Genetic Evidence for PKCδ Signaling in Thrombin‐Induced NF‐ κB Activation in Endothelial Cells

Abstract

We have shown recently that Ca2+ entry‐mediated AMPK activation promotes phosphorylation of Ca2+‐independent PKCδ at Thr505 to induce NF‐κB activation in endothelial cells. Here we utilized PKCδ knockout (PKCδ−/−) mice to show that PKCδ is indispensable for protease‐activated receptor‐1 (PAR‐1)‐induced NF‐κB and p38 MAPK activation in endothelial cells. In WT mice, administration of PAR‐1 agonist peptide (TFLLRNPNDK) through jugular vein induced NF‐κB target gene ICAM‐1 expression in lungs, whereas in PKCδ−/− mice, PAR‐1 agonist peptide failed to induce ICAM‐1 expression in lungs. Exposure of wild type (WT) mouse lung endothelial cells (MLECs) to either thrombin or TFLLRNPNDK induced Ca2+ entry, AMPK activation, IκBα phosporylation, nuclear localization of p65/RelA, p38 MAPK phosphorylation, and upregulated ICAM‐1. In PKCδ−/− MLECs, PAR‐1 activation‐induced Ca2+ entry and AMPK activation, but failed to induce IκBα phosporylation, nuclear localization of p65/RelA, p38 MAPK phosphorylation, and upregulation of ICAM‐1. Expression of WT‐PKCδ but not the PKCδ‐T505A mutant in PKCδ−/− MLECs rescued PAR‐1‐induced IκBα phosporylation, nuclear localization of p65/RelA, and p38 MAPK phosphorylation. These results provide unequivocal evidence that Ca2+ entry‐mediated PKCδ activation plays a critical role in the signaling mechanism of thrombin‐induced vascular inflammatory responses.