Gbeta gamma Mediate Differentiation of Vascular Smooth Muscle Cells*

H. Peter ReuschDagger, Michael Schaefer§, Claudia Plum, Günter Schultz§, and Martin Paul

From the Institute of Clinical Pharmacology and Toxicology and the § Institute of Pharmacology, Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin 14195, Germany

Received for publication, March 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 Galpha q, Galpha i, Galpha 12, and Galpha 13 failed to up-regulate SM-MHC transcription, whereas Gbeta gamma concentration-dependently increased the SM-MHC promoter activity. Furthermore, the Gbeta gamma scavenger beta -adrenergic receptor kinase 1 C-terminal peptide abolished the serum-mediated differentiation. We conclude that receptor-mediated differentiation of VSM cells requires Gbeta gamma and an intact Ras/Raf/MEK/ERK signaling.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). In vivo, dedifferentiated VSM cells can gradually revert toward a more contractile phenotype (2). 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. 3).

Differentiated VSM cells are characterized by high expression levels of contractile proteins such as smooth muscle alpha -actin (SM-alpha -actin) and smooth muscle myosin heavy chain (SM-MHC) (4). The expression of SM-MHC isoforms SM-1 and SM-2 is restricted to smooth muscle cells (5, 6) and is down-regulated in proliferating cells (7). 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 (8). Patterns of gene expression similar to those in cultured VSM cells from neonatal rats have been observed in neointimal cells within injured vessels (9). Neonatal VSM cells can, therefore, provide an in 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 (10). Depending on extracellular matrix composition, cultured VSM cells can either proliferate or differentiate in response to mechanical strain (11). These findings were corroborated recently by applying mechanical forces to cultured whole vessels (12). 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 (13, 14). 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 (15). 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 (16-18).

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 Gbeta gamma subunits released from the Gi subfamily of heterotrimeric G proteins mediate enhanced expression of contractile proteins in VSM cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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 Galpha i, Galpha q, and Galpha 12/Galpha 13 constructs were from S. Offermanns, Heidelberg, Germany and M. I. Simon, Pasadena, CA. Gbeta gamma expression plasmids were from L. Birnbaumer, Los Angeles, CA and M. I. Simon. beta ARK1ct was from R .J. Lefkowitz, Durham, NC.

Cell Culture, Transient Transfections, and Reporter Assays-- Primary cultures of VSM cells from newborn rats were established as described previously (19). 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) (20). 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/beta -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 (21).

Immunostaining-- 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-alpha -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.

Immunoblotting Methods-- 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 (11). 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).

RNase Protection Assay-- RNA isolation, generation of DNA templates, and hybridization conditions have been described previously (11). 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.

Single cell [Ca2+]i Measurements-- 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 (22). Rmax, Rmin, and F<UP><SUB>min</SUB><SUP>380</SUP></UP>/F<UP><SUB>max</SUB><SUP>380</SUP></UP> were 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 mM EGTA, pH 7.8.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of SM-MHC Expression by Serum Components-- 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 alpha -actin or SM-MHC revealed that after short term (24 h) exposure to serum, PCNA expression was positive, whereas SM-alpha -actin was poorly detectable (Fig. 1A). Continuous culturing in the presence of serum for an additional 4 days markedly enhanced the expression of SM-alpha -actin (Fig. 1B) 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. 1C). Serum withdrawal reduced SM-alpha -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 inducing in vitro redifferentiation of rat neonatal VSM cells.


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Fig. 1.   Effects of serum on the expression of contractile proteins in vascular smooth muscle cells. Rat neonatal VSM cells were maintained in serum-free QM for 48 h and then reexposed to serum for 24 h (panel A) or 120 h (panel B). Filamentous SM-alpha -actin and proliferating cell nuclear antigen reflecting replicating nuclei were identified with immunofluorescence applying appropriate first and secondary antibodies (see "Experimental Procedures"). Epifluorescence microphotographs were taken sequentially and then superimposed. The images represent typical viewing fields of six independent SM-alpha -actin staining experiments showing similar results. White bars depict a 50-µm scale. Panel C, cell lysates were prepared from VSM cells cultured without serum (QM) and then reexposed to serum (10%) for the indicated number of days. Whole cell lysates (20 µg of protein/lane) were separated by 4% SDS-polyacrylamide gel electrophoresis, electroblotted, and probed with a polyclonal antiserum detecting both SM-1 (204 kDa) and SM-2 (200 kDa) isoforms of SM-MHC.

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 µM angiotensin II nor 10 ng/ml PDGF-BB altered the SM-1/SM-2 expression significantly, whereas treatment with 10 ng/ml transforming growth factor-beta 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.


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Fig. 2.   Serum and receptor ligands up-regulate transcription of the SM-1/SM-2 genes in VSM cells. VSM cells were cultured either in serum-containing CM, in serum-free QM, or in QM supplemented with 10 ng/ml PDGF-BB, 1 ng/ml transforming growth factor-beta (TGF-beta ), 1 unit/ml thrombin, and 1 µM angiotensin II (Ang-II) for the indicated times. RNase protection assays were performed with 10 µg of total RNA/lane. The lengths of protected fragments correspond to the expected sizes for the SM-1 and SM-2 splice variants of SM-MHC (P, full-length probe). The abundance of a control transcript was unaltered as demonstrated by reprobing equal aliquots of total RNA with a glutaraldehyde phosphate dehydrogenase probe (data not shown).

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.


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Fig. 3.   Agonist-induced transient elevation of [Ca2+]i in single VSM cells. VSM cells were subcultured on glass coverslips, loaded with the fluorescent Ca2+ indicator fura-2, and washed in a Hepes-buffered medium containing 1 mM Ca2+. Coverslips were mounted in a monochromator-equipped fluorescence imaging system built around an inverted microscope, and fura-2 was excited alternatively at 340 and 380 nm every 0.5 s. Emitted light was recorded with a cooled CCD camera. Cells were stimulated with 0.1 unit/ml thrombin, 80 µM TRAP, 10 µM LPA, 1 µM angiotensin II (Ang-II), or 10 ng/ml PDGF-BB, as indicated. [Ca2+]i was calibrated as described (22). Time courses of [Ca2+]i in individual single cells (gray lines) are shown to indicate the number of cells responding to the respective agonist. Mean [Ca2+]i (bold black lines) was calculated from all cells selected in the experiment.

Serum, Thrombin, and TRAP Induce Biphasic ERK Phosphorylation-- 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. 4A). 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 µM A23187) induced early but not delayed ERK1/2 phosphorylation (Fig. 4B). Permanent activation of protein kinases C by 100 nM phorbol 12-myristate-13-acetate resulted in a monophasic and sustained ERK1/2 activation (Fig. 4B). Thus, in VSM cells, three distinct temporal patterns of ERK1/2 phosphorylation are elicited by different receptor ligands, Ca2+ ionophores, or phorbol esters.


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Fig. 4.   Time course of receptor-mediated ERK1/2 phosphorylation. Panel A, serum-starved VSM cells were stimulated with 10% fetal calf serum, 10 ng/ml PDGF-BB, 10 ng/ml EGF, 1 µM angiotensin II, 1 unit/ml thrombin (Thr.), 80 µg/ml TRAP, and 1 µM LPA for the indicated times. Whole cell lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis and electroblotted. Activated ERK was detected with a phospho-specific anti-ERK1/2 antiserum. Exposure times were optimized to maximize differences in band intensity within one experiment. Shown are representative experiments of three independent experiments with similar results. Each blot was reprobed with antibodies detecting total ERK1/2 demonstrating equal loading of the lanes (data not shown). Panel B, the effects of calcium elevation and of protein kinases C were assessed by 1 µM ionomycin, 1 µM calcimycin (A23187), or 100 ng/ml phorbol 12-myristate-13-acetate (PMA).

Activation of Ras/Raf/MEK/ERK Is a Prerequisite for SM-MHC Promoter Activation by Serum and Thrombin-- 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. 5A). 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).


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Fig. 5.   Receptor-mediated SM-MHC promoter activation requires the Ras/Raf/MEK/ERK signaling cascade. Panel A, VSM cells, serum-starved for 48 h, were transfected with a -1346-nucleotide SM-MHC promoter-CAT fusion construct (pCAT-1346) and then incubated in the presence of serum free medium (QM) or in QM supplemented with 10 ng/ml PDGF, 10 ng/ml EGF, 10 µM LPA, 1 unit/ml thrombin (Thr.), or 10% serum for another 48 h. Cells were lysed and assayed for CAT activity. Depicted CAT activities were normalized for protein concentrations and compared with the CAT activity of cells transfected with a reporter gene construct lacking the SM-MHC promoter. Bars represent the means ± S.E. of at least five independent transfection experiments. Panel B, to test for participation of Ras/Raf in the thrombin-mediated SM-MHC promoter induction, VSM cells were cotransfected with 0.5 µg/well pCAT-1346 and the indicated amounts (in µg) of dominant negative N17-Ras or N17-Raf expression constructs. The total amount of plasmid DNA was kept constant (1 µg/well) with promoterless pCAT-basic. Panel C, VSM cells transfected with 1 µg/ml pCAT-1346 were preincubated for 30 min with different concentrations of the MEK inhibitor PD98059 (black bars) and then stimulated with 1 unit/ml thrombin. Because dimethyl sulfoxide (the solvent of PD98059) further enhanced thrombin-stimulated SM-MHC promoter activities, controls were incubated in equivalent concentrations of the solvent (open bars). All values were normalized to the thrombin-mediated CAT activity without PD98059.

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. 5A). 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. 5B). 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. 5C). 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 IC50 to inhibit ERK1/2 phosphorylation (23). 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.

Differentiation of VSM Cells Requires Pertussis Toxin-sensitive G Proteins-- 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 (24). The putative involvement of G12/13 in the regulation of the SM-MHC promoter was tested by overexpressing constitutively active (GTPase-deficient) mutants of Galpha 12 (pCIS/Galpha 12 Q229L) and Galpha 13 (pCIS/Galpha 13 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 (25). Gq and Gi proteins couple to phospholipases Cbeta 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. 6A). PTX reduced the peak [Ca2+]i after thrombin stimulation by about 40% (Fig. 6A). The partial block of thrombin-induced [Ca2+]i transients 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. 6B).


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Fig. 6.   PTX blocks late phase ERK phosphorylation. Panel A, time courses of 0.1 unit/ml thrombin- and 1 µM LPA-induced [Ca2+]i transients in controls and in PTX-pretreated VSM cells (+PTX, 200 ng/ml for 18 h). The mean ± S.E. [Ca2+]i was computed from three independent experiments, each comprising at least 100 single cells. Panel B, effects of 10% serum, 1 unit/ml thrombin, and 1 µM LSA on ERK1/2 phosphorylation in control and PTX-pretreated (+PTX) VSM cells. Phosphorylated ERK1/2 was detected as described in Fig. 4. Blots were reprobed with anti-total ERK1/2 demonstrating equal loading of the lanes (data not shown).

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. 7A). 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.


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Fig. 7.   Gbeta gamma released from Gi proteins induce the SM-MHC promoter. Panel A, transfected VSM cells (pCAT-1346) were either left untreated or were pretreated with PTX (200 ng/ml for 18 h) and then incubated in serum-free medium (QM) or stimulated with 1 unit/ml thrombin or 10% serum in QM for 48 h. PTX was either present or absent throughout the whole experiment. Panel B, CAT activities were determined in unstimulated cells transfected with 0.5 µg/well pCAT-1346 and the indicated amounts (in µg) of expression plasmids encoding either constitutively active Galpha i (Q205L-mutant) or equal amounts of wild-type Gbeta 1 and Ggamma 2. Total plasmid DNA concentrations were adjusted to 1 µg/well with pCAT-basic. Panel C, cells were transfected with 0.5 µg/well pCAT-1346, an expression plasmid encoding the Gbeta gamma scavenger beta ARK1ct as indicated, and adjusted to 1 µg/well with pCAT-basic. CAT activities were assayed in either unstimulated (open bar) or in serum-stimulated (black bars) VSM cells. Mean CAT activities and S.E. were calculated from at least six independent transfection experiments.

Because either the alpha  or the beta gamma subunits may transmit the signal that results in SM-MHC expression, we coexpressed the constitutively active Galpha i (Q205L) together with pCAT-1346. Galpha i (Q205L), however, even reduced the SM-MHC promoter activity below base-line values (Fig. 7B). On the contrary, coexpression of Gbeta 1 and Ggamma 2 mimicked the receptor-mediated up-regulation of the SM-MHC promoter in a concentration-dependent fashion. Expression of neither Gbeta 1 nor Ggamma 2 alone was sufficient to increase the activity of the cotransfected CAT reporter. Consistent with an essential role of Gbeta gamma , coexpression of the Gbeta gamma -scavenging C-terminal peptide of the beta -adrenergic receptor kinase 1 (beta ARK1ct) concentration-dependently reverted the serum-induced activation of the SM-MHC promoter (Fig. 7C).

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-alpha -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 Gbeta gamma 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.


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Fig. 8.   PTX-sensitive induction of contractile protein expression by thrombin and serum. VSM cells were pretreated with (+PTX, 200 ng/ml for 18 h) or not treated and then cultured in serum-free medium (QM). 1 unit/ml thrombin or 10% serum was added to the medium for the indicated number of days in the continuous presence or absence of PTX. Medium was changed every 24 h. Whole cell lysates were normalized for their protein content (10 µg/lane), subjected to SDS-polyacrylamide gel electrophoresis, and probed with monoclonal antibodies detecting either SM-alpha -actin or SM-MHC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Gbeta gamma subunits mimicked and beta ARK1ct abrogated the activation of the SM-MHC promoter in response to serum components, we conclude that Gbeta gamma 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 angiotensin II or vasopressin have been shown to increase levels of steady-state mRNA and SM-alpha -actin protein expression (26-29). In both cases, CC(A/T)6GG cis-elements (CArG boxes) within the SM-alpha -actin promoter are essential for the ligand-induced promoter regulation. In addition, the same serum response factor-binding cis-elements positively regulate the SM-MHC promoter activity in VSM cells (20, 30). The upstream signaling pathways that regulate the receptor-induced expression of contractile proteins in VSM cells are poorly defined. The findings presented herein demonstrate that serum components activate the Ras/Raf/MEK/ERK cascade in VSM cells. Because dominant negative N17-Ras, N17-Raf, or the MEK inhibitor PD98059 prevented the SM-MHC promoter regulation by serum and thrombin, the entire Ras/Raf/MEK/ERK cascade appears to be required. Activated ERKs may then translocate into the nucleus to phosphorylate Elk-1 or related transcription factors (31). This signaling cascade may therefore provide a comprehensible model for activation of serum response factors in response to G protein-coupled receptors in VSM cells. Alternatively, Garat et al. (32) described that the vasopressin-mediated transcriptional regulation of SM-alpha -actin is completely blocked by pharmacological inhibition of p38 and partially sensitive to overexpressed dominant negative JNKs. The upstream signaling molecules that link the vasopressin-induced receptor activation to the MAP kinase branches p38 and c-Jun N-terminal kinase, however, remain unresolved. Moreover, RhoA-mediated cytoskeletal rearrangements have been implicated in the regulation of the transcriptional regulation of SM-22alpha and SM-alpha -actin (33). Because the authors observed an activation of these promoters by either pharmacological disruption or polymerization of actin stress fibers, RhoA-promoted cytoskeletal assembly cannot serve as the only mechanism for regulation of contractile protein expression in VSM cells. Nonetheless, these findings emphasize the importance of cytoskeletal structures for the maintenance of SM-alpha -actin expression in cultured VSM cells. Whether receptor-dependent signaling utilizes cytoskeletal dynamics to regulate the expression of contractile proteins remains to be clarified. Our data outline that the serum-induced expression of SM-MHC is transmitted by the Ras/Raf/MEK/ERK cascade. ERKs therefore either exclusively or in concert with other MAP kinases promote the expression of contractile proteins. Because thrombin stimulation activated ERKs, but neither p38 nor c-Jun N-terminal kinases in VSM cells,2 activation of ERKs appears to be both sufficient and necessary for receptor-mediated differentiation of VSM cells. The ERK pathway regulates two mutually opposing processes, cellular proliferation and differentiation, depending on the duration of activation and the cellular context (15, 34, 35). Our data indicate that a sustained rather than a short lived ERK phosphorylation is a requirement for the differentiation of VSM cells. Several other cellular models including megakaryocytes (36), thymocytes (37), and PC-12 cells (38) support the idea that the short versus long term ERK phosphorylation determines the proliferative or differentiating outcome, respectively.

Multiple upstream signaling pathways link receptor activation to phosphorylation of ERK1/2. In VSM cells, the biphasic kinetic pattern of ERK phosphorylation in response to serum, thrombin, or LPA suggests that at least two independent pathways control the early and delayed phases of ERK phosphorylation. Considering that strong Ca2+ signals result from thrombin stimulation of VSM cells, a Ca2+- and PKC-dependent formation of Ras/Raf-1 complexes (39, 40) may engage ERKs. Alternatively, Ca2+/calmodulin-dependent activation of Pyk2 (41) and subsequent Src activation may target Ras either including (42-44) or bypassing transactivated receptor tyrosine kinases (45, 46). Because Ca2+ ionophores evoked large [Ca2+]i signals but failed to induce a long lived ERK phosphorylation in VSM cells, an isolate Ca2+ elevation was not sufficient to mimic the effects of serum components. In PTX-pretreated VSM cells stimulated with serum, thrombin, or LPA, the remaining activation of Gq/11 and G12/13 induced an early ERK activation but failed to generate a sustained phospho-ERK signal. Because PTX pretreatment also abolished the contractile protein expression in response to serum and thrombin, we focused on signaling pathways that are initiated by either Galpha i or Gbeta gamma subunits released from activated Gi proteins. The inhibition of adenylyl cyclases by Galpha i lowers cAMP concentrations and subsequently protein kinase A activity. Signaling via the Ras/Raf/MEK/ERK cascade is counterregulated by protein kinase A-dependent phosphorylation and inactivation of Raf-1 (47, 48). Indeed, forskolin treatment further reduced the basal ERK phosphorylation in serum-starved VSM cells.2 Thus, a disinhibition of Ras/Raf signaling by further reducing the cAMP concentrations in quiescent cells might result in an increased activity of the c-Raf kinase as has been shown for another cell system (49). However, expression of constitutively active Galpha i (Q205L) failed to increase the SM-MHC promoter activity. Most strikingly, coexpression of Gbeta gamma subunits mimicked while the Gbeta gamma scavenger beta ARK1ct attenuated the effects of thrombin or LPA. Within the multiple Gbeta gamma effector systems, Gbeta gamma -sensitive PLC isoforms, phosphoinositide 3-kinases, or further unknown effectors bear the potential to feed into the Ras/Raf/MEK/ERK cascade. Although the molecular mechanisms are currently poorly defined, a growing body of evidence points to a role of Gbeta gamma in initiating the assembly of a multiprotein complex including beta -arrestin and c-Src in clathrin-coated pits (50, 51). Within these microdomains, a ligand-independent transactivation of receptor tyrosine kinases such as the EGF receptor may link Gbeta gamma signaling to Ras. Other concepts favor a direct association of Gbeta gamma with Raf-1 (53) or the activation of a Ras-guanine nucleotide exchange factor other than Sos1 (53). Our preliminary data demonstrating that the tyrphostin AG1478 prevents the thrombin-induced ERK phosphorylation in VSM cells point to a crucial role of an EGF receptor transactivation.

Receptors for endogenous vasoconstrictors such as endothelin-1, angiotensin II, and vasopressin or serum components such as thrombin or LPA activate the Gi, Gq, and G12/13 classes of heterotrimeric G proteins. The Galpha q-dependent pathway, via phospholipase Cbeta -catalyzed formation of inositol 1,4,5-trisphosphate, increases [Ca2+]i to activate the calmodulin-dependent myosin light chain kinase. In parallel, activated G12/13, via RhoA and Rho-kinase, inhibits a myosin phosphatase (25). Both pathways synergistically control the contraction of VSM cells by increasing the myosin light chain20 phosphorylation. The additional coupling to Gi proteins does not contribute to the acute regulation of contraction (25). It is, therefore, tempting to speculate that Gbeta gamma released from Gi proteins function to maintain the contractile phenotype by enhancing the expression of contractile proteins.

In summary, we have defined the Gi component of multiply coupling receptors as a pivotal step in the receptor-mediated expression of contractile proteins. Our data clearly indicate that Gbeta gamma subunits induce a sustained ERK phosphorylation that is critical for the differentiation of VSM cells. In the past, substantial data have been accumulated regarding the serum factor-dependent promoter regulation of contractile proteins. The addition of our data, demonstrating how receptor-mediated differentiation signals may be transmitted to the nucleus, may converge to a more clearly defined step-by-step model describing the regulation of contractile protein expression in VSM cells.

    FOOTNOTES

* This study was supported by the Sonderforschungsbereich 366 of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Institut für Klinische Pharmakologie und Toxikologie, Freie Universität Berlin, Garystr. 5, 14195 Berlin, Germany. Fax: 49-30-8445-1761; E-mail: reusch@medizin.fu-berlin.de.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101963200

2 H. P. Reusch, unpublished data.

    ABBREVIATIONS

The abbreviations used are: VSM, vascular smooth muscle; SM, smooth muscle; MHC, myosin heavy chain; MAP kinase, mitogen activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK; PTX, pertussis toxin; PDGF-BB, recombinant platelet-derived growth factor; TRAP, thrombin receptor-activating peptide; CM, complete medium; QM, quiescent medium; CAT, chloramphenicol acetyltransferase; PCNA, proliferating cell nuclear antigen; [Ca2+]i, cytosolic Ca2+ concentration; LPA, lysophosphatidic acid; EGF, epidermal growth factor; beta ARK1ct, beta -adrenergic receptor kinase 1 C-terminal peptide; PAR, proteinase-activated receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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