This program explores innate immune, pro-inflammatory, and other signaling processes dependent on deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. The prototypical NADPH oxidase complex of circulating phagocytes (Nox2-based) is well known for its functions in microbial killing. Non-phagocytic enzymes (Nox1, Nox4, Duox1, Duox2) are highly expressed in epithelial cells, notably along mucosal surfaces (lung and gastrointestinal tract), liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular tissues. ROS produced by these oxidases affect cell migration, differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, and responses to growth factors, hormones, cytokines or recognition of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In 2014, we explored the molecular basis for Nox4 induction by transforming growth factor-beta (TGF-beta) and its role in cell migration in normal and metastatic tumor cells. We initially identified Nox4 as a novel source of TGF-beta-inducible ROS in hepatitis C virus (HCV)-infected hepatocytes and suggested links between excess Nox4-derived ROS with chronic HCV infection and hallmarks of liver disease progression (fibrosis, cirrhosis, and hepatocellular carcinoma). TGF-beta induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Recent studies showed that p53 affects TGF-beta;/SMAD3-mediated signaling, cell migration, and tumorigenesis. We previously demonstrated that the Nox4 is a TGF-beta/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our current studies investigated effects of wild type p53 (WT-p53) and mutant p53 proteins on TGF-beta;-regulated Nox4 expression and cell migration. We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells overexpressing WT p53. In contrast, metastatic tumor-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-beta-dependent and TGF-beta-independent pathways. Knockdown of endogenous mutant p53 (R280K) in TGF-beta-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-beta-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal human breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. These findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signaling in TGF-beta;-mediated cell motility. The studies highlight the importance of a TGF-beta-SMAD3-mutantp53-Nox4 axis in cell migration and tumor metastasis and reveal novel targets for intervention against cancer progression and metastatic disease. In work related to our long-term interests in redox-based host-pathogen interactions, we explored release of inflammatory mediators by neutrophils exposed to Pseudomonas aeruginosa. Cystic fibrosis (CF) airways are characterized by chronic bacterial infections (typically Pseudomonas aeruginosa), robust neutrophil recruitment, excess mucus production, enhanced inflammation and oxidative stress. Neutrophil primary granule components, myeloperoxidase (MMPO) and human neutrophil elastase (HNE), are inflammatory markers in CF airways, and their increased levels are associated with poor lung function. Here, we showed that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. Bacteria are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release;NADPH oxidase inhibition suppresses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas, whereas inhibition of the cytoskeleton effectively prevents DNA release. Together these studies suggest Pseudomonas-induced NET formation is an important mechanism contributing to the enhanced inflammatory conditions and airway obstruction characteristic of advanced CF airway disease. Furthermore, they may explain why some CF patients benefit from aerosolized DNAse-based therapies, which can dissolve NET structures. We also studied involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1B;release to better understand inflammasome signaling in macrophages. Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium mobilizing agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium. Our data show that calcium is essential for IL-1B;release in human macrophages. Increases in cytosolic calcium alone lead to IL-1B;secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1B;release. Human macrophage inflammasome activation and IL-1B;secretion requires calcium but does not involve NADPH oxidases. In other studies addressing oxidative innate immune and inflammatory responses of mucosal epithelial tissues of the respiratory and gastrointestinal tracts, we are developing cell culture and knockout mouse models to explore critical roles of Nox1 and Duox isozymes. We have characterized Duox1- and Duox2-deficient mouse models with goals of examining airway epithelial responses to Influenza A infection or allergic asthmatic challenges. We showed that a Duox2 V674G mutation associated with congenital hypothyroidism leads to impaired subcellular targeting and ROS release. Immunostaining of Duox2 in salivary gland ducts showed that the defective Duox2 in these mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild type Duox2. Collaborative studies with F. Chu implicate strain-specific Duox2 polymorphisms in spontaneous colitis observed in glutathione peroxidase1/2 double knockout mice. Other work shows the cytosolic peroxidase, peroxiredoxin6, supports Nox1-based ROS generation and cell migration in colon epithelial cells, again demonstrating how mucosal ROS generators and scavengers can collaborate in processes affecting epithelial barrier formation, innate immunity, and inflammation.

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Zana, Melinda; Péterfi, Zalán; Kovács, Hajnal A et al. (2018) Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation. Free Radic Biol Med 116:41-49
Chu, Fong-Fong; Esworthy, R Steven; Doroshow, James H et al. (2017) Deficiency in Duox2 activity alleviates ileitis in GPx1- and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium. Redox Biol 11:144-156
Boudreau, Howard E; Ma, Wei Feng; Korzeniowska, Agnieszka et al. (2017) Histone modifications affect differential regulation of TGF?- induced NADPH oxidase 4 (NOX4) by wild-type and mutant p53. Oncotarget 8:44379-44397
Kwon, Jaeyul; Wang, Aibing; Burke, Devin J et al. (2016) Peroxiredoxin 6 (Prdx6) supports NADPH oxidase1 (Nox1)-based superoxide generation and cell migration. Free Radic Biol Med 96:99-115
Ueyama, Takehiko; Sakuma, Megumi; Ninoyu, Yuzuru et al. (2015) The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release. J Biol Chem 290:6495-506
Carré, Aurore; Louzada, Ruy A N; Fortunato, Rodrigo S et al. (2015) When an Intramolecular Disulfide Bridge Governs the Interaction of DUOX2 with Its Partner DUOXA2. Antioxid Redox Signal :
Rada, Balázs; Park, Jonathan J; Sil, Payel et al. (2014) NLRP3 inflammasome activation and interleukin-1? release in macrophages require calcium but are independent of calcium-activated NADPH oxidases. Inflamm Res 63:821-30
Donkó, Ágnes; Morand, Stanislas; Korzeniowska, Agnieszka et al. (2014) Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2. Free Radic Biol Med 73:190-200
Yoo, Dae-goon; Winn, Matthew; Pang, Lan et al. (2014) Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation. J Immunol 192:4728-38
Boudreau, H E; Casterline, B W; Burke, D J et al. (2014) Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-?-mediated migration of human lung and breast epithelial cells. Br J Cancer 110:2569-82

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