TY - JOUR
T1 - 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
AU - Usatyuk, Peter V.
AU - Singleton, Patrick A.
AU - Pendyala, Srikanth
AU - Kalari, Satish K.
AU - He, Donghong
AU - Gorshkova, Irina A.
AU - Camp, Sara M.
AU - Moitra, Jaideep
AU - Dudek, Steven M.
AU - Garcia, Joe G.N.
AU - Natarajan, Viswanathan
PY - 2012/3/16
Y1 - 2012/3/16
N2 - Were cently 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.
AB - Were cently 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.
UR - http://www.scopus.com/inward/record.url?scp=84863399665&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84863399665&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.294546
DO - 10.1074/jbc.M111.294546
M3 - Article
C2 - 22219181
AN - SCOPUS:84863399665
SN - 0021-9258
VL - 287
SP - 9360
EP - 9375
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -