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*

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. Conclusion: nmMLCK plays an important role in hyperoxia-induced NADPH oxidase activation and lung injury. Significance: Targeting nmMLCK may provide a novel therapeutic intervention to manage bronchopulmonary dysplasia. 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 p47phox and cortactin. Here, we demonstrate that the non-muscle ∼214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47phox 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 p47phox 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 p47phox 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.

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