UNIVERSITY OF ILLINOIS
Chemoattractant-induced phagocyte activation is an important mechanism of host defense. In phagocytes, induced activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to robust production of reactive oxygen species (ROS), which is essential for the elimination of ingested bacteria and fungi. However, extracellular ROS production by phagocyte NADPH oxidase (also termed Nox2) can be harmful to the tissue and is a major cause of vascular injury in acute inflammation. This revised competing renewal application aims to understand how chemoattractant-induced neutrophil superoxide generation is regulated at the molecular and cellular levels. Building on the systems we developed and preliminary results obtained in the previous grant cycle, the application will focus on the critical roles for p47phox in the assembly of an NADPH oxidase. Experiments are proposed in 3 specific aims to challenge existing concept and identify novel regulatory mechanisms for NADPH oxidase activation. Aim 1 will characterize Akt isoforms in chemoattractant-induced ROS production. Our preliminary study has led to an unexpected finding that the two Akt isoforms in neutrophils play different roles in NADPH oxidase activation. Experiments are proposed to determine whether p47phox phosphorylation is catalyzed differently by these Akt isoforms, and to test the hypothesis that membrane translocation of an Akt isoform is required for p47phox phosphorylation and the resulting conformational changes that lead to NADPH oxidase activation. Aim 2 is based on our recent characterization of a p47phox mutant that mediates spontaneous and potent superoxide production in the absence of physical interaction with p67phox. Experiments are designed to characterize a potentially novel mechanism for 47phox-mediated conformational change in cytochrome b558 that facilitates assembly of the NADPH oxidase complex. Aim 3 will investigate an important regulatory mechanism for p47phoxh-dependent oxidant signaling, which involves protein phosphatase in limiting neutrophil ROS production. We will examine the negative regulation of NADPH oxidase by MAP kinase phosphatase 5 (MKP5). Using in vivo and ex vivo approaches, we will investigate how MKP5 protects against LPS-induced vascular injury through suppression of neutrophil ROS production in a mouse model of vascular inflammation. Collectively, these studies are expected to provide novel insights into the activation and inactivation mechanisms of phagocyte NADPH oxidase, thereby facilitating therapeutic intervention of ROS-mediated tissue injury.