Neutrophils and other circulating phagocytes generate high levels of reactive oxygen species (ROS) in response infectious or inflammatory stimuli, in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase that produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies and suffer from enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst oxidase in phagocytes (phox system) and is characterizing related enzymes expressed in non-immune cells (Nox and Duox oxidases). We are characterizing sources of reactive oxygen species in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces, brain, and vascular tissue. In these sites, the oxidants can serve in host defense and inflammatory reactions or provide redox signals that alter gene expression patterns that mediate proliferation responses to growth factors, differentiation, cellular senescence, apoptosis (programmed cell death), or oxygen sensing. In studies on the colon oxidase, we examined expression patterns of Nox1 in colon epithelial cells and demonstrated that Nox1 is induced by terminal differentiation or by interferon-gamma treatment. Nox1 functionally replaces gp91phox, restoring stimulus-dependent superoxide release in cells co-expressing the cytosol factors p47phox and p67phox. Furthermore, we identified unique, colon-specific homologues of these cytosolic factors (Noxo1 / p41 and Noxa1 / p51), showing that Nox1 is a regulated, phox-like complex that may act in host defense and inflammatory processes in the colon epithelium. We are comparing the functions of variably spliced isoforms of Nox1 and its co-factors in several cell models. Related work is examining the sub-cellular location of Nox1 components and tracking their movement in response to cellular activation. In studies aimed at exploring the functional role of the renal oxidase (Renox or Nox4), we developed transgenic mouse lines with enhanced or deficient expression of Nox4. We identified four mouse strains in which the functional Nox4 gene is absent, which are being used to explore roles of the renal oxidase in whole animals. A current focus is on the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since Nox4 levels appear to respond to hypoxia and ROS are thought to provide negative feedback signals regulating renal erythropoietin synthesis. The renal oxidase appears to be a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Related studies are attempting to identify other functional components supporting the catalytic core of Nox4, such as Rac, p22phox, and cytosolic phox-like proteins. Finally, we have observed functional expression of thyroid dual oxidases (Duox1 and Duox2) on epithelial surfaces of airways (trachea, bronchium), salivary gland ducts, and the rectum, suggesting these enzymes serve as sources of hydrogen peroxide supporting the anti-microbial activity of lactoperoxidase on mucosal surfaces. Primary cultured human airway (bronchial) and monkey salivary gland epithelial cells were shown to produce hydrogen peroxide in a Duox-dependent (antisense-inhibited) manner in response to physiological agonists that trigger calcium release. These systems are being developed to explore Duox expression in relation to epithelial cell differentiation and to confirm roles of these oxidases in anti-microbial defenses and inflammatory processes on mucosal surfaces.
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