Bogdan Ceacareanu

Gab1, SHP2, and Protein Kinase A Are Crucial for the Activation of the Endothelial NO Synthase by Fluid Shear Stress

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress–induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress–induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress–induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

Nitric Oxide–Induced Motility in Aortic Smooth Muscle Cells: Role of Protein Tyrosine Phosphatase SHP-2 and GTP-Binding Protein Rho

Abstract— We have previously reported that SHP-2 upregulation is necessary for NO-stimulated motility in differentiated rat aortic smooth muscle cells. We now test the hypothesis that upregulation of SHP-2 is necessary and sufficient to stimulate cell motility. Overexpression of SHP-2 via recombinant adenoviral vector stimulated motility to the same extent as NO, whereas the expression of C463S-SHP-2, the dominant-negative SHP-2 allele, blocked the motogenic effect of NO. On the basis of previous studies, we next tested the hypothesis that NO decreases RhoA activity and that this event is necessary and sufficient to explain NO-induced motogenesis. We found that NO decreased RhoA activity in a concentration-dependent manner. Moreover, a dominant-negative SHP-2 allele, DSH2, blocked the NO-induced inhibition of RhoA activity, indicating that upregulation of SHP-2 is necessary for this event. Expression of G14V-RhoA, the constitutively active RhoA allele, decreased cell motility and blocked the motogenic effect of NO, whereas the expression of T19N-RhoA, the dominant-negative RhoA allele, increased cell motility to an extent similar to that induced by NO. Dominant-negative RhoA reversed the effect of dominant-negative SHP-2, indicating that RhoA functions downstream from SHP-2. To investigate events downstream from RhoA, we treated cells with fasudil, a selective Rho kinase inhibitor, and found that it increased cell motility. These results indicate that upregulation of SHP-2, leading to downregulation of RhoA, which is followed by decreased Rho kinase activity, is a sequence of events necessary and sufficient to explain NO-induced cell motility in differentiated aortic smooth muscle cells. The results may be of relevance to in vivo events such as neointimal formation, angiogenesis, and vasculogenesis.

Counter-regulatory function of protein tyrosine phosphatase 1B in platelet-derived growth factor- or fibroblast growth factor-induced motility and proliferation of cultured smooth muscle cells and in neointima formation.

Objectives—We have previously reported that vascular injury or treatment of cultured vascular smooth muscle cells with platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF2) increases the levels of protein tyrosine phosphatase (PTP)1B. The current study was designed to test the hypothesis that PTP1B attenuates PDGF- or FGF-induced motility and proliferation of cultured cells, as well as neointima formation in injured rat carotid arteries. Methods and Results—Treatment of cultured cells with adenovirus expressing PTP1B decreased PDGF-BB– or FGF2-induced cell motility and blocked PDGF-BB– or FGF2-induced proliferation, whereas expression of dominant negative PTP1B (C215S-PTP1B) uncovered the motogenic effect of subthreshold levels of PDGF-BB or FGF2, increased neointimal and medial cell proliferation, and induced neointimal enlargement after balloon injury. The inhibitory effect of PTP1B directed against PDGF in cultured cells was associated with dephosphorylation of the PDGF&bgr; receptor. Conclusions—PTP1B suppresses cell proliferation and motility in cultured smooth muscle cells treated with PDGF-BB or FGF2, and the phosphatase plays a counter-regulatory role in vascular injury-induced cell proliferation and neointima formation. Taken together with previous studies indicating increased PTP1B levels in cells treated with growth factors, the current findings are the first to report the existence of an inhibitory feedback loop involving PDGF or FGF, and PTP1B in blood vessels.

Chronic insulin treatment amplifies PDGF-induced motility in differentiated aortic smooth muscle cells by suppressing the expression and function of PTP1B.

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-beta, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)-delta and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.

Treatment With Insulin Uncovers the Motogenic Capacity of Nitric Oxide in Aortic Smooth Muscle Cells Dependence on Gab1 and Gab1-SHP2 Association

Abstract— Contrary to the antimotogenic effect of NO in dedifferentiated vascular smooth muscle cells (VSMCs), we have reported that NO stimulates the motility of differentiated cultured VSMC isolated from adult rats. This process involves upregulation of protein tyrosine phosphatase SHP2, followed by downregulation of RhoA activity. In the present study, we tested the hypothesis that insulin alters the motogenic phenotype of cultured rat aortic smooth muscle cells exposed to NO from inhibition to stimulation of cell motility. We demonstrate for the first time that NO stimulates the motility of VSMCs cultured for several days in the presence but not the absence of insulin. Moreover, we show that NO blocks PDGF-induced cell motility in insulin-naive but not in insulin-treated cells. We also demonstrate that the scaffold adapter protein Gab1, considered a physiological activator of protein tyrosine phosphatase SHP2, increases cell motility in the presence but not the absence of insulin. In cells cultured in the presence of insulin, overexpression of Gab1 mimics, whereas a dominant-negative allele of Gab1 (Gab1YF) blocks, the motility-stimulatory effect of NO. Cotransfection experiments with dominant-negative Gab1 and wild-type SHP2 or wild-type Gab1 and dominant-negative SHP2 indicate that the two proteins work together as a functional unit to induce motility. Because chronic insulin can increase the levels of phosphatidylinositol 3 (PI3) kinase in several models of hyperinsulinemia, we also tested the potential involvement of this enzyme in mechanisms leading to increased cell motility. We found that the motogenic effect of NO, Gab1, and SHP2 was blocked by the selective PI3 kinase inhibitor LY294002, suggesting a requirement of PI3 kinase in mediating motogenesis. These observations may be relevant to molecular mechanisms related to the pathogenesis of vascular disease in hyperinsulinemic diabetes. The full text of this article is available online at http://www.circresaha.org.