title: Regulation of hyperoxia-induced NADPH oxidase activation in human lung endothelial cells by the actin cytoskeleton and cortactin.
abstract: Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.
authors.affiliation: Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Usatyuk
authors.forename: Lewis H
authors.initials: LH
authors.lastname: Romer
authors.forename: Donghong
authors.initials: D
authors.lastname: He
authors.forename: Narasimham L
authors.initials: NL
authors.lastname: Parinandi
authors.forename: Michael E
authors.initials: ME
authors.lastname: Kleinberg
authors.forename: Steve
authors.initials: S
authors.lastname: Zhan
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Jacobson
authors.forename: Steven M
authors.initials: SM
authors.lastname: Dudek
authors.forename: Srikanth
authors.initials: S
authors.lastname: Pendyala
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D000199:Actins / Q000378:metabolism*; D002478:Cells, Cultured; D051356:Cortactin / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D018496:Hyperoxia; D008168:Lung / Q000378:metabolism; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000199:Actins; D051356:Cortactin; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014443:Tyrosine; D019255:NADPH Oxidases