Gβγ Mediate Differentiation of Vascular Smooth Muscle Cells
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m101963200
ISSN1083-351X
AutoresH. Peter Reusch, Michael Schaefer, Claudia Plum, Günter Schultz, Martin Paul,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoProliferation and subsequent dedifferentiation of vascular smooth muscle (VSM) cells contribute to the pathogenesis of atherosclerosis and postangioplastic restenosis. The dedifferentiation of VSM cells in vivo or in cell culture is characterized by a loss of contractile proteins such as smooth muscle-specific α-actin and myosin heavy chain (SM-MHC). Serum increased the expression of contractile proteins in neonatal rat VSM cells, indicating a redifferentiation process. RNase protection assays defined thrombin as a serum component that increases the abundance of SM-MHC transcripts. Additionally, serum and thrombin transiently elevated cytosolic Ca2+ concentrations, led to a biphasic extracellular signal-regulated kinase (ERK) phosphorylation, up-regulated a transfected SM-MHC promoter construct, and induced expression of the contractile proteins SM-MHC and α-actin. Pertussis toxin, N17-Ras/Raf, and PD98059 prevented both the serum- and thrombin-induced second phase ERK phosphorylation and SM-MHC promoter activation. Constitutively active Gαq, Gαi, Gα12, and Gα13 failed to up-regulate SM-MHC transcription, whereas Gβγ concentration-dependently increased the SM-MHC promoter activity. Furthermore, the Gβγ scavenger β-adrenergic receptor kinase 1 C-terminal peptide abolished the serum-mediated differentiation. We conclude that receptor-mediated differentiation of VSM cells requires Gβγ and an intact Ras/Raf/MEK/ERK signaling. Proliferation and subsequent dedifferentiation of vascular smooth muscle (VSM) cells contribute to the pathogenesis of atherosclerosis and postangioplastic restenosis. The dedifferentiation of VSM cells in vivo or in cell culture is characterized by a loss of contractile proteins such as smooth muscle-specific α-actin and myosin heavy chain (SM-MHC). Serum increased the expression of contractile proteins in neonatal rat VSM cells, indicating a redifferentiation process. RNase protection assays defined thrombin as a serum component that increases the abundance of SM-MHC transcripts. Additionally, serum and thrombin transiently elevated cytosolic Ca2+ concentrations, led to a biphasic extracellular signal-regulated kinase (ERK) phosphorylation, up-regulated a transfected SM-MHC promoter construct, and induced expression of the contractile proteins SM-MHC and α-actin. Pertussis toxin, N17-Ras/Raf, and PD98059 prevented both the serum- and thrombin-induced second phase ERK phosphorylation and SM-MHC promoter activation. Constitutively active Gαq, Gαi, Gα12, and Gα13 failed to up-regulate SM-MHC transcription, whereas Gβγ concentration-dependently increased the SM-MHC promoter activity. Furthermore, the Gβγ scavenger β-adrenergic receptor kinase 1 C-terminal peptide abolished the serum-mediated differentiation. We conclude that receptor-mediated differentiation of VSM cells requires Gβγ and an intact Ras/Raf/MEK/ERK signaling. vascular smooth muscle smooth muscle myosin heavy chain mitogen activated protein kinase extracellular signal-regulated kinase MAP kinase/ERK pertussis toxin recombinant platelet-derived growth factor thrombin receptor-activating peptide complete medium quiescent medium chloramphenicol acetyltransferase proliferating cell nuclear antigen cytosolic Ca2+ concentration lysophosphatidic acid epidermal growth factor β-adrenergic receptor kinase 1 C-terminal peptide proteinase-activated receptor Fully differentiated, contractile vascular smooth muscle (VSM)1 cells are major determinants of blood pressure and flow. In chronic vascular diseases such as hypertension and atherosclerosis, VSM cells proliferate and undergo a phenotypic modulation characterized by local matrix degradation and a loss of contractile function (1Ross R. Annu. Rev. Physiol. 1995; 57: 791-804Crossref PubMed Scopus (891) Google Scholar). In vivo, dedifferentiated VSM cells can gradually revert toward a more contractile phenotype (2Aikawa M. Sakomura Y. Ueda M. Kimura K. Manabe I. Ishiwata S. Komiyama N. Yamaguchi H. Yazaki Y. Nagai R. Circulation. 1997; 96: 82-90Crossref PubMed Scopus (104) Google Scholar). Interest in the underlying mechanisms and participating signal transduction pathways leading to altered phenotypes of VSM cells has led to extensive study of the VSM cell phenotype both in vivo and in vitro (for review, see Ref. 3Shanahan C.M. Weissberg P.L. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 333-338Crossref PubMed Scopus (215) Google Scholar). Differentiated VSM cells are characterized by high expression levels of contractile proteins such as smooth muscle α-actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC) (4Owens G.K. Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1382) Google Scholar). The expression of SM-MHC isoforms SM-1 and SM-2 is restricted to smooth muscle cells (5Nagai R. Larson D.M. Periasamy M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1047-1051Crossref PubMed Scopus (92) Google Scholar, 6Nagai R. Kuro-o M. Babij P. Periasamy M. J. Biol. Chem. 1989; 264: 9734-9737Abstract Full Text PDF PubMed Google Scholar) and is down-regulated in proliferating cells (7Aikawa M. Kim H.S. Kuro-o M. Manabe I. Watanabe M. Yamaguchi H. Yazaki Y. Nagai R. Ann. N. Y. Acad. Sci. 1995; 748: 578-585Crossref PubMed Scopus (21) Google Scholar). High expression levels of SM-1/2, therefore, are valuable markers for the differentiated phenotype of VSM cells. Similar to pathological proliferation during vascular disease, VSM cells down-regulate SM-1/2 expression in primary culture. Although cultured VSM cells initially retain SM-1/2 expression when cultured on laminin or under serum-free conditions, they undergo morphological changes toward a dedifferentiated phenotype within a few days (8Hedin U. Bottger B.A. Forsberg E. Johansson S. Thyberg J. J. Cell Biol. 1988; 107: 307-319Crossref PubMed Scopus (323) Google Scholar). Patterns of gene expression similar to those in cultured VSM cells from neonatal rats have been observed in neointimal cells within injured vessels (9Majesky M.W. Benditt E.P. Schwartz S.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1524-1528Crossref PubMed Scopus (169) Google Scholar). Neonatal VSM cells can, therefore, provide anin vitro model for studying phenotypic modulation processes in vascular disease. The mechanisms and signaling pathways that induce a phenotypic modulation toward the contractile phenotype of VSM cells are still largely elusive. It has been shown that application of mechanical forces can actively change the VSM phenotype (10Wilson E. Mai Q. Sudhir K. Weiss R.H. Ives H.E. J. Cell Biol. 1993; 123: 741-747Crossref PubMed Scopus (337) Google Scholar). Depending on extracellular matrix composition, cultured VSM cells can either proliferate or differentiate in response to mechanical strain (11Reusch H.P. Wagdy H. Reusch R. Wilson E. Ives H.E. Circ. Res. 1996; 79: 1046-1053Crossref PubMed Scopus (192) Google Scholar). These findings were corroborated recently by applying mechanical forces to cultured whole vessels (12Zeidan A. Nordstrom I. Dreja K. Malmqvist U. Hellstrand P. Circ. Res. 2000; 87: 228-234Crossref PubMed Scopus (57) Google Scholar). Interestingly, phenotypic modulation of VSM cells depends on the activation of mitogen- activated protein kinases (MAP kinases) in both experimental settings. In many cellular systems, the receptor-mediated proliferation and differentiation involves the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases (13Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 14Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2255) Google Scholar). ERKs are part of a multikinase module through which a variety of extracellular stimuli (growth factors, differentiation signals, and cellular stress) are transmitted into the cell (15Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4225) Google Scholar). Receptor tyrosine kinases, upon autophosphorylation and activation of adaptor proteins, recruit Ras and subsequently engage the Raf/MEK/ERK cascade. Alternatively, G protein-coupled receptors have been shown to stimulate ERKs via the Gi, Gq, or G12/13 subfamilies of heterotrimeric G proteins. In addition, transactivation of receptor tyrosine kinases has been demonstrated to participate in signaling from G protein-coupled receptors to ERKs (16Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 17Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1316) Google Scholar, 18Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1493) Google Scholar). Receptor-mediated signaling pathways that alter the phenotype of VSM cells are poorly defined. We therefore studied receptor-mediated pathways in neonatal rat VSM cells and in particular their participation during phenotypic modulation. Our findings clearly indicate that Gβγ subunits released from the Gisubfamily of heterotrimeric G proteins mediate enhanced expression of contractile proteins in VSM cells. Culture media and trypsin were purchased from Life Technologies, Inc. Fetal calf serum and phosphate-buffered saline were obtained from Biochrom. Radiochemicals were from PerkinElmer Life Sciences. Maxiscript and RPA II kits from Ambion were used for RNase protection assays. The anti-SM-1/SM-2 antiserum was kindly provided by Berlex Pharmaceuticals. Pertussis toxin (PTX), recombinant platelet derived growth factor (PDGF-BB), bisindolylmaleimide, and phorbol-12-myristate-13-acetate were obtained from Calbiochem. Thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF) was purchased from Tocris. All other reagents were obtained from Sigma. SM-MHC promoter-CAT construct was a generous gift from Cort S. Madsen, Charlottesville, VA. Dominant negative Ras/Raf constructs were kindly provided by Alan Hall, London. Constitutively active Gαi, Gαq, and Gα12/Gα13 constructs were from S. Offermanns, Heidelberg, Germany and M. I. Simon, Pasadena, CA. Gβγ expression plasmids were from L. Birnbaumer, Los Angeles, CA and M. I. Simon. βARK1ct was from R .J. Lefkowitz, Durham, NC. Primary cultures of VSM cells from newborn rats were established as described previously (19Ives H.E. Schultz G.S. Galardy R.E. Jamieson J.D. J. Exp. Med. 1978; 148: 1400-1413Crossref PubMed Scopus (74) Google Scholar). Cells were grown in minimum Eagle's medium supplemented with 10% fetal calf serum (complete medium; CM), 2% tryptose phosphate broth, 50 units/ml penicillin, and 50 units/ml streptomycin. In all experiments, cells from passages 10–15 were used. Growth arrest was induced in a serum-free quiescent medium (QM) containing 1% (w/v) bovine serum albumin and 4 mg/ml transferrin instead of serum. Prior to agonist application, cells were maintained in QM for 48–72 h. Where indicated, cells were pretreated with 200 ng/ml PTX for 12–18 h. The transcriptional regulation of SM-1/SM-2 was assessed with a chloramphenicol acetyltransferase (CAT) reporter gene expressed under the control of the MHC promoter (nucleotides −1346 to +25, pCAT-1346) (20Madsen C.S. Hershey J.C. Hautmann M.B. White S.L. Owens G.K. J. Biol. Chem. 1997; 272: 6332-6340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For transient transfection assays, cells were seeded into six-well plates at a density of 7.5 × 104 cells/well (60–80% confluence) and growth arrested in QM for 48 h prior to transfection. Transient transfections were performed in triplicate with 1 μg of plasmid DNA and 10 μl/well Superfect transfection reagent (Qiagen) for 5 h. After 48 h, cell lysates were prepared using the CAT enzyme assay system (Promega). CAT activities were normalized to the protein concentration of each sample as measured by the BCA assay. Transfection of a promoterless CAT construct served as a base-line indicator, allowing all other promoter constructs to be expressed relative to promoterless activity. All CAT activities (means ± S.E.) represent at least three independent transfection experiments with each setting tested in triplicate per experiment. Cotransfection of a viral promoter/β -galactosidase or LacZ construct to control for transfection efficiency was discontinued because variations in transfection efficiency among independent experimental samples are small (≤ 10%). Furthermore, it has been shown that such constructs interfere with SM-specific promoters, presumably because of competition for common transcription factors (21Shimizu R.T. Blank R.S. Jervis R. Lawrenz-Smith S.C. Owens G.K. J. Biol. Chem. 1995; 270: 7631-7643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). VSM cells were grown to confluence on Nunc chamber slides (Nalge Nunc International). After fixation in 1% formaline in phosphate-buffered saline and methanol, a monoclonal anti-proliferating cell nuclear antigen (anti-PCNA) antibody (1:100; DAKO) was incubated for 1 h at 20 °C in phosphate-buffered saline supplemented with 5% fetal calf serum. A secondary, biotinylated anti-mouse IgG (Sigma) and streptavidin-conjugated Texas Red (Amersham Pharmacia Biotech) were applied for detection. After the PCNA staining, SM-α-actin was detected by using a monoclonal antibody (1:150; Sigma) and a fluorescein isothiocyanate-conjugated goat anti-mouse (1:40, Dianova). Representative visual fields were exposed sequentially by applying appropriate filter sets. VSM cells were lysed directly in 1 × Laemmli buffer containing 10 mm dithiothreitol. Proteins were separated on polyacrylamide gels and electroblotted onto nitrocellulose membranes. SM myosin isoforms were separated on 4% gels and detected with a polyclonal anti-SM-1/SM-2 antiserum (1:1,000). This antiserum has been characterized previously (11Reusch H.P. Wagdy H. Reusch R. Wilson E. Ives H.E. Circ. Res. 1996; 79: 1046-1053Crossref PubMed Scopus (192) Google Scholar). ERK1/2 were separated on 10% gels and probed with affinity-purified polyclonal anti-phospho-ERK1/2 or with anti-ERK1/2 antibodies (New England Biolabs) to confirm equal loading of the gels. Primary antibodies were detected with a horseradish peroxidase-coupled secondary antibody (1:2,000, New England Biolabs) using a chemiluminescence substrate (Lumiglo, New England Biolabs). RNA isolation, generation of DNA templates, and hybridization conditions have been described previously (11Reusch H.P. Wagdy H. Reusch R. Wilson E. Ives H.E. Circ. Res. 1996; 79: 1046-1053Crossref PubMed Scopus (192) Google Scholar). In brief, 10 μg of total RNA was hybridized with a radiolabled probe covering the alternatively spliced C-terminal exons of SM-1 and SM-2 variants of rat SM-MHC. After overnight incubation at 42 °C, nonhybridized fragments were digested with a diluted RNase A/T1 mixture. The remaining protected fragments (380 nucleotides for SM-2 and 261 nucleotides for SM-1) were separated by denaturing (8% urea) polyacrylamide gel electrophoresis and exposed to Amersham Hyperfilm at −80 °C for 2–24 h. Bands were excised and counted in a liquid scintillation counter. Equal loading was controlled by hybridization of a second aliquot with a rat glutaraldehyde phosphate dehydrogenase-radiolabled probe. Cells were seeded on 24-mm glass coverslips and grown for 24 h prior to loading with 2–4 μm fura-2 in a buffer (Hepes-buffered saline) containing 135 mm NaCl, 6 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 5.5 mm glucose, 10 mm Hepes pH 7.4, and 0.2% bovine serum albumin. Coverslips were mounted in a monochromator-equipped (TILL-Photonics) inverted microscope (Carl Zeiss). Fura-2 was excited alternately at 340 nm and 380 nm. Emitted light was filtered (505 nm long pass) and recorded with a 12-bit CCD camera. After correction for background signals, intracellular [Ca2+]i was calculated as described (22Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). R max,R min, andFmin380 /Fmax380were determined in fura-2-loaded cells equilibrated for 3 h in Hepes-buffered saline supplemented with 1 μm ionomycin and either 10 mm Ca2+ or 10 mmEGTA, pH 7.8. The presence of serum is essential to grow VSM cells in primary cell culture. In addition to its mitogenic properties, we observed that fetal calf serum enhanced the expression of contractile proteins in neonatal rat VSM cells. Dual staining of PCNA and SM-specific α-actin or SM-MHC revealed that after short term (24 h) exposure to serum, PCNA expression was positive, whereas SM-α-actin was poorly detectable (Fig. 1 A). Continuous culturing in the presence of serum for an additional 4 days markedly enhanced the expression of SM-α-actin (Fig. 1 B) and SM-MHC (data not shown). Consistently, the expression of the SM-1 and SM-2 splice variants of SM-MHC were up-regulated by serum within 2–3 days as detected by immunoblotting of whole cell lysates normalized for their protein content (Fig. 1 C). Serum withdrawal reduced SM-α-actin and SM-1/SM-2 expression again within 48 h to about 20% of the expression levels in the presence of serum, demonstrating that alterations in contractile protein expression are bidirectional (data not shown). Thus, serum contains factors capable of inducingin vitro redifferentiation of rat neonatal VSM cells. Because serum contains multiple growth factors, vasoactive peptides, and other agonists mediating their responses through activation of several signaling pathways, we analyzed which receptor-coupled pathways are involved in increasing SM-MHC transcription. RNase protection assays revealed that SM-1/SM-2 transcripts are 22 ± 1.2-fold more abundant in serum-treated (CM) VSM cells compared with serum-starved controls (QM). The relatively low expression levels of SM-1/SM-2 in serum-starved VSM cells allowed us to study the effect of single compounds on SM-1/SM-2 expression. Neither 1 μmangiotensin II nor 10 ng/ml PDGF-BB altered the SM-1/SM-2 expression significantly, whereas treatment with 10 ng/ml transforming growth factor-β resulted in a further reduction of SM-1/SM-2 steady-state expression (Fig. 2). In contrast, 1 unit/ml thrombin increased SM-1/SM-2 expression by the 10 ± 0.9-fold. The substantial increase in SM-1/SM-2 steady-state expression most likely represents an up-regulation of the transcriptional activity, although changes in RNA stability and/or turnover cannot be ruled out. Because the inhomogeneous response to vasoactive peptides may rely on the presence or absence of the corresponding receptors in cultured VSM cell preparations, functional coupling of receptors in VSM cells was characterized by single cell [Ca2+]i analysis. 0.1 unit/ml thrombin induced transient elevations of [Ca2+]i in more than 95% of VSM cells after a lag phase of 10–20 s (Fig.3). To exclude unspecific, receptor-independent effects of the serine protease thrombin, we applied TRAP, which corresponds to the intramolecularly tethered ligand of the PAR-1 receptor. Indeed, 80 μm TRAP induced similar calcium transients without a typical protease lag phase (Fig.3). Addition of lysophosphatidic acid (LPA) induced calcium responses in about 60% of the cells. Only a few cells (less than 10%) were activated by 1 μm angiotensin II. Challenging VSM cells with PDGF-BB (10 ng/ml) elicited a delayed and more sustained elevation of [Ca2+]i, characteristic for ligands binding to tyrosine kinase receptors (Fig. 3). 100 μm carbachol failed to raise [Ca2+]i in VSM cells, indicating the absence of endothelial cell contaminations (data not shown). Using single cell [Ca2+]i analysis, we demonstrated that PAR receptors, endothelial differentiation gene receptors, and PDGF receptors are present and functional in the majority of VSM cells. Because both proliferative and differentiating signals can be transmitted via ERK1/2, depending on the cellular context and the transient or sustained character of their activation, we studied the kinetics of ERK phosphorylation in VSM cells. A rapid and transient phosphorylation of ERK1/2 was elicited by serum, PDGF-BB, EGF, thrombin, TRAP, LPA, and to a lower extent by angiotensin II. The early ERK1/2 phosphorylation was maximal after 3–5 min for all agonists. Only serum, thrombin, TRAP, and LPA elicited a delayed second phase ERK phosphorylation (Fig.4 A). The second phase ERK phosphorylation appeared ∼45 min after agonist application and rose continuously for another 2 h. In contrast, a delayed ERK phosphorylation was absent in response to PDGF-BB, EGF, or angiotensin II. The weak and monophasic ERK1/2 activation by angiotensin II may rely on low AT1 receptor expression in our VSM cell preparation (Fig. 3). The early ERK1/2 phosphorylation, but not the late phase ERK activation, correlated closely with the ability of agonists to raise [Ca2+]i. Consistently, Ca2+ ionophores (1 μm ionomycin or 1 μmA23187) induced early but not delayed ERK1/2 phosphorylation (Fig. 4 B). Permanent activation of protein kinases C by 100 nm phorbol 12-myristate-13-acetate resulted in a monophasic and sustained ERK1/2 activation (Fig.4 B). Thus, in VSM cells, three distinct temporal patterns of ERK1/2 phosphorylation are elicited by different receptor ligands, Ca2+ ionophores, or phorbol esters. Enhanced SM-1/SM-2 expression in response to thrombin (Fig. 2) is indicative of a G protein-mediated regulation of the SM-MHC promoter activity. To characterize signaling pathways that control transcription of contractile proteins, we studied the SM-MHC promoter activity by using a CAT reporter gene construct expressed under the control of the −1346 nucleotide promoter region of the SM-MHC gene (pCAT-1346). In the absence of serum, CAT activities in VSM cells transfected with pCAT-1346 were ∼4–6-fold higher compared with cells transfected with a promoterless pCAT-basic vector. Serum treatment further increased the CAT activity by 5.1 ± 0.5-fold (Fig. 5 A). Expression of CAT driven by an SV40 promoter (pCAT-control) was about 4-fold higher compared with the SM-MHC promoter in the presence of serum (data not shown). Transfection of pCAT-1346 in Swiss 3T3 fibroblasts did not significantly induce CAT activity irrespective of the absence or presence of serum (data not shown). In serum-starved VSM cells transfected with pCAT-1346, the addition of 10 ng/ml PDGF-BB, 10 ng/ml EGF, 10 μm LPA, or 1 unit/ml thrombin resulted in a 1.1 ± 0.1-, 1.2 ± 0.1-, 2.1 ± 0.2-, and 2.0 ± 0.1-fold increase in CAT activity over control cells incubated in serum-free QM (Fig. 5 A). These data confirm that increases in SM-1/SM-2 mRNA steady-state concentrations (Fig. 2) indeed result from transcriptional activation. Furthermore, the reporter gene assay allows for analysis of signaling cascades by applying genetically encoded modulators. To define participation of members of the Ras/Raf/MEK/ERK cascade, the reporter gene construct pCAT-1346 was cotransfected with expression plasmids encoding dominant negative N17-Ras or N17-Raf. In all cotransfection experiments the total amount of transfected plasmid cDNA was kept constant by adding cDNA encoding promoterless pCAT-basic. The thrombin-stimulated CAT activity was abrogated by coexpression of dominant negative N17-Ras or N17-Raf in a concentration-dependent manner (Fig. 5 B). Conversely, coexpression of the Raf C terminus increased CAT activity about 2-fold in the absence of agonists (data not shown). Additionally, the MEK inhibitor PD98059 largely reduced the thrombin-stimulated SM-MHC promoter activity (Fig. 5 C). Because PD98059 was dissolved in dimethyl sulfoxide, the effect of the solvent on the SM-MHC promoter activity was assessed in parallel. Dimethyl sulfoxide (up to 0.5%) further increased the thrombin-stimulated CAT activity almost 1.8-fold, an effect that was also blocked by PD98059. The observed half-maximal inhibitory concentration of PD98059 (3–5 μm) is well in line with its described IC50to inhibit ERK1/2 phosphorylation (23Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3249) Google Scholar). Correspondingly, in VSM cells the serum- or thrombin-mediated ERK1/2 phosphorylation was largely reduced by 5–20 μm PD98059 and abolished by 50 μm MEK inhibitor (data not shown). These higher concentrations, however, exhibited a toxic effect during long term incubation of VSM, thereby precluding a subsequent determination of SM-MHC promoter activity. Both modulations, expression of dominant negative Ras/Raf and pretreatment of VSM cells with PD98059, also impaired the serum-mediated up-regulation of the SM-MHC promoter (data not shown). These data strongly suggest that the thrombin- and serum-induced increase in SM-1/SM-2-expression depends on an intact Ras/Raf/MEK/ERK signaling cascade. Furthermore, the ability of different agonists to up-regulate the SM-MHC promoter activity correlated closely with a biphasic and sustained ERK1/2 phosphorylation. The transient elevation of [Ca2+]i and biphasic ERK1/2 phosphorylation induced by thrombin could be mimicked with the tethered ligand of the PAR-1 receptor, TRAP. PAR-1 receptors couple to the Gi, Gq, and G12/13 subfamilies of heterotrimeric G proteins (24Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar). The putative involvement of G12/13 in the regulation of the SM-MHC promoter was tested by overexpressing constitutively active (GTPase-deficient) mutants of Gα12 (pCIS/Gα12 Q229L) and Gα13 (pCIS/Gα13 Q226L). Both constructs and their combination failed to induce SM-MHC promoter activity significantly over a wide range of transfected cDNA concentrations (data not shown). The biological activity of these constructs has been demonstrated previously by their ability to induce contraction of VSM cells (25Gohla A. Schultz G. Offermanns G. Circ. Res. 2000; 87: 221-227Crossref PubMed Scopus (198) Google Scholar). Gq and Gi proteins couple to phospholipases Cβ to release [Ca2+]i from inositol 1,4,5-trisphosphate-sensitive stores. The possible role of the Gi class of heterotrimeric G proteins was assessed by pretreating cells with 200 ng/ml PTX for at least 18 h. Inactivation of Gi proteins by this protocol was demonstrated by a more than 80% reduction of [Ca2+]i signals in response to LPA (Fig. 6 A). PTX reduced the peak [Ca2+]i after thrombin stimulation by about 40% (Fig. 6 A). The partial block of thrombin-induced [Ca2+]itransients in PTX-pretreated VSM cells reflects coupling to both Gi and Gq/11. To test further whether the Gi subfamily also participates in the prolonged ERK1/2 activation, serum-starved VSM cells were pretreated with PTX. Subsequent addition of serum, thrombin, and LPA left early ERK1/2 phosphorylation almost unaltered, whereas the second phase of ERK1/2 activation was completely abrogated in PTX-pretreated cells (Fig.6 B). Because sustained ERK activation may be required for the regulation of transcriptional activity, we tested whether PTX pretreatment affects the ligand-induced SM-MHC promoter activity. In addition to the modulation of ERK1/2 signaling, PTX treatment abolished the thrombin-induced up-regulation of the SM-MHC promoter activity (Fig.7 A). Moreover, even the strong induction of the SM-MHC promoter by serum was reverted completely in PTX-pretreated VSM cells. This indicates that all serum components that are involved in the up-regulation of the SM-MHC promoter depend on the presence of functional Gi proteins. Because either the α or the βγ subunits may transmit the signal that results in SM-MHC expression, we coexpressed the constitutively active Gαi (Q205L) together with pCAT-1346. Gαi (Q205L), however, even reduced the SM-MHC promoter activity below base-line values (Fig. 7 B). On the contrary, coexpression of Gβ1 and Gγ2 mimicked the receptor-mediated up-regulation of the SM-MHC promoter in a concentration-dependent fashion. Expression of neither Gβ1 nor Gγ2 alone was sufficient to increase the activity of the cotransfected CAT reporter. Consistent with an essential role of Gβγ, coexpression of the Gβγ-scavenging C-terminal peptide of the β-adrenergic receptor kinase 1 (βARK1ct) concentration-dependently reverted the serum-induced activation of the SM-MHC promoter (Fig.7 C). Finally, the Gi protein-dependent redifferentiation in response to thrombin and serum was confirmed by analyzing the expression of contractile proteins in untransfected cells. In whole cell lysates from VSM cells stimulated with thrombin or serum and normalized for protein content, an increased expression of SM-α-actin and of SM-MHC was detected (Fig.8). When incubated in the continuous presence of PTX, thrombin and serum failed to increase the expression of both contractile proteins (Fig. 8). Hence, these data demonstrate that Gβγ released from Gi proteins link proximal signaling to the Ras/Raf/MEK/ERK cascade to mediate the in vitro redifferentiation of vascular smooth muscle cells shown in Fig. 1. In this study we describe a receptor-mediated signaling pathway leading to differentiation of VSM cells. The thrombin-induced SM-MHC expression is transmitted via the Ras/Raf signaling cascade and leads to a biphasic temporal pattern of ERK1/2 phosphorylation. Pertussis toxin abrogated both the second phase ERK1/2 phosphorylation and the up-regulation of contractile proteins in response to serum, thrombin, and LPA. Because coexpression of Gβγ subunits mimicked and βARK1ct abrogated the activation of the SM-MHC promoter in response to serum components, we conclude that Gβγ mediate the agonist-induced differentiation of VSM cells. A limited number of reports describe a phenotypic modulation of mature VSM cells toward a more contractile phenotype. Vasoconstrictors such as
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