Monocyte Pathogenesis: Regulation of NADPH Oxidase

Cleveland Clinic Lerner Com-Cwru, Cleveland OH

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): The production of superoxide anion (02-) by activated monocytes and neutrophils is an essential element of host defense and inflammation. 02- is generated via the enzyme complex, NADPH oxidase. In addition to its role in killing microorganisms, 02- production contributes to the oxidation of lipids. Lipid oxidation, mediated by monocyte NADPH oxidase, is believed to significantly contribute to atherogenesis. Understanding of the regulation and function of the monocyte NADPH oxidase and 02- production is limited. We are finding that the monocyte NADPH oxidase is regulated very differently than the neutrophil enzyme complex. Since neutrophils are not present in the artery wall, either in early or late lesion development, understanding the regulation of the activity of this enzyme complex in monocytes is paramount. Particular importance of NADPH oxidase in atherogenesis is derived from recent studies showing that the development of atherosclerosis is less in animals deficient in this enzyme. NADPH oxidase is comprised of several components. In resting monocytes some components are membrane-bound while others are cytosolic. The latter must form a membrane-associated complex with other oxidase components to allow enzyme activity. Three critical cytosolic components of NADPH oxidase that translocate to form the active enzyme complex are the focus of this application. They are p47phox, p67phox and Racl. Prior studies from our laboratory have delineated an activation-induced pathway involving increased intracellular calcium, PKCalpha translocation, PKCalpha-dependent phosphorylation and activation of cPLA2, generation of arachidonic acid (AA) and activation of NADPH oxidase. In Aim 1 we propose to identify the critical, PKCalpha-dependent phosphorylation sites on cPLA2 and examine their effects on cPLA2 activity. Comparisons will be made between in vitro generated and monocyte-mediated phosphorylation sites. Studies in Aim 2 explore regulation of NADPH oxidase activity by cPLA2-derived AA and how AA can regulate translocation of NADPH oxidase components. We have new evidence that PKCdelta is involved in the phosphorylation of p47phox. Therefore in the third aim we propose studies to examine the PKCdelta-dependent phosphorylation sites on p47phox and determine their effects on p47phox interaction with other NADPH oxidase components. We will also initiate studies to identify the kinase responsible for phosphorylating p67phox. Studies in Aim 4 will test the hypothesis that Racl is critical for mediating p47phox and p67phox interaction and formation of the active NADPH oxidase complex. Due to the tools that we have developed to probe signal transduction pathways in monocytes, we are in a unique position to specifically identify these pathways and discover novel approaches for influencing inflammatory responses that have developed into pathogenic processes.