Neutrophils and other circulating phagocytes generate high levels of reactive oxygen species (ROS) in response to a variety of 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 oxidative responses of 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 may serve in host defense and inflammatory reactions or provide redox """"""""second messengers"""""""" that affect gene expression patterns (proliferation responses to growth factors, differentiation, cellular senescence, apoptosis or programmed cell death, and oxygen sensing). In studies on the colon-specific oxidase, we have examined expression patterns of Nox1 in colon epithelial cells and demonstrated that Nox1 is induced by terminal differentiation or by interferon-gamma treatment. Transduction with a retrovirus encoding 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 homologs of these cytosolic factors, suggesting that Nox1 is part of a regulated, phox-like complex acting in host defense and inflammation in the colon epithelium. In studies aimed at exploring the functional role of the renal oxidase (renox or Nox4), we have developed transgenic mouse lines for renal-specific Nox4 over-expression or suppression (anti-sense) under the control of dietary doxycycline. We also identified four viable mouse strains in which the functional Nox4 gene is absent, which will be used to explore to role of the renal oxidase in whole animals. Finally, we have documented functional expression of thyroid dual oxidases (Duox1 and Duox2) on epithelial surfaces of airways (trachea, bronchium), salivary gland ducts, and the rectum, suggesting that these enzymes serve as sources of hydrogen peroxide supporting the anti-microbial activity of the lactoperoxidase on mucosal surfaces. Cultured primary human airway (bronchial) epithelial cells were shown to produce hydrogen peroxide in response to calcium signals in a Duox1-dependent (antisense-inhibited) manner. This system is being developed to confirm roles of this oxidase in airway anti-microbial defense and inflammatory diseases.
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