Neutrophils comprise the first line of host defense, killing invading microbes by generating reactive oxygen species (ROS) through an activatable NADPH oxidase system. This respiratory burst oxidase is genetically absent in patients with chronic granulomatous disease, resulting in recurring severe infections with high morbidity and mortality. The phagocyte oxidase features membrane-bound catalytic components (gp91phox/p22phox) and cytosolic co-factors (p47phox, p67phox, Rac1/2) that undergo stimulus-dependent translocation during assembly of the active NADPH oxidase. Recently, it has been found that gp91phox is actually the founder of the 7-member NOX/DUOX gene family, each homologue having its characteristic pattern of tissue expression and functional role. We have recently demonstrated a novel feed-forward regulation of NOX5 by hydrogen peroxide (H2O2) acting through a signaling pathway involving calcium and the non-receptor tyrosine kinase c-Abl. Especially relevant to the current proposal are our preliminary data suggesting that a related pathway exists for the phagocyte NADPH oxidase (NOX2) and that this pathway is dysregulated with aging, a biological process associated with ROS and oxidative stress. Our main objectives are to characterize the regulation of NOX2 in human phagocytic cells by H2O2 and to assess the relevance of such regulation to chronic inflammation and aging. Our hypothesis is that H2O2 is a common regulator of the NOX enzymes through specific redox-mediated signaling pathways and that this mechanism is dysregulated during aging.
Three specific aims will guide the work.
Aim 1 is to dissect the proximal elements of this signal transduction pathway as a means of defining the early targets of H2O2, focusing on the H2O2-activated Ca2+ channels involved, the mechanisms of channel regulation, and the signaling intermediates, especially protein tyrosine kinases (PTKs), that are directly regulated by Ca2+ influx.
Aim 2 is to investigate the intermediates that lead from H2O2-activated PTKs (c-Abl, c-Src) to assembly and activation of NOX2, considering the role of specific PKC isotypes, the mechanism of activation of Rac GTPase, and the direct tyrosine phosphorylation of NOX2.
Aim 3 is to examine the status and role of H2O2-NOX2 regulation in human phagocytic leukocytes as a function of age, including both cross-sectional and longitudinal assessment of NOX2-mediated superoxide generation, correlation with biochemical and functional markers of the aging phenotype, and mechanistic exploration of the signaling pathways defined in Aims 1 and 2. The studies proposed are innovative because they capitalize on our novel observations on redox regulation of NOX, and take advantage of key reagents, constructs, and cells, as well as extensive preliminary data and the broad-based expertise of our research team. The experiments will use state-of-the-art techniques in cell biology, protein chemistry, and molecular biology. The scientific significance of our proposal is that it has the potential for establishing broadly applicable precedents for the cell biology of ROS generation, host defenses, and cell signaling, as well as aging-associated ROS dysregulation.
Project Narrative The veterans health relevance of this proposal is that the results are likely to have significant implications for a variety of diseases that are highly prevalent in veterans, as well as in the general population. NOX2- derived toxic reactive oxygen species are presently known to be involved in the pathogenesis of a number of disorders, including among others atherosclerosis, myocardial infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and ischemic stroke. Moreover, there is convincing evidence that NOX-derived toxic oxygen products are involved in the aging process. Since this project will address regulation of the production of the damaging forms of oxygen that mediate such disorders, there is considerable potential for improved understanding, as well as the identification of new targets for therapeutic intervention for the benefit of patients with these diseases, as well as for the amelioration of certain aspects of the aging process.