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 PKCaδ isoform promotes thrombininduced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCaδ inhibitory studies (rottlerin), dominant negative PKCaδ construct and PKCaδ silencing (siRNA). In addition, we identified PKCaδ as a signaling mediator upstream of both thrombininduced 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 PKCaδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCaδ targets, were found to be activated by PKCaδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCaδ in EC cytoskeleton regulation, and highlight PKCaδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.his work was supported by National Institutes of Health/ National Heart, Lung, and Blood Institute (NHLBI) P01HL58064 and R01HL91889 (JGNG), P01HL98050 (VN), R01HL96887 (JRJ) and T32HL007249 (GTK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.>