This subsection of the Names and taxonomy section indicates the name (s) of the gene (s) that code for the protein sequence (s) described in … NADPH oxidase can be inhibited by apocynin, nitric oxide (NO), and diphenylene iodonium. 2009 Sep 15;47(6):825-34. doi: 10.1016/j.freeradbiomed.2009.06.025.  Nitro blue tetrazolium is used in a diagnostic test, in particular, for chronic granulomatous disease, a disease in which there is a defect in NADPH oxidase; therefore, the phagocyte is unable to make the reactive oxygen species or radicals required for bacterial killing, resulting in bacteria thriving within the phagocyte. 16, No. NOX4 or PAI-1 mRNA levels were normalized to actin levels. All animal procedures were approved by the local legislation on protection of animals (Regierung von Oberbayern, Munich, Germany). 7, No. 90, No. 9, No. 1, 1 August 2014 | Biochemistry, Vol. 20, No. (A and B) Pulmonary artery smooth-muscle cells (PASMCs) were treated with actinomycin D (Act, 5 μM) or DMSO (Ctr) for 1 h and exposed to hypoxia for 4 h. (A) Northern blot analyses were performed using a specific probe for NOX4, 18S staining served as loading control. 2017, Antioxidants & Redox Signaling, Vol. THE NADPH OXIDASEis a multi-subunit enzyme that cat- alyzes the reduction of molecular oxygen and oxidation of NADPH to generate superoxide radicals (O 2 ¥) (5, 53). As background control, chromatin immunoprecipitation (ChIP) without antibody was performed. (D) HepG2 cells were exposed to hypoxia for 3 h. Chromatin immunoprecipitation (ChIP) was performed with an antibody against HIF-1α.  Atherosclerosis is caused by the accumulation of macrophages containing cholesterol (foam cells) in artery walls (in the intima). Control cells (Ctr, siCtr) were set to 100%, and the relative change in HIF-1α–overexpressing cells is displayed (n = 3; *p < 0.05 vs. control). ROS levels of hypoxic control cells (siCtr) were set to 100% (n = 3; *p < 0.05 vs. hypoxic control). Data represent % change of protein levels versus normoxic control (100%; n = 3, *p < 0.05 vs. control). 117, Antioxidants & Redox Signaling, Vol. 128, No. Figure 2. 8, No. NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) is a membrane-bound enzyme complex that faces the extracellular space. Hypoxia increases NOX4 expression in mouse lungs. NADPH oxidases—do they play a role in TRPC regulation under hypoxia? 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. Furthermore, our findings may provide a molecular link for previous reports describing enhanced HIF-α levels as well as NOX4 levels in pulmonary hypertension induced by chronic hypoxia (Yu et al., 1998, 1999; Brusselmans et al., 2003; Mittal et al., 2007), although our findings clearly demonstrate that up-regulation of NOX4 by HIF-1α takes place already after 4 h of hypoxia. 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. NADPH oxidases have been identified as important regulators of ROS in vascular cells and also in other cell types, and the NADPH oxidase subunit NOX4 has been associated with systemic and pulmonary smooth-muscle function (Brown and Griendling, 2009). 124, No. Northern hybridizations were carried out with digoxigenin-labeled antisense RNA probes for NOX4, which were transcribed from a NOX4 antisense construct using digoxigenin-labeled nucleotides and T7 polymerase (Roche) at 65°C for 16 h. Detection was performed after incubation with a digoxigenin antibody conjugated with alkaline phosphatase by using the chemiluminescent substrate CDPStar (Roche). 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. Compared with NOX2, the other family members, which are often coexpressed in the same cells, differ in their subunit requirements and subcellular localization (Lambeth et al., 2007; Ushio-Fukai, 2009). 10, Antioxidants & Redox Signaling, Vol. Because hypoxia increased the levels of NOX4, we next tested the contribution of NOX4 to ROS generation after hypoxia. A., Black S. M. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. In contrast to our findings of an immediate effect of hypoxia on NOX4 expression, however, the effect of chronic hypoxia on NOX4 expression may not be a direct one, but may be due to secondary effects mediated by growth factors, vasoactive peptides, or cytokines induced by chronic hypoxia as has been suggested earlier (Ismail et al., 2009). Gorin Y., Ricono J. M., Kim N. H., Bhandari B., Choudhury G. G., Abboud H. E. Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells. PASMCs were plated to a density of 70% and cultured for 24 h. Transfection efficiency was on average 40%. Thereby, the family of NADPH oxidases represents the only known enzyme system whose primary biological function is to produce ROS (Babior, 1999). 8, 5 November 2010 | Cell and Tissue Research, Vol. Interestingly, NOX4 protein levels were also strongly enhanced after 1 d of hypoxia, whereby NOX4 was primarily localized in the α-actin–positive media but also in the endothelial layer of small pulmonary arteries. Expression of vectors encoding two different shRNAs against NOX4 decreased not only NOX4 induction by hypoxia (Figure 6C), but also the proliferative activity of PASMCs under these conditions (Figure 6, A and B), suggesting that HIF-1α–dependent NOX4 induction was important for the proliferative activity of PASMCs in response to hypoxia. NADPH oxidase subunit gp91phox: A proton pathway Henderson, Lydia 2005-02-24 00:00:00 Protoplasma (2001) 217:37-42 PROTOPLASMA 9 Springer-Verlag 2001 Printed in Austria NADPH oxidase subunit gp91Ph~ a proton pathway Lydia M. Henderson Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol Received May 28, 2000 Accepted October 28, … ROS mediate proliferation by hypoxia, NOX4, and HIF-1α. The hypoxia-inducible factor-2alpha is stabilized by oxidative stress involving NOX4. , In animals, NADPH oxidase is found in two types: one in white blood cells (neutrophilic) and the other in vascular cells, differing in biochemical structure and functions. 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. Pulmonary artery smooth-muscle cells (PASMCs) were transfected with vectors encoding different shRNAs against NOX4 (siN4I, siN4II) or control shRNA (siCtr) and were either cotransfected with a plasmid coding for HIF-1α or were exposed to hypoxia for 4 h. Proliferative activity was determined by (A) BrdU incorporation or (B) determination of cell numbers using a hemocytometer. This protein is one part (subunit) of a group of proteins that forms an enzyme complex called NADPH oxidase, which plays an essential role in the immune system. 12, No. 302, No. Petry A., Djordjevic T., Weitnauer M., Kietzmann T., Hess J., Gorlach A. NOX2 and NOX4 mediate proliferative response in endothelial cells. The mice were killed, and lungs were dissected and either snap-frozen in liquid nitrogen or inflated and Formalin-fixed. 4, 1 October 2015 | Pflügers Archiv - European Journal of Physiology, Vol. 3, Antioxidants & Redox Signaling, Vol. , More advanced NADPH oxidase inhibitors include GKT-831 (Formerly GKT137831), a dual Inhibitor of isoforms NOX4 and NOX1 which was patented in 2007. Cells were exposed to hypoxia (Hyp) or were cotransfected with a plasmid coding for HIF-1α. Mitochondrial complex III: An essential component of universal oxygen sensing machinery? 6, No. HIF-1α binding to chromatin was revealed after background subtraction as relative amount of the input used. Compared with normoxia, exposure to hypoxia increased NOX4 promoter activity (Figure 3C), further indicating that hypoxia induces NOX4 transcription. Cell migration was measured using a 35-mm gridded μ-dish (Ibidi, Martinsried, Germany). 216, No. 1, 20 February 2015 | Clinical Science, Vol. 5, 1 June 2015 | Toxicological Sciences, Vol. Djordjevic T., Pogrebniak A., BelAiba R. S., Bonello S., Wotzlaw C., Acker H., Hess J., Gorlach A. , The membrane-bound vascular enzyme is composed of five parts: two cytosolic subunits (p47phox and p67phox), a cytochrome b558 which consists of gp91phox, p22phox and a small G protein Rac. 5, 15 October 2020 | Antioxidants, 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. Before measurement, the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH; Noxygen, Elzach, Germany) was added to a final concentration of 100 μM. RT-PCR (A) or real-time PCR (B) were performed using specific primers for NOX4 or 18S. 23, No. A 2355-base pair fragment from the NOX4 gene (gene Id: 50507) upstream from the start codon was amplified by PCR using primers with appropriate restriction sites (forward: 5′-GTA AAG CAA CAT AAA CAG ATC TCA GTC CCC TC-3′; reverse: 5′-ACA GCC ATG GCG CCG GCC C-3′) and digested with KpnI to reveal a 968-base pair fragment containing 730 base pairs of the NOX4 promoter and 238 base pairs of the first exon. Cell suspension was scanned 20 times at 37°C for a total of 10 min in an EPR spectrometer with temperature control (e-scan, Noxygen) with the following parameters: microwave power = 23.89 mW; center field = 3459–3466 G; modulation frequency = 86 kHz; scan time 10.49 s per scan; and modulation amplitude = 2.93 G. Superoxide generation rate was calculated using linear regression and normalized to the protein content. Real-time PCR analysis was performed as described (Diebold et al., 2010a) using the Perfecta SYBR Green FastMix (VWR, Darmstadt, Germany) in a Rotor-Gene 6000 (Corbett, Wasserburg, Germany). 1, 10 November 2015 | Journal of Biological Chemistry, Vol. In this regard, we could demonstrate that NOX4 is involved in the up-regulation of HIF-1α (Bonello et al., 2007). Superoxides are crucial in killing foreign bacteria in the human body. 1, 22 July 2015 | Journal of the American Society of Nephrology, Vol. MOX-1. Figure 7. 25, No. Hemalum was used for counterstaining. 31, No. 96, 7 June 2016 | Journal of Toxicology and Environmental Health, Part A, Vol. These findings show that NOX4 is a new target gene of HIF-1α involved in the response to hypoxia. Djordjevic T., BelAiba R. S., Bonello S., Pfeilschifter J., Hess J., Gorlach A. Prabhakar N. R., Kumar G. K., Nanduri J., Semenza G. L. ROS signaling in systemic and cellular responses to chronic intermittent hypoxia. Ismail S., Sturrock A., Wu P., Cahill B., Norman K., Huecksteadt T., Sanders K., Kennedy T., Hoidal J. NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-β1 and insulin-like growth factor binding protein-3, Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes, Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression, Transcriptional regulation of serine/threonine kinase-15 (STK15) expression by hypoxia and HIF-1, Chronic hypoxia activates a local angiotensin-generating system in rat carotid body, Regulation of Nox and Duox enzymatic activity and expression, NADPH oxidases: functions and pathologies in the vasculature, Redox signaling, vascular function, and hypertension.  Conversely, assembly of the complex can be inhibited by apocynin and diphenylene iodonium. Data represent % increase of NOX4 mRNA levels compared with control (n = 3; *p < 0.05 vs. Ctr). As negative control to analyze unspecific binding and precipitation, real-time PCR using primers amplifying a region within the third intron of the β-actin gene (gene ID: 60) not containing a putative HRE (5′-ACGTG-3′) was performed (forward, 5′-AAC ACT GGC TCG TGT GAC AA-3′ and reverse, 5′-AAA GTG CAA AGA ACA CGG CT-3′). 52, No. NOX1, NOX3, and NOX4 share some similarities with NOX2, such as structural organization and molecular weight. To determine, whether hypoxia up-regulates NOX4 also in vivo, lung tissue samples were obtained from mice exposed to 10% oxygen for 1 d. Compared with lung tissue from normoxic mice, NOX4 mRNA levels were up-regulated in samples from hypoxic mice as was determined by semiquantitative RT-PCR and real-time PCR (Figure 2, A and B). catalytic subunit of the NADPH oxidase and is also called NOX2. The epigenetic landscape related to reactive oxygen species formation in the cardiovascular system, The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis, The Role of NOX4 and TRX2 in Angiogenesis and Their Potential Cross-Talk, Mechanisms of N-acetylcysteine in reducing monocrotaline-induced pulmonary hypertension in rats: Inhibiting the expression of Nox1 in pulmonary vascular smooth muscle cells, Deletion of hypoxia-inducible factor-1α in myeloid lineage exaggerates angiotensin II-induced formation of abdominal aortic aneurysm, Metabolic zonation of the liver: The oxygen gradient revisited, Glutaminase and poly(ADP-ribose) polymerase inhibitors suppress pyrimidine synthesis and VHL-deficient renal cancers, Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics, Metabolic Reprogramming and Redox Signaling in Pulmonary Hypertension, NADPH Oxidase Deficiency: A Multisystem Approach, Novel Therapeutic Effects of Leonurine On Ischemic Stroke: New Mechanisms of BBB Integrity, NADPH oxidases and vascular remodeling in cardiovascular diseases, Renal Carcinogenesis, Tumor Heterogeneity, and Reactive Oxygen Species: Tactics Evolved, Differential transcriptional regulation of hypoxia-inducible factor-1α by arsenite under normoxia and hypoxia: involvement of Nrf2, Grape seed procyanidin extract attenuates hypoxic pulmonary hypertension by inhibiting oxidative stress and pulmonary arterial smooth muscle cells proliferation, Peroxisome proliferator-activated receptor δ inhibits GFP or NOX4 protein levels in siCtr-expressing cells were set to 100%. (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. Western blot analyses were performed with antibodies against GFP and NOX4 (Figure 1D). The higher the blue score the better the cell is at producing reactive oxygen species. (A) Pulmonary artery smooth-muscle cells (PASMCs) were transfected with plasmids coding for NOX4 or for HIF-1α and were cotransfected with shRNA against NOX4 (siN4I) or with control shRNA (siCtr). In addition, this putative NOX4 HRE was not only in its sequence, but also in its relative position in the NOX4 promoter similar to several known HREs in HIF target genes. Blots were scanned and analyzed using GelDoc software (Bio-Rad, Munich, Germany). 452, No. 5, 1 October 2012 | Pulmonary Circulation, Vol. * These authors contributed equally to this work. Because NOX4-dependent NADPH oxidases have been shown to generate ROS, we hypothesized that enhanced levels of HIF-1α should be able to increase ROS levels via NOX4. In both cases, more than 50% reduction of protein levels was observed. Rhoades R. A., Packer C. S., Roepke D. A., Jin N., Meiss R. A. Reactive oxygen species alter contractile properties of pulmonary arterial smooth muscle, Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. Data represent % change of NOX4 mRNA levels versus hypoxic control (n = 3, *p < 0.05 vs. hypoxic control). Apocynin acts by preventing the assembly of the NADPH oxidase subunits. Thereafter, cells were quickly washed with HBSS to remove excess dye, and DHE fluorescence was monitored using 480-nm excitation and 620-nm emission wavelength in a microplate reader (Safire, Tecan, Crailsheim, Germany) for a total time of 3 min at normoxic conditions. Statistical analysis was performed using a standard curve of the input. 2, 31 May 2012 | Cellular and Molecular Life Sciences, Vol. PASMCs have been suggested to be particularly sensitive to oxygen availability and to be responsible for acute hypoxic vasoconstriction and the development of pulmonary hypertension due to chronic hypoxia (Weir et al., 2005; Aaronson et al., 2006; Stenmark et al., 2006; Gupte and Wolin, 2008). 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. 1, Experimental & Translational Stroke Medicine, Vol. These findings complement earlier reports demonstrating NOX4 expression in the media of remodeled pulmonary vessels in mice exposed to hypoxia for 3 wk (Mittal et al., 2007). Luciferase activities under the respective control conditions (Ctr) for each reporter plasmid were set equal to 100%. Real-time PCR was performed with primers for the NOX4 promoter (forward, 5′-GAT AAA GAA ACT GGC GGC TG-3′ and reverse, 5′-GTA ACG AAA TTT GAG CCG GA-3′) flanking the potential HRE −391 to −387 bp and with primers for the PAI-1 promoter containing known HREs as positive control (forward, 5′-GCT CTT TCC TGG AGG TGG TC-3′ and reverse, 5′-GGG CAC AGA GAG AGT CTG GA-3′) using a Rotor-Gene 6000 (Corbett). NADPH oxidases gained increasing interest and importance as sources of ROS in the vasculature (Griendling et al., 2000). In support, NOX4 mRNA levels have been reported to be up-regulated by hypoxia for 24 h in PASMCs (Mittal et al., 2007; Ismail et al., 2009).  It is also stimulated by agonists and arachidonic acid. 31, Antioxidants & Redox Signaling, Vol. 2, Antioxidants & Redox Signaling, Vol. Weir E. K., Lopez-Barneo J., Buckler K. J., Archer S. L. Integration of oxygen signaling at the consensus HRE. 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