Abstract
Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.
Highlights
Vascular endothelial cells, which form the inner lining of the blood vessel wall, are exposed to fluid shear stress, the dragging force generated by blood flow
We have found that flow rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligandindependent but Src-kinase-dependent manner, which leads to activation of Akt and endothelial nitric-oxide synthase (eNOS) in endothelial cells [18]
The major findings of the present study are that flow stimulates tyrosine phosphorylation of Gab1 in a Src kinase-dependent and VEGFR2-dependent manner, and that tyrosinephosphorylated Gab1 is required for flow-induced activation of Akt and eNOS in endothelial cells
Summary
Vascular endothelial cells, which form the inner lining of the blood vessel wall, are exposed to fluid shear stress, the dragging force generated by blood flow. Steady laminar flow has beneficial effects on endothelial cell function, including inhibition of platelet aggregation, low density lipoprotein uptake, adhesion molecule expression, and vascular smooth muscle cell proliferation, as well as enhancing endothelial cell survival [3] Many of these effects are related to increased nitric oxide (NO) production by endothelial cells exposed to flow [4]. Tyrosine phosphorylation of Gab is functionally important, because flow stimulates association of Gab with p85 and inhibiting Gab function and expression decreases flow-induced Akt and eNOS activation. These data demonstrate a critical role of Gab in flow-mediated PI3K/Akt/ eNOS signaling in endothelial cells
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