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Role of Phospholipase D in Hyperoxia‐Mediated Myosin Light Chain Kinase Activation, Reactive Oxygen Species Formation and Cytoskeleton Organization
Author(s) -
Usatyuk Peter V,
Gorshkova Irina A,
Pendyala Srikanth,
Kalari Satish K,
Kamp Sara M,
Dudek Steven M,
Garcia Joe G.N.,
Natarajan Viswanathan
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a818-d
Subject(s) - myosin light chain kinase , microbiology and biotechnology , cortactin , nadph oxidase , chemistry , paxillin , phosphorylation , reactive oxygen species , tyrosine phosphorylation , cytoskeleton , biochemistry , biology , focal adhesion , cell
Exposure of lung to supra‐physiological concentrations of oxygen causes extensive damage to the alveolar‐capillary barrier resulting in increased permeability and decreased pulmonary function. Recently we demonstrated that exposure of human pulmonary artery endothelial cells (HPAECs) to hyperoxia (HO) resulted in enhanced phosphorylation of myosin light chain (MLC) and increased phospholipase D (PLD)‐dependent generation of reactive oxygen species (ROS) via NADPH oxidase activation. The mechanism(s) by which PLD regulates endothelial NADPH oxidase is not well defined. Here we examined the role of PLD in HO‐mediated myosin light chain kinase (MLCK) activation and ROS production. Transfection of HPAECs with siRNA MLCK (50 nM/ml) attenuated HO‐induced ROS and superoxide production. Additionally, MLCK siRNA, but not scrambled siRNA, attenuated HO‐mediated MLC phosphorylation and actin and cortactin rearrangement to cell periphery. Transient expression HPAECs with catalytically inactive mutants hPLD1‐K898R and mPLD2‐K758R blocked HO‐induced ROS production, phosphorylation of MLC, cytoskeletal (actin, cortactin) and focal adhesion and adherent junction proteins (VE‐cadherin, b‐catenin, paxillin) redistribution. These results suggest a role for PLD in HO‐mediated MLCK activation, cytoskeletal reorganization and ROS/superoxide production in endothelium. This work was supported by NIH grants RO1 HL085553 and PO1 HL058064 (V.N. and J.G.N.G).