Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS.
The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2 for 5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-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 a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.
Yeh
L H
LH
Vascular Bioengineering Laboratory, Department of Biomedical Engineering, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Park
Y J
YJ
Hansalia
R J
RJ
Ahmed
I S
IS
Deshpande
S S
SS
Goldschmidt-Clermont
P J
PJ
Irani
K
K
Alevriadou
B R
BR
D000818:Animals; D001011:Aorta; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000502:physiology*; D015398:Signal Transduction; D013314:Stress, Mechanical; D020744:rac GTP-Binding Proteins
D017382:Reactive Oxygen Species; D019000:Phosphotyrosine; D006861:Hydrogen Peroxide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins