Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 μM H2O2. This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Cα (PKCα) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm2 laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCα or the PKCα inhibitors, Rö-32-0432 or Gö-6976. Phosphorylation of PKCαThr638, 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 μM H2O2. The anti-VCAM-1-stimulated phosphorylation of PKCαThr38 was blocked by scavenging of H2O2 and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCα. Oxidized PKCα is a transiently active form of PKCα 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 PKCα activation that is necessary for VCAM-1-dependent transendothelial cell migration.
ASJC Scopus subject areas
- Immunology and Allergy