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Endocrinology Vol. 140, No. 10 4659-4668
Copyright © 1999 by The Endocrine Society

ARTICLES

Endothelin-Mediated Vascular Growth Requires p42/p44 Mitogen-Activated Protein Kinase and p70 S6 Kinase Cascades via Transactivation of Epidermal Growth Factor Receptor1

Hiroaki Iwasaki, Satoru Eguchi, Hikaru Ueno, Fumiaki Marumo and Yukio Hirata

Division of Endocrinology and Metabolism (H.I., S.E., F.M., Y.H.), the Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; and Molecular Cardiology Unit (H.U.), Research Institute of Angiocardiology and Cardiovascular Clinic, Kyusyu University School of Medicine, Fukuoka 812-8582, Japan

Address all correspondence and requests for reprints to: Dr. Yukio Hirata, Division of Endocrinology and Metabolism, the Second Department of Internal Medicine, Tokyo Medical and Dental University, 1–5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY GROWTH stimuli cause tyrosine phosphorylation of various cellular proteins through receptors, either directly or indirectly coupled to tyrosine kinases, which is believed to play an essential role in mitogenesis and cell differentiation (1). Epidermal growth factor receptor (EGFR) is a single-transmembrane receptor tyrosine kinase. EGF, after binding to the extracellular domain of EGFR, induces receptor dimerization and clustering to activate the intrinsic tyrosine kinase in the intracellular domain (2). The activated EGFR provides binding sites for cellular proteins containing Src homology-2 domain of adaptor proteins, such as Shc and Grb2 (1, 2). Both EGFR and Shc, when tyrosine-phosphorylated, bind to each other as well as Grb2-Sos complex, which brings Sos to the plasma membrane, thus enabling to catalyzation of the conversion of p21ras-GDP to p21ras-GTP. The p21ras-GTP is then able to bind to and activate Raf-1 kinase, a serine/threonine protein kinase, which, in turn, activates mitogen-activated protein (MAP) kinase kinase/ERK kinase (MEK), a dual-specificity kinase that phosphorylates threonine and tyrosine residue of p42/p44 MAP kinases (ERK1/2) (3). The activation of p42/p44 MAP kinase results in phosphorylation of numerous cellular proteins, including a nuclear transcription factor, Elk1, which binds to the serum response element of the immediate early protooncogene c-fos gene promoter to stimulate its expression. Recently, it has been reported that increased MAP kinase activity contributes to the proliferation of rat vascular smooth muscle cells (VSMC) after balloon injury (4, 5), suggesting its pathogenic role in vascular remodeling.

In addition to activation by receptor and nonreceptor tyrosine kinases, MAP kinase is also activated by stimulation of GTP-binding protein-coupled receptors (GPCR) (6). Although the mechanism of MAP kinase activation by GPCR agonists is poorly understood, recent accumulating lines of evidence suggest that signal transduction by GPCR uses many of the same intermediates shared by receptor and nonreceptor tyrosine kinases (1, 6).

The p70 S6 kinase (p70S6K), an ubiquitous mitogen-activated serine/threonine kinase, plays a critical role in progression of cell cycle and protein synthesis by regulating the translation of a class of messenger RNA (mRNA) transcripts (7, 8). Although the direct signaling component by which receptor tyrosine kinase increases p70S6K activity remains largely unclear, it involves phosphatidylinositol 3 (PI 3)-kinase (9), independent of p21ras (10). Recently, it has been reported that activation of p70S6K is required for angiotensin II-induced growth of VSMC (11) and cardiac myocytes (12).

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide, originally isolated from porcine endothelial cells (13). Subsequent complementary DNA cloning of the human genomic library uncovered three isopeptides (ET-1, ET-2, and ET-3), whose actions are mediated by at least two receptor subtypes, ET-1-selective receptor (ETA) and nonisopeptide-selective receptor (ETB), both of which belong to the GPCR superfamily (14). Gq-coupled ETA receptor (predominantly expressed in VSMC) mediates vasoconstriction, while Gi-coupled ETB receptor (predominantly expressed in vascular endothelial cells) mediates vasodilatation via nitric oxide production (15). ET-1 has been shown to stimulate proliferation of a variety of cell types, including VSMC (16, 17), implicating its pathogenic role in vascular remodeling (14). In this regard, ET-1 activates several protein kinases, such as protein kinase C (PKC) (18), MAP kinase (19), and protein tyrosine kinases (PTK) (20). Recently, it has been shown that certain GPCR agonists (including ET-1) stimulate tyrosine phosphorylation of EGFR via a ligand-independent transactivation mechanism in the rat fibroblast cell line, thereby leading to MAP kinase activation, c-fos expression, and mitogenesis (21). However, it remains unknown whether the growth-promoting effect by ET-1 on VSMC also involves receptor tyrosine kinase-derived signaling machinery.

In the present study, we examine whether transactivation of EGFR participates in ET-1-induced growth of cultured rat VSMC and whether both MAP kinase and p70S6K cascades are linked to growth-promoting signaling by ET-1 via EGFR transactivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM were purchased from Life Technologies (Rockville, MD); FCS from Life Technologies/BRL (Grand Island, NY); ET-1 from Peptide Institute (Osaka, Japan); phorbol-12-myristate-13-acetate (PMA), AG1478, AG1296, pertussis toxin (PTX), 8-(diethylamino)octyl-3, 4, 5-trimethoxybenzoate (TMB-8), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), bisindolylmaleimide-1 (GF109203X), calphostin C, and rapamycin from Calbiochem-Nobabiochem (La Jolla, CA); l-{alpha}-lysophosphatidic acid (LPA) from Sigma-Aldrich Co. (St. Louis, MO); indomethacin from Wako Pure Chemical Industries Ltd. (Osaka, Japan); PD98059, polyclonal antiphospho p42/p44 MAP kinase antibody, and antiphospho-p70S6K antibody from New England Biolabs, Inc. (Beverly, MA); polyclonal anti-p42 MAP kinase antibody, polyclonal anti-p70S6K antibody, polyclonal anti-EGFR antibody, monoclonal anti-H-Ras antibody, antirat horseradish peroxidase (HRP)-conjugated second antibody, and agarose-conjugated glutathione-S-transferase (GST)-Grb2 (1–217) fusion protein, protein A/G-agarose from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); HRP-conjugated recombinant antibody fragment specific for phosphotyrosine (RC20H) from Transduction Laboratories, Inc. (Lexington, KY); human EGF, monoclonal antiphosphotyrosine antibody (4G10), and polyclonal anti-Shc antibody from Upstate Biotechnology, Inc. (Lake Placid, NY); antimouse and antirabbit HRP-conjugated second antibody and [32P]{gamma}ATP from Amersham International (Buckinghamshire, UK); and [3H]leucine and [3H]thymidine from NEN Life Science Products (Boston, MA).

Cell culture
Rat VSMCs were prepared from the thoracic aorta of 12-week-old male Sprague Dawley rats by the explant method and cultured in DMEM containing 10% FCS at 37 C in a humidified atmosphere of 95% air-5% CO2, as described (22). Quiescent VSMC (5–15th passages), after 72 h of serum starvation, were used in the following experiments.

MAP kinase activity
Quiescent VSMCs, pretreated with various agents for 30 min, were usually stimulated with ET-1 (100 nM) for 5 min at 37 C in serum-free DMEM unless otherwise stated. The reaction was terminated by the replacement of medium with the ice-cold lysis buffer (10 mM Tris-HCl, 20 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluorid, 10 µg/ml leupeptin, and 10 µg/ml aprotinin), pH 7.4. After brief sonication (10 sec), the samples were centrifuged for 5 min at 14,000 x g, and the supernatant was assayed for MAP kinase activity using an assay kit (Amersham International) as described (22).

Immune-complex p70S6K assay
Quiescent VSMCs, pretreated with or without AG1478 (250 nM) for 30 min, were stimulated with ET-1 (100 nM) for 20 min at 37 C under serum-free condition. The reaction was terminated by the replacement of medium with ice-cold lysis buffer. After brief sonication, the lysates were clarified by centrifugation for 5 min at 14,000 x g and immunoprecipitated with anti-p70S6K antibody (2 µg) preabsorbed to protein A/G agarose beads at 4 C for 2 h. The immune complexes were washed two times with lysis buffer and once with kinase assay buffer (20 mM 3-[N-morpholino]propanesulfonic acid, 25 mM ß-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol), pH 7.2. The beads were then resuspended in kinase assay buffer containing 50 µM specific S6 kinase substrate AKRRRLSSLRA (Upstate Biotechnology, Inc.), 15 mM MgCl2, 100 µM ATP, 5 µCi 32P-ATP (3,000 Ci/mmol), and protein kinase inhibitor cocktail, which blocks the activity of other serine/threonine kinases (Upstate Biotechnology, Inc.). After incubation at 30 C for 10 min, the reaction mixtures were applied onto P-81 phosphocellulose membrane (Whatmann International, Maidstone, UK) and were extensively washed in 1% acetic acid and then in deionized water. The radioactivity trapped on the membrane was measured in a liquid scintillation counter.

Immunoprecipitation and immunoblotting
Western blotting was performed as previously described (22). Quiescent VSMCs were stimulated with ET-1 (100 nM) at 37 C for the indicated times under a serum-free condition. The reaction was terminated by the replacement of medium with 100 µl SDS-PAGE buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue), pH6.8. After brief sonication, samples were boiled for 5 min at 95 C and centrifuged, and aliquots of the supernatants were subjected to 10% SDS-PAGE. Proteins in the gel were transferred to a nitrocellulose membrane by electroblotting. The membrane was treated with the first antibodies [polyclonal antiphospho-p42/44 MAP kinase antibody (dilution ratio; 1:20,000), polyclonal anti-p42 MAP kinase (1:10,000), monoclonal anti-H-Ras antibody (1:5,000), and polyclonal antiphospho-p70S6K (1:2,000)], and then with the second antibodies (antimouse, antirat, and antirabbit HRP-conjugated second antibody (1:2,000). After incubation, immunoreactive proteins were detected by an ECL system (Amersham Pharmacia Biotech).

For immunoblot analysis of EGFR- or Grb2-associable proteins, quiescent VSMCs were stimulated with ET-1 (100 nM) for the indicated times and lysed in 0.8 ml lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM EDTA, 1.0% Triton-X, 0.1% SDS, 10% glycerol, 50 mM NaF, 10 mM Na3P2O7, 1.0% deoxycholic acid, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluorid, and 10 µg/ml aprotinin), pH7.4. Lysates were sonicated and centrifuged at 14,000 x g for 5 min, and the supernatant was rocked with polyclonal anti-EGFR antibody (2 µg) with protein A/G agarose or agarose-conjugated GST-Grb2 fusion protein (3 µg) for 16 h at 4 C. Samples were centrifuged at 14,000 x g for 5 sec, and the beads were washed three times with lysis buffer, solubilized in Laemmli sample buffer, and subjected to immunoblotting. After the membrane was initially treated with mouse monoclonal antiphosphotyrosine antibody (RC20H) (1:2,000), polyclonal anti-EGFR antibody (1:5,000) or polyclonal anti-Shc antibody (1:5,000), and then with secondary antibodies (1:2,000), immunoreactive proteins were detected by the ECL system.

Transfection of a dominant negative H-Ras mutant
A replication-defection E1- and E3- adenoviral vector, containing CA promoter comprising a cytomegalovirus enhancer and chicken ß-actin promoter, was ligated to a dominant negative mutant of H-Ras (AdRasY57), in which tyrosine replaces asparatic acid at residue 57, or bacterial ß-galactosidase (AdLacZ) as reported (23). VSMCs grown on 12-well plates in serum-free medium were incubated with DMEM containing either AdRasY57 or AdLacZ at 37 C for 2 h, washed with fresh medium, further incubated for 3 days under a serum-free condition, and used for the experiments.

DNA and protein syntheses
DNA and protein syntheses were assessed by incorporation of [3H]thymidine and [3H]leucine into cells, respectively, as reported (22). In brief, quiescent VSMCs, pretreated with or without AG1478 or PD98059 for 30 min and with AdRasY57 or AdLacZ for 3 days, were incubated with ET-1 (100 nM) at 37 C for 20 h in serum-free DMEM, after which 1 µCi each of [3H]thymidine and [3H]leucine was added, and the cells were further incubated for 4 h. After completion, trichloroacetic acid-insoluble radioactivity was measured in a liquid scintillation counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ET-1 induced MAP kinase activation via transactivation of EGFR in VSMC
To examine whether EGFR tyrosine kinase is involved in growth-promoting signaling by ET-1 in rat VSMC, we first studied protein tyrosine phosphorylation after stimulation with ET-1, by immunoblot analysis using antiphosphotyrosine antibody (Fig. 1Go). ET-1 (100 nM) rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins with different molecular masses (~185, 180, 80, 60, 45, and 40 kDa), which were then decreased by 10 min (Fig. 1Go, upper panel). The tyrosine-phosphorylated protein ~180 kDa) was assumed to be EGFR, as recognized by the anti-EGFR antibody (Fig. 1Go, lower panel).



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  		Figure 1. ET-1 stimulated tyrosine phosphorylation of several proteins in rat VSMC. Cells were stimulated with ET-1 (100 nM) for the indicated times; cell lysates were immunoblotted with antiphosphotyrosine antibody (Ab) (upper panel) and anti-EGFR antibody (lower panel), respectively, as described in Materials and Methods. Molecular size markers (kDa) are shown on the left; arrows in the right denote tyrosine-phosphorylated proteins. pTyr, Phosphotyrosine.

 
Because the association of adaptor proteins (Shc and Grb2) with phosphorylated PTKs plays a central role in recruiting Sos, an activator of p21ras, and thereby initiates sequential activation of MAP kinase cascade (6), we examined whether ET-1 stimulates the association of EGFR and Shc with GST-Grb2 fusion protein. ET-1 rapidly (within 2 min) increased association of the tyrosine-phosphorylated 180-kDa protein with GST-Grb2 fusion protein (Fig. 2Go, upper panel). The phosphorylated 180-kDa protein was identified as EGFR, as recognized by the anti-EGFR antibody (Fig. 2Go, middle panel). The rapid association of two Shc isoforms (p52 and p46) with GST-Grb2 fusion protein was also observed upon ET-1 stimulation (Fig. 2Go, lower panel). These data suggest that the trans-phosphorylated EGFR by ET-1 complexes with adaptor proteins (Shc and Grb2) in rat VSMC, which could recruit Sos, thereby leading to sequential activation of p21ras-dependent MAP kinase cascade.



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  		Figure 2. ET-1 stimulated association of tyrosine-phosphorylated EGFR and Shc with Grb2 in rat VSMC. Cells were stimulated with ET-1 (100 nM) for 2 min; cell lysates were coprecipitated with GST-Grb2 fusion protein and then immunoblotted with antiphosphotyrosine antibody (upper panel), anti-EGFR antibody (middle panel), and anti-Shc antibody (lower panel), respectively. An arrow denotes phosphorylated EGFR (upper and middle panels) and two Shc isoforms (lower panel), respectively.

 
To determine whether ET-1-induced transactivation of EGFR is indeed required for MAP kinase activation, we next studied the effect of tyrophostin AG1478, a selective EGFR tyrosine kinase inhibitor (24), on MAP kinase activation, as measured by immunoblotting with an antibody that selectively recognizes enzymatically active Tyr204-phosphorylated MAP kinases and by a MAP kinase assay. ET-1 caused a rapid phosphorylation of p42/p44 MAP kinases that was detectable at 2 min, peaked at 5 min, and decreased at 10 min; this effect was markedly decreased by pretreatment with AG1478 (250 nM) (Fig. 3AGo). AG1478 dose-dependently (25–2500 nM) decreased ET-1-induced MAP kinase activation with a maximal inhibition (~60%) at 250 nM, as assessed by the kinase activity (Fig. 3BGo). However, tyrophostin AG1296 (25 µM), a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (25), failed to inhibit ET-1-induced MAP kinase phosphorylation and activation (data not shown). Collectively, these data suggest that a selective transactivation of EGFR plays an important role in ET-1-induced MAP kinase activation in rat VSMC. Subsequent studies were usually performed by using AG1478 at a dose of 250 nM unless otherwise stated.



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  		Figure 3. Effect of EGFR kinase inhibitor (AG1478) on ET-1-induced MAP kinase activation in rat VSMC. A, After pretreatment with or without AG1478 (250 nM) for 30 min, cells were stimulated with ET-1 (100 nM) for the indicated times; cell lysates were subjected to immunoblotting with antiphospho MAP kinase antibody (upper panel) and anti-p42 MAP kinase antibody (lower panel), respectively. B, After pretreatment with or without AG1478 in the concentrations indicated, cells were stimulated with (•) or without ({circ}) ET-1 (100 nM) for 5 min for measurement of MAP kinase activity. Each point with a bar represents the mean ± SEM (n = 4). DMSO, Dimethylsulfoxide.

 
ET-1-induced tyrosine phosphorylation of EGFR and MAP kinase activation is mediated via ETA receptor and a Ca2+-sensitive mechanism
To determine which receptor subtype is involved in the EGFR and MAP kinase activation by ET-1, the effects of selective receptor antagonists for ETA (BQ123) and for ETB (BQ788) were tested (Fig. 4Go). Pretreatment with BQ123 (1 µM), but not with BQ788 (1 µM), inhibited ET-1-induced tyrosine-phosphorylation of EGFR as well as MAP kinase activation, indicating that ET-1-stimulated activation of EGFR and MAP kinase is mainly mediated through ETA receptor in rat VSMC.



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  		Figure 4. Effects of ETA (BQ123) and ETB (BQ788) receptor antagonists on ET-1-induced EGFR phosphorylation and MAP kinase activation in rat VSMC. A, Cells pretreated with or without BQ123 (1 µ[M) for 30 min were stimulated with ET-1 (100 nM) for 2 min; cell lysates were immunoprecipitated with anti-EGFR antibody and then immunoblotted with antiphosphotyrosine antibody (upper panel) and anti-EGFR antibody (lower panel), respectively. B, After pretreatment with or without BQ123 (1 µM) and BQ788 (1 µM), cells were stimulated with or without ET-1 (100 nM) for 5 min, for measurement of MAP kinase activity. Each column with a bar represents the mean ± SEM (n = 4).

 
It has been shown that ET-1 activates phospholipase Cß through Gq-coupled ETA receptor in VSMC, to generate inositol 1,4,5-triphosphate and diacylglycerol, which mobilizes intracellular calcium from the store sites and activates PKC, respectively (14). To determine whether these second messengers are involved in EGFR and MAP kinase activation by ET-1 in rat VSMC, the effects of intracellular calcium antagonists (TMB-8 and BAPTA/AM) and selective PKC inhibitors (GF109203X and calphostin C) were tested. Both tyrosine-phosphorylation of EGFR and activation of MAP kinase by ET-1 were completely inhibited by 1 µM TMB-8 (Fig. 5Go) and 10 µM BAPTA/AM (data not shown). However, neither GF109203X (2 µM) nor calphostin C (1 µM) had any effect on ET-1-induced EGFR phosphorylation or MAP kinase activation (Fig. 6AGo), whereas GF109203X completely blocked PMA-induced MAP kinase activation (Fig. 6BGo). These data suggest that ETA receptor-mediated increase in intracellular Ca2+, but not activation of PKC, constitutes a critical signal for EGFR transactivation and subsequent MAP kinase activation.



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  		Figure 5. Effect of intracellular calcium antagonist (TMB-8) on ET-1-induced EGFR phosphorylation and MAP kinase activation in rat VSMC. A, Cells pretreated with or without TMB-8 (1 µM) for 30 min were stimulated with ET-1 (100 nM) for 2 min; cell lysates were immunoprecipitated with anti-EGFR antibody and then immunoblotted with antiphosphotyrosine antibody (upper panel) and anti-EGFR antibody (lower panel), respectively. B, After pretreatment with or without TMB-8 (1 µM), cells were stimulated with or without ET-1 (100 nM) for 5 min, for measurement of MAP kinase activity. Data are shown as in Fig. 4Go (n = 4).

 


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  		Figure 6. Effect of PKC inhibitor (GF109203X) on ET-1-induced EGFR phosphorylation and MAP kinase activation in rat VSMC. A, Cells pretreated with or without GF109203X (2 µM) for 30 min were stimulated with ET-1 (100 nM) for 2 min; cell lysates were immunoprecipitated with anti-EGFR antibody and then immunoblotted with antiphosphotyrosine antibody (upper panel) and anti-EGFR antibody (lower panel), respectively. B, After pretreatment with or without GF109203X (2 µM), cells were stimulated with or without ET-1 (100 nM) and PMA (1 µM) for 5 min, for measurement of MAP kinase activity. Data are shown as in Fig. 4Go (n = 4). GFX, GF109203X.

 
Several GPCR agonists, such as LPA, thrombin, and {alpha}2A-agonist, use PTX-sensitive Gi/Go proteins to activate PTK and MAP kinase (6). Therefore, we studied whether PTX-sensitive G proteins are involved in EGFR and MAP kinase activation by ET-1. Pretreatment with PTX (1 µg/ml) markedly inhibited LPA-induced EGFR phosphorylation as well as MAP kinase activation, whereas it had no effects on ET-1-induced EGFR phosphorylation or MAP kinase activation (Fig. 7Go). Thus, the ET-1-induced EGFR and MAP kinase activation occurs independent of PTX-sensitive G proteins.



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  		Figure 7. Effect of PTX on ET-1-induced EGFR phosphorylation and MAP kinase activation in rat VSMC. A, Cells pretreated with or without PTX (1 µg/ml) for 24 h were stimulated with ET-1 (100 nM) or LPA (1 µM) for 2 min; cell lysates were immunoprecipitated with anti-EGFR antibody and then immunoblotted with antiphosphotyrosine antibody (upper panel) and anti-EGFR antibody (lower panel), respectively. B, After treatment with or without PTX (1 µg/ml) for 24 h, cells were stimulated with or without ET-1 (100 nM) and LPA (1 µM) for 5 min, for measurement of MAP kinase activity. Data are shown as in Fig. 4Go (n = 4).

 
Activation of EGFR and MAP kinase is essential for ET-1-induced growth-promoting effect
To determine whether the ET-1-induced EGFR and MAP kinase activation are indeed essential for its growth-promoting effect on VSMC, we examined the effects of AG1478 and PD98059, a MEK-1 inhibitor (26), on ET-1-stimulated protein and DNA synthesis. AG1478 dose-dependently (25–250 nM) inhibited the ET-1-stimulated incorporation of both [3H]leucine and [3H]thymidine, whereas it had no effect on their basal levels (Fig. 8Go). PD98059 (25 µM) also completely blocked the ET-1-stimulated incorporation of both [3H]-leucine and [3H]thymidine (Fig. 9Go); it also decreased their basal levels. These data suggest that activation of both EGFR and MAP kinase is essential for the ET-1-mediated vascular growth.



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  		Figure 8. Dose-response inhibition of AG1478 on ET-1-induced protein and DNA synthesis in rat VSMC. After treatment with or without AG1478 (25–250 nM) for 30 min, cells were incubated with (•) or without ({circ}) ET-1 (100 nM) for 20 h; [3H]leucine (A) and [3H]thymidine (B), incorporated during 4 h, were measured. Each point with a bar represents the mean ± SEM (n = 3).

 


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  		Figure 9. Effects of MEK-1 inhibitor (PD98059) on ET-1-induced protein and DNA synthesis in rat VSMC. After treatment with or without PD98059 (25 µM) for 30 min, cells were incubated with or without ET-1 (100 nM) for 20 h; [3H]leucine (A) and [3H]thymidine (B), incorporated during 4 h, were measured. Each column with a bar represents the mean ± SEM (n = 3).

 
p21ras-dependent pathway contributes to MAP kinase activation and growth-promoting effect by ET-1
It has been recently reported that certain GPCR agonists initiate MAP kinase cascade by GTP-loaded p21ras (6). To elucidate whether p21ras-dependent pathway contributes to MAP kinase activation and growth-promoting effects by ET-1, we tested the effect of adenovirus-mediated transfection of a dominant negative mutant of H-Ras (AdRasY57) (Fig. 10Go). Adenovirus-mediated transfection of RasY57, but not of LacZ, resulted in dose-dependent (40–120 moi) increases in H-Ras protein levels and reciprocal decreases in the ET-1-induced Tyr204-phosphorylated MAP kinase (Fig. 10AGo); the expression of p42 MAP kinase was comparable between AdRasY57- and AdLacZ-infected cells. A similar inhibition was observed in the EGF (100 ng/ml)-stimulated MAP kinase activation after transfection of RasY57 (data not shown). The ET-1-stimulated protein synthesis (Fig. 10BGo) and DNA synthesis (Fig. 10CGo) were completely inhibited after transfection of AdRasY57, but not of AdLacZ, whereas basal levels were not affected by either transfection. These data suggest that p21ras-dependent MAP kinase activation is required for growth-promoting effect by ET-1 in rat VSMC.



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  		Figure 10. Effect of a dominant negative mutant of H-Ras on ET-1-induced MAP kinase activation, protein, and DNA synthesis in rat VSMC. A, Cells transfected with or without AdRasY57 (40–120 moi) or AdLacZ (120 moi) for 3 days were stimulated with ET-1 (100 nM) for 5 min; cell lysates were immunoblotted with antiphospho-MAP kinase antibody (upper panel), anti-p42 MAP kinase antibody (middle panel), and anti-H-Ras antibody (lower panel), respectively. Cells transfected with or without AdRasY57 (100 moi) or AdLacZ (100 moi) were incubated with ET-1 (100 nM) for 20 h. [3H]leucine (B) and [3H]thymidine (C), incorporated during 4 h, were measured. Data are shown as in Fig. 9Go (n = 3).

 
p70S6K is involved in ET-1-induced growth-promoting effect via EGFR transactivation
p70S6K plays a critical role for the initiation of protein synthesis by phosphorylation of 40S ribosomal protein S6 (8). To elucidate whether p70S6K is also involved in the ET-1-induced growth-promoting signaling via EGFR transactivation, the effects of AG1478 and PD98059 were tested by immunoblotting with antiphospho-p70S6K antibody. ET-1 (100 nM) caused a transient phosphorylation of p70S6K protein, which peaked at 10–20 min and decreased at 40 min; this effect was decreased by pretreatment with AG1478 (250 nM) (Fig. 11AGo) and PD98059 (25 µM) (data not shown). AG1478 also completely inhibited ET-1-induced p70S6K activation, as assessed by immune-complex kinase assay (Fig. 11BGo). Furthermore, rapamycin (10 nM), a selective mammalian target of rapamycin (mTOR) inhibitor (27), completely blocked ET-1-stimulated incorporation of both [3H]leucine and [3H]thymidine (Fig. 12Go). Rapamycin, added alone, had no effect on basal [3H]leucine uptake, but it decreased basal [3H]thymidine uptake. These data suggest that activation of both MAP kinase and p70S6K, via transactivation of EGFR, is required for the ET-1-mediated vascular growth.



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  		Figure 11. Effect of AG1478 on ET-1-induced p70S6K activation in rat VSMC. A, After pretreatment with or without AG1478 (250 nM) for 30 min, cells were stimulated with ET-1 (100 nM) for the indicated times; cell lysates were immunoblotted with antiphospho p70S6K antibody (upper panel) and anti-p42 MAP kinase antibody (lower panel), respectively. B, After pretreatment with or without AG1478 (250 nM) for 30 min, cells were stimulated with or without ET-1 (100 nM) for 20 min, for measurement of p70S6K activity. Data are shown as in Fig. 4Go (n = 4).

 


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  		Figure 12. Effect of mTOR inhibitor (rapamycin) on protein and DNA synthesis in cultured rat VSMC. After pretreatment with or without rapamycin (100 nM) for 30 min, cells were incubated with or without ET-1 (100 nM) for 20 h; [3H]leucine (A) and [3H]thymidine (B), incorporated during 4 h, were measured. Data are shown as in Fig. 9Go (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ET-1, a potent endothelium-derived vasoconstrictor peptide, stimulates proliferation of a variety of cell types, including VSMC (16, 17). A growing body of evidence suggests that endogenous ET-1 is involved in vascular remodeling. For example, local production of ET-1 has been shown to be increased in the vascular lesions, such as in hypercholesterolemia (28), atherosclerosis (29, 30, 31), neointima after balloon angioplasty (32, 33), and hypertension (34); VSMCs and macrophages (in addition to endothelial cells) abundantly express ET-1 transcripts and secrete mature ET-1. Likewise, enhanced expression of both ETA and ETB receptors, as well as endothelin-converting enzyme 1, has been demonstrated in the vascular lesions (32, 33). Moreover, administration of ET receptor antagonists reduced neointimal formation after balloon injury in rats (35, 36), and prevented vascular remodeling in deoxycorticosterone acetate-salt hypertensive rats (37). Although ET-1 has been shown to stimulate MAP kinase and VSMC growth, its underlying molecular signaling mechanism in VSMC is poorly characterized. Here, we have demonstrated that ET-1-induced activation of both MAP kinase and p70S6K, via the transactivation of EGFR, is required for its hypertrophic and hyperplastic response in cultured rat VSMC.

In the present study, ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins with different molecular sizes (~185, 180, 80, 60, 45, 40 kDa) in rat VSMC. The apparent molecular masses of these tyrosine-phosphorylated proteins are almost comparable to those of the previous observations in rat astrocytes (20) and mesangial cells (38). Previous study has shown that angiotensin II, a GPCR agonist, induced MAP kinase activation through a PTK-dependent mechanism in rat VSMC (39). Recently, Daub et al. (21) have demonstrated that certain GPCR agonists, such as ET-1, thrombin, and LPA, induced tyrosine phosphorylation of EGFR in rat fibroblast cell line, and that treatment with a selective EGFR tyrosine kinase inhibitor (AG1478) or transfection of a dominant-negative EGFR mutant suppressed MAP kinase activation, c-fos expression, and DNA synthesis stimulated by these agonists. We have also recently shown that angiotensin II-induced MAP kinase activation is mediated though EGFR transactivation in rat VSMC (40). The present study clearly revealed that the tyrosine-phosphorylated 180-kDa protein, induced by ET-1, represents the trans-phosphorylated EGFR. Moreover, EGFR tyrosine kinase inhibitor (AG1478) caused a marked and dose-dependent inhibition of MAP kinase activation, with a concomitant inhibition of both protein and DNA synthesis stimulated by ET-1. Taken together, our data suggest that transactivation of EGFR is a common early signaling event for the growth-promoting effect on VSMC shared by certain vasoconstrictive agonists, like angiotensin II and ET-1.

AG1478 is a highly selective inhibitor for EGFR, but it may nonspecifically affect other nonreceptor PTKs or signaling intermediates in higher doses. However, we have chosen the appropriate dose (250 nM) that effectively inhibited the ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake from the dose-response experiments and other studies showing that AG1478 (100–250 nM) selectively inhibits GCPR agonists-induced EGFR transactivation without affecting other nonreceptor PTK in other cell types (21, 41). Furthermore, we have demonstrated that AG1478 (250 nM) selectively and completely inhibits MAP kinase activation by EGF as well as by angiotensin II- and Ca2+ ionophore, but not by PDGF or PMA (40), and more recently that AG1478 (250 nM) had no effect on angiotensin II-induced nonreceptor PTK, such as prolin-rich tyrosine kinase 2 (PYK2) or its association with pp60c-src (42). Thus, it seems most likely that AG1478, at a dose used in the present studies, is highly selective, to inhibit EGFR tyrosine kinase over other nonreceptor PTKs.

It should be noted that AG1478 completely blocked the ET-1-stimulated protein and DNA synthesis, whereas MAP kinase activation by ET-1 was incompletely suppressed by AG1478. These data suggest that there exists a EGFR-independent signal(s) for MAP kinase activation and/or MAP kinase-independent growth-promoting signal(s) in the downstream of EGFR. For example, p185neu and pp60c-src may be responsible for EGFR-independent signal(s) for MAP kinase activation. Alternatively, PI 3-kinase, p70S6K, and other MAP kinases, such as c-Jun NH2-terminal protein kinase (JNK) and p38 kinase, may be responsible for MAP kinase-independent growth-promoting signal(s). In fact, it has recently been shown that ET-1 sequentially activates MAP kinase and PI 3-kinase in rat mesangial cells (43), the wartmannin-sensitive PI 3-kinase activation is required for ET-1-induced mitogenic response in CHO cells (44), and ET-1 activates JNK and p38 kinase other than MAP kinase in rat cardiac myocytes (45, 46). Therefore, the ET-1-induced EGFR transactivation and MAP kinase activation linked to its growth-promoting signal(s) seems to be more complex, depending on cell types given.

Several tyrosine-phosphorylated proteins, other than 180 kDa by ET-1 (as demonstrated in this study) are currently unidentified. The tyrosine-phosphorylated 185-kDa protein might be p185neu (ErbB2/HER2), an orphan receptor tyrosine kinase of EGFR family (47), because it has been shown to be trans-phosphorylated by LPA, thrombin, and ET-1 in fibroblasts (21). ET-1 has been shown to activate pp60c-src in rat mesangial cells (48). The Src tyrosine kinase family has been shown to mediate p21ras-dependent MAP kinase activation by angiotensin II in rat cardiomyocytes (49) and VSMC (50). We have recently shown that pp60c-src is also involved in angiotensin II-induced EGFR transactivation in rat VSMC (40). The question as to whether these receptor and nonreceptor tyrosine kinases other than EGFR are also involved in the ET-1-induced MAP kinase activation remains to be determined.

Recently, Shc proteins have been implicated in p21ras-dependent MAP kinase activation by several GPCR agonists (6). The present study clearly showed that ET-1 rapidly increased the amounts of two Shc isoforms (p46 and p52) and tyrosine-phosphorylated EGFR associated with GST-Grb2 fusion protein. In rat astrocytes, ET-1 induced tyrosine phosphorylation of Shc and its association with Grb2 through its Src homology-2 domain (51). In rat mesangial cells, the ET-1-induced p21ras activation also involves association of phosphorylated Shc with Grb2-Sos complex (43). Taken together, it is suggested that the ET-1-induced MAP kinase activation in rat VSMC is partly mediated through Shc, recruited and phosphorylated by transactivated EGFR in rat VSMC.

Several PTX-sensitive GPCR agonists seem to use ß{gamma} subunits of Gi/Go proteins for PTK-dependent p21ras activation (6). Recently, it has been demonstrated that LPA, a Gi-coupled receptor agonist, induced EGFR transactivation in COS-7 cells (52). However, our result showed that PTX had no effects on the ET-1-induced EGFR transactivation and MAP kinase activation, but it completely blocked LPA-induced MAP kinase activation. These data suggest that PTX-insensitive G protein is involved in ET-1-induced EGFR and MAP kinase activation in VSMC.

Activation of Gq-coupled receptors by certain agonists seems to induce EGFR transactivation in a variety of cells; LPA, thrombin, and ET-1 in fibroblasts (21), angiotensin II in VSMC (40), thrombin in COS-7 cells (53), and bradykinin in PC12 cells (54). Recently, it has been reported that the activation of m1 muscarinic receptor results in GF109203X-sensitive EGFR transactivation in human embryonic kidney cells (41), whereas both PKC-dependent and EGFR-dependent pathways contribute to MAP kinase activation by angiotensin II in rat liver epithelial cells (55). In the present study, both EGFR phosphorylation and MAP kinase activation were blocked by intracellular calcium antagonists (TMB-8 and BAPTA), but not by PKC inhibitors (GF109203X and calphostin C), whereas PMA-induced MAP kinase activation was completely blocked by GF109203X. Although the pharmacological inhibitors used in the present experiments were not specific, the results using the appropriate low doses and at least two structurally different compounds strongly suggest that the ET-1-induced EGFR transactivation and MAP kinase activation involves a Ca2+-dependent (but PKC-independent) mechanism in rat VSMC. In fact, our present data are consistent with those of angiotensin II in rat VSMC (21) and bradykinin in PC12 cells (54) using the same inhibitors as in this study.

The present study clearly demonstrated that the MAP kinase cascade constitutes a pivotal signal transduction for VSMC growth. ET-1 has been shown to activate p21ras/Raf-1 for c-fos activation in rat mesangial cells (56). The present study revealed that MAP kinase activation, as well as protein and DNA synthesis by ET-1 in rat VSMC, was completely blocked after transfection of a dominant negative H-Ras mutant (AdRasY57). Furthermore, pretreatment with a highly selective MEK-1 inhibitor (PD98059) also blocked ET-1-stimulated MAP kinase activation and cell growth. Although PD98059 has recently been shown to block eicosanoid production by direct inhibition of cyclooxygenase (57), indomethacin, a cyclooxygenase inhibitor, failed to affect ET-1-induced MAP kinase activation (unpublished observation), suggesting that eicosanoid is not involved in ET-1-stimulated MAP kinase activation and VSMC growth. Collectively, our data provide strong evidence that the ET-1-induced vascular growth requires p21ras-dependent MAP kinase cascade.

The present study also revealed that both AG1478 and PD98059 completely inhibited the ET-1-induced phosphorylation and activation of a 70-kDa ribosomal serine/threonine kinase (p70S6K), as demonstrated by immunoblotting and immune-complex kinase assay. Whereas most studies indicate that MAP kinase activation leads to activation of 90-kDa ribosomal protein kinase (p90S6K), p70S6K is also stimulated in response to insulin by an as-yet-unknown mechanism, possibly involving a mTOR and PI 3-kinase-dependent pathway (7, 58). Despite the blockade of p70S6K activation by PD98059, the ability of dominant-negative p21ras to block MAP kinase activation would not indicate that p70S6K is activated via MAP kinase pathway because dominant-negative p21ras can interfere with the activation of both MAP kinase and PI 3-kinase in response to insulin (59). Rapamycin, an immunosuppressant antibiotic, that selectively inhibits mTOR (27), a putative upstream positive regulator of p70S6K, also completely blocked ET-1-stimulated protein and DNA synthesis in this study. This is consistent with previous observations that rapamycin, in a dose comparable with that in this study, inhibited both p70S6K activation and protein synthesis by angiotensin II in rat VSMC (11). Although the blockade of rapamycin on ET-1-stimulated cell growth suggests the possible involvement of the p70S6K pathway, other mTOR-dependent pathways may also be responsible. In fact, it has been reported that rapamycin also inhibits insulin-induced dissociation of phosphorylated heat- and acid-stable protein (PHAS-1) from eukaryotic initiation factor 4E (eIF-4E) complex, to promote protein synthesis (58, 59). These data suggest that a rapamycin-sensitive factor(s) other than p70S6K pathway may also be involved in the regulation of cell growth. Our results suggest that the ET-1-induced VSMC growth also requires p70S6K and/or other mTOR-dependent pathways, possibly in the downstream of EGFR transactivation.

In conclusion, we have demonstrated herein, for the first time, that the ETA receptor-mediated growth-promoting effect by ET-1 requires both p21ras-dependent MAP kinase and p70S6K cascades partly mediated via Ca2+-dependent EGFR transactivation. In this regard, PYK2, a novel nonreceptor PTK, is a possible upstream signaling candidate because of its Ca2+-dependency. Thus, the identification and characterization of the putative transducer(s) that directly induces Ca2+-sensitive EGFR transactivation by ET-1 remain to be determined.


   Footnotes
 
1 This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, of Japan. Back

Received December 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. van der Geer P, Hunter T 1994 Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10:251–337[CrossRef]
  2. Lemmon MA, Schlessinger J 1994 Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem Sci 19:459–463[CrossRef][Medline]
  3. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185[CrossRef][Medline]
  4. Hu Y, Cheng L, Hochleitner B-W, Xu Q 1997 Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after ballon injury. Arterioscler Thromb Vasc Biol 17:2808–2816[Abstract/Free Full Text]
  5. Koyama H, Olson NE, Dastvan F, Reidy MA 1998 Cell replication in the arterial wall: activation of signaling pathway following in vivo injury. Circ Res 82:713–721[Abstract/Free Full Text]
  6. van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ 1996 Mitogenic signaling via G protein-coupled receptors. Endocr Rev 17:698–714[CrossRef][Medline]
  7. Brown EJ, Schreiber SL 1996 A signal pathway to translational control. Cell 86:517–520[CrossRef][Medline]
  8. Pullen N, Thomas G 1997 The molecular phosphorylation and activation of p70S6K. FEBS Lett 410:78–82[CrossRef][Medline]
  9. Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J 1994 PDGF- and insulin-dependent pp70S6K activation mediated by phosphatidylinositol-3-OH kinase. Nature 370:71–75[CrossRef][Medline]
  10. Ming X-F, Burgering BMT, Wennström S, Claesson-Welsh L, Heldin C-H, Bos JL, Kozma SC, Thomas G 1994 Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature 371:426–429[CrossRef][Medline]
  11. Giasson E, Meloche S 1995 Role of p70 S6 kinase in angiotensin II-induced protein synthesis in vascular smooth muscle cells. J Biol Chem 270:5225–5231[Abstract/Free Full Text]
  12. Takano H, Komuro I, Zou Y, Kudoh S, Yamazaki T, Yazaki Y 1996 Activation of p70 S6 kinase is necessary for angiotensin II-induced hypertrophy in neonatal rat cardiac myocytes. FEBS Lett 379:255–259[CrossRef][Medline]
  13. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yamazaki Y, Goto K, Masaki T 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415[CrossRef][Medline]
  14. Masaki T 1993 Endothelins: homeostatic and compensatory actions in the circulatory and endocine systems. Endocr Rev 14:256–268[CrossRef][Medline]
  15. Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, Marumo F 1993 Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest 91:1367–1373
  16. Hirata Y, Takagi Y, Fukuda Y, Marumo F 1989 Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 78:225–228[CrossRef][Medline]
  17. Eguchi S, Hirata Y, Ihara M, Yano M, Marumo F 1992 A novel ETA receptor antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells. FEBS Lett 302:243–246[CrossRef][Medline]
  18. Marsden PA, Danthuluri NR, Brenner BM, Ballermann BJ, Brock TA 1989 Endothelin action on vascular smooth muscle involves inositol triphosphate and calcium mobilization. Biochem Biophys Res Commun 158:86–93[CrossRef][Medline]
  19. Wang Y, Simonson MS, Pouyssègur J, Dunn MJ 1992 Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells. Biochem J 287:589–594
  20. Cazaubon S, Parker PJ, Strosberg AD, Couraud PO 1993 Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes. Biochem J 293:381–386
  21. Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of transactivation of the EGF receptor in signaling by G-protein-coupled receptors. Nature 379:557–560[CrossRef][Medline]
  22. Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 139:3432–3441[Abstract/Free Full Text]
  23. Ueno H, Yamamoto H, Ito S, Li J, Takeshita A 1997 Adenovirus-mediated transfer of a dominant-negative H-ras suppresses neointimal formation in balloon-injured arteries in vivo. Arterioscler Thromb Vasc Biol 17:898–904[Abstract/Free Full Text]
  24. Levitzki A, Gazit A 1995 Tyrosine kinase inhibition: an approach to drug development. Science 267:1782–1787[Abstract/Free Full Text]
  25. Kovalenko M, Gazit A, Böhmer A, Rorsman C, Rönnstrand L, Heldin C-H, Waltenberger J, Böhmer F-D, Levitzki A 1994 Selective platelet-derived growth factor receptor kinase blockers reverse sis-transformation. Cancer Res 54:6106–6114[Abstract/Free Full Text]
  26. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270:27489–27494[Abstract/Free Full Text]
  27. Chung J, Kuo CJ, Crabtree GR, Blenis J 1992 Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69:1227–1236[CrossRef][Medline]
  28. Lerman A, Webster MWI, Chesebro JH, Edwards WD, Wei C-M, Fuster V, Burnett JC 1993 Circulating and tissue endothelin immunoreactivity in hypercholesterolemic pigs. Circulation 88:2923–2928[Abstract/Free Full Text]
  29. Lerman A, Edwards BS, Hallett JW, Heublein DM, Sanberg SM, Burnett JC 1991 Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N Engl J Med 325:997–1001[Abstract]
  30. Zeiher AM, Goebel H, Schächinger V, Ihling C 1995 Tissue endothelin-1 immunoreactivity in the active coronary atherosclerotic plaque. Circulation 91:941–947[Abstract/Free Full Text]
  31. Ravalli S, Szabolcs M, Albala A, Michler RE, Cannon PJ 1996 Increased immunoreactive endothelin-1 in human transplant coronary artery disease. Circulation 94:2096–2102[Abstract/Free Full Text]
  32. Minamino T, Kurihara H, Takahashi M, Shimada K, Maemura K, Oda H, Ishikawa T, Uchiyama T, Tanazawa K, Yazaki Y 1997 Endothelin-converting enzyme expression in the rat vascular injury model and human coronary artherosclerosis. Circulation 95:221–230[Abstract/Free Full Text]
  33. Wang X, Douglas SA, Louden C, Vickery-Clark LM, Feuerstein GZ, Ohlstein EH 1996 Expression of endothelin-1, endothelin-3, endothelin-converting enzyme-1, and endothelin-A and endothelin-B receptor mRNA after angioplasty-induced neointimal formation in the rat. Circ Res 78:322–328[Abstract/Free Full Text]
  34. Larivière R, Thibault G, Schiffrin EL 1993 Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension 21:294–300[Abstract/Free Full Text]
  35. Douglas SA, Louden C, Vickery-Clark LM, Storer BL, Hart T, Feuerstein GZ, Elliott JD, Ohlstein EH 1994 A role for endogenous endothelin-1 in neointimal formation after rat carotid artery balloon angioplasty: protective effects of the novel nonpeptide endothelin receptor antagonist SB 209670. Circ Res 75:190–197[Abstract/Free Full Text]
  36. Tsujino M, Hirata Y, Eguchi S, Watanabe T, Chatani F, Marumo F 1995 Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty. Life Sci 56:PL449–PL454
  37. Li JS, Larivière R, Schiffrin EL 1994 Effect of a nonselective endothelin antagonist on vascular remodeling in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 24:183–188[Abstract/Free Full Text]
  38. Force T, Kyriakis JM, Avruch J, Bonventre JV 1991 Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. J Biol Chem 266:6650–6656[Abstract/Free Full Text]
  39. Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T 1996 Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. J Biol Chem 271:14169–14175[Abstract/Free Full Text]
  40. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T 1998 Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273:8890–8896[Abstract/Free Full Text]
  41. Tsai W, Morielli AD, Peralta EG 1997 The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity. EMBO J 16:4597–4605[CrossRef][Medline]
  42. Eguchi S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa T, Motoley ED, Owada KM, Marumo F, Hirata Y 1999 Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension [Part 2] 33:201–206
  43. Foschi M, Chiari S, Dunn MJ, Sorokin A 1997 Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16:6439–6451[CrossRef][Medline]
  44. Sugawara F, Ninomiya H, Okamoto Y, Miwa S, Mazda O, Katsura Y, Masaki T 1996 Endothelin-1-induced mitogenic responses of Chinese hamster ovary cells expressing human endothelin A: the role of a wartmannin-sensitive signaling pathway. Mol Pharmacol 49:447–457[Abstract]
  45. Bogoyevitch MA, Ketterman AJ, Sugden PH 1995 Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem 270:29710–29717[Abstract/Free Full Text]
  46. Clerk A, Michael A, Sugden PH 1998 Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy? J Cell Biol 142:523–535[Abstract/Free Full Text]
  47. Peles E, Yarden Y 1993 Neu and its ligands: from an oncogene to neural factors. Bioessays 15:815–824[CrossRef][Medline]
  48. Simonson MS, Herman WH 1993 Protein kinase C and protein tyrosine kinase activity contribute to mitogenic signaling by endothelin-1: cross-talk between G protein-coupled receptors and pp60c-src. J Biol Chem 268:9347–9357[Abstract/Free Full Text]
  49. Sadoshima J, Izumo S 1996 The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J 15:775–787[Medline]
  50. Ishida M, Ishida T, Thomas SM, Berk BC 1998 Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circ Res 82:7–12[Abstract/Free Full Text]
  51. Cazaubon SM, Ramos-Morales F, Fischer S, Schweighoffer F, Strosberg AD, Couraud P-O 1994 Endothelin induces tyrosine phosphorylation and GRB2 association of Shc in astrocytes. J Biol Chem 269:24805–24809[Abstract/Free Full Text]
  52. Luttrell LM, Della Rocca GJ, van Biesen T, Luttrell DK, Lefkowitz RJ 1997 Gß{gamma} subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. J Biol Chem 272:4637–4644[Abstract/Free Full Text]
  53. Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A 1997 Signal characteristics of G protein-transactivated EGF receptor. EMBO J 16:7032–7044[CrossRef][Medline]
  54. Zwick E, Daub H, Aoki N, Yamaguchi-Aoki Y, Tinhofer I, Maly K, Ullrich A 1997 Critical role of calcium-dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem 272:24767–24770[Abstract/Free Full Text]
  55. Li X, Lee JW, Graves LM, Earp HS 1998 Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled-EGF receptor transactivation pathway. EMBO J 17:2574–2583[CrossRef][Medline]
  56. Herman WH, Simonson MS 1995 Nuclear signaling by endothelin-1. J Biol Chem 270:11654–11661[Abstract/Free Full Text]
  57. Börsch-Haubold AG, Pasquet S, Watson SP 1998 Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. J Biol Chem 273:28766–28772[Abstract/Free Full Text]
  58. Lawrence JC, Abraham RT 1997 PHAS/4E-BPs as regulators of mRNA translation and cell proliferation. Trends Biochem Sci 22:345–349[CrossRef][Medline]
  59. Surtherland C, Waltner-Law M, Gnudi L, Kahn BB, Granner DK 1998 Activation of the ras mitogen-activated protein kinase-ribosomal protein kinase pathway is not required for the repression of phosphoenolpyruvate carboxykinase gene transcription by insulin. J Biol Chem 273:3198–3204[Abstract/Free Full Text]



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