Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells

Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H₂O₂) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H₂O₂ (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H₂O₂-induced tyrosine phosphorylation of FAK, and the micro filament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM attenuated H₂O₂-induced increases in intracellular Ca²⁺ but did not show any consistent effect on H₂O₂-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H₂O₂-induction of mitogen-activated protein kinase activity. We conclude that H₂O₂-induced increases in FAK tyrosine phosphorylation may be important in H₂O₂-mediated endothelial cell activation.