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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL061971-05A1
Application #
6772279
Study Section
Pathology A Study Section (PTHA)
Program Officer
Wassef, Momtaz K
Project Start
1999-09-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
5
Fiscal Year
2004
Total Cost
$344,250
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Thiagarajan, Praveena S; Akbasli, Ayse C; Kinter, Michael T et al. (2013) Vimentin is a target of PKC* phosphorylation in MCP-1-activated primary human monocytes. Inflamm Res 62:991-1001
Elsori, Deena H; Yakubenko, Valentin P; Roome, Talat et al. (2011) Protein kinase C? is a critical component of Dectin-1 signaling in primary human monocytes. J Leukoc Biol 90:599-611
Cathcart, Martha K (2009) Signal-activated phospholipase regulation of leukocyte chemotaxis. J Lipid Res 50 Suppl:S231-6
Mishra, Ravi S; Carnevale, Kevin A; Cathcart, Martha K (2008) iPLA2beta: front and center in human monocyte chemotaxis to MCP-1. J Exp Med 205:347-59
Bhattacharjee, Ashish; Mishra, Ravi S; Feldman, Gerald M et al. (2008) In vivo validation of signaling pathways regulating human monocyte chemotaxis. J Immunol Methods 330:86-95
Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M et al. (2007) PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes. Cell Signal 19:359-66
Zhao, Xiaoxian; Xu, Bo; Bhattacharjee, Ashish et al. (2005) Protein kinase Cdelta regulates p67phox phosphorylation in human monocytes. J Leukoc Biol 77:414-20
Bey, Erik A; Xu, Bo; Bhattacharjee, Ashish et al. (2004) Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes. J Immunol 173:5730-8
Carnevale, Kevin A; Cathcart, Martha K (2003) Protein kinase C beta is required for human monocyte chemotaxis to MCP-1. J Biol Chem 278:25317-22
Zhao, Xiaoxian; Carnevale, Kevin A; Cathcart, Martha K (2003) Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex. J Biol Chem 278:40788-92

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