This program explores innate anti-microbial defense and inflammatory mechanisms involving the host's ability to deliberately produce reactive oxygen species (ROS). Circulating phagocytes generate high levels of ROS in response to infectious or inflammatory stimuli. This is attributed to NADPH oxidase-mediated production of superoxide, a precursor of ROS that are important microbicidal agents. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the phagocytic NADPH oxidase (phox or Nox2-based system) and is characterizing related Nox Family NADPH oxidases expressed in non-phagocytic cells (Nox1, Nox3, Nox4, Nox5, Duox1, Duox2). We are studying sources of ROS in several non-myeloid tissues, notably colon, kidney, liver, thyroid and salivary glands, mucosal surfaces (lung and gastrointestinal tract), brain, and vascular tissues. Several of these non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, since they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by pro-inflammatory cytokines or recognition of microbial factors. ROS produced by these enzymes can also provide redox signals that affect gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to infection, growth factors, hormones or cytokines. In 2009, we have advanced our understanding of processes involved in the biosynthesis, subcellular targeting and activation of the dual oxidases (Duox1 and Duox2) as we have developed systems for efficient reconstitution of the recombinant enzymes by co-expressing newly-identified Duox maturation factors (a.k.a. Duox activators (Duoxa)). These studies have provided insight on the function of these oxidases as dedicated hydrogen peroxide generators, whereas all other Nox enzymes generate superoxide. We identified several Duoxa1 splice variants, showed that the alpha isoform efficiently targets Duox to the plasma membrane, while the gamma variant targets Duox to intracellular vesicles, where it may serve intracellular redox-related functions (signaling, proliferation, migration). Golgi-based carbohydrate modifications in Duox and DuoxA proteins suggest that Duox becomes active within post-Golgi compartments and the detection of stable Duox-Duoxa dimeric complexes suggests that the Duox maturation factors function as part of the hydrogen peroxide-generating complex. Predominant expression of DuoxA1-alpha isoform in airway epithelial cells is consistent with its detection on the apical plasma membrane, where Duox provides extracellular hydrogen peroxide to support lactoperoxidase-based antimicrobial activity in the airway surface layer. Our advances in Duox reconstitution technology are being used to screen effects of various Duox or DuoxA single nucleotide polymorphisms (SNPs) and putative mutations for alterations in oxidase function or cellular targeting that may relate to altered genetic susceptibility to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection). In related studies, we developed polarized human airway epithelial models (air-liquid interface cultures) to examine Duox-dependent antimicrobial responses to viral and bacterial pathogens. We have shown that mature primary human bronchial epithelial cells produce sufficient Duox-derived hydrogen peroxide to kill several airway pathogens (Pseudomonas aeruginosa, Burkholderia Cepacia, and Staphylococcus aureus). The primary and reconstituted airway models are being used to examine Duox-related responses to pathogen exposure, including Duox subcellular targeting, ROS production, and downstream redox-related cellular changes. These experiments on airway epithelial cell-pathogen interactions will be complemented by infection studies in Duox-deficient animal models. In efforts aimed at exploring functional roles of Nox4 (or Renox), we are characterizing mice in which the Nox4 gene is deleted. Nox4-deficient mice exhibit a normal life-span and phenotype in the unstressed state. Gene microarray studies are focused on identifying alterations in other oxidant generating or scavenging systems to explore mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. Nox4 is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. We are investigating the proposed role of Nox4 in renal oxygen sensing and hematopoiesis, since ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. We have shown that Nox4 levels respond directly to Transforming Growth Factor-beta (TGF-beta) or hypoxia in renal cells and to hepatitis C virus (HCV) in hepatic cells, and we are exploring the mechanistic basis for these effects. Future work will examine responses of Nox4-deficient mice to these factors to assess potential roles of Nox4 in fibrotic disease (cirrhosis) and redox homeostasis related to hypoxia (angiogenesis, anemia, vascular regulation). Finally, our interests in the multi-component Nox1-based oxidase are aimed at characterizing functional partners of Nox activator 1 (Noxa1) identified in yeast two-hybrid screening experiments. Site-directed mutagenesis experiments are aimed at establishing involvement of Noxa1 partners in Nox1 activation in whole cell models.
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