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J. Biol. Chem., Vol. 282, Issue 32, 23284-23295, August 10, 2007

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Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin^*

Peter V. Usatyuk, Lewis H. Romer, Donghong He, Narasimham L. Parinandi^¶, Michael E. Kleinberg^||, Steve Zhan^||, Jeffrey R. Jacobson, Steven M. Dudek, Srikanth Pendyala, Joe G. N. Garcia, and Viswanathan Natarajan¹

From the Department of Medicine, University of Chicago, Chicago, Illinois 60637, the Department of Anesthesiology and Critical Care, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the ^¶Department of Medicine, Ohio State University, Columbus, Ohio 43210, and the ^||University of Maryland School of Medicine, Baltimore, Maryland 21201

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.

Received for publication, January 19, 2007 , and in revised form, April 26, 2007.

^* This work was supported by National Institutes of Health Grants RO1 HL 69909 (to V. N.), PO1 HL 58064 (to V. N. and J. G. N. G.) and DE13079-01 and AI061042 (to L. H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Integrative Science Bldg., Rm. 408B, 929 East 57th St., Chicago, IL 60637. Tel.: 773-834-2638; Fax: 773-834-2687; E-mail: vnataraj{at}medicine.bsd.uchicago.edu.

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