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• W2171256006 abstract "The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of anin vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser259, resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser259 phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells. The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of anin vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser259, resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser259 phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells. platelet-derived growth factor chloramphenicol acetyltransferase extracellular signal-regulated kinase mitogen-activated protein kinase ERK kinase phosphatidylinositol 3-kinase smooth muscle α-actin smooth muscle myosin heavy chain vascular smooth muscle glutathione S-transferase polyacrylamide gel electrophoresis quiescent medium complete medium nerve growth factor phospholipase C insulin-like growth factor Vascular smooth muscle cells determine blood pressure and flow-through modulation of the vascular tone. The contractility depends on the expression of proteins such as smooth muscle α-actin and smooth muscle myosin, and their expression levels vary depending on developmental and/or differentiation stage. During progression of vascular diseases or vascular injury following balloon dilatation, the release of growth factors such as PDGF,1 epidermal growth factor, or IGF has been shown to increase the smooth muscle cell proliferation and migration (1Jawien A. Bowen-Pope D.F. Lindner V. Schwartz S.M. Clowes A.W. J. Clin. Invest. 1992; 89: 507-511Crossref PubMed Scopus (590) Google Scholar, 2Jackson C.L. Raines E.W. Ross R. Reidy M.A. Arterioscler. Thromb. 1993; 13: 1218-1226Crossref PubMed Scopus (229) Google Scholar, 3Arnqvist H.J. Bornfeldt K.E. Chen Y. Lindstrom T. Metabolism. 1995; 44: 58-66Abstract Full Text PDF PubMed Scopus (64) Google Scholar). This de-differentiation is characterized by a decreased expression of contractile proteins. Following ligand binding, tyrosine kinase receptors undergo dimerization which allows transphosphorylation at multiple tyrosine residues. The intracellular signal transduction involves direct interaction of effector molecules via specific domains, e.g.Src homology 2 domains and phosphotyrosine-binding domains. More than 10 different Src homology 2 domain-containing molecules have been shown to bind to different autophosphorylation sites in the PDGF receptors, including signal transduction molecules with enzymatic activity like phosphatidylinositol 3-kinases (PI 3-kinases), phospholipases Cγ, or Src as well as adaptor molecules such as Grb2 and Shc (4Heldin C.H. Ostman A. Ronnstrand L. Biochim. Biophys. Acta. 1998; 1378: 79-113PubMed Google Scholar). Binding of Grb2/Sos or Shc in turn activates the small GTP-binding protein Ras which couples to the Raf/MEK/ERK cascade. The cellular response of receptor tyrosine kinase signaling is influenced by the strength and the duration of ERK1/2 phosphorylation. Depending on the cellular context, either proliferation or differentiation may result (5Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4225) Google Scholar). Other signaling cascades initiated by PDGF receptors and their potential cross-talk is currently under extensive investigation. Phosphorylated tyrosine residues (Tyr740 and Tyr751) on the PDGF β-receptor recruit the PI 3-kinases α and β to the plasma membrane via docking of the common p85 regulatory subunit (6Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2222) Google Scholar, 7Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1428) Google Scholar). Upon activation, the lipid kinase activity of PI 3-kinases catalyzes the formation of PI(3,4,5)-P3, a well defined plasma membrane anchor for the pleckstrin homology domains of 3-phosphoinositide-dependent kinase I and protein kinase B/Akt (8Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (791) Google Scholar). The plasma membrane recruitment exposes Akt to subsequent activation by 3-phosphoinositide-dependent kinase I and related kinases that phosphorylate Akt at Thr308 and Ser473 (9Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2498) Google Scholar). Akt is a major participant in growth factor-mediated transcription and promotes cell survival by inhibiting apoptosis. These processes appear to involve phosphorylation and inactivation of several targets including Bad (10Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4919) Google Scholar), forkhead transcription factors (11Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar), and caspase-9 (12Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2722) Google Scholar). Recent reports by Rommel et al. (13Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar) and Zimmerman and Moelling (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar) demonstrated that Akt negatively regulates the Ras/Raf/MEK/ERK pathway via phosphorylation and inactivation of Raf at Ser259. Both PDGF and thrombin receptors qualitatively engage phospholipases C, ERKs, and PI 3-kinases. Nonetheless, in VSM cells, these agonists exert virtually opposite effects regarding the phenotypic modulation. Whereas thrombin via protease-activated receptors and Gβγ released from activated Gi proteins up-regulates the expression of contractile proteins, PDGF treatment exerted no differentiating effect. Vice versa, thrombin stimulation was without significant mitogenic potential, while PDGF almost reconstituted the proliferative effect of serum. To evaluate the contribution of the MAP kinase and PI 3-kinase pathways to the phenotypic modulation of VSM cells, we studied the coupling of PDGF and thrombin receptors to the Ras/Raf/MEK/ERK and the PI 3-kinase/Akt cascades and their cross-regulation. Our results demonstrate that PDGF and thrombin activate both pathways but exerted substantial differences in signal intensity and their kinetic patterns. Biochemical analysis revealed an interaction between Akt and Raf in PDGF-stimulated VSM cells that modulates the late-phase ERK1/2 phosphorylation. Abrogation of the PI 3-kinase/Akt signaling changed the PDGF-induced proliferative response in VSM cells toward enhanced expression of contractile proteins. Culture media and trypsin were purchased from Life Technologies. Fetal calf serum and phosphate-buffered saline were obtained from Biochrom. Radiochemicals were from PerkinElmer Life Sciences. The anti-Raf monoclonal antibody was purchased from Transduction Laboratories. Unless otherwise stated, all other antibodies were from New England Biolabs. LY294002, wortmannin, recombinant growth factors PDGF-BB, IGF-I, and epidermal growth factor were obtained from Calbiochem, and recombinant GST-MEK-His6was from Upstate Biotechnology. All other reagents were obtained from Sigma. Primary cultures of VSM cells from newborn rats were established as previously described (15Ives 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 minimal essential medium supplemented with 10% fetal calf serum (complete medium, CM), 2% tryptose phosphate broth, penicillin (50 units/ml), and streptomycin (50 units/ml). 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. The transcriptional regulation of SM-1/SM-2 was assessed with a chloramphenicol acetyltransferase (CAT) reporter gene expressed under the control of the myosin heavy chain promoter (nucleotides −1346 to +25, pCAT-1346) as described (16Madsen 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 6-well plates at a density of 7.5 × 104 cells/well (60–80% confluency) and growth arrested in QM for 48 h prior to transfection. Transient transfections were performed in triplicates with 1 μg of plasmid DNA and 10 μl/well Superfect transfection reagent (Qiagen) for 5 h. After 36–48 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 baseline indicator, allowing all other promoter constructs to be expressed relative to promoterless activity. VSM cells were grown to confluency on Nunc Chamber Slides (Nalge Nunc International). After fixation in 1% formaline in phosphate-buffered saline, smooth muscle α-actin was detected by using a monoclonal primary antibody (1:150; Sigma) and a fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:40, Dianova). Representative visual fields were photographed in an epifluorescence microscope (Nikon Diaphot) applying a fluorescein isothiocyanate filter set (Chroma). VSM cells were directly lysed in Laemmli buffer containing 10 mm dithiothreitol. Proteins were separated on polyacrylamide gels and electroblotted to nitrocellulose membranes. Akt, ERK1/2, MEK, or Raf were separated on 10% gels and probed with affinity purified polyclonal anti-phospho-Akt, phospho-ERK1/2, phospho-MEK, and phospho-Raf or with anti-Akt, -ERK1/2, MEK (New England Biolabs), and -Raf antibodies (Transduction Laboratories) to confirm equal loading of the gels. Primary antibodies were detected with a horseradish peroxidase-coupled secondary antibody (1:2000, New England Biolabs) using a chemiluminescence substrate (Lumiglo, New England Biolabs). RNA isolation, generation of DNA templates, and hybridization conditions were described previously (17Reusch H.P. Wagdy H. Reusch R. Wilson E. Ives H.E. Circ. Res. 1996; 79: 1046-1053Crossref PubMed Scopus (192) Google Scholar). The Maxiscript and RPA II kits from Ambion were used for RNase protection assays. In brief, 10 μg of total RNA was hybridized with radiolabeled probes overnight at 42 °C. Non-hybridized fragments were digested with RNase A/T1. The remaining protected fragments 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 with a rat glutaraldehyde-3-phosphate dehydrogenase probe. VSM cells were serum starved for 48 h in serum-free medium. After stimulation, cells were lysed in RIPA buffer (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar), and Raf protein was immunoprecipitated with an anti-Raf monoclonal antibody (Transduction Laboratories) as described previously (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar). In vitro kinase assays were performed by incubating the immunocomplexes in 30 μl of kinase buffer containing 1 μg of recombinant GST-MEK-His6 (Upstate Biotechnology) and 10 μCi of [γ-32P]ATP in kinase buffer for 30 min at 30 °C. Proteins were separated by SDS-PAGE, and their phosphorylation was visualized and quantified with a phosphorimaging system (Fuji Bas-1500). VSM cells were lysed in a 0.25% Nonidet P-40 containing lysis buffer as described previously (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar). Cleared lysates (350 μg of protein in 800 μl) were immunoprecipitated overnight at 4 °C with 2 μg of monoclonal anti-Raf-1 antibodies (Transduction Laboratories) coupled to suspended Protein A-coupled Sepharose beads (Sigma). The pelleted beads were washed three times in 400 μl of lysis buffer. Immunoprecipitates were boiled in SDS-Laemmli buffer and subjected to Western blot analysis with anti-Raf (Transduction Laboratories), anti-Akt and anti-phospho-Akt antibodies (New England Biolabs). We have recently demonstrated that serum, in addition to its mitogenic properties, increases the expression of contractile proteins in neonatal rat vascular smooth muscle (VSM) cells (18Reusch H.P. Schaefer M. Plum C. Schultz G. Paul M. J. Biol. Chem. 2001; 276: 19540-19547Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To evaluate the proliferative effects of serum, PDGF, and thrombin, VSM cells were cultured in serum-free QM supplemented with the respective agonist. The initial cell counts were assessed at the beginning of the experiment (day 0, Fig.1 A) and every following day. To account for potential degradation of the agonists, media were replaced every day. Whereas cell counts remained almost constant in QM, doubling rates in the presence of serum were about 1.5 days. Unlike thrombin (1 unit/ml), PDGF-BB (10 ng/ml) was a powerful mitogen that reconstituted about 80% of the serum-mediated cell proliferation (Fig.1 A). The abundant expression of the contractile protein SM-α-actin in VSM cells cultured in serum-containing medium (Fig. 1 B), however, was not maintained when serum was replaced by PDGF (10 ng/ml; Fig. 1 C). The quantitative analysis of SM-α-actin steady-state expression applying RNase protection assays confirmed that SM-α-actin transcripts are highly abundant in VSM cells maintained in serum-containing CM compared with serum-starved (QM) controls. Re-exposure to serum increased the SM-α-actin steady-state expression within 24 h by 15-fold, whereas PDGF failed to significantly up-regulate the SM-α-actin expression within up to 3 days (Fig. 1,D and E). To define signaling pathways involved in PDGF-induced mitogenesis, we analyzed the activation of ERK1/2 and Akt, a downstream effector of the PDGF-induced PI 3-kinase signaling. Addition of PDGF (10 ng/ml) to serum-starved VSM cells led to a complete ERK1/2 phosphorylation which peaked within 5–10 min and returned to baseline levels at 30–60 min (a representative example of at least three independent experiments showing similar results is shown in Fig. 2). Equivalent results were obtained when lysates were probed for activated MEK applying phospho-specific anti-MEK antibodies (data not shown). Probing the same cell lysates with phospho-S473-Akt antibodies revealed a strong PDGF-induced Akt phosphorylation. Of note, antibodies raised against the amino acids 466–479 of Akt preferentially recognized the unphosphorylated state and consistently showed weaker signals when their epitope was phosphorylated at Ser473. The almost complete mobility shift of Akt in response to PDGF stimulation correlates with the reduction of anti-Akt signal intensities and gives a means for a semi-quantitative analysis of the phosphorylation state of Akt. In between 10 min and 1 h, the PDGF-induced Akt phosphorylation was almost complete (n = 12) and gradually declined within the following 2 h (Fig. 2). Thus, the phosphorylation of ERK1/2 appeared to decline when Akt is activated. On the other hand, thrombin stimulation resulted in similar early phosphorylation of ERK1/2, but additionally induced a delayed second-phase ERK1/2 phosphorylation. Regarding Akt, thrombin exerted only a weak and transient phosphorylation as compared with the PDGF-stimulated samples that were processed on the same blot (Fig. 2). To estimate the phosphorylated, slower migrating fraction of ERK1/2 and to ensure equal loading of the lanes, blots were reprobed with antibodies detecting total ERK1/2. A comparable extent and kinetic pattern of Akt- and ERK1/2 phosphorylation was achieved when VSM cells were challenged with IGF-I (10 ng/ml). The addition of epidermal growth factor (10 and 100 ng/ml), however, led to a more transient phosphorylation of Akt (data not shown). Because cross-regulation of the PI 3-kinase/Akt and Ras/Raf/MEK/ERK cascades has been shown to influence proliferation or differentiation of myoblasts and HEK 293 cells by an Akt-dependent association and phosphorylation of Raf (13Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar, 14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar), we analyzed whether Raf and Akt physically interact in VSM cells. Serum-starved VSM cells were stimulated with PDGF for 15 and 60 min, and Raf was immunoprecipitated from cell lysates applying anti-Raf-1 antibodies. Western blotting of the immunoprecipitates with anti-Akt antibodies revealed an increased interaction between Akt and Raf after 15 and 60 min of PDGF-BB stimulation (Fig. 3). The relative weak signals may be due to a specific interaction of the Ser473-phosphorylated form of Akt that is poorly recognized by the antibody. The anti-Raf-1 immunoprecipitates was, therefore, repeated and probed with anti-phospho-Akt antibodies. Indeed, after PDGF treatment, Ser473-phosphorylated Akt could be co-immunoprecipitated with anti-Raf-1 antibodies (Fig. 3). Thus, as a consequence of PDGF receptor signaling, the PI 3-kinase/Akt and Raf/MEK/ERK cascades interact at the level of Akt and Raf. The functional consequence of this interaction was therefore investigated. The PDGF-induced Akt phosphorylation is most likely due to PI 3-kinases that are docked and activated by the tyrosine-phosphorylated receptor. To analyze whether the interaction between phospho-Akt and Raf affects the phosphorylation-state of Raf, MEK, and ERK in living VSM cells, we studied the temporal phosphorylation pattern of these molecules in the absence and presence of the PI 3-kinase inhibitor LY294002 (20 μm). Aliquots of cell lysates were first probed with phospho-S473-Akt and Akt antibodies demonstrating a more than 80% reduction of the PDGF-induced Akt phosphorylation by LY294002 (Fig.4 A). Consistent with the findings shown in Fig. 2, thrombin induced a weak Akt phosphorylation at the 10-min time point that was sensitive to the PI 3-kinase inhibitor. At later time points, Akt phosphorylation was not detectable irrespective of the absence or presence of LY294002. A second set of aliquots from the same cell lysates were probed for phospho-S259-Raf and total Raf. After 10, 30, and, most strikingly, after 60 min of PDGF stimulation, the Ser259 phosphorylation of Raf was less intense in samples from VSM cells that were pretreated with LY294002 compared with controls without pretreatment. Since it is known that phospho-Ser259 serves a docking site for the inhibitory 14-3-3 protein, PI 3-kinase/Akt signaling may thereby reduce Raf kinase activity. By contrast, the thrombin induced moderate increase in Ser259 phosphorylation of Raf was further increased in the presence of the PI 3-kinase inhibitor (Fig. 4 B). At the level of MEK, the PDGF-induced phosphorylation was prolonged in the presence of LY294002, particularly after 60 min, suggesting an altered Raf kinase activity. The enhanced late-phase MEK phosphorylation was of importance since the resulting ERK1/2 phosphorylation was comparable to the peak signals induced by thrombin (Fig. 4, C andD). Similar results were obtained in a second experiment and in two additional experiments applying wortmannin (100 nm) instead of LY294002 (data not shown). The marked effect of PI 3-kinase inhibition on ERK1/2 and MEK phosphorylation in conjunction with an increased Ser259 phosphorylation of Raf points to a regulatory role of PI 3-kinase/Akt on the Raf kinase activity. We therefore determined the in vitro Raf kinase activity by co-incubating immunoprecipitated Raf protein and recombinant GST-MEK in the presence of [γ-32P]ATP. The formed [32P]GST-MEK was separated by SDS-PAGE, blotted, and visualized by autoradiography (Fig.5 A). The recovery of Raf was assessed by immunoblot analysis of the precipitates (Fig.5 A). In the absence of LY294002, the PDGF-induced Raf activity increased about 2-fold (n = three independent experiments) as compared with unstimulated cells at 10 min, 1.4-fold at 30 min, and returned to baseline at 60 min (Fig. 5 B). In contrast, in the presence of LY294002, Raf activity was about 1.5-fold at 3–10 min, but further increased to 1.7-fold at 30 min and 1.9-fold at 45–60 min (n = 6). Thus, the PI 3-kinase/Akt pathway attenuates the Raf kinase activity and the resulting phosphorylations of MEK and ERK in PDGF-stimulated VSM cells. The role of PI 3-kinase signaling on the activity of the PDGF and thrombin mediated activity of the Ras/Raf/MEK/ERK cascade was further analyzed by monitoring the extended time course of ERK1/2 phosphorylation in the presence of different concentrations of LY294002. The pretreatment of VSM cells with 20 or 50 μmLY294002 led to a slightly delayed but long-lasting ERK phosphorylation as compared with solvent-pretreated cells (Fig.6 A). Similar alterations in ERK1/2 kinetics were obtained when VSM cells were pretreated with 100 nm wortmannin (data not shown). Thus, by inhibiting PI 3-kinases, the PDGF-induced, short-lived ERK1/2 phosphorylation kinetic was converted into a sustained ERK1/2 activity which is almost comparable to the kinetic of ERK1/2 phosphorylation in response to thrombin (0.1 unit/ml) treatment (Fig. 6 B). At 50 μm concentrations, LY294002 diminished the thrombin-induced ERK1/2 phosphorylation, an effect that is consistent with the observed PI 3-kinase-dependent increase in Ser259 phosphorylation of Raf (Fig. 4 B). Considering that a sustained ERK1/2 phosphorylation in response to thrombin was a prerequisite for the agonist-induced de novo synthesis of SM-MHC (18Reusch H.P. Schaefer M. Plum C. Schultz G. Paul M. J. Biol. Chem. 2001; 276: 19540-19547Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), one may speculate that after disruption of the PI 3-kinase signaling, PDGF-induced phenotypic modulation of VSM cells may shift toward differentiation. This hypothesis was addressed by transient transfection of a CAT-reporter construct expressed under the control of the −1346 nucleotide promoter region of the SM-MHC gene (pCAT-1346). VSM cells maintained in QM showed an about 4-fold increased CAT activity as compared with controls transfected with a promoterless pCAT-basic vector. Under these conditions, the addition of PDGF for 36 h did not further increase the promoter activity (Fig.7 A). However, the pretreatment with LY294002 (50 μm) for 30 min prior to the addition of 10 ng/ml PDGF resulted in a more than 2-fold increase in CAT activity as compared with the absence of the PI 3-kinase inhibitor (n = 6). Similar results were obtained with wortmannin (100 nm). In the absence of PDGF, neither LY294002 nor wortmannin alone were sufficient to increase the promoter activity (data not shown). To further substantiate that the effect of PI 3-kinase inhibition on the PDGF-induced SM-MHC promoter activity is mediated via Akt, genetically encoded modulators were applied. VSM cells were co-transfected with pCAT-1346 and different amounts of expression plasmids encoding dominant negative Akt (K179A mutant). In all co-transfection experiments, the total amount of transfected plasmid DNA was kept constant (1 μg/well) by addition of promoterless pCAT-basic. The CAT activity in PDGF-stimulated VSM cells was concentration dependently increased by coexpression of dominant negative Akt (Fig. 7 B) corroborating the results with PI 3-kinase inhibitors. Thus, in PDGF-stimulated VSM cells, inhibition of PI 3-kinase/Akt extended ERK1/2 activity and up-regulated the SM-MHC promoter activity. Conversely, constitutively active Akt should disrupt the differentiating signal of thrombin stimulation. The coexpression of Akt N-terminal fused to the myristoylation/palmitylation motif from the Lck tyrosine kinase (19Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar) concentration dependently disrupted the thrombin-induced promoter CAT activation by more than 90% (Fig.7 C) similar to the action of dominant-negative Raf (18Reusch H.P. Schaefer M. Plum C. Schultz G. Paul M. J. Biol. Chem. 2001; 276: 19540-19547Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). These data demonstrate that sustained Raf/MEK/ERK signaling correlates with in vitro re-differentiation of VSM cells and is negatively controlled by the PI 3-kinase/Akt-pathway. Consistent with the hypothesis that the expression of contractile proteins may be controlled by a sustained ERK activation irrespective of the kind of the input signal, permanent activation of protein kinases C by phorbol 12-myristate 13-acetate (1–100 nm) induced a sustained ERK1/2 activation and increased the SM-MHC promoter activity in a concentration-dependent fashion up to the 2.3-fold at 10 nm (data not shown). Similarly, heterologous expression of constitutively active Raf (C-terminal fragment) increased the SM-MHC promoter activity by about 2-fold (data not shown). Hence, expression of contractile proteins in VSM cells is increased either by ligands inducing a sustained ERK activation or by suppression of PI 3-kinase/Akt that blocks sustained signaling through the Ras/Raf/MEK/ERK cascade at the level of Raf. In neonatal rat VSM cells, serum treatment induces proliferation but also increases the expression of contractile proteins. Although the serum components PDGF and thrombin activate similar signal transduction pathways, they have a divergent impact on mitogenesis and differentiation. Our results in VSM cells demonstrate that the balance of the PI 3-kinase/Akt and Ras/Raf/MEK/ERK activation corresponds to the proliferative and differentiating potential of the respective agonist. The PI 3-kinase-dependent activation of Akt results in an interaction with Raf that is accompanied by a phosphorylation at Ser259, a decrease in Raf kinase activity, and subsequent reduced MEK and ERK1/2 phosphorylation. Thrombin induced a partial and temporary phosphorylation of Akt that was not" @default.
• W2171256006 created "2016-06-24" @default.
• W2171256006 creator A5049872530 @default.
• W2171256006 creator A5056415759 @default.
• W2171256006 creator A5056434779 @default.
• W2171256006 creator A5060041272 @default.
• W2171256006 creator A5090198573 @default.
• W2171256006 date "2001-09-01" @default.
• W2171256006 modified "2023-10-16" @default.
• W2171256006 title "Regulation of Raf by Akt Controls Growth and Differentiation in Vascular Smooth Muscle Cells" @default.
• W2171256006 cites W1487207306 @default.
• W2171256006 cites W1585517331 @default.
• W2171256006 cites W1594020010 @default.
• W2171256006 cites W1646296237 @default.
• W2171256006 cites W1667204010 @default.
• W2171256006 cites W1675086682 @default.
• W2171256006 cites W1726025741 @default.
• W2171256006 cites W1971727526 @default.
• W2171256006 cites W1974379534 @default.
• W2171256006 cites W1979012323 @default.
• W2171256006 cites W1981930770 @default.
• W2171256006 cites W1982186905 @default.
• W2171256006 cites W1982479809 @default.
• W2171256006 cites W1994957003 @default.
• W2171256006 cites W1995819749 @default.
• W2171256006 cites W2001895687 @default.
• W2171256006 cites W2008940139 @default.
• W2171256006 cites W2017292900 @default.
• W2171256006 cites W2018006603 @default.
• W2171256006 cites W2024220711 @default.
• W2171256006 cites W2024306318 @default.
• W2171256006 cites W2024895303 @default.
• W2171256006 cites W2033473463 @default.
• W2171256006 cites W2040061450 @default.
• W2171256006 cites W2044766743 @default.
• W2171256006 cites W2045876058 @default.
• W2171256006 cites W2052208096 @default.
• W2171256006 cites W2055330386 @default.
• W2171256006 cites W2055387349 @default.
• W2171256006 cites W2057939121 @default.
• W2171256006 cites W2063277519 @default.
• W2171256006 cites W2063373358 @default.
• W2171256006 cites W2064242629 @default.
• W2171256006 cites W2074860444 @default.
• W2171256006 cites W2078102222 @default.
• W2171256006 cites W2087061858 @default.
• W2171256006 cites W2097767970 @default.
• W2171256006 cites W2098916975 @default.
• W2171256006 cites W2099583647 @default.
• W2171256006 cites W2103043702 @default.
• W2171256006 cites W2109283410 @default.
• W2171256006 cites W2110487056 @default.
• W2171256006 cites W2118678876 @default.
• W2171256006 cites W2123290226 @default.
• W2171256006 cites W2125966163 @default.
• W2171256006 cites W2128623551 @default.
• W2171256006 cites W2137035181 @default.
• W2171256006 cites W2150597600 @default.
• W2171256006 cites W2160761045 @default.
• W2171256006 cites W2161810908 @default.
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