved VSMC were treated with vehicle or indicated concentrations of PDGF-BB or ET-1 for 2 h. Northern blotting was as described in Fig. 1. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.

Role of PKC activation on the regulation of ets-1 mRNA. PMA activates PKC through binding to the C₁ region of its regulatory domain (18). PMA stimulation by itself caused a significant increase in ets-1 mRNA: it was maximal at 2 h and returned completely to basal levels after 12 h (data not shown). Cells were pretreated with PMA (200 ng/ml) for 24 h to deplete PKC activity. The downregulation of PKC isozymes by chronic PMA treatment almost completely abrogated the induction of ets-1 mRNA stimulated by PDGF-BB, ET-1, and PMA (Fig. 4A). H-7 inhibits PKC activity via a direct interaction on the catalytic site of the enzyme and suppresses PKC-mediated phosphorylation. Preexposure to 20 µM H-7 attenuated the maximal ets-1 mRNA levels induced by PDGF-BB (to 50% of stimulated levels), ET-1 (43%; P = 0.07), and PMA (10%, P < 0.01) (Fig. 4B). In contrast, herbimycin A, a protein tyrosine kinase inhibitor, blunted the ets-1 mRNA stimulation by ET-1 (to 6% of stimulated levels; P < 0.01) but had a marginal effect on PDGF-BB- or PMA-induced ets-1 mRNA (Fig. 4C).

View larger version (42K): [in this window] [in a new window]   View larger version (47K): [in this window] [in a new window]   View larger version (39K): [in this window] [in a new window]   Fig. 4.   Effects of inhibition of protein kinase C or protein tyrosine kinase activity on the regulation of ets-1 mRNA. A: VSMC were serum deprived for 24 h and then treated with or without 200 ng/ml of PMA for a further 24 h, before addition of one of the following agonists: PDGF-BB (25 ng/ml), ET-1 (100 ng/ml), or PMA (100 ng/ml) for 2 h. B: VSMC were serum deprived for 48 h. Cells were then pretreated with or without 20 µM H-7 for 1.5 h before addition of the indicated agonists for 2 h. C: VSMC were serum deprived for 48 h. Cells were then pretreated with or without herbimycin A (0.75 µg/ml) for 18 h before addition of the indicated agonists for 2 h. Northern blotting was as described in Fig. 1. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb (and 4 kb, second arrow from top in B) and GAPDH of 1.4 kb.

Inhibition of protein synthesis is associated with superinduction of ets-1 mRNA. Activation of PKC results in the rapid stimulation of biosynthesis of the c-Jun and c-Fos proteins, which after assembling into the AP-1 complex modulate the transcription of a repertoire of genes. Because the ets-1 promoter contains a putative AP-1 binding site, we examined whether general inhibition of protein synthesis (and hence of generation of AP-1 proteins) would impair ET-1 or PDGF-BB induction of ets-1 mRNA content. Pretreatment with cycloheximide alone was associated with a gradual accumulation of ets-1 mRNA (Fig. 5A). In the presence of the protein synthesis inhibitor, ET-1 and PDGF-BB superinduced ets-1 mRNA content (Fig. 5, A and B). The pattern of superinduction was similar to that of c-jun, an immediate early response mRNA that is not dependent on newly generated transcription factor synthesis for expression. Thus ets-1 gene expression in VSMC is consistent with that of an immediate early response mRNA and does not appear to be dependent on AP-1 synthesis.

View larger version (33K): [in this window] [in a new window]   View larger version (45K): [in this window] [in a new window]   Fig. 5.   Effect of inhibition of either protein synthesis or transcriptional activity on the regulation of ets-1 mRNA by ET-1 (A) or PDGF-BB (B). VSMC were serum deprived for 48 h. One hour before stimulation with agonists, cells were treated with or without 10 µg/ml cycloheximide (Cx) or 5 µg/ml actinomycin D (AcD). Cells were harvested at indicated times after addition of ET-1, PDGF-BB, or vehicle. Northern blots were sequentially hybridized with the indicated cDNAs. Arrows point to ets-1 mRNA transcripts of ~5.3 and 2.5 kb, c-jun mRNA of 2.7 kb, and GAPDH of ~1.4 kb.

Inhibition of ets-1 mRNA by adenosine 3',5'-cyclic monophosphate-mediated signal transduction. Forskolin stimulates the production of adenosine 3',5'-cyclic monophosphate (cAMP) and leads to the activation of protein kinase A (PKA). There is ample documentation that activation of PKA impairs phospholipase C (PLC)-mediated signal transduction. Forskolin (100 µM) markedly inhibited ets-1 mRNA induction by PDGF-BB, ET-1, or PMA (Fig. 6). As shown in Fig. 7, addition of either ET-1 or PDGF-BB significantly increased inositol phosphate generation. Pretreatment with forskolin abrogated the ET-1- and PDGF-BB-stimulated inositol phosphate production (Fig. 7), but, in these experimental conditions, pretreatment did not appear to impair intracellular Ca²⁺ mobilization (see Fig. 10).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Effect of forskolin on agonist-stimulated ets-1 mRNA expression. VSMC were serum deprived for 48 h. Cells were treated with or without 100 µM forskolin 5 min before addition of vehicle, PDGF-BB, ET-1, or PMA for 2 h. Northern blotting was as described in Fig. 1. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of forskolin on ET-1 or PDGF-BB stimulation of inositol phosphate formation. VSMC were treated with or without 100 µM forskolin for 5 min before addition of 100 ng/ml ET-1 (A) or 25 ng/ml PDGF-BB (B) for 1 min. Levels of inositol phosphates were determined as described in EXPERIMENTAL PROCEDURES. Values are expressed as means ± SD (n = 3 separate experiments). cpm, Counts/min. * P < 0.05.

Role of intracellular Ca²⁺ concentration on the regulation of ets-1 mRNA. A primary consequence of inositol 1,4,5-trisphosphate (IP₃) formation is the stimulation of IP₃-sensitive Ca²⁺ channels in the ER and release of Ca²⁺ stores. To explore the role of intracellular Ca²⁺ concentration ([Ca²⁺]_i) on regulation of ets-1 gene expression, cells were treated with either EGTA, a potent chelator of extracellular Ca²⁺, or the [Ca²⁺]_i chelator BAPTA-AM (19). Pretreatment with EGTA did not modify basal, ET-1-, PDGF-BB-, or PMA-induced ets-1 mRNA levels. In contrast, [Ca²⁺]_i chelation with BAPTA-AM resulted in almost complete abrogation of ets-1 mRNA production in response to these stimuli (Fig. 8). Acute treatment with thapsigargin, a selective inhibitor of the ER Ca²⁺-ATPase, results in an increase in [Ca²⁺]_i, followed by sustained inhibition of Ca²⁺ uptake and mobilization (see Fig. 10). Interestingly, thapsigargin was not associated with an increase in ets-1 mRNA, indicating that release of [Ca²⁺]_i was not sufficient by itself to induce ets-1 gene expression. However, pretreatment with thapsigargin for 8 h to deplete intracellular Ca²⁺ content of the ER markedly inhibited the induction of ets-1 mRNA by PDGF-BB, ET-1, and PMA (Fig. 9).

View larger version (47K): [in this window] [in a new window]   Fig. 8.   Effect of EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) pretreatment on agonist-stimulated ets-1 mRNA expression. VSMC were serum deprived for 48 h. EGTA (5 mM) pretreatment was performed at 5 min before addition of vehicle, PDGF-BB, ET-1, or PMA for 2 h. BAPTA-AM (30 µM) pretreatment was initiated 30 min before addition of agonists. Northern blotting was as described in Fig. 1. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.

View larger version (43K): [in this window] [in a new window]   Fig. 9.   Effect of thapsigargin (Tg) pretreatment on agonist-stimulated ets-1 mRNA expression. VSMC were serum deprived for 48 h and treated with either 100 nM thapsigargin or indicated agonists for 2 h. To examine the effects of chronic exposure to the endoplasmic reticulum Ca2+-ATPase inhibitor, serum-starved cells were pretreated with 100 nM thapsigargin for 8 h before addition of vehicle or indicated agonists. Northern blotting was as described in Fig. 1. Arrows point to ets-1 mRNA transcripts of 5.3 and 2.5 kb and GAPDH of 1.4 kb.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   Effect of forskolin, PMA, EGTA, BAPTA-AM, or thapsigargin on ET-1-induced intracellular Ca2+ concentration mobilization in VSMC. Serum-starved cells were grown on coverslips and loaded with fura 2-AM, and changes in intracellular Ca2+ were measured after addition of indicated stimuli, as described in EXPERIMENTAL PROCEDURES. Arrows indicate time of addition of the drugs. The following conditions are shown: 100 ng/ml ET-1 (A); cells pretreated with 100 µM forskolin before stimulation with 100 ng/ml ET-1 (B); cells exposed to 100 ng/ml PMA for 24 h before ET-1 (C); EGTA (5 mM) added before ET-1 (D); BAPTA-AM (30 µM) added before ET-1 (E); 100 nM thapsigargin (Tg, F); treatment with 100 nM thapsigargin for 8 h before addition of ET-1 (G). Data are representative of at least 7 separate cell tracings obtained from 2 independent experiments for each condition.

Effect of H-7 and herbimycin A on sequential induction of expression of ets-1 and collagenase I mRNA by ET-1. There is growing evidence that ets-1 is involved in transcriptional control of matrix-degrading proteinases such as collagenase I, stromelysin 1, and the urokinase-type plasminogen activator. Inhibitors of ets-1 mRNA expression in response to ET-1 such as herbimycin A and H-7 (Fig. 11A) also resulted in abrogation of expression of collagenase I mRNA (Fig. 11B). Interestingly, expression of stromelysin mRNA was abundant even in serum-deprived SMC and only modestly induced by ET-1 (data not shown).

View larger version (K): [in this window] [in a new window]   Fig. 11.   Sequential activation of ets-1 and collagenase mRNA levels by ET-1 in VSMC: effects of H-7 and herbimycin A (HA). Time course of regulation of ets-1 (A) or collagenase I mRNA (B) by ET-1 in the presence or absence of either H-7 or herbimycin A. Abundance of indicated transcripts was calculated by densitometry relative to that of GAPDH mRNA, used as an internal control for uniformity of loading and transfer.

	DISCUSSION

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The c-ets-1 protooncogene is believed to transactivate the promoters of a number of cellular genes coding for growth factors, proteins involved in the immune response, and extracellular matrix-degrading proteinases (4). Because of its relatively restricted pattern of expression during embryogenesis, in which it is preferentially detected in lymphoid tissues, there has been considerable attention focused on its possible role in T cell maturation in the fetal thymus and T cell activation in adults (4). However, c-ets-1 expression is also seen in mesenchymal and endothelial cells of the developing embryo (27). Its expression has also been noted in stromal cells and capillary vessels at the margins of tumor invasion (30). Recently, c-ets-1 expression was observed in periluminal arterial SMC following balloon injury (12). The pattern of expression in human cancer fibroblasts and VSMC is consistent with induction of expression mediated by extracellular stimuli, presumably by growth factors or cytokines. Notably, there is scant information on which factors may be involved in regulating ets-1 gene expression and of the signal transduction pathways implicated. The transcriptional activity of ets-1 is modulated in part through a ras-mediated threonine phosphorylation in its conserved amino-terminal domain, resulting in superactivation of reporter systems driven by promoters containing ets/AP-1 binding sites (31). However, although the activity of ets-1 is subject to regulation through ras-mediated posttranslational modifications of the protein, before this study it was not clear whether the overall abundance of ets-1 was also subject to regulation by growth factors. In this study, we describe that ets-1 fulfills the characteristics of an early response gene in arterial SMC and that it is subject to regulation by a PKC-mediated signal transduction pathway activated by growth factors such as PDGF-BB and ET-1.

Treatment of VSMC with the protein synthesis inhibitor cycloheximide resulted in clear superinduction of ets-1 mRNA in response to both ET-1 and PDGF-BB. This indicates that neither of these growth factors requires synthesis of an intermediary protein(s) to induce ets-1 mRNA. The accumulation of ets-1 mRNA in the presence of cycloheximide alone may be due to either the abrogation of synthesis of a repressor of ets-1 transcription or of a factor accelerating its degradation. While this paper was in preparation, Gilles et al. (8) reported that ets-1 mRNA was also superinduced in human fibroblasts in response to tumor necrosis factor-. To our knowledge, the categorization of ets-1 as an early response gene is novel and has significant implications. Structural analysis of the human ets-1 promoter identified one binding site each for AP-1, AP-2, and ets-1 itself (17). Functional studies demonstrated that exogenous expression of c-jun, AP-2, and ets-1 were associated with activation of the ets-1 promoter (17). The evidence shown here that PDGF-BB and ET-1 induction of ets-1 mRNA does not require new protein synthesis indicates that generation of intermediary transcription factors such as ets-1, c-jun, and AP-2 is not essential for expression of the ets-1 gene.

Of the various growth factors tested, PDGF-BB and ET-1 were the most powerful stimuli of ets-1 mRNA in quiescent SMC. Interestingly, PDGF-BB and ET-1 interact with receptors belonging to two distinct families (and couple to particular sets of signaling intermediates) that converge on PKC through alternative routes. After PDGF binds and activates its specific receptor tyrosine kinase, it induces tyrosine phosphorylation of PLC-. The stimulation of PLC- results in phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis, with consequent generation of inositol 1,4,5-trisphosphate and diacylglycerol (DAG), leading to [Ca²⁺]_i mobilization and PKC activation, respectively. On the other hand, ET-1, originally isolated as a vasoconstrictor peptide, interacts with at least two subtypes of membrane-anchored serpentine proteins belonging to the family of G protein-coupled receptors. ET-1 stimulates PLC- through G protein activation and PLC- through autophosphorylation of nonreceptor tyrosine kinases such as pp60^c-src (14, 24). Indeed, PDGF-BB and ET-1 are known to evoke the expression of c-fos and c-jun mRNA levels through PKC and/or protein tyrosine kinase activity in mesangial cells, fibroblasts, and VSMC (19, 21, 23, 25, 29). There is increasing evidence that AP-1 cooperates with ets-1 in the transcriptional stimulation of genes for matrix-degrading enzymes, which heightens the significance of the coactivation of c-ets-1, c-jun, and c-fos gene expression by PKC-mediated pathways.

In contrast to the extensive investigations on the role of ets transcription factors on the function of various gene promoters, the signal transduction pathways involved in the control of ets-1 gene expression are poorly understood. We showed that H-7, a PKC antagonist, inhibited ets-1 mRNA expression by both PDGF-BB and ET-1. Because the action of H-7 is not entirely specific to PKC, other lines of evidence were pursued to substantiate signaling through this pathway in the control of ets-1 mRNA abundance. The role of PKC activation in this process was even more apparent after chronic PMA pretreatment, which leads to PKC isozyme downregulation and degradation, which was associated with almost complete abrogation of PDGF-BB-, ET-1-, or acute PMA-stimulated ets-1 gene expression. We observed that inhibition of tyrosine kinase activity by herbimycin A, an antibiotic that irreversibly inhibits the auto- and transphosphorylation of pp60^c-src and abolishes PLC- tyrosine phosphorylation in VSMC (22, 26, 29), strongly blocked ets-1 mRNA expression by ET-1. The inhibition of tyrosine phosphorylation of PLC-, with consequent impairment in the hydrolysis of PIP₂ to IP₃ and DAG, may play an essential role in the activation of downstream components in the signal transduction for ets-1 gene expression by ET-1. Herbimycin A had an effect of lesser magnitude on PDGF-BB-induced ets-1 mRNA. These data indicate that PDGF-BB-stimulated ets-1 gene expression might not be entirely dependent on PLC-, since this enzyme is known to be functionally inactivated in SMC at the range of herbimycin A concentrations used, suggesting that PKC may be partially activated by PDGF-BB through alternative routes involving other components of glycerolipid metabolism, such as arachidonic acid (6). In summary, the acute stimulatory effects of phorbol esters, as well as the inhibition by chronic PMA pretreatment and H-7, strongly point to a key role for PKC activation in the induction of ets-1 gene expression by both PDGF-BB and ET-1 in VSMC.

Pharmacological treatment with forskolin activates adenylate cyclase, and the resulting increase in cellular concentration of cAMP leads to stimulation of PKA activity. Forskolin inhibited basal ets-1 mRNA in SMC and almost completely abrogated the phorbol ester, PDGF, and ET-1 induction of ets-1 mRNA. cAMP regulation of gene expression is chiefly considered to occur via CREB binding to target regulatory elements in the promoter region of the target gene. The human ets-1 promoter contains binding sites for PEA3, AP-1, AP-2, and Sp1; however, CREB binding sites have not been detected, suggesting that the impairment of ets-1 gene expression may occur through interference with more proximal signaling events (11, 17). Activation of PKA is thought to lead to the inhibition of stimulated PIP₂ hydrolysis (1, 2) and inhibition of [Ca²⁺]_i released from the ER, probably via phosphorylation of IP₃ receptors (13). Under the experimental conditions used here, we observed that forskolin partially inhibited inositol phosphate generation by ET-1, although this did not appear to reduce the magnitude of ET-1-induced intracellular Ca²⁺ release. These findings indicate that the cAMP/PKA-mediated inhibition of ets-1 mRNA expression by ET-1 is not likely to result from reduction of IP₃-mediated Ca²⁺ release. Consistent with what has been previously reported, the decrease in DAG production through inhibition of PIP₂ breakdown may have resulted in partial inhibition of PKC activity and consequently in inhibition of ets-1 gene expression.

Rothman et al. (21) observed that acute thapsigargin stimulation caused a significant increase in a subset of c-jun and c-fos mRNAs in pulmonary VSMC, implicating a direct role of intracellular Ca²⁺ release from the ER in transcriptional control of a set of early response genes, effects that were independent of PKC activation (21). Furthermore, they showed that the depletion of [Ca²⁺]_i stores by chronic thapsigargin pretreatment blocked egr-1 and fra-1 mRNA induction by thrombin, but not by PDGF, whereas chelation of extracellular Ca²⁺ with EGTA inhibited the induction of a subset of c-jun and c-fos mRNAs by both thrombin and PDGF. In the present study, short-term treatment with thapsigargin itself did not induce ets-1 mRNA in VSMC. However, chelation of [Ca²⁺]_i by BAPTA or depletion of ER Ca²⁺ stores by prolonged thapsigargin treatment inhibited ets-1 mRNA expression by PDGF-BB, ET-1, and PMA. These data suggest that the ets-1 gene is regulated through a signaling pathway distinct from that controlling c-jun and c-fos gene expression. They indicate that release of [Ca²⁺]_i from ER stores is required for appropriate ets-1 gene expression in response to ET-1 and PDGF-BB. However, the lack of effect of acute treatment with thapsigargin shows that, as opposed to the regulation of c-fos and c-jun in pulmonary SMC, [Ca²⁺]_i release alone is not sufficient to induce ets-1 mRNA. This is further supported by the fact that, whereas downregulation of PKC by chronic PMA treatment strongly inhibited ets-1 mRNA expression by ET-1, it did not have a significant effect on the [Ca²⁺]_i increase caused by ET-1. It is possible that the modest effects of acute treatment with thapsigargin on ets-1 mRNA may be due to the slow elevation of intracellular Ca²⁺ elicited by this agent and that a more abrupt rise that more closely mimics the pattern seen after exposure to ET-1 would have a more robust effect. Alternatively, a theoretical basis for these results can be proposed on the basis of recent evidence indicating that depletion of Ca²⁺ from the lumen of the ER and nuclear envelope with ionophores or thapsigargin rapidly and potently inhibits passive diffusion and signal-mediated transport of proteins into the nucleus (16). Intracellular Ca²⁺ stored in the ER may be a major component required for nuclear transport of signals generated after PKC activation, although the precise mechanisms of how this may occur are unclear.

As mentioned above, ets-1 is believed to play a role in the transcriptional activation of matrix metalloproteinases. Agents that interfered with ets-1 gene expression in response to ET-1 (i.e., H-7, herbimycin A) also impaired stimulation of collagenase I. In contrast, stromelysin mRNA was not induced by either PDGF-BB or ET-1, irrespective of a brisk stimulation of ets-1 gene expression. Although they do not prove causality, these results are consistent with a role for ets-1 in the regulation of collagenase gene expression. Because the promoter regions of both the collagenase I and stromelysin genes have ets-1 binding sites (PEA3 motifs), it is possible that stromelysin activation requires other cofactors not present after ET-1 stimulation or that its expression is only subject to ets-1 regulation in other cell types. The same pattern of collagenase I and stromelysin gene regulation was also observed after treatment with PDGF-BB (not shown).

In conclusion, the present studies demonstrate that ets-1 functions as an early response gene after mitogen stimulation. PKC activation and intracellular Ca²⁺ stored in ER are pivotal components of the signal transduction pathway for activation of ets-1 gene expression by PDGF-BB and ET-1. In turn, ets-1 may play a key role in VSMC migration or in arterial remodeling through control of expression of matrix-degrading metalloproteinases.