Regulation of Raf by Akt Controls Growth and Differentiation in Vascular Smooth Muscle Cells

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 an in 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. 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 (1-3). 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 domaincontaining 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 C␥, or Src as well as adaptor molecules such as Grb2 and Shc (4). 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 (5). Other signaling cascades initiated by PDGF receptors and their potential cross-talk is currently under extensive investigation. Phosphorylated tyrosine residues (Tyr 740 and Tyr 751 ) on the PDGF ␤-receptor recruit the PI 3-kinases ␣ and ␤ to the plasma membrane via docking of the common p85 regulatory subunit (6, 7). Upon activation, the lipid kinase activity of PI 3-kinases catalyzes the formation of PI(3,4,5)-P 3 , a well defined plasma membrane anchor for the pleckstrin homology domains of 3-phosphoinositide-dependent kinase I and protein kinase B/Akt (8). The plasma membrane recruitment exposes Akt to subsequent activation by 3-phosphoinositide-dependent kinase I and related kinases that phosphorylate Akt at Thr 308 and Ser 473 (9). Akt is a major participant in growth factor-mediated transcription and promotes cell survival by inhibiting apoptosis. These processes appear to invol...

Thrombin Stimulation of Vascular Adhesion Molecule-1 in Endothelial Cells Is Mediated by Protein Kinase C (PKC)-δ-NF-κB and PKC-ζ-GATA Signaling Pathways

“…It is interesting to speculate that VSMCs express low levels of the GPCR-coupled PI3K-␥ isoform or lack the appropriate repertoire of G proteins to activate PKC-␦-NF-B and PKC--GATA-2 pathways. In support of the former hypothesis, thrombin has been shown to induce only a weak and transient phosphorylation of Akt compared with PDGF in VSMC (65).…”

Constitutive activation of the Raf–MAPK pathway causes negative feedback inhibition of Ras–PI3K–AKT and cellular arrest through the EphA2 receptor

“…This cross-talk inhibition of the Raf-MAPK pathway by PI3K-AKT was required for the differentiation of muscle cells and provides the first cellular process exhibiting cross-talk signaling between these two pathways. PI3K-AKT inhibition of Raf-MAPK signaling is also required for vascular smooth muscle and intestinal epithelial cell differentiation (Reusch et al, 2001; Laprise et al, 2004).…”

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy

“…Serine-phosphorylated Raf molecules then become inactivated by binding to the 14-3-3 adaptor protein. [113][114][115] The inhibitory effect of Akt on Raf activity appears to be dependent upon cell type and stage of differentiation 116,117 because some investigators have not observed this effect. Certain differentially expressed mediators may be essential for association of Akt and Raf.…”