RESUMO
OBJECTIVE: To determine whether the ß1 integrin/caveolin-1 signaling complex plays a role in shear stress regulation of RhoA activity . METHODS AND RESULTS: Hemodynamic shear stress influences the phenotype of the endothelium. Integrins and RhoA are essential components in the process that allows endothelial cells to adapt to flow. However, the signaling mechanisms that relay from integrins to RhoA are not well defined. Bovine aortic endothelial cells were subjected to laminar shear stress (10 dyne/cm(2)) for up to 6 hours. ß1 integrin blockade inhibited Src family kinases and p190RhoGAP tyrosine phosphorylation observed after the immediate onset of shear stress. Depletion of caveolin-1 blocked the decline in p190RhoGAP tyrosine phosphorylation observed at later points by sustaining Src family kinase activity. The manipulation of ß1 integrin and caveolin-1 also altered shear regulation of RhoA activity. More importantly, cells depleted of p190RhoGAP showed faulty temporal regulation of RhoA activity. Each of these treatments attenuated actin reorganization induced by flow. Similarly, stress fibers failed to form in endothelial cells exposed to enhanced blood flow in caveolin-1 knockout mice. CONCLUSIONS: Our studies demonstrate that p190RhoGAP links integrins and caveolin-1/caveolae to RhoA in a mechanotransduction cascade that participates in endothelial adaptation to flow.
Assuntos
Actinas/metabolismo , Artérias Carótidas/metabolismo , Caveolina 1/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Integrina beta1/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Fenômenos Biomecânicos , Bovinos , Caveolina 1/genética , Células Cultivadas , Endotélio Vascular/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Estresse MecânicoRESUMO
We previously reported that caveolin-1 is a key component in a beta1 integrin-dependent mechanotransduction pathway suggesting that caveolae organelles and integrins are functionally linked in their mechanotransduction properties. Here, we exposed BAEC monolayers to shear stress then isolated caveolae vesicles form the plasma membrane. While little beta1 integrin was detected in caveolae derived from cells kept in static culture, shear stress induced beta1 integrin transposition to the caveolae. To evaluate the significance of shear-induced beta1 integrin localization to caveolae, cells were pretreated with cholesterol sequestering compounds or caveolin-1 siRNA to disrupt caveolae structural domains. Cholesterol depletion attenuated integrin-dependent caveolin-1 phosphorylation, Src activation and Csk association with beta1 integrin. Reduction of both caveolin-1 protein and membrane cholesterol inhibited downstream shear-induced, integrin-dependent phosphorylation of myosin light chain. Taken together with our previous findings, the data supports the concept that beta1 integrin-mediated mechanotransduction is mediated by caveolae domains.