title: Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.
abstract: Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.
authors.affiliation: Department of Medicine, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637, USA. psinglet@medicine.bsd.uchicago.edu
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Singleton
authors.forename: Srikanth
authors.initials: S
authors.lastname: Pendyala
authors.forename: Irina A
authors.initials: IA
authors.lastname: Gorshkova
authors.forename: Nurbek
authors.initials: N
authors.lastname: Mambetsariev
authors.forename: Jaideep
authors.initials: J
authors.lastname: Moitra
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D018496:Hyperoxia / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D016315:Proto-Oncogene Proteins c-abl; D034285:Dynamin II