This program explores innate anti-microbial defense and inflammatory mechanisms involving the body's deliberate production of reactive oxygen species (ROS). Neutrophils and other circulating phagocytes generate high levels of ROS in response to infectious or inflammatory stimuli in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. 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 respiratory burst oxidase in phagocytes (phox system) and is characterizing related oxidant-generating NADPH oxidases expressed in non-immune cells (Nox/ Duox family of NADPH oxidases). We are studying sources of reactive oxygen species in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces (lung and GI tract), brain, and vascular tissues. Recent evidence from this laboratory indicates that several of these oxidases 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 by recognition of microbial factors. The reactive oxidants produced by these enzymes also provide redox signals that can affect gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to growth factors, cytokines, or hormones. ? ? Nox1 and Nox3, the closest homologues of the phagocytic (Nox2-based) oxidase, were shown to function as multi-component phox-like enzymes. We systematically examined structural and functional requirements for localization, assembly and activation of these novel oxidases. Both oxidases involve p22phox as a subunit that stabilizes the catalytic chains and is required for their targeting to the plasma membrane. We recently identified colon-specific Nox1-supportive cofactors homolgous to p47phox and p67phox, and showed that this oxidase is a regulated, phox-like complex that can act in host defense and inflammatory processes in the colon epithelium. We showed that both Nox1 and Nox3 function as Rac1-dependent enzymes, in which Rac1 acts in a GTP-dependent manner through its binding partner, Nox activator 1 (Noxa1). Membrane targeting of the Noxa1 was shown to be dependent on the Nox organizer (Noxo1), homologous to p47phox. Targeting of Noxo1, in turn, depends on its variably spliced PX domain, thereby affecting localization at different cellular sites in different tissues. ? ? In efforts aimed at exploring functional roles of the renal oxidase (renox or Nox4), we are characterizing several mouse strains in which the Nox4 gene is deleted. We are investigating the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. The renal oxidase is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Nox4 levels were shown to respond directly to hypoxia in renal cells. Furthermore, the Nox4 promoter fused to Nox4 cDNA or to other reporters demonstrated direct responsiveness to hypoxia. Surprisingly, Nox4-/- mice exhibit a normal phenotype in the unstressed state. The hematology as well as serum and urine chemistries (i.e., urine hydrogen peroxide levels) are normal in these animals. Related studies are focused on alterations in other oxidant generating or scavenging systems to explain the mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. ? ? We are exploring functional expression of dual oxidases (Duox1 and Duox2) in epithelial cells of airwways, salivary gland ducts, and the gastro-intestinal tract and in transfected cell models. Their expression on apical surfaces of bronchial epithelium cells indicates these oxidases serve as sources of extracellular hydrogen peroxide supporting the well-documented anti-microbial (bacterial, viral, and fungal) activities of lactoperoxidase. We are examining primary airway epithelial cell Duox expression in response to differentiation, pathogen exposure, and pro-inflammatory cytokines. We demonstrated Duox- and lactoperoxidase-dependent microbial killing (S. aureus and P. aeruginosa), and we are exploring host-pathogen interactions triggering host cell oxidative responses, and adaptive microbial counter-defenses to these oxidants. We have confirmed that airway Duox isozymes are induced by interferon-gamma, IL-4, and IL-13, suggesting roles in airway viral and microbial infection and in inflammatory disease (i.e., asthma). Active recombinant forms of Duox, co-expressed along with essential maturation factors, have been produced in whole transfected cells, which secrete hydrogen peroxide in response to intracellular calcium signals. We are exploring structural determinants for delivery of active Duox to the plasma membrane, where it supports extracellular peroxidases. This recombinant system is also being used to screen for Duox and maturation factor genetic polymorphisms associated with altered oxidase function. ? ? Finally, our long-standing interests in the phagocytic (Nox2-based or phox) oxidase are focused on the activating roles of cytosolic regulators, p40phox and cytosolic phospholipase A2 (cPLA2). cPLA2 is recruited to the membrane during cellular activation through direct interactions with the oxidase complex, as it appears to serve an essential role in releasing arachidonic acid within the immediate environment of the newly assembled oxidase. We are also exploring the role of p40phox as an adaptor that links other essential phox regulators to phagosomes and showed that recruitment of p40phox to phagosomal membranes involves arachidonate-regulated exposure of its membrane-binding PX domain.
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