Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

Abstract

Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKC delta isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKC delta inhibitory studies (rottlerin), dominant negative PKC delta construct and PKC delta silencing (siRNA). In addition, we identified PKC delta as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKC delta activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKC mu) and CPI-17, two known PKC delta targets, were found to be activated by PKC delta in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKC delta in EC cytoskeleton regulation, and highlight PKC delta as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.