Reactive oxygen species (ROS) initiate activation of the transcription factor NF-KB in a variety of cell systems. Perhaps the most potent biological source of ROS is the NADPH respiratory burst oxidase of phagocytic cells -neutrophils, eosinophils, monocytes, and macrophages. This multi- component enzyme catalyzes the formation of superoxide anion, which undergoes further reactions to generate a spectrum of ROS. Although phagocytes use this oxidase to kill ingested microorganisms, the products also mediate a broad range of biological oxidation reactions and some evidence exists for activation of NF-KB through this mechanism. It appears that the components of the phagocyte NADPH oxidase are also expressed in certain non-phagocytic cells. Moreover, we have recently discovered and cloned a widely expressed homologue (NOH-1) of the major catalytic component of the system. The basis of this proposal is a hypothesis that the products of NADPH oxidases of both the phagocyte and homologous types cause the activation of NF-KB, which in turn contributes to the changes in gene expression associated with aging.
Our specific aims are: 1) To explore using model systems and stimulated phagocytes, the ability of ROS generated by NADPH oxidase to activate NF-KB in various target cells, as assessed by gel mobility shift, nuclear translocation of GFP-tagged NF-KB, changes in transcription of specific genes, and expression of a heterologous reporter construct. The mechanisms responsible for major differences in susceptibility to ROS-dependent NF-KB activation among cell types will be explored. 2) To characterize the NAD(P)H oxidase activity of the recently- discovered homologue of the phagocyte catalytic subunit. NOH-1 will be expressed in the membranes of K562 human leukemia cells and other cell lines. Membrane fractions will be tested in a broken-cell oxidase system, along with cytosols from cells likely to express oxidase components/co- factors. This approach should permit a definition of the enzymatic activity of NOH-1 and its co-factor requirements. 3) To determine the role of the NOH-1 nonphagocyte oxidase system in cellular generation of ROS and activation of NF-KB. Cells that exhibit stable expression of functionally active NOH-1 will be developed and studied in parallel with cells that naturally express this protein. Mechanisms of oxidase activation, rates of ROS formation, and ability to activate NF-KB and modify gene expression will be determined. Collectively, these studies will establish a scientific basis for the potential role in aging of the activation of NF-KB by the products of NADPH oxidases of phagocytic and non-phagocytic cells.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG019519-02
Application #
6372553
Study Section
Special Emphasis Panel (ZAG1-PKN-2 (J2))
Program Officer
Finkelstein, David B
Project Start
2000-09-30
Project End
2005-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
2
Fiscal Year
2001
Total Cost
$227,604
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
El Jamali, Amina; Valente, Anthony J; Clark, Robert A (2010) Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca(2+)/c-Abl signaling pathway. Free Radic Biol Med 48:798-810
El Jamali, Amina; Valente, Anthony J; Lechleiter, James D et al. (2008) Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med 44:868-81
Valente, Anthony J; Zhou, Qing; Lu, Zhenhua et al. (2008) Regulation of NOX1 expression by GATA, HNF-1alpha, and Cdx transcription factors. Free Radic Biol Med 44:430-43
Valente, Anthony J; El Jamali, Amina; Epperson, Terry Kay et al. (2007) NOX1 NADPH oxidase regulation by the NOXA1 SH3 domain. Free Radic Biol Med 43:384-96
Qin, Bin; Cartier, Laetitia; Dubois-Dauphin, Michel et al. (2006) A key role for the microglial NADPH oxidase in APP-dependent killing of neurons. Neurobiol Aging 27:1577-87
Banfi, Botond; Malgrange, Brigitte; Knisz, Judit et al. (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem 279:46065-72
Banfi, Botond; Tirone, Fabiana; Durussel, Isabelle et al. (2004) Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). J Biol Chem 279:18583-91
Clark, Robert A; Valente, Anthony J (2004) Nuclear factor kappa B activation by NADPH oxidases. Mech Ageing Dev 125:799-810
Banfi, Botond; Clark, Robert A; Steger, Klaus et al. (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem 278:3510-3
Banfi, B; Molnar, G; Maturana, A et al. (2001) A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem 276:37594-601