Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium

Peter V Usatyuk,Patrick A Singleton,Srikanth Pendyala,Satish K Kalari,Donghong He,Irina A Gorshkova,Sara M Camp,Jaideep Moitra,Steven M Dudek,Joe G.N Garcia,Viswanathan Natarajan

doi:10.1074/jbc.m111.294546

Abstract

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

Highlights

Hyperoxia activates lung endothelial cell NADPH oxidase and generates oxidants
Our data indicate that the association between nmMLCK and cortactin and between nmMLCK and p47phox at the cell periphery was enhanced in human lung endothelial cells
Our results on regulation of hyperoxia-mediated reactive oxygen species (ROS) production by nmMLCK in human pulmonary artery endothelial cells (HPAECs) are consistent with the adult in vivo model of hyperoxia-induced lung injury wherein nmMLCKϪ/Ϫ mice exhibited less inflammation, ROS production, and pulmonary leak compared with nmMLCK wild type mice

Summary

Background

Hyperoxia activates lung endothelial cell NADPH oxidase and generates oxidants. Results: nmMLCK modulates hyperoxia-induced interaction between cortactin and p47phox, oxidant production, and vascular leak. In vascular smooth muscle cells the angiotensin II-mediated activation of NADPH oxidase and ROS generation is regulated in part by p47phox/actin interaction through cortactin [5] It seems that agonist-mediated cortactin and p47phox phosphorylation, translocation to cell periphery, and cortactin/p47phox co-localization to the plasma membrane [5, 9, 13,14,15] are all critically important events in human lung EC NADPH oxidase activation and ROS production. Production is attenuated by downregulation or inhibition of nmMLCK in lung ECs, (ii) nmMLCK is essential for the translocation and association of cortactin and p47phox, (iii) down-regulation of nmMLCK expression with siRNA blocks recruitment of Src, cortactin, and p47phox and phosphorylated Src, cortactin, and MLC to CEMs, and (iv) nmMLCK knock-out mice show decreased ROS production, phosphorylation of Src, cortactin, and MLC and pulmonary leak These results provide strong evidence for the involvement of ϳ214-kDa non-muscle MLCK in the assembly and activation of non-phagocytic NADPH oxidase and ROS/(O2. These results provide strong evidence for the involvement of ϳ214-kDa non-muscle MLCK in the assembly and activation of non-phagocytic NADPH oxidase and ROS/(O2. ) production in response to hyperoxia in lung endothelium

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION