MAP KINASE ACTIVATION BY FLOW IN ENDOTHELIAL-CELLS - ROLE OF BETA-1 INTEGRINS AND TYROSINE KINASES

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activ ated protein (MAP) kinase is rapidly stimulated by flow in bovine aort ic endothelial cells. Integrin receptors may act as mechanotransducers , as suggested by rapid remodeling of focal adhesion complexes in resp onse to flow. To study the role of integrins in flow-mediated MAP kina se activation, we compared the effects of beta 1 integrin activation ( with 8A2 antibody) and flow in cultured human umbilical vein endotheli al cells (HUVECs). Both 8A2 (3 mu g/mL) and flow (shear stress, 12 dyn es/cm(2)) stimulated MAP kinase, although the flow response was faster and greater. TO characterize flow-activated tyrosine kinases, tyrosin e-phosphorylated proteins were immunoprecipitated and identified by We stern blot. There was a time-dependent increase in phosphotyrosine con tent in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD prote ins. A 125-kD protein was identified as focal adhesion kinase (FAK), s uggesting that flow activates integrins. In comparison with how, 8A2 c aused less tyrosine phosphorylation of fewer proteins, although FAK wa s tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 a nd flow caused additive increases in MAP kinase. Antibody 8A2 increase d binding of the beta 1 affinity-sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a beta 1-activati ng antibody and flow stimulate tyrosine kinases, leading to activation of FAK and-MAP kinase signal-transduction pathways. However, the cell ular responses elicited by 8A2 represent only a portion of those stimu lated by flow, suggesting that ''costimulatory'' events such as calciu m mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.