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.
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.
Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.
Peter V
PV
Usatyuk
Patrick A
PA
Singleton
Srikanth
S
Pendyala
Satish K
SK
Kalari
Donghong
D
He
Irina A
IA
Gorshkova
Sara M
SM
Camp
Jaideep
J
Moitra
Steven M
SM
Dudek
Joe G N
JG
Garcia
Viswanathan
V
Natarajan
D000818:Animals; D002478:Cells, Cultured; D051356:Cortactin / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D018496:Hyperoxia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism*
D051356:Cortactin; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009219:Myosin-Light-Chain Kinase