Regulation of Raf by Akt Controls Growth and Differentiation in 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. 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 that PDGF and thrombin pathways but exerted in signal and their analysis revealed interaction between Akt and Raf in VSM cells that the ERK1/2 phosphorylation. of the PI 3-kinase/Akt signaling the PDGF-induced proliferative response in VSM cells toward expression of contractile proteins. and from and from from The was from from growth factors and epidermal growth factor from and from from of VSM cells from as G.S. J. Exp. PubMed Scopus Google Scholar). in medium with and cells from was induced in a quiescent medium and of serum. to cells in for The of was with a chloramphenicol acetyltransferase under the of the myosin heavy chain promoter to as J.C. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cells at a of and growth in for to in with of and 10 for cell the to the protein of as by the of a as a promoter to relative to activity. VSM cells to on in in smooth muscle α-actin was by a and a in a VSM cells in 10 on polyacrylamide and to Akt, or Raf on and with and or with MEK and to of the with a a of and H. R. E. Res. 1996; PubMed Scopus Google Scholar). The and from for 10 of was with at with The by polyacrylamide gel electrophoresis and to at for and in a was by with a VSM cells for in cells in (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar), and Raf protein was with as (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar). vitro kinase by the in of kinase of and 10 of in kinase for at by and their phosphorylation was and with a VSM cells in a as (14Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar). of protein in at with 2 of to The in of in and to analysis with and have demonstrated that in to its mitogenic the expression of contractile proteins in vascular smooth muscle (VSM) cells M. C. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). To evaluate the proliferative effects of PDGF, and VSM cells in with the The cell at the of the and following To for potential of the Whereas cell almost in in the of thrombin was a mitogen that reconstituted of the cell proliferation The expression of the contractile protein in VSM cells in medium was not was by PDGF The analysis of expression that in VSM cells in with to increased the expression by whereas PDGF to the expression to and To signaling pathways in PDGF-induced we the activation of ERK1/2 and Akt, a effector of the PDGF-induced PI 3-kinase of PDGF to VSM cells to a complete ERK1/2 phosphorylation which and to levels at of at is shown in for activated MEK not the same cell with revealed a strong PDGF-induced Akt phosphorylation. the of Akt the and their was phosphorylated at The almost complete of Akt in response to PDGF stimulation with the of signal intensities and a for a analysis of the phosphorylation of Akt. between 10 and the PDGF-induced Akt phosphorylation was almost complete and the following 2 Thus, the phosphorylation of ERK1/2 to Akt is the thrombin stimulation in phosphorylation of but induced a ERK1/2 phosphorylation. Akt, thrombin exerted only a and phosphorylation as with the that on the same To the of ERK1/2 and to of the with A and pattern of and ERK1/2 phosphorylation was VSM cells with The of epidermal growth factor and to a more phosphorylation of Akt not cross-regulation of the PI 3-kinase/Akt and Ras/Raf/MEK/ERK cascades has been shown to proliferation or differentiation of and cells by 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, S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (905) Google Scholar), we Raf and Akt physically in VSM cells. VSM cells with PDGF for and and Raf was from cell of the with revealed increased interaction between Akt and Raf and of stimulation The relative may to a specific interaction of the form of Akt that is by the The and with PDGF Akt with Thus, as a of PDGF receptor the PI 3-kinase/Akt and Raf/MEK/ERK cascades at the of Akt and Raf. The of interaction was The PDGF-induced Akt phosphorylation is to PI 3-kinases that and activated by the To the interaction between and Raf the of and ERK in VSM cells, we studied the temporal phosphorylation pattern of these molecules in the and of the PI 3-kinase of cell with and Akt a more than of the PDGF-induced Akt phosphorylation by with the shown in thrombin induced a Akt phosphorylation at the that was to the PI 3-kinase Akt phosphorylation was not of the or of A of from the same cell for and Raf. of PDGF the phosphorylation of Raf was in from VSM cells that with with without is that a docking for the PI 3-kinase/Akt signaling may thereby Raf kinase activity. By contrast, the thrombin induced increase in phosphorylation of Raf was increased in the of the PI 3-kinase the of the PDGF-induced phosphorylation was in the of Raf kinase activity. The MEK phosphorylation was of the resulting ERK1/2 phosphorylation was to the induced by thrombin C in a and in of not The marked effect of PI 3-kinase on ERK1/2 and MEK phosphorylation in with increased phosphorylation of Raf to a regulatory of PI 3-kinase/Akt on the Raf kinase activity. the in vitro Raf kinase activity by Raf protein and in the of The was by and by The of Raf was by analysis of the the of the PDGF-induced Raf activity increased as with cells at 10 at and to at contrast, in the of Raf activity was at but increased to at and at Thus, the PI 3-kinase/Akt pathway the Raf kinase activity and the resulting of MEK and ERK in VSM cells. The of PI 3-kinase signaling on the activity of the PDGF and thrombin activity of the Ras/Raf/MEK/ERK was by the of ERK1/2 phosphorylation in the of different of The of VSM cells with or to a but ERK phosphorylation as with cells in ERK1/2 VSM cells with not Thus, by inhibiting PI the short-lived ERK1/2 phosphorylation was a sustained ERK1/2 activity which is almost to the of ERK1/2 phosphorylation in response to thrombin treatment the ERK1/2 effect that is with the PI increase in phosphorylation of Raf that a sustained ERK1/2 phosphorylation in response to thrombin was a for the of SM-MHC M. C. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar), may that of the PI 3-kinase PDGF-induced phenotypic modulation of VSM cells may toward This was by of a under the of the promoter of the SM-MHC VSM cells in increased activity as with with a Under these conditions, the of PDGF for not increase the promoter activity the with for to the of 10 PDGF in a more than increase in activity as with the of the PI 3-kinase with the of PDGF, to increase the promoter activity not To that the effect of PI 3-kinase on the PDGF-induced SM-MHC promoter activity is via Akt, VSM cells with and different of expression dominant negative Akt the of was by of The activity in VSM cells was increased by of dominant negative Akt the with PI 3-kinase Thus, in VSM cells, of PI 3-kinase/Akt ERK1/2 activity and the SM-MHC promoter activity. constitutively active Akt the differentiating signal of thrombin The of Akt to the from the tyrosine kinase M. D.R. R. A. M. Cron P. Cohen P. Hemmings B.A. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) the promoter activation by more than to the of Raf M. C. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). These that sustained Raf/MEK/ERK signaling with in vitro re-differentiation of VSM cells and is negatively by the PI with the that the expression of contractile proteins may by a sustained ERK activation of the of the activation of protein kinases C by induced a sustained ERK1/2 activation and increased the SM-MHC promoter activity in a to the at 10 not expression of constitutively active Raf increased the SM-MHC promoter activity by not expression of contractile proteins in VSM cells is increased either by a sustained ERK activation or by of PI 3-kinase/Akt that sustained signaling the Ras/Raf/MEK/ERK at the of Raf. VSM cells, treatment proliferation but the expression of contractile proteins. the PDGF and thrombin signal transduction have a on and Our in VSM cells that the balance of the PI 3-kinase/Akt and Ras/Raf/MEK/ERK activation to the proliferative and differentiating potential of the The PI activation of Akt in interaction with Raf that is by a phosphorylation at Ser259, a in Raf kinase and subsequent reduced MEK and ERK1/2 phosphorylation. Thrombin induced a and phosphorylation of Akt that was not to the ERK By contrast, the PDGF-induced strong and phosphorylation of Akt Raf which the coupling to MEK and Disruption of the PI 3-kinase/Akt signaling the PDGF-induced Raf kinase activity resulting in a sustained ERK1/2 phosphorylation and subsequent activation of the SM-MHC receptor tyrosine kinases and receptors at common signaling that increase the and protein kinases C, the ERK of MAP and the PI 3-kinase/Akt The balance of signal and potential between these pathways may the of phenotypic modulation in VSM cells. PDGF and thrombin induced a and duration of M. C. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). receptors initiated a and almost complete phosphorylation of ERK1/2 as by shifts of protein cell with a ERK1/2 phosphorylation was only cells with More Akt was only and transiently phosphorylated by thrombin whereas PDGF induced a and Akt phosphorylation. 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