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10, 11 March 2015 | The Journal of Physiology, Vol. Griendling K. K., Sorescu D., Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease, Oxidant and redox signaling in vascular oxygen sensing: implications for systemic and pulmonary hypertension. 1, 22 July 2015 | Journal of the American Society of Nephrology, Vol. 33, No. 1, 27 October 2014 | Journal of Enzyme Inhibition and Medicinal Chemistry, Vol. Western blot analyses for HIF-1α or aryl hydrocarbon receptor nuclear translocator (ARNT) were performed with 50 μg of protein, separated by 8% SDS-PAGE, and transferred to nitrocellulose membranes. 94, No. 3, Free Radical Biology and Medicine, Vol. classical neutrophil NADPH oxidase comprises a catalytic subunit, gp91phox (91kDa apparent molecular mass on gels when glycosylated; where phox is phagocyte oxidase), which in conjunction with a p22phox subunit forms a membrane-bound heterodimeric flavo-cytochrome b 558. Reactive oxygen species (ROS) such as superoxide anion radicals (O2−) and hydrogen peroxide (H2O2) have been for a long time recognized as unwanted byproducts of oxidative ATP generation in the mitochondria. Compared with control cells, proliferative activity was significantly enhanced (Figure 6, A and B). 288, No. Our novel findings that NOX4 is a target gene of HIF-1, and possibly also of HIF-2, together with previous data that NOX4 regulates HIF-1α and HIF-2α levels (Bonello et al., 2007; Block et al., 2007; Diebold et al., 2010b) suggest a positive-feedback loop whereby NOX4 would induce HIF-α proteins and vice versa. 65, 22 April 2013 | Brain Sciences, Vol. 1, 20 February 2015 | Clinical Science, Vol. 1, 7 May 2015 | Journal of Drug Targeting, Vol. Toll-like receptor signalling in liver disease: ER stress the missing link? 10, Folia Pharmacologica Japonica, Vol. Similar responses were observed in HEK293 cells and human microvascular endothelial cells (data not shown). Mutations in the NADPH oxidase subunit genes cause several Chronic Granulomatous Diseases (CGD), characterized by extreme susceptibility to infection. Wingler K., Wunsch S., Kreutz R., Rothermund L., Paul M., Schmidt H. H. Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo, Reactive oxygen species and vascular signal transduction mechanisms. catalytic subunit of the NADPH oxidase and is also called NOX2. (B) Proliferative activity was determined by BrdU incorporation. 6, No. [11] Atherosclerosis is caused by the accumulation of macrophages containing cholesterol (foam cells) in artery walls (in the intima). 24, No. 67, 3 December 2013 | International Journal of Molecular Medicine, Vol. 14, No. Western blot analyses were performed with antibodies against NOX4 and GFP. 468, No. 6, No. 7, Free Radical Biology and Medicine, Vol. 9, Stem Cells and Development, Vol. First-strand cDNA was synthesized from 1 μg RNA using reverse transcriptase (Invitrogen, Karlsruhe, Germany). This work was supported by Deutsche Forschungsgemeinschaft Grant GO709/4-4, Fondation Leducq, and Metoxia (HEALTH-F2-2009-222741) under the 7th research framework program of the European Union. Hypoxia increases NOX4 expression in vitro. In line, NOX4 protein levels were increased in these samples to a similar extent than HIF-1α protein levels (Figure 2C). Human isoforms of the catalytic component of the complex include NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2.[1]. (C) Pulmonary artery smooth-muscle cells (PASMCs) were transfected with vectors encoding shRNA against NOX4 (siN4I) or control shRNA (siCtr) and were cotransfected with an expression plasmid for HIF-1α or were exposed to hypoxia for 4 h after wounding the cell layer with a 10-μl tip. ROS generation was detected using the fluoroprobe dihydroethidium (DHE; Invitrogen) as described previously (BelAiba et al., 2007). These effects are in part responsible for inducing pre-eclampsia in pregnant women[16]. The protein is a super-oxide generating enzyme which forms reactive oxygen species (ROS). Humbs, Valley, Germany) for 1 d. Age-matched control animals breathed room air. It is the terminal component of a respiratory chain that transfers single electrons from cytoplasmic NADPH across the plasma membrane to molecular oxygen on the exterior (By similarity).By similarity Molecular Biology of the Cell is published by the American Society for Cell Biology. Prabhakar N. R., Kumar G. K., Nanduri J., Semenza G. L. ROS signaling in systemic and cellular responses to chronic intermittent hypoxia. 216, No. Abstract Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. 49, No. The p47 phox subunit of the NADPH oxidase system is a critical determinant of the Ang II-mediated oxidative stress in the cardiovascular system. Human PASMCs were obtained from Cambrex (Verviers, Belgium), cultured in the medium provided as recommended, and used from passages 3–11. During this process O2 is transported from the extracellular space to the cell interior and the H+ is exported[citation needed]. NADPH oxidases—do they play a role in TRPC regulation under hypoxia? 10, No. 18, Free Radical Biology and Medicine, Vol. [8] Moreover, in white blood cells, superoxide has been found to transfer electrons across the membrane to extracellular oxygen, while in vascular cells, the radical anion appears to be released mainly intracellularly.[9][10]. (A/B) HEK293 cells were transfected with vectors encoding shRNA against NOX4 (siN4I) or control shRNA (siCtr) and were cotransfected with an expression plasmid for HIF-1α or were exposed to hypoxia for 4 h. (A) Proliferative activity of HEK293 cells was determined by BrdU incorporation. 4, No. 3, Antioxidants & Redox Signaling, Vol. Chromatin was then precipitated with an antibody against HIF-1α (Novus) overnight at 4°C. On the other hand, a link between HIF-1α and NOX4 levels has not been established. Finally, to analyze whether ROS mediate NOX4-dependent proliferation under conditions of elevated HIF-1α levels, PASMCs were pretreated with vitamin C which can act as an antioxidant. In fact, vitamin C decreased ROS production following 4 h of hypoxia, and prevented induction of ROS levels by NOX4 or HIF-1α (Figure 8, A and B). This indicates that the relatively low amounts of p47 phox protein in remaining viable cells at 5 days were sufficient to support significant per cell correction of NADPH oxidase function for almost the duration of the neutrophil lifespan between 5 and 90 hours in circulation. 10, Antioxidants & Redox Signaling, Vol. Total RNA from PASMCs (10–15 μg) was separated on 1.3% agarose gels, transferred to nylon membranes, and cross-linked by UV irradiation. Mutation of a putative hypoxia-responsive element in the NOX4 promoter abolished hypoxic and HIF-1α–induced activation of the NOX4 promoter. 3, Respiratory Physiology & Neurobiology, Vol. 2, 8 June 2017 | Antioxidants, Vol. Chronic Granulomatous Disease (CGD) manifests as a predisposition to infection as a result of defective function of the NADPH oxidase of phago‐cytic cells. In this study, we showed that the NADPH oxidase subunit NOX4 is up-regulated by hypoxia at the mRNA and protein levels in several cell types including PASMCs and HEK293 cells, but also in human microvascular endothelial cells, as well as in human A549 lung carcinoma cells (data not shown) within 4 h of exposure, indicating that NOX4 expression is sensitive to oxygen availability independently of the cell type. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47 phox, which results in NADPH oxidase activation. Cells were seeded in 10-cm dishes and exposed to hypoxia for 4 h. After washing with phosphate-buffered saline (PBS), cells were scraped in PBS, spun down (2000 rpm, 4°C, 5 min) and resuspended in 80 μl of Krebs HEPES buffer, pH 7.35 (99 mM NaCl, 4.69 mM KCl, 25 mM NaHCO3, 1.03 mM KH2PO4, 5.6 mM d-glucose, 20 mM Na-HEPES, 2.5 mM CaCl2, 1.2 mM MgSO4) supplemented with 25 μM desferoxamine and 5 μM o,o-diethyl-thio-phosphoryl-chloride (DETC). All reagents were from Sigma (Taufkirchen, Germany) unless otherwise stated. Statistical analysis was performed using a standard curve of the input. GFP or NOX4 protein levels in siCtr-expressing cells were set to 100%. Thus, these findings clearly show that activation of HIF-1 by hypoxia results in up-regulation of NOX4, which contributes to adaptation of ROS levels and proliferation and migration of PASMCs. Quantification was performed using ΔCT calculation. Critical component of the membrane-bound oxidase that generates superoxide. 14, 4 February 2013 | Clinical Science, Vol. Data represent % change of NOX4 mRNA levels versus normoxic control (100%; n = 3, *p < 0.05 vs. control). ROS levels of cells transfected with control vectors under normoxic conditions were set to 100% (n = 3; *p < 0.05 vs. normoxic controls, #p < 0.05 vs. hypoxic control). Data represent % change of protein levels versus hypoxic control (n = 3, *p < 0.05 vs. hypoxic control). Reactive oxygen species and the brain in sleep apnea, NOX/NADPH Oxidase, the Superoxide-Generating Enzyme: Its Transcriptional Regulation and Physiological Roles. ROS levels were evaluated by DHE fluorescence. 291, No. [14] Similar loss is observed in schizophrenia, and the results may point at the NADPH oxidase as a possible player in the pathophysiology of the disease. We then evaluated the proliferative activity of PASMCs upon exposure to hypoxia for 4 h. Interestingly, PASMC proliferation was enhanced in hypoxic cells compared with normoxic cells to a similar degree as in HIF-1α–overexpressing cells (Figure 6, A and B). As expected, the levels of HIF-1α were markedly increased in hypoxic pulmonary vessels. 21, No. Jacob CO, Yu N, Yoo D-G et al (2017) Haploinsufficiency of NADPH oxidase subunit neutrophil cytosolic factor 2 is sufficient to accelerate full-blown lupus in NZM 2328 mice. This sequence contained the essential core sequence for HIF binding and was also very similar to other HIF target genes in the flanking sequences (Wenger et al., 2005), and mutation of this HRE abolished NOX4 promoter activation by hypoxia. Site-directed mutagenesis was performed at a putative hypoxia-responsive element (HRE) at −391 to −387 base pairs upstream of the transcription start by inserting a G→T base exchange at −387 base pairs using the QuickChange mutagenesis kit (Promega) and the following primers: forward: 5′-GAG GAA GGG TGG GAG AAA CGT TAA CTA GCA CAC-3′; reverse: 5′-CAG CCT TTT GTG TGC TAG TTA ACG TTT CTC CCA CCC TTC CTC-3′ revealing pglNOX4-730m. Data represent % change of protein levels versus normoxic control (100%; n = 3, *p < 0.05 vs. control). It has also been shown that NADPH oxidase plays a role in the mechanism that induces the formation of sFlt-1, a protein that deactivates certain proangiogenic factors that play a role in the development of the placenta, by facilitating the formation of reactive oxygen species, which are suspected intermediaries in sFlt-1 formation. p < 0.05 was considered statistically significant. This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-12-1003) on April 28, 2010. aryl hydrocarbon receptor nuclear translocator. Phagocyte NADPH oxidase (PHOX, often referred to as NOX2), an activity-dependent enzyme complex, is widely expressed in various immune cells including microglia, macrophages, and neutrophils. This group of multicomponent enzymes generates superoxide anion radicals in a regulated manner by allowing the transfer of electrons from NADPH via a catalytic core protein (NOX) containing flavin and heme moieties to molecular oxygen. 5, 1 October 2012 | Pulmonary Circulation, Vol. To verify the specificity of the NOX4 antibody used, a plasmid coding for a GFP-NOX4 fusion protein was coexpressed together with a vector encoding an shRNA against NOX4. In contrast, NOX4 has been suggested to act constitutively and not to require cytosolic regulators for its function (Martyn et al., 2006). The active enzyme is assembled from a membrane-bound flavocytochrome B, a heterodimer composed of gp91phox and p22phox subunits, and cytosolic regulatory components, including p47phox, p67phox, p40phox, and Rac GTPase. 69, Trends in Cardiovascular Medicine, Vol. Arthritis Rheumatol 69:1647–1660 PubMed Google Scholar Figure 8. In fact, when ROS levels were determined in PASMCs after removal from hypoxic incubation for 4 h, a condition where NOX4 levels were increased, ROS levels not only returned to baseline, but were even higher than in the normoxic control cells (Figure 5B). Interestingly, a putative HRE was found at base pairs −387 to −391. Because ROS are important regulators of vascular tone and function (Rhoades et al., 1990; Demiryurek and Wadsworth, 1999; Cai and Harrison, 2000; Lee and Griendling, 2008) and increased expression of NADPH oxidase subunits correlated with enhanced vascular superoxide production in various cardiovascular diseases (Brennan et al., 2003; Selemidis et al., 2008; Lassegue and Griendling, 2010), we tested whether the induction of NOX4 by HIF-1α has functional relevance for ROS generation in PASMCs. 22, Free Radical Biology and Medicine, Vol. In contrast, our findings that NOX4 is rapidly up-regulated by hypoxia in vitro and in vivo clearly point to a direct regulatory mechanism responsible for these effects. In this regard, however, it is of note that NOX4 itself cannot only up-regulate HIF-1α in PASMCs (Bonello et al., 2007), but also HIF-2α in several cell lines including von Hippel-Lindau protein (VHL)-deficient RCC4 cells (Block et al., 2007) and PASMCs (Diebold et al., 2010b). 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