VCAM-1 signals activate endothelial cell protein kinase Cα via oxidation

Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 mu M H2O2. This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase C alpha (PKC alpha) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm(2) laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKC alpha or the PKC alpha inhibitors, Ro-32-0432 or Go-6976. Phosphorylation of PKC alpha(Thr638), an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 mu M H2O2. The anti-VCAM-1-stimulated phosphorylation of PKCThr638 was blocked by scavenging of H2O2 and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKC alpha. Oxidized PKC alpha is a transiently active form of PKC alpha that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKC alpha activation that is necessary for VCAM-1-dependent transendothelial cell migration.