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

Lishi Xie,Tingting Zhou,Patrick A Singleton,Viswanathan Natarajan,Ting Wang,Xiaomin Wu,Jeffrey R Jacobson,Joe G N Garcia,Prasad Kanteti,Gabriel T Kelly,Steven M Dudek,Eddie T Chiang,Steven M Black,Sara M Camp,Anna A Birukova

doi:10.1371/journal.pone.0158865

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 PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ 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 PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

Highlights

The lung endothelium serves as a dynamic, semi-permeable barrier between the interstitial alveolar spaces and the circulating blood, with the integrity of this cellular barrier vital to pulmonary homeostasis
The Role of protein kinase C (PKC) delta in Endothelial cell (EC) Barrier Function studies have shown that activators of PKC such as phorbol esters and diacylglycerol increase the permeability of the endothelial monolayer, whereas PKC inhibitors reduce the cell barrier disruption caused by thrombin, bradykinin, platelet derived growth factor (PDGF), hydrogen peroxide and vascular endothelial growth factor (VEGF) on neutrophils [4]
To analyze the participation of PKC isoforms in EC barrier regulation, human pulmonary artery EC were treated with thrombin, an edemagenic agent, and the activation status of 10 PKC isoforms were determined based on their translocation from the cytosol to the membrane

Summary

Introduction

The lung endothelium serves as a dynamic, semi-permeable barrier between the interstitial alveolar spaces and the circulating blood, with the integrity of this cellular barrier vital to pulmonary homeostasis. Endothelial cell (EC) barrier function is highly regulated by incompletely defined stimulus–coupling pathways. We have previously shown that protein kinase C (PKC), a Ser/Thr kinase plays an important role in the regulation of EC barrier function [1,2,3]. The Role of PKC delta in EC Barrier Function studies have shown that activators of PKC such as phorbol esters and diacylglycerol increase the permeability of the endothelial monolayer, whereas PKC inhibitors reduce the cell barrier disruption caused by thrombin, bradykinin, platelet derived growth factor (PDGF), hydrogen peroxide and vascular endothelial growth factor (VEGF) on neutrophils [4]. A full understanding of the contribution of PKC to EC barrier function is limited as PKC is a superfamily that comprises at least 10 members whose expression and functions are differentially regulated [5]. Each cell type expresses a particular combination of PKC isozymes that elicit diverse and sometimes opposing responses [6], thereby making it difficult to infer the exact role of PKC in physiological and pathological conditions

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Conclusion