Abstract 539: Atypical PKc Zeta Mediates TNF Induced Endothelial Dysfunction
TNF-a stimulates pro-atherogenic signaling events including activation of JNK, expression of ICAM-1, VCAM-1 and apoptosis. Recently, atypical PKCz, an abundant PKC isoform in endothelial cells (EC), was shown to mediate NFkB activation and ICAM-1 expression. Because, PKCz contains a protein-protein interaction domain termed “PB1”, that is common to several critical EC signaling molecules downstream of the TNF-a receptor, we assessed the role of the PKCz PB1 domain in TNF-a signaling. Inhibiting PKCz function with PKCz pseudosubstrate (20uM), siRNA (50nM) or dominant-negative (DN) PKCz significantly decreased TNF-a (10ng/ml, 30 min) dependent JNK activation in EC. In contrast, overexpressing wild-type PKCz enhanced JNK activity showing that PKCz is a positive regulator of JNK. Since both MEKK3, an upstream regulator of JNK, and PKCz are PB1 domain containing proteins, we hypothesized they interact with each other. Immunoprecipitated MEKK3 pulled down wild type PKCz but not CAT-z, a form of PKCz lacking the PB1 domain. Both PKCz siRNA and the PB1 domain of PKCz blocked the stimulatory effects of constitutively active MEKK3 on JNK activity in EC. These observations establish that MEKK3 and PKCz , via their PB1 domains, cooperate to regulate JNK. The positive effect of PKCz on JNK is functionally important since DN-PKCz or PKCz siRNA significantly decreased EC apoptosis (measured by caspase-3 activity) and VCAM-1 expression by 50%. These data show that PKCz is a novel mediator of TNF-a signaling in EC that may contribute to endothelial dysfunction associated with inflammation.