Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway invascular endothelial cells involves tyrosine phosphorylation andactivation of mitogen-activated protein (MAP) kinase, which may beresponsible for the sustained release of nitric oxide. MAP kinase isknown to be activated by reactive oxygen species (ROS), such asH₂O₂,in several cell types. ROS production in ligand-stimulatednonphagocytic cells appears to require the participation of aRas-related small GTP-binding protein, Rac1. We hypothesized that Rac1might serve as a mediator for the effect of shear stress on MAP kinaseactivation. Exposure of bovine aortic endothelial cells to laminarshear stress of 20 dyn/cm² for5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidantsN-acetylcysteine and pyrrolidinedithiocarbamate inhibited in a dose-dependent manner theshear-stimulated increase in total cytosolic and, specifically, MAPkinase tyrosine phosphorylation. Hence, the onset of shear stresscaused an enhanced generation of intracellular ROS, as evidenced by anoxidized protein detection kit, which were required for theshear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing adominant negative Rac1 gene product (N17rac1). We concluded that theGTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAPkinase via regulation of the flow-dependent redox changes inendothelial cells in physiological and pathological circumstances.