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. Recent evidence from this lab indicates that several of these oxidases also serve in host defense and inflammatory processes, since these oxidase homologues are predominately expressed on apical surfaces of epithelial cells. The reactive oxidants produced by these enzymes can also provide redox signals that alter gene expression patterns that mediate proliferation responses to growth factors, differentiation, cellular senescence, apoptosis or programmed cell death, and 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. We identified unique, colon-specific homologues of these cytosolic factors, showing that Nox1 is a regulated, phox-like complex that can act in host defense and inflammatory processes in the colon epithelium. We have compared the functions of variably spliced isoforms of Nox1 and its co-factors in several cell models. Related work is examining the structural requirements and sub-cellular location of Nox1-supportive components and tracking their movement in response to cellular activation. Nox1 and Nox3, the closest homologues of the phagocytic oxidase that both function as multi-component enzymes, also appear to involve p22phox as a subunit that stabilizes these oxidases and affects their sub-cellular localization. Furthermore, we have evidence suggesting both Nox1 and Nox3 function as Rac-dependent enzymes, like the phagocytic system. In a major effort this year aimed at exploring the functional role of the renal oxidase (renox or Nox4), we are characterizing four mouse strains in which the Nox4 gene is deleted. We are investigating 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. Surprisingly, the Nox4-/- mice exhibit a normal phenotype in the unstressed state; the hematology as well as serum and urine chemistries (i.e., urine peroxide levels) are normal in these animals. Current work is focusing on alterations in other redox generating or scavenging systems to understand mechanisms maintaining redox homeostasis in Nox4-deficient animals. 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 are exploring the functional expression of dual oxidases (Duox1 and Duox2) in epithelial cells of airways (trachea, bronchium), salivary gland ducts, and the rectum. Their expression on apical surfaces of epithelial cells suggests these oxidases serve as sources of hydrogen peroxide supporting the anti-microbial activity of lactoperoxidase on mucosal surfaces. Recombinant forms of Duox2 have been produced and purified to examine the activity of the peroxidase homology domain in vitro. Primary cultured human airway (bronchial) and monkey salivary gland epithelial cells were shown to produce extracellular hydrogen peroxide in a Duox-dependent manner in response to physiological agonists that trigger calcium release. These primary culture 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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