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Sample records for nadph oxidase activity

  1. Regulation of NADPH Oxidase Activity in Phagocytes

    PubMed Central

    Debeurme, Franck; Picciocchi, Antoine; Dagher, Marie-Claire; Grunwald, Didier; Beaumel, Sylvain; Fieschi, Franck; Stasia, Marie-Jos

    2010-01-01

    The X+-linked chronic granulomatous disease (X+-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X+-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X+-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, ?507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2. PMID:20724480

  2. Peroxiredoxin-6 and NADPH oxidase activity.

    PubMed

    Ambruso, Daniel R

    2013-01-01

    Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2. Peroxiredoxin 6 (Prdx6), a protein belonging to the class of 1-cys Prdxs, was identified in polymorphonuclear leukocytes or neutrophils, defined by amino acid sequence and activity, and found associated with a component of the NADPH oxidase (Nox2), p67(phox). Prdx6 plays an important role in neutrophil function and supports the optimal activity of Nox2. In this chapter, methods are described for determining the Prdx activity of Prdx6. In addition, the approach for assessing the effect of Prdx6 on Nox2 in the SDS-activated, cell-free system of NADPH oxidase activity is presented. Finally, the techniques for suppressing Prdx6 expression in phox-competent K562 cells and cultured myeloid cells with siRNA and shRNA methods are described. With these approaches, the role of Prdx6 in Nox2 activity can be explored with intact cells. The biochemical mechanisms of the Prdx6 effect on the NADPH oxidase can be investigated with the experimental strategies described. PMID:23830630

  3. Activation of antibacterial autophagy by NADPH oxidases.

    PubMed

    Huang, Ju; Canadien, Veronica; Lam, Grace Y; Steinberg, Benjamin E; Dinauer, Mary C; Magalhaes, Marco A O; Glogauer, Michael; Grinstein, Sergio; Brumell, John H

    2009-04-14

    Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcgamma receptors (FcgammaRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy. PMID:19339495

  4. NADPH oxidase and neurodegeneration.

    PubMed

    Hernandes, Marina S; Britto, Luiz R G

    2012-12-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  5. NADPH Oxidase and Neurodegeneration

    PubMed Central

    Hernandes, Marina S; Britto, Luiz R G

    2012-01-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  6. HIF-1α activation by intermittent hypoxia requires NADPH oxidase stimulation by xanthine oxidase.

    PubMed

    Nanduri, Jayasri; Vaddi, Damodara Reddy; Khan, Shakil A; Wang, Ning; Makarenko, Vladislav; Semenza, Gregg L; Prabhakar, Nanduri R

    2015-01-01

    Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO. PMID:25751622

  7. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  8. Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

    PubMed Central

    Masoud, Rawand; Bizouarn, Tania; Houée-Levin, Chantal

    2014-01-01

    The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase. PMID:25462061

  9. Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells.

    PubMed

    Pal, Rituraj; Monroe, Tanner O; Palmieri, Michela; Sardiello, Marco; Rodney, George G

    2014-01-31

    Neurodegenerative diseases are attributed to impairment of the ubiquitin-proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity. PMID:24374334

  10. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice.

    PubMed

    Nagasu, Hajime; Satoh, Minoru; Kiyokage, Emi; Kidokoro, Kengo; Toida, Kazunori; Channon, Keith M; Kanwar, Yashpal S; Sasaki, Tamaki; Kashihara, Naoki

    2016-01-01

    Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes. PMID:26552047

  11. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

    PubMed Central

    Nagasu, Hajime; Satoh, Minoru; Kiyokage, Emi; Kidokoro, Kengo; Toida, Kazunori; Channon, Keith M; Kanwar, Yashpal S; Sasaki, Tamaki; Kashihara, Naoki

    2016-01-01

    Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes. PMID:26552047

  12. Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

    PubMed Central

    Agwu, D E; McPhail, L C; Sozzani, S; Bass, D A; McCall, C E

    1991-01-01

    Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway. Images PMID:1864964

  13. NADPH Oxidases and Angiotensin II Receptor Signaling

    PubMed Central

    Garrido, Abel Martin; Griendling, Kathy K.

    2010-01-01

    Over the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in the development of different diseases in which Ang II is a central component. In this review, we summarize the mechanism of activation of NADPH oxidases by Ang II and describe the molecular targets of ROS in Ang II signaling in the vasculature, kidney and brain. We also discuss the effects of genetic manipulation of NADPH oxidase function on the physiology and pathophysiology of the renin angiotensin system. PMID:19059306

  14. Adenosine A3 Receptor Suppresses Prostate Cancer Metastasis by Inhibiting NADPH Oxidase Activity12

    PubMed Central

    Jajoo, Sarvesh; Mukherjea, Debashree; Watabe, Kounosuke; Ramkumar, Vickram

    2009-01-01

    Prostate cancer is the most commonly diagnosed and second most lethal malignancy in men, due mainly to a lack of effective treatment for the metastatic disease. A number of recent studies have shown that activation of the purine nucleoside receptor, adenosine A3 receptor (A3AR), attenuates proliferation of melanoma, colon, and prostate cancer cells. In the present study, we determined whether activation of the A3AR reduces the ability of prostate cancer cells to migrate in vitro and metastasize in vivo. Using severe combined immunodeficient mice, we show that proliferation and metastasis of AT6.1 rat prostate cancer cells were decreased by the administration of A3AR agonist N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide. In vitro studies show that activation of A3AR decreased high basal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity present in these cells, along with the expression of Rac1 and p47phox subunits of this enzyme. Inhibition of NADPH oxidase activity by the dominant-negative RacN17 or short interfering (si)RNA against p47phox reduced both the generation of reactive oxygen species and the invasion of these cells on Matrigel. In addition, we show that membrane association of p47phox and activation of NADPH oxidase is dependent on the activity of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase pathway. We also provide evidence that A3AR inhibits ERK1/2 activity in prostate cancer cells through inhibition of adenylyl cyclase and protein kinase A. We conclude that activation of the A3AR in prostate cancer cells reduces protein kinase A-mediated stimulation of ERK1/2, leading to reduced NADPH oxidase activity and cancer cell invasiveness. PMID:19881949

  15. Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate

    SciTech Connect

    Umeki, Shigenobu )

    1990-01-01

    Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg{sup 2+} and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.

  16. Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

    PubMed Central

    Gan, Xiaoliang; Xing, Dandan; Su, Guangjie; Li, Shun; Luo, Chenfang; Irwin, Michael G.; Xia, Zhengyuan; Li, Haobo; Hei, Ziqing

    2015-01-01

    Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and β-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation. PMID:26246867

  17. Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

    PubMed

    Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U

    2012-03-15

    Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection. PMID:22423966

  18. Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

    PubMed

    Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

    2015-05-01

    Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. PMID:25615792

  19. Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

    PubMed Central

    Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

    2014-01-01

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

  20. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

    PubMed Central

    Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A.; Chrzanowska-Wodnicka, Magdalena; Wittchen, Erika S.; Li, Dean Y.; Hartnett, M. Elizabeth

    2014-01-01

    Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2?-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b?/? mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.Wang, H., Jiang, Y., Shi, D., Quilliam, L. A., Chrzanowska-Wodnicka, M., Wittchen, E. S., Li, D. Y., Hartnett, M. E. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization. PMID:24043260

  1. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    SciTech Connect

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-04-15

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

  2. NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy.

    PubMed

    Zhu, Xinjian; Shen, Kai; Bai, Ying; Zhang, Aifeng; Xia, Zhengrong; Chao, Jie; Yao, Honghong

    2016-05-01

    Growing evidence indicates that alterations in autophagy are present in a variety of neurological disorders, ranging from neurodegenerative diseases to acute neurological insults. Only recently has the role of autophagy in epilepsy started to be recognized. In this study, we used pentylenetetrazole (PTZ) kindling, which provides a model of chronic epilepsy, to investigate the involvement of autophagy in the hippocampus and the possible mechanisms involved. Our western blot results showed that autophagy-related proteins were significantly increased after the mice were fully kindled. In addition, immunofluorescence studies revealed a significant increase in the punctate accumulation of LC3 in the hippocampal CA1 region of fully PTZ-kindled mice. Consistent with the upregulation of ATG proteins and punctate accumulation of LC3 in the hippocampal CA1 region, autophagosomal vacuole formation was observed by an ultrastructural analysis, verifying the presence of a hippocampal autophagic response in PTZ-kindled mice. Increased oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurological diseases, including epilepsy. In this study, we demonstrate that PTZ kindling induced reactive oxygen species (ROS) production and lipid peroxidation, which were accompanied by mitochondrial ultrastructural damage due to the activation of NADPH oxidase. Pharmacological inhibition of NADPH oxidase by apocynin significantly suppressed the oxidative stress and ameliorated the hippocampal autophagy in PTZ-kindled mice. Interestingly, pharmacological induction of autophagy suppressed PTZ-kindling progress and reduced PTZ-kindling-induced oxidative stress while inhibition of autophagy accelerated PTZ kindling progress and increased PTZ-kindling-induced oxidative stress. These results suggest that the oxidative stress induced by NADPH oxidase activation may play a pivotal role in PTZ-kindling process as well as in PTZ kindling-induced hippocampal CA1 autophagy. PMID:26969791

  3. Intermittent Hypoxia-Induced Cognitive Deficits Are Mediated by NADPH Oxidase Activity in a Murine Model of Sleep Apnea

    PubMed Central

    Nair, Deepti; Dayyat, Ehab A.; Zhang, Shelley X.; Wang, Yang; Gozal, David

    2011-01-01

    Background In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction. Methods and Findings The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox_/Y) and wild-type littermates. On a standard place training task, gp91phox_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA) controls, while no changes emerged in gp91phox_/Y mice. Additionally, wild-type mice, but not gp91phox_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures. Conclusions The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing. PMID:21625437

  4. SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: implications for vascular aging.

    PubMed

    Zarzuelo, María José; López-Sepúlveda, Rocío; Sánchez, Manuel; Romero, Miguel; Gómez-Guzmán, Manuel; Ungvary, Zoltan; Pérez-Vizcaíno, Francisco; Jiménez, Rosario; Duarte, Juan

    2013-05-01

    Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD(+)-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22(phox) and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation. PMID:23422569

  5. Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

    SciTech Connect

    Ahluwalia, Jatinder

    2008-01-11

    Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

  6. Targeting NADPH oxidases in vascular pharmacology

    PubMed Central

    Schramm, Agata; Matusik, Paweł; Osmenda, Grzegorz; Guzik, Tomasz J

    2012-01-01

    Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the selective inhibition of dysfunctional NADPH oxidase homologs. This appears to be the most reasonable approach, potentially much more efficient than non-selective scavenging of all ROS by the administration of antioxidants. PMID:22405985

  7. Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase

    PubMed Central

    Serezani, Carlos H. C.; Aronoff, David M.; Jancar, Sonia; Mancuso, Peter; Peters-Golden, Marc

    2005-01-01

    Leukotrienes (LTs) are lipid mediators that participate in inflammatory diseases and innate immune function. We sought to investigate the importance of LTs in regulating the microbicidal activity of alveolar macrophages (AMs) and the molecular mechanisms by which this occurs. The role of LTs in enhancing AM microbicidal activity was evaluated pharmacologically and genetically using in vitro challenge with Klebsiella pneumoniae. Exogenous LTs increased AM microbicidal activity in a dose- and receptor-dependent manner, and endogenous production of LTs was necessary for optimal killing. Leukotriene B4 (LTB4) was more potent than cysteinyl LTs. An important role for nicotinamide adenine dinucleotide (NADPH) oxidase in LT-induced microbicidal activity was indicated by the fact that bacterial killing was abrogated by the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 μM) and in AMs derived from gp91phox-deficient mice. By contrast, LT-induced microbicidal activity was independent of the generation of nitric oxide. LTs increased H2O2 production, and LTB4 was again the more potent agonist. Both classes of LTs elicited translocation of p47phox to the cell membrane, and LTB4 induced phosphorylation of p47phox in a manner dependent on protein kinase C-δ (PKC-δ) activity. In addition, the enhancement of microbicidal activity by LTs was also dependent on PKC-δ activity. Our results demonstrate that LTs, especially LTB4, enhanceAM microbicidal activity through the PKC-δ-dependent activation of NADPH oxidase. PMID:15718414

  8. Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.

    PubMed Central

    Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi

    2004-01-01

    VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase. PMID:14594451

  9. Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase

    PubMed Central

    Loehr, James A.; Abo-Zahrah, Reem; Pal, Rituraj; Rodney, George G.

    2015-01-01

    Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function. PMID:25653619

  10. Exploiting algal NADPH oxidase for biophotovoltaic energy.

    PubMed

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K; Bombelli, Paolo; Howe, Christopher J; Merchant, Sabeeha S; Davies, Julia M; Smith, Alison G

    2016-01-01

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices. PMID:25641364

  11. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

    PubMed

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  12. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  13. Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase.

    PubMed

    Moe, Kyaw Thu; Khairunnisa, Katwadi; Yin, Nwe Oo; Chin-Dusting, Jaye; Wong, Philip; Wong, Meng Cheong

    2014-09-01

    An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes. PMID:25059721

  14. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  15. Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

    PubMed Central

    González-Perilli, Lucía; Álvarez, María Noel; Prolo, Carolina; Radi, Rafael; Rubbo, Homero; Trostchansky, Andrés

    2013-01-01

    Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species (ROS) under pro-inflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2·−), due to the NADPH dependent univalent reduction of oxygen to O2·− by the phagocytic NADPH-oxidase isoform (NOX2), being the activity of NOX2 the main source of O2·− in monocytes/macrophages. Since NOX2 and AA pathways are connected, we propose that AANO2can modulate macrophage activation by inhibiting O2·− formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex Red fluorescence and flow cytometry; this process also occurs in physiological mimic conditions within the phagosomes. AANO2 decreased O2·− production in a dose-(IC50= 4.1 ± 1.8 μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g. p40phox and p47phox), as analyzed by immunoprecipitation and western blot. However, a reduction of the migration to the membrane of p47phox was obtained suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, where subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate treated mice. PMID:23318789

  16. Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

    PubMed

    Rezende, Flávia; Löwe, Oliver; Helfinger, Valeska; Prior, Kim-Kristin; Walter, Maria; Zukunft, Sven; Fleming, Ingrid; Weissmann, Norbert; Brandes, Ralf P; Schröder, Katrin

    2016-03-01

    NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is, in part, based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol, or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. In this study, we demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in the aorta, heart, and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo angiotensin II pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells, whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations, we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity. Antioxid. Redox Signal. 24, 392-399. PMID:25906178

  17. NADPH Oxidase- and Mitochondria-derived Reactive Oxygen Species in Proinflammatory Microglial Activation: A Bipartisan Affair?

    PubMed Central

    Bordt, Evan A.; Polster, Brian M.

    2014-01-01

    Microglia are the resident immune cells of the brain and play major roles in central nervous system development, maintenance, and disease. Brain insults cause microglia to proliferate, migrate, and transform into one or more activated states. Classical M1 activation triggers the production of proinflammatory factors such as tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), and reactive oxygen species which, in excess, can exacerbate brain injury. The mechanisms underlying microglial activation are not fully understood, yet reactive oxygen species are increasingly implicated as mediators of microglial activation. In this review, we highlight studies linking reactive oxygen species, in particular hydrogen peroxide derived from NADPH oxidase-generated superoxide, to the classical activation of microglia. In addition, we critically evaluate controversial evidence suggesting a specific role for mitochondrial reactive oxygen species in the activation of the NLRP3 inflammasome, a multiprotein complex that mediates the production of IL-1β and IL-18. Finally, the limitations of common techniques used to implicate mitochondrial ROS in microglial and inflammasome activation, such as the use of the mitochondrially-targeted ROS indicator MitoSOX and the mitochondrially-targeted antioxidant MitoTEMPO, are also discussed. PMID:25091898

  18. p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase.

    PubMed

    Watanabe, Hiroshi; Miyamoto, Yohei; Honda, Daisuke; Tanaka, Hisae; Wu, Qiong; Endo, Masayuki; Noguchi, Tsuyoshi; Kadowaki, Daisuke; Ishima, Yu; Kotani, Shunsuke; Nakajima, Makoto; Kataoka, Keiichiro; Kim-Mitsuyama, Shokei; Tanaka, Motoko; Fukagawa, Masafumi; Otagiri, Masaki; Maruyama, Toru

    2013-04-01

    The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-β1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate. PMID:23325087

  19. Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress

    PubMed Central

    Wang, Xin; Ke, Zunji; Chen, Gang; Xu, Mei; Bower, Kimberly A.; Frank, Jacqueline A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

    2012-01-01

    It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47phox and p67phox, the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47phox and p67phox, which were prerequisites for NOX assembly and activation. Knocking down p47phox with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons. PMID:22662267

  20. Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

    PubMed

    Kassan, Modar; Ait-Aissa, Karima; Ali, Maha; Trebak, Mohamed; Matrougui, Khalid

    2015-10-01

    We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2. PMID:26036345

  1. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  2. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  3. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis

    PubMed Central

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  4. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis.

    PubMed

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  5. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  6. Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

    PubMed Central

    Chu, Chun-Hsien; Qian, Li; Chen, Shih-Heng; Wilson, Belinda; Oyarzabal, Esteban; Jiang, Lulu; Ali, Syed; Robinson, Bonnie; Kim, Hyoung-Chun

    2014-01-01

    Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1−/−), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose–response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91phox and inducing membrane translocation of the cytosolic subunits p47phox and p67phox. The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD. PMID:25209287

  7. Current status of NADPH oxidase research in cardiovascular pharmacology

    PubMed Central

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

    2013-01-01

    The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new inhibitors. All this knowledge and the research presently underway will likely result in development of new drugs for inhibition of NADPH oxidase and application of therapeutic approaches based on their action, for the treatment of cardiovascular disease in the next few years. PMID:23983473

  8. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts

    PubMed Central

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A.

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  9. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts.

    PubMed

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  10. NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats

    PubMed Central

    Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.

    2010-01-01

    Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague–Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.− production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.− production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.− generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975–986. PMID:20406136

  11. Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

    PubMed Central

    Casbon, Amy-Jo; Long, Matthew E.; Dunn, Kenneth W.; Allen, Lee-Ann H.; Dinauer, Mary C.

    2012-01-01

    Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated. PMID:22822009

  12. Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    PubMed Central

    Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

    2012-01-01

    The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.−) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

  13. NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

    PubMed Central

    Sancho, Patricia; Mainez, Jèssica; Crosas-Molist, Eva; Roncero, César; Fernández-Rodriguez, Conrado M.; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat, Isabel

    2012-01-01

    A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes. PMID:23049784

  14. Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity

    PubMed Central

    RADA, B K; GEISZT, M; VAN BRUGGEN, R; NÉMET, K; ROOS, D; LIGETI, E

    2003-01-01

    The relation of O2.−-production and Ca2+ homeostasis was investigated in PLB-985 cell lines and neutrophilic granulocytes from peripheral blood. In differentiated wild-type PLB-985 cells, a high level of O2.−-production was associated with a significant decrease in the membrane potential and the inhibition of capacitative Ca2+ entry. These correlations were not observed in gp91phox –/– cells or in cells transfected with a non-functional mutant of gp91phox (Thr341Lys). Membrane depolarization and inhibition of Ca2+ entry reappeared in cells transfected with wild-type gp91phox. These experiments demonstrate that inhibition of Ca2+ entry depends on the presence of a functional NADPH oxidase. The Ca2+ signal induced by stimulation of chemotactic receptors also showed remarkable differences: [Ca2+]ic in the sustained phase was higher in gp91phox–/– than in wild-type cells. Alteration of the Ca2+ signal was reproduced by treating peripheral blood neutrophils with the NADPH oxidase inhibitor diphenylene-iodonium. It is concluded that the deficiency in O2.−-production is accompanied by significant alterations of Ca2+ homeostasis in myeloid cells. PMID:12653836

  15. Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

    PubMed Central

    Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

  16. Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation

    PubMed Central

    Dubiella, Ullrich; Seybold, Heike; Durian, Guido; Komander, Eileen; Lassig, Roman; Witte, Claus-Peter; Schulze, Waltraud X.; Romeis, Tina

    2013-01-01

    In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid–mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant. PMID:23650383

  17. NADPH-oxidase 2 activation promotes opioid-induced antinociceptive tolerance in mice

    PubMed Central

    Doyle, Timothy; Esposito, Emanuela; Bryant, Leesa; Cuzzocrea, Salvatore; Salvemini, Daniela

    2013-01-01

    The analgesic effectiveness of long-term opioid therapies is compromised by the development of antinociceptive tolerance linked to the overt production of peroxynitrite (ONOO−, PN), the product of the interaction between superoxide (O2˙−, SO) and nitric oxide (NO), and to neuroinflammatory processes. We have recently reported that in addition to post-translational nitration and inactivation of mitochondrial MnSOD, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme (NOX) in the spinal cord is a major source for the overt production of superoxide-derived PN during the development of morphine-induced antinociceptive tolerance. However, the NOX complex involved in these processes is not known. The objective of these studies is to identify a potential role for the NOX2 complex, an enzyme involved in inflammation. Mice lacking the catalytic subunit of NOX2 (Nox2−/−) or its regulatory subunit, p47phox (p47phox−/−), developed antinociceptive tolerance similar to wildtype (wt) mice after three days of continuous morphine. However, while wt mice continue to develop tolerance by day six, morphine analgesia was restored in both Nox2−/− and p47phox−/− mice. Moreover, the loss of Nox2 or p47 did not affect acute morphine analgesia in naïve mice. In wt mice, antinociceptive tolerance was associated with increased activation of NOX, nitration of MnSOD, and proinflammatory cytokines production in spinal cord. These events were markedly attenuated in Nox2−/− and p47phox−/− mice and instead, there was enhanced formation of antiinflammatory cytokine (IL4 and IL10) production. These results suggest that NOX2 activity provides a significant source of superoxide-derived PN to undertake post-translational modifications of mitochondrial MnSOD and to engage neuroinflammatory signaling in the spinal cord associated with opioid-induced antinociceptive tolerance. Thus, NOX2 may provide a potential target for adjuvant therapy to protect opioid analgesia. PMID:23454539

  18. The antioxidant activity of soursop decreases the expression of a member of the NADPH oxidase family.

    PubMed

    Zamudio-Cuevas, Y; Díaz-Sobac, R; Vázquez-Luna, A; Landa-Solís, C; Cruz-Ramos, M; Santamaría-Olmedo, M; Martínez-Flores, K; Fuentes-Gómez, A J; López-Reyes, A

    2014-02-01

    Cellular oxidative stress produced by an increase in free radicals is one of the factors that promote the development of chronic degenerative diseases; therefore, consuming natural antioxidants helps minimize their negative effects. This study evaluated the cytotoxicity of the soursop extract (Annona muricata), its cytoprotective capacity against oxidative stress induced by hydrogen peroxide, the inhibitory potential of reactive oxygen species (ROS), the molecular mechanism of its antioxidant action, and its capacity to repair cellular damage in the fibroblast cell line. The soursop extract proved not to be cytotoxic in fibroblast cultures and showed cytoprotective capacity against hydrogen peroxide-induced stress; in cell culture it reduced the generation of ROS significantly by inhibiting a sub-unit of the NADPH oxidase enzyme (p47phox). The soursop extract can prevent damage caused by cellular oxidants. PMID:24337133

  19. Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity.

    PubMed

    Roca, M Gabriela; Weichert, Martin; Siegmund, Ulrike; Tudzynski, Paul; Fleissner, Andr

    2012-03-01

    In many filamentous ascomycete species, the early steps of colony development include fusion between germinating vegetative spores (conidial germlings). Often these fusion events are mediated by specialized hyphal structures, so-called conidial anastomosis tubes (CATs). Here, we show that germling fusion in the grey mould Botrytis cinerea is mediated by hyphal structures possessing the typical features of CATs. Formation of these structures is delayed when spores are germinating on complex media compared to growth on poor substrates. Fusion frequency is also influenced by the growth conditions of the precultures from which spores were obtained. During germination on hydrophobic plant surfaces, which induce pathogenic development, CAT formation is significantly suppressed. Screening of existing B. cinerea gene knockout mutants identified strains lacking the NADPH oxidase BcNoxA or the potential Nox regulator BcNoxR as fusion deficient, suggesting a potential role of reactive oxygen species (ROS) signalling in CAT formation and fusion. PMID:22385620

  20. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    SciTech Connect

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin; Park, Ji-hoon; Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook; Kim, Soon Ha

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

  1. Role of NADPH Oxidase and Xanthine Oxidase in Mediating Inducible VT/VF and Triggered Activity in a Canine Model of Myocardial Ischemia

    PubMed Central

    Martins, James B.; Chaudhary, Ashok K.; Jiang, Shuxia; Kwofie, Michael; Mackie, Prescott; Miller, Francis J.

    2014-01-01

    Background: Ventricular tachycardia or fibrillation (VT/VF) of focal origin due to triggered activity (TA) from delayed afterdepolarizations (DADs) is reproducibly inducible after anterior coronary artery occlusion. Both VT/VF and TA can be blocked by reducing reactive oxygen species (ROS). We tested the hypothesis that inhibition of NADPH oxidase and xanthine oxidase would block VT/VF. Methods: 69 dogs received apocynin (APO), 4 mg/kg intraveneously (IV), oxypurinol (OXY), 4 mg/kg IV, or both APO and OXY (BOTH) agents, or saline 3 h after coronary occlusion. Endocardium from ischemic sites (3-D mapping) was sampled for Rac1 (GTP-binding protein in membrane NADPH oxidase) activation or standard microelectrode techniques. Results (mean ± SE, * p < 0.05): VT/VF originating from ischemic zones was blocked by APO in 6/10 *, OXY in 4/9 *, BOTH in 5/8 * or saline in 1/27; 11/16 VT/VFs blocked were focal. In isolated myocardium, TA was blocked by APO (10−6 M) or OXY (10−8 M). Rac1 levels in ischemic endocardium were decreased by APO or OXY. Conclusion: APO and OXY suppressed focal VT/VF due to DADs, but the combination of the drugs was not more effective than either alone. Both drugs inhibited ischemic Rac1 with inhibition by OXY suggesting ROS-induced ROS. The inability to totally prevent VT/VF suggests that other mechanisms also contribute to ischemic VT. PMID:25375191

  2. NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1β secretion but not in inflammasome activation.

    PubMed

    Gabelloni, María Laura; Sabbione, Florencia; Jancic, Carolina; Fuxman Bass, Juan; Keitelman, Irene; Iula, Leonardo; Oleastro, Matías; Geffner, Jorge R; Trevani, Analía S

    2013-12-01

    Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role. PMID:23963575

  3. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology. Antioxid. Redox Signal. 20, 2755–2775. PMID:24386930

  4. The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages

    PubMed Central

    Liu, Honglei; Zhang, Hongqiao; Iles, Karen E.; Rinna, Alessandra; Merrill, Gary; Yodoi, Junji; Torres, Martine; Forman, Henry Jay

    2009-01-01

    The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of O2•− and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx). ADP induced the dissociation of ASK1/Trx complex and thus resulted in ASK1 activation, as assessed by phosphorylation at Thr845, which was enhanced after treatment with aurothioglucose (ATG), an inhibitor of Trx reductase. While dissociation of the complex implies Trx oxidation, protein electrophoretic mobility shift assay detected oxidation of Trx only after bolus H2O2 but not after ADP stimulation. These results demonstrate that the ADP-stimulated respiratory burst activated the ASK1-MKK4-JNK1/c-Jun signaling pathway in AM and suggest that transient and localized oxidation of Trx by the NADPH oxidase-mediated generation of H2O2 may play a critical role in ASK1 activation and the inflammatory response. PMID:17015265

  5. Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease

    PubMed Central

    Pushkaran, Suvarnamala; Konstantinidis, Diamantis G.; Koochaki, Sebastian; Malik, Punam; Mohandas, Narla; Zheng, Yi; Joiner, Clinton H.; Kalfa, Theodosia A.

    2013-01-01

    Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca2+ signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor β1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD. PMID:23349388

  6. NADPH Oxidases in Chronic Liver Diseases

    PubMed Central

    Jiang, Joy X.; Török, Natalie J.

    2015-01-01

    Oxidative stress is a common feature observed in a wide spectrum of chronic liver diseases including viral hepatitis, alcoholic, and nonalcoholic steatohepatitis. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are emerging as major sources of reactive oxygen species (ROS). Several major isoforms are expressed in the liver, including NOX1, NOX2, and NOX4. While the phagocytic NOX2 has been known to play an important role in Kupffer cell and neutrophil phagocytic activity and inflammation, the nonphagocytic NOX homologues are increasingly recognized as key enzymes in oxidative injury and wound healing. In this review, we will summarize the current advances in knowledge on the regulatory pathways of NOX activation, their cellular distribution, and their role in the modulation of redox signaling in liver diseases. PMID:26436133

  7. Mono-O-methylated flavanols and other flavonoids as inhibitors of endothelial NADPH oxidase.

    PubMed

    Steffen, Yvonne; Gruber, Claudia; Schewe, Tankred; Sies, Helmut

    2008-01-15

    The dietary flavan-3-ol (-)-epicatechin improves the bioactivity of nitric oxide in arterial vessels in vivo. Moreover, it effectively protects cultured vascular endothelial cells from signs of oxidative stress and elevates intracellular nitric oxide in vitro. We addressed the effects of (-)-epicatechin, its metabolic conversion products and structurally related compounds on NADPH oxidase activity in intact human umbilical vein endothelial cells (HUVEC) and in cell lysates. (-)-Epicatechin proved to be an O2*(-)-scavenger but did not inhibit NADPH oxidase activity, whereas the converse pattern was observed for the metabolites 3'- and 4'-O-methyl epicatechin. The dimer procyanidin B2 and (-)-epicatechin glucuronide were O2*(-)-scavengers and inhibited NADPH oxidase. Analysis of structure-activity relations with 45 compounds suggests an apocynin-like mode of NADPH oxidase inhibition. Notably, HUVEC converted (-)-epicatechin to NADPH oxidase-inhibitory methyl ethers. These data identify endothelial NADPH oxidase as candidate target of dietary flavonoids and particularly of their metabolites. PMID:17996190

  8. Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

    PubMed Central

    Pugin, A.; Frachisse, J. M.; Tavernier, E.; Bligny, R.; Gout, E.; Douce, R.; Guern, J.

    1997-01-01

    Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date. PMID:12237354

  9. Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

    PubMed Central

    Sokolovska, Anna; Becker, Christine E.; Eddie Ip, WK; Rathinam, Vijay A.K.; Brudner, Matthew; Paquette, Nicholas; Tanne, Antoine; Vanaja, Sivapriya K.; Moore, Kathryn J.; Fitzgerald, Katherine A.; Lacy-Hulbert, Adam; Stuart, Lynda M.

    2013-01-01

    Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense. PMID:23644505

  10. NADPH Oxidase-Induced NALP3 Inflammasome Activation Is Driven by Thioredoxin-Interacting Protein Which Contributes to Podocyte Injury in Hyperglycemia

    PubMed Central

    Gao, Pan; He, Fang-Fang; Tang, Hui; Lei, Chun-Tao; Chen, Shan; Meng, Xian-Fang; Su, Hua; Zhang, Chun

    2015-01-01

    Diabetic nephropathy (DN) is one of the major causes of end-stage renal disease, and previously we demonstrated that NALP3 inflammasome was involved in the pathogenesis of DN. Here we investigated the mechanisms of NALP3 inflammasome activation in podocyte injury during DN. We found that, besides the activation of NALP3 inflammasome and upregulated thioredoxin-interacting protein (TXNIP), the glomerular expression of gp91phox, a subunit of NADPH oxidase, was enhanced in DN mice simultaneously. Inhibiting NADPH oxidase abrogated NALP3 inflammasome activation, and IL-1β production and eventually protected podocytes from high glucose- (HG-) induced injury. TXNIP, an inhibitor of thioredoxin, acts as a suppressor for antioxidant defense system. Our observation indicated that in HG-exposed podocytes genetic deletion of TXNIP by shRNA reversed gp91phox overexpression and alleviated the injury of podocyte. Collectively, our findings proposed that HG-induced NADPH oxidase activation was driven by TXNIP which subsequently triggered NALP3 inflammasome activation in podocytes and ultimately led to podocyte injury, and blocking TXNIP/NADPH oxidase signaling may be a promising treatment for DN. PMID:25834832

  11. Essential role of nuclear factor-κB for NADPH oxidase activity in normal and anhidrotic ectodermal dysplasia leukocytes

    PubMed Central

    Luengo-Blanco, Marcos; Prando, Carolina; Bustamante, Jacinta; Aragão-Filho, Walmir Cutrim; Pereira, Paulo Vitor Soeiro; Rehder, Jussara; Padden, Carolyn; Casanova, Jean-Laurent; Condino-Neto, Antonio

    2008-01-01

    This work investigated the functional role of nuclear factor–κB (NF-κB) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-κB (IκBα-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorder of NF-κB function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X910 CGD). NCF1 gene expression in EDA-ID S32I cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A470) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-κB site 5′ to the CYBB gene in U937 cells treated with NF-κB inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-κB repressor. These studies show that NF-κB is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system. PMID:18523147

  12. Synaptic localization of a functional NADPH oxidase in the mouse hippocampus

    PubMed Central

    Tejada-Simon, Maria V.; Serrano, Faridis; Villasana, Laura E.; Kanterewicz, Beatriz I.; Wu, Gang-Yi; Quinn, Mark T.; Klann, Eric

    2007-01-01

    Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function. PMID:15866050

  13. Oxidative stress, NADPH oxidases, and arteries.

    PubMed

    Sun, Qi-An; Runge, Marschall S; Madamanchi, Nageswara R

    2016-05-10

    Atherosclerosis and its major complications - myocardial infarction and stroke - remain major causes of death and disability in the United States and world-wide. Indeed, with dramatic increases in obesity and diabetes mellitus, the prevalence and public health impact of cardiovascular diseases (CVD) will likely remain high. Major advances have been made in development of new therapies to reduce the incidence of atherosclerosis and CVD, in particular for treatment of hypercholesterolemia and hypertension. Oxidative stress is the common mechanistic link for many CVD risk factors. However, only recently have the tools existed to study the interface between oxidative stress and CVD in animal models. The most important source of reactive oxygen species (and hence oxidative stress) in vascular cells are the multiple forms of enzymes nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Recently published and emerging studies now clearly establish that: 1) NADPH oxidases are of critical importance in atherosclerosis and hypertension in animal models; 2) given the tissue-specific expression of key components of NADPH oxidase, it may be possible to target vascular oxidative stress for prevention of CVD. PMID:25649240

  14. Exposure of Madin-Darby Canine Kidney (MDCK) Cells to Oxalate and Calcium Oxalate Crystals Activates Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-Oxidase

    PubMed Central

    Khan, Aslam; Byer, Karen; Khan, Saeed R.

    2014-01-01

    OBJECTIVE To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. METHODS Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 μmol, 1 mmol Ox or 33, 66, 132 μg/cm2 CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. RESULTS Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. CONCLUSION Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells. PMID:24360063

  15. Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

    SciTech Connect

    Pan Xinjuan; Dai Yujie; Li Xing; Niu Nannan; Li Wenjie; Liu Fangli; Zhao Yang; Yu Zengli

    2011-08-01

    Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.

  16. NADPH oxidases in the arbuscular mycorrhizal symbiosis.

    PubMed

    Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

    2016-04-01

    Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

  17. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism.

    PubMed

    Yang, Ting; Peleli, Maria; Zollbrecht, Christa; Giulietti, Alessia; Terrando, Niccolo; Lundberg, Jon O; Weitzberg, Eddie; Carlström, Mattias

    2015-06-01

    Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response. PMID:25724690

  18. Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.

    PubMed Central

    Geiszt, M; Dagher, M C; Molnár, G; Havasi, A; Faure, J; Paclet, M H; Morel, F; Ligeti, E

    2001-01-01

    We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase. PMID:11311150

  19. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Dhar, Deepanshi; Panigrahi, Gati K; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P; Oberlies, Nicholas H; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  20. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    PubMed Central

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  1. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

  2. Modulation of cellular redox status by thiamine-activated NADPH oxidase confers Arabidopsis resistance to Sclerotinia sclerotiorum.

    PubMed

    Zhou, Jun; Sun, Aizhen; Xing, Da

    2013-08-01

    Sclerotinia sclerotiorum can initially suppress host oxidative burst to aid infection establishment, but later promotes reactive oxygen species (ROS) generation as proliferation advances. Here, it was shown that the cellular redox status can be modulated by thiamine to protect Arabidopsis thaliana against Sclerotinia at the early stages of infection. The initial inhibition of host ROS generation by Sclerotinia-secreted oxalate could effectively be alleviated by thiamine. Thiamine pre-treatment and subsequent wild-type Sclerotinia invasion induced an increase of ascorbate peroxidase activity concomitant with decreased ascorbate/dehydroascorbate ratios, which led to the cellular transition towards oxidative status in infected tissues. Particularly, it was observed that wild-type Sclerotinia, but not oxalate-deficient A2 mutant, could suppress the activity of NADPH oxidase (NOX), which might be an important mechanism underlying the early inhibition of ROS burst. Nevertheless, thiamine pre-treatment followed by wild-type Sclerotinia infection promoted NOX-derived ROS accumulation. Further studies showed that cytosolic Ca(2+) and staurosporine-sensitive protein kinase(s) participated in thiamine-induced activation of NOX. Moreover, thiamine-induced tissue defence responses including callose/lignin deposition and stomatal closure were closely correlated with NOX-derived ROS generation. Additionally, studies with Brassica species indicated that the regulation of thiamine is largely conserved upon Sclerotinia infection. Collectively, it was concluded that thiamine reverses the initial reducing status through activating NOX-dependent ROS signalling to perturb the disease progress of Sclerotinia. PMID:23814275

  3. NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases.

    PubMed

    Potocký, Martin; Pejchar, Přemysl; Gutkowska, Małgorzata; Jiménez-Quesada, María José; Potocká, Andrea; Alché, Juan de Dios; Kost, Benedikt; Žárský, Viktor

    2012-11-01

    Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca(2+) and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway. PMID:22762791

  4. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets

    PubMed Central

    Drummond, Grant R.; Selemidis, Stavros; Griendling, Kathy K.; Sobey, Christopher G.

    2012-01-01

    NADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS). The NOX1 (NADPH oxidase 1) and NOX2 oxidases are the major sources of ROS in the artery wall in conditions such as hypertension, hypercholesterolaemia, diabetes and ageing, and so they are important contributors to the oxidative stress, endothelial dysfunction and vascular inflammation that underlies arterial remodelling and atherogenesis. In this Review, we advance the concept that compared to the use of conventional antioxidants, inhibiting NOX1 and NOX2 oxidases is a superior approach for combating oxidative stress. We briefly describe some common and emerging putative NADPH oxidase inhibitors. In addition, we highlight the crucial role of the NADPH oxidase regulatory subunit, p47phox, in the activity of vascular NOX1 and NOX2 oxidases, and suggest how a better understanding of its specific molecular interactions may enable the development of novel isoform-selective drugs to prevent or treat cardiovascular diseases. PMID:21629295

  5. Bone Morphogenetic Protein-2 Activates NADPH Oxidase to Increase Endoplasmic Reticulum Stress and Human Coronary Artery Smooth Muscle Cell Calcification

    PubMed Central

    Liberman, Marcel; Johnson, Rebecca C.; Handy, Diane E.; Loscalzo, Joseph; Leopold, Jane A.

    2011-01-01

    Bone morphogenetic protein-2 (BMP-2) increases oxidant stress and endoplasmic reticulum (ER) stress to stimulate differentiation of osteoblasts; however, the role of these signaling pathways in the transition of smooth muscle cells to a calcifying osteoblast-like phenotype remains incompletely characterized. We, therefore, treated human coronary artery smooth muscle cells (HCSMC) with BMP-2 (100 ng/ml) and found an increase in NADPH oxidase activity and oxidant stress that occurred via activation of the bone morphogenetic protein receptor 2 and Smad 1 signaling. BMP-2-mediated oxidant stress also increased endoplasmic reticulum (ER) stress demonstrated by increased expression of GRP78, phospho-IRE1α, and the transcription factor XBP1. Analysis of a 1 kb segment of the Runx2 promoter revealed an XBP1 binding site; electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that XBP1 bound to the Runx2 promoter at this site in BMP-2-treated HCSMC. Inhibition of oxidant stress or ER stress decreased Runx2 expression, intracellular calcium deposition, and mineralization of BMP-2-treated HCSMC. Thus, in HCSMC, BMP-2 increases oxidant stress and ER stress to increase Runx2 expression and promote vascular smooth muscle cell calcification. PMID:21907184

  6. NADPH oxidase activity is essential for Keap1/Nrf2-mediated induction of GCLC in response to 2-indol-3-yl-methylenequinuclidin-3-ols.

    PubMed

    Sekhar, Konjeti R; Crooks, Peter A; Sonar, Vijayakumar N; Friedman, David B; Chan, Jeff Y; Meredith, Michael J; Starnes, Joseph H; Kelton, Kathy R; Summar, Samantha R; Sasi, Soumya; Freeman, Michael L

    2003-09-01

    Glutamate cysteine ligase, the rate-limiting enzyme for the synthesis of glutathione, represents an important component of chemoprevention paradigms. GCLC and GCLM, the genes encoding glutamate cysteine ligase subunits, are induced by indoles, such as indomethacin. Novel functionalized indole analogues and other structurally related compounds were synthesized and used for a comparative structure analysis of GCLC induction. Use of mouse embryo fibroblasts null for Nrf2 (nuclear factor-erythroid 2p45-related transcription factor) and HepG2 cells overexpressing Keap1 demonstrated that indole analogue-mediated GCLC expression was regulated by Nrf2-Keap1 interactions. Indole analogues capable of inducing GCLC were found to increase NADPH oxidase activity. Indole analogues unable to induce GCLC did not increase oxidase activity. HepG2 cells transfected with FLAG/Keap1 were exposed to indomethacin, and the redox state of Keap1 cysteine residues was assessed. The data indicated that Keap1 exhibited several oxidation states that were sensitive to indomethacin treatment. These indomethacin-mediated changes in thiol oxidation states were suppressed by diphenyleneiodonium, a NADPH oxidase inhibitor. Diphenyleneiodonium also suppressed indole analogue-mediated increases in GCLC mRNA. In summary, the use of the indole analogues identified NADPH oxidase activity as a novel upstream activity regulating Nrf2/Keap1 signaling of GCLC, provided data supporting the hypothesis that Keap1 is a downstream effector for oxidase activity, and afforded in vivo data to support the hypothesis that Keap1 thiols can act as molecular sensors of reactive oxygen species. Finally, the comparative structure analysis suggests that 2-indol-3-yl-methylenequinuclidin-3-ols may represent a prototype for the development of novel chemopreventative agents able to activate Keap1/Nrf2 signaling. PMID:14500406

  7. Impaired Priming and Activation of the Neutrophil NADPH Oxidase in Patients with IRAK4- or NEMO-deficiency *

    PubMed Central

    Singh, Anjali; Zarember, Kol A.; Kuhns, Douglas B.; Gallin, John I.

    2013-01-01

    The NADPH oxidase (NOX), an oligomeric enzyme, plays a key role in polymorphonuclear neutrophil (PMN)-mediated host defense by producing cytotoxic superoxide anion (O2.−). Whereas in vitro and biochemical studies have examined the assembly and activation of this important host immune defense system, few studies have examined the function of NOX in human patients with primary immunodeficiencies other than chronic granulomatous disease. We studied the activation of NOX in PMN from patients with two distinct immunodeficiencies, interleukin-1 receptor associated kinase 4 (IRAK4) deficiency and nuclear factor kappa (NF-κB) essential modulator (NEMO or IKKγ) deficiency. We observed impaired O2.− generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4. NEMO-deficient PMN generated significantly less O2.− in response to LPS-primed fMLP and translocated less p67phox than normal PMN although p47phox and Rac2 translocation were normal. Generally, responses of NEMO-deficient cells were intermediate between IRAK4-deficient cells and normal cells. Decreased LPS and fMLP induced phosphorylation of p38 MAPK in both IRAK4- and NEMO-deficient PMN implicates additional signal transduction pathways in regulating PMN activation by LPS and fMLP. Decreased activation of NOX may contribute to the increased risk of infection seen in patients with IRAK4- and NEMO-deficiency. PMID:19414794

  8. A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse LungS⃞

    PubMed Central

    Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Zagorski, John; Mesaros, Clementina; Blair, Ian A.; Feinstein, Sheldon I.; Jain, Mahendra

    2013-01-01

    1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02–0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67–87% of the injected dose for i.t. and 23–42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24–72 hours. Mice treated with MJ33 at 12.5–25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation. PMID:23475902

  9. NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney.

    PubMed

    Zhu, Kai; Kakehi, Tomoko; Matsumoto, Misaki; Iwata, Kazumi; Ibi, Masakazu; Ohshima, Yoichi; Zhang, Jia; Liu, Junjie; Wen, Xiaopeng; Taye, Ashraf; Fan, Chunyuan; Katsuyama, Masato; Sharma, Kumar; Yabe-Nishimura, Chihiro

    2015-06-01

    Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16(INK4A) and p21(Cip1), were equivalent between the genotypes, increased levels of p27(Kip1) and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27(Kip1) signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy. PMID:25701431

  10. NADPH Oxidase 4 Induces Cardiac Fibrosis and Hypertrophy through Activating Akt/mTOR and NFκB Signaling Pathways

    PubMed Central

    Zhao, Qingwei David; Viswanadhapalli, Suryavathi; Williams, Paul; Shi, Qian; Tan, Chunyan; Yi, Xiaolan; Bhandari, Basant; Abboud, Hanna E.

    2015-01-01

    Background NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived ROS in the myocardium. We investigated the role of Nox4 and Nox4 associated signaling pathways in the development of cardiac remodeling. Methods and Results Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: 1) control mice (CTL): littermates that are negative for hNox4 transgene but Cre positive; 2) c-hNox4 Tg mice; 3) angiotensin II (AngII)-infused CTL mice and 4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited approximately 10-fold increase in Nox4 protein expression and 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII-infusion to CTL mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least partially by enhancing Nox4 expression. Moreover, hNox4 transgene and/or AngII-infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed Akt-mTOR and NFκB signaling pathway and attenuated cardiac remodeling. Conclusion Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling. PMID:25589557

  11. Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation.

    PubMed

    Awasthi, Deepika; Nagarkoti, Sheela; Kumar, Amit; Dubey, Megha; Singh, Abhishek Kumar; Pathak, Priya; Chandra, Tulika; Barthwal, Manoj Kumar; Dikshit, Madhu

    2016-04-01

    Neutrophil extracellular traps (NETs) formation was initially linked with host defence and extracellular killing of pathogens. However, recent studies have highlighted their inflammatory potential. Oxidized low density lipoprotein (oxLDL) has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including systemic inflammatory response syndrome (SIRS). In the present study we investigated effect of oxLDL on NETs formation and elucidated the underlying signalling mechanism. Treatment of oxLDL to adhered PMNs led to a time and concentration dependent ROS generation and NETs formation. OxLDL induced free radical formation and NETs release were significantly prevented in presence of NADPH oxidase (NOX) inhibitors suggesting role of NOX activation in oxLDL induced NETs release. Blocking of both toll like receptor (TLR)-2 and 6 significantly reduced oxLDL induced NETs formation indicating requirement of both the receptors. We further identified Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), mitogen-activated protein kinase (MAPK) pathway as downstream intracellular signalling mediators involved in oxLDL induced NETs formation. OxLDL components such as oxidized phospholipids (lysophosphatidylcholine (LPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)) were most potent NETs inducers and might be crucial for oxLDL mediating NETs release. Other components like, oxysterols, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were however less potent as compared to oxidized phospholipids. This study thus demonstrates for the first time that treatment of human PMNs with oxLDL or its various oxidized phopholipid component mediated NETs release, implying their role in the pathogenesis of inflammatory diseases such as SIRS. PMID:26774674

  12. Targeting NADPH Oxidases for the Treatment of Cancer and Inflammation

    PubMed Central

    Bonner, Michael Y.; Arbiser, Jack L

    2015-01-01

    NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these Highly reactive and short lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in subunit composition, cellular location, and tissue specific expression. While the complexity of the nox family (Nox1–5, Duox1,2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. This review will discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of nox inhibitors. PMID:22581366

  13. Farrerol can attenuate the aortic lesion in spontaneously hypertensive rats via the upregulation of eNOS and reduction of NAD(P)H oxidase activity.

    PubMed

    Qin, Xiaojiang; Hou, Xiaomin; Liang, Taigang; Chen, Lijun; Lu, Taotao; Li, Qingshan

    2015-12-15

    Farrerol, a typical natural flavanone, is the major active component of Rhododendron dauricum L. The objective of this study was to evaluate the attenuation effect of farrerol against the aortic lesions in spontaneously hypertensive rats (SHR) for the first time. Twelve-week-old male SHR were orally administered with farrerol (50mg/kg/day), verapamil (50mg/kg/day, positive control), or vehicle for 8 weeks (n=10 in each group). Age-matched Wistar-Kyoto rats (WKY) served as normal controls (n=10). Our results revealed that farrerol significantly reduced the systolic blood pressure in SHR (from 177±4mmHg to 158±5mmHg) and also dramatically attenuated the aortic lesion, which is characterized by decreased media thickness, wall area, media-lumen ratio, nuclei size and an increased nuclei number (P<0.05). Moreover, the levels of O2(-) along with NAD(P)H oxidase activity were reduced (P<0.05), while the activity of endothelial nitric oxide synthase (eNOS) was elevated (P<0.05) in aortic homogenates after the intervention of farrerol. Furthermore, farrerol upregulated the expression of eNOS in both of mRNA and protein levels, accompanied by the downregulated mRNA and protein expression of p22(phox) (P<0.05), an essential subunit for NADPH oxidase activity. Our findings indicated that farrerol has a significant protective effect against the aortic lesion in SHR, which may be related to the enhanced eNOS activity and reduced NAD(P)H oxidase activity. PMID:26593430

  14. NADPH oxidases are critical targets for prevention of ethanol-induced bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms through which chronic alcohol consumption induce bone loss and osteoporosis are largely unknown. Ethanol increases expression and activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzymes (Nox) in osteoblasts leading to accumulation of reactive oxygen spe...

  15. NADPH-oxidase activity and lipid peroxidation in neutrophils from rats fed fat-rich diets.

    PubMed

    Lopes, L R; Laurindo, F R; Mancini-Filho, J; Curi, R; Sannomiya, P

    1999-03-01

    In order to investigate the effect of fat-rich diets on neutrophil functions, 21 day-aged rats were fed for 6 weeks with a control diet consisting of a regular laboratory rodent chow (4 per cent final fat content), a control diet supplied with soybean oil (15 per cent final fat content), or a control diet supplied with coconut oil (15 per cent final fat content). Glycogen-elicited peritoneal neutrophils from rats fed soybean and coconut oil-enriched diets presented a reduction in spontaneous and PMA-stimulated H2O2 generation relative to neutrophils from rats fed the control diet. The activity of superoxide dismutase, glutathione peroxidase and catalase did not change in animals fed fat-rich diets. In addition, the capacity to generate O2-, spontaneously or in response to PMA, did not change in neutrophils from animals fed fat-rich diets. Values attained matched those observed in animals fed the control diet, regardless of the method used to measure O2-, the superoxide dismutase-inhibitable reduction of cytochrome c or the lucigenin-dependent chemiluminescence. However, the initial rate of O2- generation both in resting neutrophils and in PMA-stimulated cells was significantly reduced when animals were fed with coconut or soybean oil-enriched diets due, at least in part, to a reduction in the activity of glucose-6-phosphate dehydrogenase. The concentration of thiobarbituric acid reactive substances, an index of lipid peroxidation, was increased in animals fed both fat-rich diets. This was accompanied by an increase in arachidonic acid content in these cells. Results presented suggest that lipid peroxidation in neutrophils from animals fed fat-rich diets may be associated with a consumption of H2O2 yielding more reactive oxygen-derived species such as the hydroxyl radical. PMID:10191509

  16. Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells.

    PubMed

    Lin, Chih-Chung; Lee, I-Ta; Wu, Wan-Ling; Lin, Wei-Ning; Yang, Chuen-Mao

    2012-09-01

    Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5'-O-(3-thiotriphosphate) (ATPγS) led to an increase in COX-2 gene expression and prostaglandin E(2) (PGE(2)) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATPγS induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)-κB cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-κB and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-κB cascade stimulated by ATPγS. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE(2) expression in ATPγS-treated A549 cells. Taken together, we demonstrated that ATPγS stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/NF-κB/p300 cascade to regulate the expression of COX-2 and synthesis of PGE(2) in A549 cells. Understanding the regulation of COX-2 expression and PGE(2) release by ATPγS on A549 cells may provide potential therapeutic targets of NSCLC. PMID:22773695

  17. A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor-α

    PubMed Central

    Gustafson, Sally J.; Dunlap, Kriya L.; McGill, Colin M.; Kuhn, Thomas B.

    2012-01-01

    Inflammation and oxidative stress are key to the progressive neuronal degeneration common to chronic pathologies, traumatic injuries, and aging processes in the CNS. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) orchestrates cellular stress by stimulating the production and release of neurotoxic mediators including reactive oxygen species (ROS). NADPH oxidases (NOX), ubiquitously expressed in all cells, have recently emerged as pivotal ROS sources in aging and disease. We demonstrated the presence of potent NOX inhibitors in wild Alaska bog blueberries partitioning discretely into a nonpolar fraction with minimal antioxidant capacity and largely devoid of polyphenols. Incubation of SH-SY5Y human neuroblastoma cells with nonpolar blueberry fractions obstructed the coalescing of lipid rafts into large domains disrupting NOX assembly therein and abolishing ROS production characteristic for TNF-α exposure. These findings illuminate nutrition-derived lipid raft modulation as a novel therapeutic approach to blunt inflammatory and oxidative stress in the aging or diseased CNS. PMID:22530077

  18. NADPH oxidase limits lipopolysaccharide-induced lung inflammation and injury in mice through redox regulation of NF-κB activity

    PubMed Central

    Han, Wei; Li, Hui; Cai, Jiyang; Gleaves, Linda A.; Polosukhin, Vasiliy V.; Segal, Brahm H.; Yull, Fiona E.; Blackwell, Timothy S.

    2013-01-01

    Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type and p47phox deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-κB pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47phox-/- mice while these mice had increased neutrophilic alveolitis and greater lung injury compared to wild-type controls. In mice interbred with transgenic NF-κB reporters (HLL), we found exaggerated LPS-induced NF-κB activation and increased expression of pro-inflammatory cytokines in lungs of p47phox-/-/HLL mice compared to controls. Both lung macrophages and bone marrow-derived macrophages (BMDMs) isolated from p47phox-/-/HLL mice showed enhanced LPS-stimulated NF-κB activity compared to controls. While nuclear translocation of NF-κB proteins was similar between genotypes, electrophoretic mobility shift assays under non-reducing conditions showed increased DNA binding in p47phox-/-/HLL BMDMs, suggesting that ROS production reduces NF-κB binding to DNA without affecting nuclear translocation. Increased intracellular GSH/GSSG ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47phox-/-/HLL compared to wild-type BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1 specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-κB activation in p47phox-/-/HLL BMDMs but not in wild-type/HLL cells. Consistent with these findings, siRNA against Ref-1 selectively reduced NF-κB activity in LPS-treated p47phox-/-/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-κB transcriptional activity through regulation of intracellular redox state. PMID:23530143

  19. NADPH oxidase mediates the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage.

    PubMed

    Tang, Xiangqi; Zhong, Wei; Tu, Qiuyun; Ding, Binrong

    2014-02-01

    Oxygen free radicals and their reactive lipid peroxidation are known to be elements promoting ischemia-reperfusion damage. NADPH oxidase is a major factor in peroxide production. Excessive production of oxygen free radicals is considered as an important mechanism in the expression of matrix metalloproteinase (MMP)-9 and in damage to the blood-brain barrier (BBB). In this study, we evaluated changes in the expression of the NADPH oxidase catalytic subunit gp91(phox) and oxidase activity, as well as the involvement of NADPH oxidase catalysis in the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage. A middle cerebral artery occlusion (MCAO) model was established using male Sprague-Dawley (SD) rats. Brain tissue was isolated for triphenyltetrazolium chloride (TTC) staining, gp91(phox) mRNA quantitative PCR analysis, western blot analysis, NADPH oxidase activity determination (detection), and MMP-9 gelatin zymography analysis. In the MCAO rats, gp91(phox) and MMP-9 expression was upregulated in the ischemic hemisphere of the brain tissue after 90 minutes of MCAO with 22·5 hours of reperfusion. Inhibition of NADPH oxidase with apocynin reduced the increase in MMP-9. These results suggest that NADPH oxidase is a major precipitating factor for the expression of MMP-9 in the ischemic brain tissue. PMID:24131725

  20. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    PubMed Central

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  1. Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species.

    PubMed

    Goyal, Parag; Weissmann, Norbert; Grimminger, Friedrich; Hegel, Cornelia; Bader, Lucius; Rose, Frank; Fink, Ludger; Ghofrani, Hossein A; Schermuly, Ralph T; Schmidt, Harald H H W; Seeger, Werner; Hänze, Jörg

    2004-05-15

    Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways. PMID:15110393

  2. Oxalate-induced activation of PKC-α and -δ regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

    PubMed Central

    Menon, Mani; Thamilselvan, Sivagnanam

    2009-01-01

    Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-α and -δ from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-α or -δ significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-α and -δ. Silencing of PKC-α expression by PKC-α-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-α- and -δ-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers. PMID:19692488

  3. Cannabinoids prevent lipopolysaccharide-induced neurodegeneration in the rat substantia nigra in vivo through inhibition of microglial activation and NADPH oxidase.

    PubMed

    Chung, Eun Sook; Bok, Eugene; Chung, Young Cheul; Baik, Hyung Hwan; Jin, Byung Kwan

    2012-04-27

    We investigated the effects of synthetic cannabinoids, WIN55,212-2 and HU210, on LPS-injected rat substantia nigra in vivo. Intranigral injection of LPS resulted in a significant loss of nigral dopaminergic (DA) neurons, as determined by Nissl staining and TH immunohistochemistry. LPS-induced neurotoxicity was accompanied by microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis, ELISA assay and hydroethidine histochemistry revealed activation of NADPH oxidase, as demonstrated by increased translocation of the cytosolic proteins p47(phox) and p67(phox), generation of reactive oxygen species (ROS) and increased level of proinflammatory cytokines (TNF-α and IL-1β), where degeneration of nigral DA neurons was evident. Interestingly, WIN55,212-2 and HU210 increased the survival of nigral DA neurons at 7days post-LPS treatment. Consistent with these results, cannabinoids inhibited activation of NADPH oxidase, ROS production and production of proinflammatory cytokines in the rat SN. The present data suggest that cannabinoids may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglial activation. PMID:22436849

  4. Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

    PubMed Central

    Grimm, Melissa J.; Lewandowski, David C.; Pham, Christine T. N.; Blackwell, Timothy S.; Petraitiene, Ruta; Petraitis, Vidmantas; Walsh, Thomas J.; Urban, Constantin F.; Segal, Brahm H.

    2011-01-01

    NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens. PMID:22163282

  5. NADPH Oxidase 4-Derived H2O2 Promotes Aberrant Retinal Neovascularization via Activation of VEGF Receptor 2 Pathway in Oxygen-Induced Retinopathy

    PubMed Central

    Li, Jingming; Wang, Joshua J.; Zhang, Sarah X.

    2015-01-01

    NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidase in retinal endothelial cells. Our previous study suggests that upregulation of Nox4 in retinal endothelial cells contributes to retinal vascular leakage in diabetes. In the current study, we investigated the role and mechanism of Nox4 in regulation of retinal neovascularization (NV), a hallmark of proliferative diabetic retinopathy (PDR), using a mouse model of oxygen-induced retinopathy (OIR). Our results confirmed that Nox4 was expressed predominantly in retinal vasculature of mouse retina. Retinal expression of Nox4 was markedly increased in OIR, in parallel with enhanced phosphorylation of ERK. In human retinal microvascular endothelial cells (HRECs), overexpression of Nox4 by adenovirus significantly increased extracellular H2O2 generation, resulting in intensified VEGFR2 activation and exacerbated angiogenesis upon VEGF stimulation. In contrast, silencing Nox4 expression or scavenging H2O2 by polyethylene glycol- (PEG-) conjugated catalase inhibited endothelial migration, tube formation, and VEGF-induced activation of VEGFR2 signaling. Importantly, knockdown of retinal Nox4 by adenovirus-delivered siRNA significantly reduced ERK activation and attenuated retinal NV formation in OIR. Taken together, our data indicate that Nox4 promotes retinal NV formation through H2O2/VEGFR2/ERK signaling pathway. Reducing retinal Nox4 expression may represent a promising therapeutic approach for neovascular retinal diseases such as PDR. PMID:25866826

  6. Differential effects of NADPH oxidase and xanthine oxidase inhibition on sympathetic reinnervation in postinfarct rat hearts.

    PubMed

    Lee, Tsung-Ming; Chen, Chien-Chang; Hsu, Yu-Jung

    2011-06-01

    Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway. PMID:21295134

  7. Role of NADPH Oxidases in Liver Fibrosis

    PubMed Central

    Paik, Yong-Han; Kim, Jonghwa; Aoyama, Tomonori; De Minicis, Samuele; Bataller, Ramon

    2014-01-01

    Abstract Significance: Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. Recent Advances: In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. Critical Issues: Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. Future Directions: A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antioxid. Redox Signal. 20, 2854–2872. PMID:24040957

  8. Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems.

    PubMed

    Fernandes, Denise C; Wosniak, João; Pescatore, Luciana A; Bertoline, Maria A; Liberman, Marcel; Laurindo, Francisco R M; Santos, Célio X C

    2007-01-01

    Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies. PMID:16971501

  9. Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke-prone SHR.

    PubMed

    Chen, X; Touyz, R M; Park, J B; Schiffrin, E L

    2001-09-01

    Ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) have antioxidant properties that could improve redox-sensitive vascular changes associated with hypertension. We determined whether vitamins C and E influence vascular function and structure in hypertension by modulating activity of NADPH oxidase and superoxide dismutase (SOD). Adult stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 3 groups: control (C; n=6), vitamin C-treated (vit C, 1000 mg/day; n=7), and vitamin E-treated (vit E, 1000 IU/day; n=8). All rats were fed 4% NaCl. Blood pressure was measured weekly. After 6 weeks of treatment, the rats were killed, and mesenteric arteries were mounted as pressurized preparations. Vascular O(2)(-) generation and NADPH oxidase activity were measured by chemiluminescence. Vascular SOD activity and plasma total antioxidant status (TAS) were determined spectrophotometrically. Blood pressure increased from 212+/-7 to 265+/-6 mm Hg in controls. Treatment prevented progression of hypertension (vit C, 222+/-6 to 234+/-14 mm Hg; vit E, 220+/-9 to 227+/-10 mm Hg). Acetylcholine-induced vasodilation was improved (P<0.05), and media-to-lumen ratio was reduced (P<0.05) in the treated rats. O(2)(-) was lower in vitamin-treated groups compared with controls (vit C, 10+/-4 nmol. min(-1). g(-1) dry tissue weight; vit E, 9.6+/-3.5 nmol. min(-1). g(-1) dry tissue weight; C, 21+/-9 nmol. min(-1). g(-1) dry tissue weight; P<0.05). Both vitamin-treated groups showed significant improvement (P<0.01) in TAS. These effects were associated with decreased activation of vascular NADPH oxidase (vit C, 46+/-10; vit E, 50+/-9; C, 70+/-16 nmol. min(-1). g(-1) dry tissue weight, P<0.05) and increased activation of SOD (vit C, 12+/-2; vit E, 8+/-1; C, 4.6+/-1 U/mg; P<0.05). Our results demonstrate that vitamins C and E reduce oxidative stress, improve vascular function and structure, and prevent progression of hypertension in SHRSP. These effects may be mediated via modulation of enzyme systems that generate free radicals. PMID:11566940

  10. Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).

    PubMed

    Ahmed, S; Prigmore, E; Govind, S; Veryard, C; Kozma, R; Wientjes, F B; Segal, A W; Lim, L

    1998-06-19

    Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase. PMID:9624165

  11. Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

    PubMed Central

    Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

    2013-01-01

    Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

  12. A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF.

    PubMed

    Kawarazaki, Tomoko; Kimura, Sachie; Iizuka, Ayako; Hanamata, Shigeru; Nibori, Hitomi; Michikawa, Masataka; Imai, Aya; Abe, Mitsutomo; Kaya, Hidetaka; Kuchitsu, Kazuyuki

    2013-12-01

    Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses. PMID:23872431

  13. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  14. Female mice lacking active nadph-oxidase enzymes are protected against “western diet”--induced obesity and metabolic syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADPH oxidase (Nox) enzymes have been implicated in regulation of adipocyte differentiation and inflammation in a variety of tissues. We examined the effects of feeding AIN-93G or a “Western diet” (WD) (45% fat, 0.5% cholesterol) on development of obesity and “metabolic syndrome” in wild type (WT) m...

  15. Bile Acid Reflux Contributes To Development of Esophageal Adenocarcinoma Via Activation Of Phosphatidylinositol-Specific Phospholipase Cγ2 And NADPH Oxidase NOX5-S

    PubMed Central

    Hong, Jie; Behar, Jose; Wands, Jack; Resnick, Murray; Wang, Li Juan; DeLellis, Ronald A.; Lambeth, David; Cao, Weibiao

    2009-01-01

    Gastroesophageal reflux disease complicated by Barrett’s esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, H2O2 production and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibiter of PI-PLC. PI-PLCβ1, β3, β4, γ1 and γ2, but not β2 and δ1 were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCγ2 or ERK-2 MAP kinase with siRNAs significantly decreased TDCA-induced increase in NOX5-S expression, H2O2 production and cell proliferation. In contrast, knockdown of PI-PLC β1, β3, β4, γ1 or ERK-1 MAP kinase had no significant effect. TDCA significantly increased ERK-2 phosphorylation, an increase which was reduced by U73122 or PI-PLCγ2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCγ2 and ERK-2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with Barrett’s esophagus may increase ROS production and cell proliferation via activation of PI-PLCγ2, ERK-2 MAP kinase and NADPH oxidase NOX5-S, thereby contributing to the development of EA. PMID:20086178

  16. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells.

    PubMed

    Prasad, Ram; Kappes, John C; Katiyar, Santosh K

    2016-02-16

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  17. Human Recombinant Cytochrome P450 Enzymes Display Distinct Hydrogen Peroxide Generating Activities During Substrate Independent NADPH Oxidase Reactions

    PubMed Central

    Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as “oxidase” activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the “oxidase” activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5–7), and when CPR was present in excess (CYP/CPR = 0.2–0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

  18. Trimer hydroxylated quinone (IIIHyQ) derived from apocynin targets cysteine residues of p47phox preventing the activation of human vascular NADPH oxidase

    PubMed Central

    Mora-Pale, Mauricio; Joon-Kwon, Seok; Linhardt, Robert J.; Dordick, Jonathan S.

    2012-01-01

    Enzymatic derived oligophenols from apocynin can be effective inhibitors of human vascular NADPH oxidase. An isolated IIIHyQ has been shown to inhibit endothelial NADPH oxidase with an IC50 ~30 nM. In vitro studies demonstrated that IIIHyQ is capable on disrupting the interaction between p47phox and p22phox, thereby blocking the activation of the Nox2 isoform. Herein, we report the role of key cysteine residues in p47phox as targets for the IIIHyQ. Incubation of p47phox with IIIHyQ results in a decrease of ~80% of the protein free cysteine residues; similar results were observed using 1,2- and 1,4-naphthoquinoes, while apocynin was unreactive. Mutants of p47phox, where each Cys was individually replaced by Ala (at residues 111, 196 and 378) and Gly (at residue 98), were generated to evaluate their individual importance in IIIHyQ-mediated inhibition of p47phox interaction with p22phox. Specific Michael addition on Cys196, within the N-SH3 domain, by the IIIHyQ is critical for disrupting the p47phox-p22phox interaction. When a C196A mutation was tested, the IIIHyQ was unable to disrupt the p47phox-p22phox interaction. However, the IIIHyQ was effective at disrupting this interaction with the other mutants, displaying IC50 values (4.9, 21.0, and 2.3 ?M for the C111A, C378A, and C98G mutants, respectively) comparable to that of wild type p47phox. PMID:22240153

  19. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    SciTech Connect

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-10-15

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces MMP-9 expression via a NADPH oxidase/ROS-dependent NF-κB signaling. • TNF-α activates MAPK phosphorylation through NADPH oxidase/ROS generation.

  20. Localization of NADPH Oxidase in Sympathetic and Sensory Ganglion Neurons and Perivascular Nerve Fibers

    PubMed Central

    Cao, Xian; Demel, Stacie L.; Quinn, Mark T.; Galligan, James J.; Kreulen, David L.

    2009-01-01

    Superoxide anion (O2−•) production was previously reported to be increased in celiac ganglia (CG) during DOCA-salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons is not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22phox and neuropeptide Y (NPY) were colocalized in all neurons. P22phox was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22phox and p47phox were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22phox and p47phox was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O2−• – generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control. PMID:19716351

  1. High-glucose-increased expression and activation of NADPH oxidase in human vascular smooth muscle cells is mediated by 4-hydroxynonenal-activated PPARα and PPARβ/δ.

    PubMed

    Manea, Adrian; Manea, Simona-Adriana; Todirita, Andra; Albulescu, Irina Cristina; Raicu, Monica; Sasson, Shlomo; Simionescu, Maya

    2015-08-01

    High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 μM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 μM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARβ/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARβ/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes. PMID:25722086

  2. Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme

    PubMed Central

    Katsuyama, Masato; Matsuno, Kuniharu; Yabe-Nishimura, Chihiro

    2012-01-01

    NADPH oxidase is a superoxide (O2•−)-generating enzyme first identified in phagocytes, essential for their bactericidal activities. Later, in non-phagocytes, production of O2•− was also demonstrated in an NADPH-dependent manner. In the last decade, several non-phagocyte-type NADPH oxidases have been identified. The catalytic subunit of these oxidases, NOX, constitutes the NOX family. There are five homologs in the family, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. Transgenic or gene-disrupted mice of the NOX family have also been established. NOX/DUOX proteins possess distinct features in the dependency on other components for their enzymatic activities, tissue distributions, and physiological functions. This review summarized the characteristics of the NOX family proteins, especially focused on their functions clarified through studies using gene-modified mice. PMID:22247596

  3. Thapsigargin Triggers Cardiac Contractile Dysfunction via NADPH Oxidase -Mediated Mitochondrial Dysfunction: Role of Akt Dephosphorylation

    PubMed Central

    Zhang, Yingmei; Ren, Jun

    2011-01-01

    ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in the ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. WT and transgenic mice with cardiac-specific overexpression of active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3 mg/kg, i.p. for 48 hr). Thapsigargin compromised echocardiographic parameters including elevated LVESD and reduced fractional shortening, suppressed cardiomyocyte contractile function, intracellular Ca2+ handling and cell survival, along with enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3β (leading to activation of GSK3β), the effect of which was abrogated in MyAkt hearts. Knockdown of cytosolic subunit of NADPH oxidase p47phox using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure of thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca2+ uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeation pore opening, similar to those induced by chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca2+ homeostasis, cell survival and mitochondrial integrity through an Akt dephosphorylation and NADPH oxidase-dependent mechanism. PMID:21996563

  4. Characterisation of electron currents generated by the human neutrophil NADPH oxidase

    SciTech Connect

    Ahluwalia, Jatinder

    2008-04-11

    Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I{sub e}) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I{sub e} when the NADPH oxidase is activated by NADPH and GTP{gamma}S. The neutrophil I{sub e} was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I{sub e} measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I{sub e} depolarises the neutrophil plasma membrane at a rate of 2.3 V s{sup -1} and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl{sub 2} depolarised the membrane potential to +97.8 {+-} 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.

  5. A leading role for NADPH oxidase in an in-vitro study of experimental autoimmune encephalomyelitis.

    PubMed

    Seo, Ji-Eun; Hasan, Mahbub; Rahaman, Khandoker Asiqur; Kang, Min-Jung; Jung, Byung-Hwa; Kwon, Oh-Seung

    2016-04-01

    Myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG35-55) is a major autoantigen inducing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis that is characterized by blood-brain barrier (BBB) disruption. Various experimental approaches have employed MOG35-55 in vivo; however, in vitro BBB models using MOG35-55 are rarely reported. We investigated MOG35-55 exposure effects with complete Freund's adjuvant (CFA) and pertussis toxin (PTX) on brain endothelial cells and elucidated the relationships among NADPH oxidase, MMP-9, ICAM-1, and VCAM-1. These 4 factors significantly increased in MOG35-55+CFA+PTX-exposed endothelial cells compared with the control cells. NADPH oxidase inhibition using apocynin reduced MMP-9 activity, ICAM-1, and VCAM-1. MMP-9 inhibitor I decreased expression of ICAM-1 and VCAM-1, and both anti-ICAM-1 and anti-VCAM-1 inhibited MMP-9 activity. Inhibitions of MMP-9, ICAM-1, and VCAM-1 did not change NADPH oxidase activity. Although inhibition of these 4 factors decreased BBB permeability in cells, inhibition of NADPH oxidase exhibited the highest decrease among these. NADPH oxidase directly influenced MMP-9, ICAM-1, and VCAM-1, but not vice versa. MMP-9 and the cell adhesion molecules reversibly affected each other. In conclusion, NADPH oxidase-derived superoxide elevated expression of MMP-9, ICAM-1, and VCAM-1, and these interactions can finally result in increases of BBB permeability in MOG35-55+CFA+PTX-exposed endothelial cells. PMID:26928315

  6. NOX3 NADPH Oxidase Couples Transient Receptor Potential Vanilloid 1 to Signal Transducer and Activator of Transcription 1-Mediated Inflammation and Hearing Loss

    PubMed Central

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P.

    2011-01-01

    Abstract Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. Antioxid. Redox Signal. 14, 999–1010. PMID:20712533

  7. NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss.

    PubMed

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P; Ramkumar, Vickram

    2011-03-15

    Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. PMID:20712533

  8. Glucose-6-phosphate dehydrogenase and NADPH oxidase 4 control STAT3 activity in melanoma cells through a pathway involving reactive oxygen species, c-SRC and SHP2

    PubMed Central

    Cai, Tianchi; Kuang, Yingmin; Zhang, Chunhua; Zhang, Zheng; Chen, Long; Li, Bo; Li, Yuqian; Wang, Yanling; Yang, Huixin; Han, Qiaoqiao; Zhu, Yuechun

    2015-01-01

    Background: Glucose-6-phosphate dehydrogenase (G6PD) participates in glucose utilization by catalysing the first step of the pentose-phosphate pathway in mammalian cells. Previous studies have shown that changes in G6PD levels can promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in a human melanoma xenograft model. G6PD cooperates with NADPH oxidase 4 (NOX4) in the cellular metabolism of reactive oxygen species (ROS) and in maintaining the intracellular redox state. Methods: In this study, the effect of G6PD or NOX4 silencing in the melanoma line A375 was examined in terms of redox state, proto-oncogene tyrosine-protein kinase Src (c-Src) and the tyrosine-specific protein phosphatase SHP2 expression as well as cell cycle progression. Results: The results demonstrate that: (1) Downregulation of cyclin D1 and CDK4 and up-regulation of p53 and p21 occurred in response to silencing of G6PD and NOX4 thus resulting in G1/S cell cycle arrest and inhibition of A375 cell proliferation. (2) The blockade of cell proliferation is primarily due to a reduced DNA-binding activity of STAT3. (3) The DNA-binding activity of STAT3 was regulated by the upstream factors, c-SRC and SHP2. Silencing of NOX4 in A375 cells inhibited c-SRC and SHP2 regulated STAT3 activity. Conclusion: The data are consistent with a novel G6PD-NOX4-NADPH-ROS-c-SRC/SHP2 pathway controlling STAT3 activity in A375 melanoma cells. PMID:26175932

  9. Cross-talk between IRAK-4 and the NADPH oxidase1

    PubMed Central

    Pacquelet, Sandrine; Johnson, Jennifer L.; Ellis, Beverly A.; Brzezinska, Agnieszka A.; Lane, William S.; Munafo, Daniela B.; Catz, Sergio D.

    2007-01-01

    Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation. PMID:17217339

  10. A subset of N-substituted phenothiazines inhibits NADPH oxidases.

    PubMed

    Seredenina, Tamara; Chiriano, Gianpaolo; Filippova, Aleksandra; Nayernia, Zeynab; Mahiout, Zahia; Fioraso-Cartier, Laetitia; Plastre, Olivier; Scapozza, Leonardo; Krause, Karl-Heinz; Jaquet, Vincent

    2015-09-01

    NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors. PMID:26013584

  11. A wheat superoxide dismutase gene TaSOD2 enhances salt resistance through modulating redox homeostasis by promoting NADPH oxidase activity.

    PubMed

    Wang, Mengcheng; Zhao, Xin; Xiao, Zhen; Yin, Xunhao; Xing, Tian; Xia, Guangmin

    2016-05-01

    Superoxide dismutase (SOD) is believed to enhance abiotic stress resistance by converting superoxide radical (O2 (-)) to H2O2 to lower ROS level and maintain redox homeostasis. ROS level is controlled via biphasic machinery of ROS production and scavenging. However, whether the role of SOD in abiotic stress resistance is achieved through influencing the biophasic machinery is not well documented. Here, we identified a wheat copper-zinc (Cu/Zn) SOD gene, TaSOD2, who was responsive to NaCl and H2O2. TaSOD2 overexpression in wheat and Arabidopsis elevated SOD activities, and enhanced the resistance to salt and oxidative stress. TaSOD2 overexpression reduced H2O2 level but accelerated O2 (-) accumulation. Further, it improved the activities of H2O2 metabolic enzymes, elevated the activity of O2 (-) producer NADPH oxidase (NOX), and promoted the transcription of NOX encoding genes. The inhibition of NOX activity and the mutation of NOX encoding genes both abolished the salt resistance of TaSOD2 overexpression lines. These data indicate that Cu/Zn SOD enhances salt resistance, which is accomplished through modulating redox homeostasis via promoting NOX activity. PMID:26869262

  12. Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

    PubMed Central

    Trepout, Sylvain; Wien, Frank; Marco, Sergio

    2015-01-01

    Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development. PMID:26714308

  13. A novel superoxide-producing NAD(P)H oxidase in kidney.

    PubMed

    Shiose, A; Kuroda, J; Tsuruya, K; Hirai, M; Hirakata, H; Naito, S; Hattori, M; Sakaki, Y; Sumimoto, H

    2001-01-12

    During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney. PMID:11032835

  14. Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous in vitro data suggest that ethanol (EtOH) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption obse...

  15. Regulation of NAD(P)H oxidases by AMPK in cardiovascular systems

    PubMed Central

    Song, Ping; Zou, Ming-Hui

    2012-01-01

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are ubiquitously produced in cardiovascular systems. Under physiological conditions, ROS/RNS function as signaling molecules that are essential in maintaining cardiovascular function. Aberrant concentrations of ROS/RNS have been demonstrated in cardiovascular diseases due to increased production or decreased scavenging, which have been considered as common pathways for the initiation and progression of cardiovascular diseases such as atherosclerosis, hypertension, (re)stenosis, and congestive heart failure. NAD(P)H oxidases are primary sources of ROS and can be induced or activated by all known cardiovascular risk factors. Stresses, hormones, vasoactive agents, and cytokines via different signaling cascades control the expression and activity of these enzymes and of their regulatory subunits. But the molecular mechanisms by which NAD(P)H oxidase is regulated in cardiovascular systems remain poorly characterized. Investigations by us and others suggest that adenosine monophosphate-activated protein kinase (AMPK), as an energy sensor and modulator, is highly sensitive to ROS/RNS. We have also obtained convincing evidence that AMPK is a physiological suppressor of NAD(P)H oxidase in multiple cardiovascular cell systems. In this review, we summarize our current understanding of how AMPK functions as a physiological repressor of NAD(P)H oxidase. PMID:22357101

  16. NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

    PubMed Central

    Cave, Alison; Grieve, David; Johar, Sofian; Zhang, Min; Shah, Ajay M

    2005-01-01

    Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease. PMID:16321803

  17. A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

    PubMed Central

    Matute, Juan D.; Arias, Andres A.; Wright, Nicola A. M.; Wrobel, Iwona; Waterhouse, Christopher C. M.; Li, Xing Jun; Marchal, Christophe C.; Stull, Natalie D.; Lewis, David B.; Steele, MacGregor; Kellner, James D.; Yu, Weiming; Meroueh, Samy O.; Nauseef, William M.

    2009-01-01

    Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease. PMID:19692703

  18. NADPH Oxidase-2: Linking Glucose, Acidosis, and Excitotoxicity in Stroke

    PubMed Central

    Brennan-Minnella, Angela M.; Won, Seok Joon

    2015-01-01

    Abstract Significance: Neuronal superoxide production contributes to cell death in both glutamate excitotoxicity and brain ischemia (stroke). NADPH oxidase-2 (NOX2) is the major source of neuronal superoxide production in these settings, and regulation of NOX2 activity can thereby influence outcome in stroke. Recent Advances: Reduced NOX2 activity can rescue cells from oxidative stress and cell death that otherwise occur in excitotoxicity and ischemia. NOX2 activity is regulated by several factors previously shown to affect outcome in stroke, including glucose availability, intracellular pH, protein kinase ζ/δ, casein kinase 2, phosphoinositide-3-kinase, Rac1/2, and phospholipase A2. The newly identified functions of these factors as regulators of NOX2 activity suggest alternative mechanisms for their effects on ischemic brain injury. Critical Issues: Key aspects of these regulatory influences remain unresolved, including the mechanisms by which rac1 and phospholipase activities are coupled to N-methyl-D-aspartate (NMDA) receptors, and whether superoxide production by NOX2 triggers subsequent superoxide production by mitochondria. Future Directions: It will be important to establish whether interventions targeting the signaling pathways linking NMDA receptors to NOX2 in brain ischemia can provide a greater neuroprotective efficacy or a longer time window to treatment than provided by NMDA receptor blockade alone. It will likewise be important to determine whether dissociating superoxide production from the other signaling events initiated by NMDA receptors can mitigate the deleterious effects of NMDA receptor blockade. Antioxid. Redox Signal. 22, 161–174. PMID:24628477

  19. Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

    PubMed Central

    Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y.; Wood, W. Gibson; Weisman, Gary A.

    2011-01-01

    Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A2 (PLA2) and secretory PLA2. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA2 can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease. PMID:20195796

  20. Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana.

    PubMed

    Andronis, Efthimios A; Moschou, Panagiotis N; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A

    2014-01-01

    Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions ([Formula: see text] ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and [Formula: see text] . These results suggest that the ratio of [Formula: see text] /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of [Formula: see text] by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed. PMID:24765099

  1. Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    PubMed Central

    Andronis, Efthimios A.; Moschou, Panagiotis N.; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A.

    2014-01-01

    Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2•− ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2•− . These results suggest that the ratio of O2•− /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2•− by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed. PMID:24765099

  2. Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis

    PubMed Central

    McCaffrey, Ramona L.; Schwartz, Justin T.; Lindemann, Stephen R.; Moreland, Jessica G.; Buchan, Blake W.; Jones, Bradley D.; Allen, Lee-Ann H.

    2010-01-01

    Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence. PMID:20610796

  3. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones

    PubMed Central

    Munnamalai, Vidhya; Weaver, Cory J.; Weisheit, Corinne E.; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T.; Suter, Daniel M.

    2014-01-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NOX2-type NADPH oxidase complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40phox. p40phox itself exhibited co-localization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91phox and p40phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in co-localization of p40phox with NOX2/gp91phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. PMID:24702317

  4. Upregulation of Intermediate-Conductance Ca2+-Activated K+ Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)

    PubMed Central

    Gole, Hope K. A.; Tharp, Darla L.; Bowles, Douglas K.

    2014-01-01

    Aims NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors. Methods and Results Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration. Conclusions Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis. PMID:25144362

  5. NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells.

    PubMed

    Joo, Jung Hee; Oh, Hyunjin; Kim, Myungjin; An, Eun Jung; Kim, Rae-Kwon; Lee, So-Young; Kang, Dong Hoon; Kang, Sang Won; Keun Park, Cheol; Kim, Hoguen; Lee, Su-Jae; Lee, Daekee; Seol, Jae Hong; Bae, Yun Soo

    2016-02-15

    The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis. Cancer Res; 76(4); 855-65. ©2016 AACR. PMID:26781991

  6. NADPH oxidases, reactive oxygen species, and the kidney: friend and foe.

    PubMed

    Sedeek, Mona; Nasrallah, Rania; Touyz, Rhian M; Hébert, Richard L

    2013-10-01

    Reactive oxygen species (ROS) play an important role in normal cellular physiology. They regulate different biologic processes such as cell defense, hormone synthesis and signaling, activation of G protein-coupled receptors, and ion channels and kinases/phosphatases. ROS are also important regulators of transcription factors and gene expression. On the other hand, in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. NADPH oxidases are one of the many sources of ROS in biologic systems, and there are seven isoforms (Nox1-5, Duox1, Duox2). Nox4 is the predominant form in the kidney, although Nox2 is also expressed. Nox4 has been implicated in the basal production of ROS in the kidney and in pathologic conditions such as diabetic nephropathy and CKD; upregulation of Nox4 may be important in renal oxidative stress and kidney injury. Although there is growing evidence indicating the involvement of NADPH oxidase in renal pathology, there is a paucity of information on the role of NADPH oxidase in the regulation of normal renal function. Here we provide an update on the role of NADPH oxidases and ROS in renal physiology and pathology. PMID:23970124

  7. Role of NADPH Oxidase-Mediated Reactive Oxygen Species in Podocyte Injury

    PubMed Central

    Chen, Shan; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Proteinuria is an independent risk factor for end-stage renal disease (ESRD) (Shankland, 2006). Recent studies highlighted the mechanisms of podocyte injury and implications for potential treatment strategies in proteinuric kidney diseases (Zhang et al., 2012). Reactive oxygen species (ROS) are cellular signals which are closely associated with the development and progression of glomerular sclerosis. NADPH oxidase is a district enzymatic source of cellular ROS production and prominently expressed in podocytes (Zhang et al., 2010). In the last decade, it has become evident that NADPH oxidase-derived ROS overproduction is a key trigger of podocyte injury, such as renin-angiotensin-aldosterone system activation (Whaley-Connell et al., 2006), epithelial-to-mesenchymal transition (Zhang et al., 2011), and inflammatory priming (Abais et al., 2013). This review focuses on the mechanism of NADPH oxidase-mediated ROS in podocyte injury under different pathophysiological conditions. In addition, we also reviewed the therapeutic perspectives of NADPH oxidase in kidney diseases related to podocyte injury. PMID:24319690

  8. Endothelial actin depolymerization mediates NADPH oxidase-superoxide production during flow reversal.

    PubMed

    Choy, Jenny S; Lu, Xiao; Yang, Junrong; Zhang, Zhen-Du; Kassab, Ghassan S

    2014-01-01

    Slow moving blood flow and changes in flow direction, e.g., negative wall shear stress, can cause increased superoxide (O2(·-)) production in vascular endothelial cells. The mechanism by which shear stress increases O2(·-) production, however, is not well established. We tested the hypothesis that actin depolymerization, which occurs during flow reversal, mediates O2(·-) production in vascular endothelial cells via NADPH oxidase, and more specifically, the subunit p47(phox). Using a swine model, we created complete blood flow reversal in one carotid artery, while the contralateral vessel maintained forward blood flow as control. We measured actin depolymerization, NADPH oxidase activity, and reactive oxygen species (ROS) production in the presence of various inhibitors. Flow reversal was found to induce actin depolymerization and a 3.9 ± 1.0-fold increase in ROS production as compared with forward flow. NADPH oxidase activity was 1.4 ± 0.2 times higher in vessel segments subjected to reversed blood flow when measured by a direct enzyme assay. The NADPH oxidase subunits gp91(phox) (Nox2) and p47(phox) content in the vessels remained unchanged after 4 h of flow reversal. In contrast, p47(phox) phosphorylation was increased in vessels with reversed flow. The response caused by reversed flow was reduced by in vivo treatment with jasplakinolide, an actin stabilizer (only a 1.7 ± 0.3-fold increase). Apocynin (an antioxidant) prevented reversed flow-induced ROS production when the animals were treated in vivo. Cytochalasin D mimicked actin depolymerization in vitro and caused a 5.2 ± 3.0-fold increase in ROS production. These findings suggest that actin filaments play an important role in negative shear stress-induced ROS production by potentiating NADPH oxidase activity, and more specifically, the p47(phox) subunit in vascular endothelium. PMID:24186098

  9. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    SciTech Connect

    Riganti, Chiara . E-mail: dario.ghigo@unito.it

    2006-05-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.

  10. Role of the NADPH oxidases in the subfornical organ in angiotensin II-induced hypertension.

    PubMed

    Lob, Heinrich E; Schultz, David; Marvar, Paul J; Davisson, Robin L; Harrison, David G

    2013-02-01

    Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22(phox). SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22(phox), Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min(-1) × 2 weeks) was blunted in adenovirus encoding cre-recombinase-treated mice, as detected by dihydroethidium fluorescence. Deletion of p22(phox) in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97 ± 15 versus 154 ± 6 mm Hg, respectively; P=0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22(phox). These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling in hypertension. PMID:23248154

  11. The PPARgamma ligand, rosiglitazone, reduces vascular oxidative stress and NADPH oxidase expression in diabetic mice.

    PubMed

    Hwang, Jinah; Kleinhenz, Dean J; Rupnow, Heidi L; Campbell, Adam G; Thul, Peter M; Sutliff, Roy L; Hart, C Michael

    2007-06-01

    Oxidative stress plays an important role in diabetic vascular dysfunction. The sources and regulation of reactive oxygen species production in diabetic vasculature continue to be defined. Because peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduced superoxide anion (O(2)(-.)) generation in vascular endothelial cells in vitro by reducing NADPH oxidase and increasing Cu/Zn superoxide dismutase (SOD) expression, the current study examined the effect of PPARgamma ligands on vascular NADPH oxidase and O(2)(-.) generation in vivo. Lean control (db(+)/db(-)) and obese, diabetic, leptin receptor-deficient (db(-)/db(-)) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7-days. Compared to controls, db(-)/db(-) mice weighed more and had metabolic derangements that were not corrected by treatment with rosiglitazone for 1-week. Aortic O(2)(-.) generation and mRNA levels of the NADPH oxidase subunits, Nox-1, Nox-2, and Nox-4 as well as Nox-4 protein expression were elevated in db(-)/db(-) compared to db(+)/db(-) mice, whereas aortic Cu/Zn SOD protein and PPARgamma mRNA levels were reduced in db(-)/db(-) mice. Treatment with rosiglitazone for 1-week significantly reduced aortic O(2)(-.) production and the expression of Nox-1, 2, and 4 but failed to increase Cu/Zn SOD or PPARgamma in aortic tissue from db(-)/db(-) mice. These data demonstrate that the vascular expression of Nox-1, 2, and 4 subunits of NADPH oxidase is increased in db(-)/db(-) mice and that short-term treatment with the PPARgamma agonist, rosiglitazone, has the potential to rapidly suppress vascular NADPH oxidase expression and O(2)(-.) production through mechanisms that do not appear to depend on correction of diabetic metabolic derangements. PMID:17337254

  12. Amyloid-? and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons

    PubMed Central

    Kane, Sarah J.; Shaw, Alisa E.; Brown, David R.; Pulford, Bruce; Zabel, Mark D.; Lambeth, J. David; Bamburg, James R.

    2014-01-01

    Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1?1 cofilin?actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (530 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ?6 h) occurs in a subpopulation (?20%) of hippocampal neurons upon exposure to soluble human amyloid-? dimer/trimer (A?d/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNF?, IL-1?, IL-6) also induce rods at the same rate and within the same neuronal population as A?d/t. Neurons from prion (PrPC)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or A?d/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrPC is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrPC-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrPC-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse A?-binding membrane proteins induce synaptic dysfunction. PMID:24760020

  13. Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

    PubMed Central

    Thamilselvan, Vijayalakshmi; Menon, Mani

    2013-01-01

    Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

  14. Isoproterenol induced stressful reactions in the brain are characterized by inflammation due to activation of NADPH oxidase and ER stress: attenuated by Apocynin, Rehmannia complex and Triterpene acids.

    PubMed

    Mo, Guo-Long; Li, Ying; Du, Rong-Hui; Dai, De-Zai; Cong, Xiao-Dong; Dai, Yin

    2014-04-01

    Inflammatory changes in the cerebral network are present in early mechanisms involved in neurodegenerative disease, Alzheimer disease (AD), and aging brain. We intended to verify that these are likely due to an activation of NADPH oxidase (NOX) and endoplasmic reticulum (ER) stress. Apocynin (APO) an inhibitor of NOX is potential to ameliorate these changes. Rehmannia complex (Reh) a famous prescription in China and the triterpene acids (TTA) isolated from Reh may relieve the isoproterenol (ISO) induced chronic inflammation in the brain, compared with APO. Rats were administered with ISO for 10 days and astrocytes were incubated with ISO for 24 h. Changes in neural MMP (matrix metalloproteinase), Cx43, AQP4 (aquaporin 4), NFκB, IκBβ, and p-PERK (PKB like kinase) were conducted and intervened with APO, Reh and TTA, in vivo and in vitro, respectively. An increased MDA and upregulated NOX subunit p47phox, ETA, PERK in association with abnormal MMP-2/9 and Cx40/43 were found in cerebral tissue of ISO-injected rats. Astrocytes incubated with ISO exhibited upregulated APQ4, IκBβ, NFκB and p-PERK/PERK and downregulated Cx43. These were significantly abrogated by APO and Reh, in vivo, and APO and TTA in vitro. In conclusion, neural damages induced by ISO were characterized by inflammatory changes in cerebral tissue and astrocytes, which were blunted significantly by APO, Reh and TTA, respectively. Reh and TTA are potential in alleviating the early pathogenesis in neurodegenerative changes in AD in the clinical settings through suppressing NOX and ER stress in the brain. PMID:24570113

  15. Versatile roles of plant NADPH oxidases and emerging concepts.

    PubMed

    Kaur, Gurpreet; Sharma, Ashutosh; Guruprasad, Kunchur; Pati, Pratap Kumar

    2014-01-01

    NADPH oxidase (NOX) is a key player in the network of reactive oxygen species (ROS) producing enzymes. It catalyzes the production of superoxide (O2(-)), that in turn regulates a wide range of biological functions in a broad range of organisms. Plant Noxes are known as respiratory burst oxidase homologs (Rbohs) and are homologs of catalytic subunit of mammalian phagocyte gp91(phox). They are unique among other ROS producing mechanisms in plants as they integrate different signal transduction pathways in plants. In recent years, there has been addition of knowledge on various aspects related to its structure, regulatory components and associated mechanisms, and its plethora of biological functions. This update highlights some of the recent developments in the field with particular reference to important members of the plant kingdom. PMID:24561450

  16. NADPH Oxidase and the Cardiovascular Toxicity Associated with Smoking

    PubMed Central

    Kim, Mikyung; Han, Chang-ho

    2014-01-01

    Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and α,β-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD. PMID:25343008

  17. [The Nox/Duox family of ROS-generating NADPH oxidases].

    PubMed

    Guichard, Cécile; Pedruzzi, Eric; Fay, Michèle; Ben Mkaddem, Sanae; Coant, Nicolas; Daniel, Fanny; Ogier-Denis, Eric

    2006-11-01

    Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes. PMID:17101097

  18. Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species

    PubMed Central

    Alili, Lirija; Sack, Maren; Puschmann, Katharina; Brenneisen, Peter

    2013-01-01

    Tumour–stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGFβ (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGFβ1, is involved in the fibroblast-to-MF switch. The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGFβ1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a ‘stromal therapy’ which was described earlier. PMID:24299025

  19. Reactive Oxygen Species and Angiogenesis: NADPH Oxidase as Target for Cancer Therapy

    PubMed Central

    Ushio-Fukai, Masuko; Nakamura, Yoshimasa

    2009-01-01

    Angiogenesis is essential for tumor growth, metastasis, arteriosclerosis as well as embryonic development and wound healing. Its process is dependent on cell proliferation, migration and capillary tube formation in endothelia cells (ECs). High levels of reactive oxygen species (ROS) such as superoxide and H2O2 are observed in various cancer cells. Accumulating evidence suggests that ROS function as signaling molecules to mediate various growth-related responses including angiogenesis. ROS-dependent angiogenesis can be regulated by endogenous antioxidant enzymes such as SOD and thioredoxin. Vascular endothelial growth factor (VEGF), one of the major angiogenesis factor, is induced in growing tumors and stimulates EC proliferation and migration primarily through the VEGF receptor type2 (VEGFR2, Flk1/KDR). Major source of ROS in ECs is a NADPH oxidase which consists of Nox1, Nox2, Nox4, Nox5, p22phox, p47phox and the small G protein Rac1. NADPH oxidase is activated by various growth factors including VEGF and angiopoietin-1 as well as hypoxia and ischemia, and ROS derived from this oxidase are involved in VEGFR2 autophosphorylation, and diverse redox signaling pathways leading to induction of transcription factors and genes involved in angiogenesis. Dietary antioxidants appear to be effective for treatment of tumor angiogenesis. The aim of this review is to provide an overview of the recent progress on role of ROS derived from NADPH oxidase and redox signaling events involved in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for tumor angiogenesis. PMID:18406051

  20. Cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NF-κB-MT1MMP in activating proMMP-2 by ET-1 in pulmonary artery smooth muscle cells.

    PubMed

    Sarkar, Jaganmay; Chowdhury, Animesh; Chakraborti, Tapati; Chakraborti, Sajal

    2016-04-01

    Treatment of bovine pulmonary artery smooth muscle cells with endothelin-1 (ET-1) caused an increase in the expression and activation of proMMP-2 in the cells. The present study was undertaken to determine the underlying mechanisms involved in this scenario. We demonstrated that (i) pretreatment with NADPH oxidase inhibitor, apocynin; PKC-α inhibitor, Go6976; p(38)MAPK inhibitor SB203580 and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by ET-1; (ii) ET-1 treatment to the cells stimulated NADPH oxidase and PKCα activity, p(38)MAPK phosphorylation as well as NF-κB activation by translocation of NF-κBp65 subunit from cytosol to the nucleus, and subsequently by increasing its DNA-binding activity; (iii) ET-1 increases MT1-MMP expression, which was inhibited upon pretreatment with apocynin, Go6976, SB293580, and Bay 11-7082; (iv) ET-1 treatment to the cells downregulated TIMP-2 level. Although apocynin and Go6976 pretreatment reversed ET-1 effect on TIMP-2 level, yet pretreatment of the cells with SB203580 and Bay 11-7082 did not show any discernible change in TIMP-2 level by ET-1. Overall, our results suggest that ET-1-induced activation of proMMP-2 is mediated via cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NFκB-MT1MMP signaling pathways along with a marked decrease in TIMP-2 expression in the cells. PMID:26910780

  1. Reduced NADPH oxidase type 2 activity mediates sleep fragmentation-induced effects on TC1 tumors in mice

    PubMed Central

    Zheng, Jiamao; Almendros, Isaac; Wang, Yang; Zhang, Shelley X; Carreras, Alba; Qiao, Zhuanhong; Gozal, David

    2015-01-01

    The molecular mechanisms underlying how sleep fragmentation (SF) influences cancer growth and progression remain largely elusive. Here, we present evidence that SF reduced ROS production by downregulating gp91phox expression and activity in TC1 cell tumor associated macrophages (TAMs), while genetic ablation of phagocytic Nox2 activity increased tumor cell proliferation, motility, invasion, and extravasation in vitro. Importantly, the in vivo studies using immunocompetent syngeneic murine tumor models suggested that Nox2 deficiency mimics SF-induced TAMs infiltration and subsequent tumor growth and invasion. Taken together, these studies reveal that perturbed sleep could adversely affect innate immunity within the tumor by altering Nox2 expression and activity, and indicate that selective potentiation of Nox2 activity may present a novel therapeutic strategy in the treatment of cancer. PMID:25949873

  2. Differential roles of NADPH oxidases in vascular physiology and pathophysiology

    PubMed Central

    Amanso, Angelica M.; Griendling, Kathy K.

    2012-01-01

    Reactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur in discrete subcellular locations, allowing for specific, compartmentalized signaling. Among the many sources of ROS in the vessel wall, NADPH oxidases are implicated in physiological functions such as control of vasomotor tone, regulation of extracellular matrix and phenotypic modulation of vascular smooth muscle cells. They are involved in the response to injury, whether as an oxygen sensor during hypoxia, as a regulator of protein processing, as an angiogenic stimulus, or as a mechanism of wound healing. These enzymes have also been linked to processes leading to disease development, including migration, proliferation, hypertrophy, apoptosis and autophagy. As a result, NADPH oxidases participate in atherogenesis, systemic and pulmonary hypertension and diabetic vascular disease. The role of ROS in each of these processes and diseases is complex, and a more full understanding of the sources, targets, cell-specific responses and counterbalancing mechanisms is critical for the rational development of future therapeutics. PMID:22202108

  3. Nrf2 regulates ROS production by mitochondria and NADPH oxidase

    PubMed Central

    Kovac, Stjepana; Angelova, Plamena R.; Holmstrm, Kira M.; Zhang, Ying; Dinkova-Kostova, Albena T.; Abramov, Andrey Y.

    2015-01-01

    Background Nuclear factor (erythroid-derived 2) factor 2 (Nrf2) is a crucial transcription factor mediating protection against oxidants. Nrf2 is negatively regulated by cytoplasmic Kelch-like ECH associated protein 1 (Keap1) thereby providing inducible antioxidant defence. Antioxidant properties of Nrf2 are thought to be mainly exerted by stimulating transcription of antioxidant proteins, whereas its effects on ROS production within the cell are uncertain. Methods Live cell imaging and qPCR in brain hippocampal glio-neuronal cultures and explants slice cultures with graded expression of Nrf2, i.e. Nrf2-knockout (Nrf2-KO), wild-type (WT), and Keap1-knockdown (Keap1-KD). Results We here show that ROS production in Nrf2-KO cells and tissues is increased compared to their WT counterparts. Mitochondrial ROS production is regulated by the Keap1Nrf2 pathway by controlling mitochondrial bioenergetics. Surprisingly, Keap1-KD cells and tissues also showed higher rates of ROS production when compared to WT, although with a smaller magnitude. Analysis of the mRNA expression levels of the two NOX isoforms implicated in brain pathology showed, that NOX2 is dramatically upregulated under conditions of Nrf2 deficiency, whereas NOX4 is upregulated when Nrf2 is constitutively activated (Keap1-KD) to a degree which paralleled the increases in ROS production. Conclusions These observations suggest that the Keap1Nrf2 pathway regulates both mitochondrial and cytosolic ROS production through NADPH oxidase. General significance Findings supports a key role of the Keap1Nrf2 pathway in redox homeostasis within the cell. PMID:25484314

  4. Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death.

    PubMed

    Kim, Jin Hee; Jang, Bong Geom; Choi, Bo Young; Kim, Hyeong Seop; Sohn, Min; Chung, Tae Nyoung; Choi, Hui Chul; Song, Hong Ki; Suh, Sang Won

    2013-03-01

    The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. Apocynin (30mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction. PMID:23313582

  5. Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress

    PubMed Central

    Natarajan, Sathish Kumar; Becker, Donald F

    2012-01-01

    Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted. PMID:22593641

  6. NADPH oxidases in lung biology and pathology: host defense enzymes, and more.

    PubMed

    van der Vliet, Albert

    2008-03-15

    The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, in cell proliferation, differentiation, and in regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology. PMID:18164271

  7. NADPH OXIDASES IN LUNG BIOLOGY AND PATHOLOGY. HOST DEFENSE ENZYMES, AND MORE

    PubMed Central

    van der Vliet, Albert

    2008-01-01

    The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, including cell proliferation, differentiation, and regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology. PMID:18164271

  8. NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension.

    PubMed

    Zalba, Guillermo; San Jos, Gorka; Moreno, Mara U; Fortuo, Ana; Dez, Javier

    2005-01-01

    Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress. PMID:16115038

  9. A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.

    PubMed

    Movitz, C; Sjölin, C; Dahlgren, C

    1997-10-01

    Neutrophil production of reactive oxygen species is dependent on an assembly process that involves a translocation of the cytosolic NADPH-oxidase components (p47phox; p67phox; Rac2) to a b cytochrome containing membrane. Based on the fact that an intracellular Ca2+ rise can activate the oxidase without any extracellular release of reactive oxygen species, we suggest that the oxidase can be assembled in a membrane distinct from the plasma membrane. Disintegrated cells were used to monitor Ca2+ dependent membrane binding of neutrophil cytosolic proteins. Membranes containing the b cytochrome part of the oxidase, i.e., specific granules and plasma membranes/secretory vesicles, were used in the translocation experiments. Several cytosolic proteins were found to translocate to specific granules as well as the plasma membranes/secretory vesicles, one of them being annexin I. Using antibodies in the blotting assay against the cytosolic oxidase components p47phox and p67phox, we could show that no Ca2+ dependent translocation of these cytosolic proteins occur to neither of the b cytochrome containing membranes. PMID:9343750

  10. The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice.

    PubMed

    Nisbet, Rachel E; Graves, Anitra S; Kleinhenz, Dean J; Rupnow, Heidi L; Reed, Alana L; Fan, Tai-Hwang M; Mitchell, Patrick O; Sutliff, Roy L; Hart, C Michael

    2009-05-01

    Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH. PMID:18952568

  11. The Role of NADPH Oxidase in Chronic Intermittent Hypoxia-Induced Pulmonary Hypertension in Mice

    PubMed Central

    Nisbet, Rachel E.; Graves, Anitra S.; Kleinhenz, Dean J.; Rupnow, Heidi L.; Reed, Alana L.; Fan, Tai-Hwang M.; Mitchell, Patrick O.; Sutliff, Roy L.; Hart, C. Michael

    2009-01-01

    Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2?) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidasederived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor ? and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidasederived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH. PMID:18952568

  12. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.

    PubMed

    Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

    2011-10-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. PMID:21605929

  13. Novel Role of NADPH Oxidase in Angiogenesis and Stem/Progenitor Cell Function

    PubMed Central

    Urao, Norifumi

    2009-01-01

    Abstract Neovascularization is involved in normal development and wound repair as well as ischemic heart disease and peripheral artery disease. Both angiogenesis and vasculogenesis [de novo new vessel formation through mobilization of stem/progenitor cells from bone marrow (BM) and their homing to the ischemic sites] contribute to the formation of new blood vessels after tissue ischemia. Angiogenesis is dependent on cell proliferation, migration, and capillary tube formation in endothelial cells (ECs). Stem/progenitor cells have been used for cell-based therapy to promote revascularization after peripheral or myocardial ischemia. Excess amounts of reactive oxygen species (ROS) are involved in senescence and apoptosis of ECs and stem/progenitor cells, causing defective neovascularization. ROS at low levels function as signaling molecules to mediate cell proliferation, migration, differentiation, and gene expression. NADPH oxidase is one of the major sources of ROS in ECs and stem/progenitor cells, and is activated by various growth factors, cytokines, hypoxia, and ischemia. ROS derived from NADPH oxidase play an important role in redox signaling linked to angiogenesis ECs, as well as stem/progenitor cell mobilization, homing, and differentiation, thereby promoting neovascularization. Understanding these mechanisms may provide insight into NADPH oxidase and its mediators as potential therapeutic targets for ischemic heart and limb disease. Antioxid. Redox Signal. 11, 2517–2533. PMID:19309262

  14. Traumatic Brain Injury and NADPH Oxidase: A Deep Relationship

    PubMed Central

    Prata, Cecilia; Vieceli Dalla Sega, Francesco; Piperno, Roberto; Hrelia, Silvana

    2015-01-01

    Traumatic brain injury (TBI) represents one of the major causes of mortality and disability in the world. TBI is characterized by primary damage resulting from the mechanical forces applied to the head as a direct result of the trauma and by the subsequent secondary injury due to a complex cascade of biochemical events that eventually lead to neuronal cell death. Oxidative stress plays a pivotal role in the genesis of the delayed harmful effects contributing to permanent damage. NADPH oxidases (Nox), ubiquitary membrane multisubunit enzymes whose unique function is the production of reactive oxygen species (ROS), have been shown to be a major source of ROS in the brain and to be involved in several neurological diseases. Emerging evidence demonstrates that Nox is upregulated after TBI, suggesting Nox critical role in the onset and development of this pathology. In this review, we summarize the current evidence about the role of Nox enzymes in the pathophysiology of TBI. PMID:25918580

  15. NADPH Oxidase: A Potential Target for Treatment of Stroke

    PubMed Central

    Zhang, Li; Wu, Jie; Duan, Xiaochun; Tian, Xiaodi; Shen, Haitao; Sun, Qing; Chen, Gang

    2016-01-01

    Stroke is the third leading cause of death in industrialized nations. Oxidative stress is involved in the pathogenesis of stroke, and excessive generation of reactive oxygen species (ROS) by mitochondria is thought to be the main cause of oxidative stress. NADPH oxidase (NOX) enzymes have recently been identified and studied as important producers of ROS in brain tissues after stroke. Several reports have shown that knockout or deletion of NOX exerts a neuroprotective effect in three major experimental stroke models. Recent studies also confirmed that NOX inhibitors ameliorate brain injury and improve neurological outcome after stroke. However, the physiological and pathophysiological roles of NOX enzymes in the central nervous system (CNS) are not known well. In this review, we provide a comprehensive summary of our current understanding about expression and physiological function of NOX enzymes in the CNS and its pathophysiological roles in the three major types of stroke: ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage. PMID:26941888

  16. NADPH oxidase 2 plays a role in experimental corneal neovascularization.

    PubMed

    Chan, Elsa C; van Wijngaarden, Peter; Chan, Elsie; Ngo, Darleen; Wang, Jiang-Hui; Peshavariya, Hitesh M; Dusting, Gregory J; Liu, Guei-Sheung

    2016-05-01

    Corneal neovascularization, the growth of new blood vessels in the cornea, is a leading cause of vision impairment after corneal injury. Neovascularization typically occurs in response to corneal injury such as that caused by infection, physical trauma, chemical burns or in the setting of corneal transplant rejection. The NADPH oxidase enzyme complex is involved in cell signalling for wound-healing angiogenesis, but its role in corneal neovascularization has not been studied. We have now analysed the role of the Nox2 isoform of NADPH oxidase in corneal neovascularization in mice following chemical injury. C57BL/6 mice aged 8-14 weeks were cauterized with an applicator coated with 75% silver nitrate and 25% potassium nitrate for 8 s. Neovascularization extending radially from limbal vessels was observed in corneal whole-mounts from cauterized wild type mice and CD31+ vessels were identified in cauterized corneal sections at day 7. In contrast, in Nox2 knockout (Nox2 KO) mice vascular endothelial growth factor-A (Vegf-A), Flt1 mRNA expression, and the extent of corneal neovascularization were all markedly reduced compared with their wild type controls. The accumulation of Iba-1+ microglia and macrophages in the cornea was significantly less in Nox2 KO than in wild type mice. In conclusion, we have demonstrated that Nox2 is implicated in the inflammatory and neovascular response to corneal chemical injury in mice and clearly VEGF is a mediator of this effect. This work raises the possibility that therapies targeting Nox2 may have potential for suppressing corneal neovascularization and inflammation in humans. PMID:26814205

  17. Subcellular Localization of NADPH Oxidase Subunits in Neurons and Astroglia of the Rat medial Nucleus Tractus Solitarius: Relationship with Tyrosine Hydroxylase Immunoreactive Neurons

    PubMed Central

    Glass, Michael J.; Huang, Jie; Oselkin, Martin; Tarsitano, M. Jacqueline; Wang, Gang; Iadecola, Costantino; Pickel, Virginia M.

    2007-01-01

    Superoxide produced by the enzyme NADPH oxidase mediates crucial intracellular signaling cascades in the mNTS, a brain region populated by catecholaminergic neurons, as well as astroglia that play an important role in autonomic function. The mechanisms mediating NADPH oxidase (phox) activity in the neural regulation of cardiovascular processes are incompletely understood, however the subcellular localization of superoxide produced by the enzyme is likely to be an important regulatory factor. We used immunogold electron microscopy to determine the phenotypic and subcellular localization of the NADPH oxidase subunits p47phox, gp91phox, and p22phox in the mNTS. The mNTS contains a large population of neurons that synthesize catecholamines. Significantly, catecholaminergic signaling can be modulated by redox reactions. Therefore, the relationship of NADPH oxidase subunit labeled neurons or glia with respect to catecholaminergic neurons was also determined by dual labeling for the superoxide producing enzyme and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the mNTS, NADPH oxidase subunits were present primarily in somatodendritic processes and astrocytes, some of which also contained TH, or were contacted by TH labeled axons, respectively. Immunogold quantification of NADPH oxidase subunit localization showed that p47phox and gp91phox were present on the surface membrane, as well as vesicular organelles characteristic of calcium storing smooth endoplasmic reticula in dendritic and astroglial processes. These results indicate that NADPH oxidase assembly and consequent superoxide formation are likely to occur near the plasmalemma, as well as on vesicular organelles associated with intracellular calcium storage within mNTS neurons and glia. Thus, NADPH oxidase-derived superoxide may participate in intracellular signaling pathways linked to calcium regulation in diverse mNTS cell types. Moreover, NADPH oxidase-derived superoxide in neurons and glia may directly or indirectly modulate catecholaminergic neuron activity in the mNTS. PMID:17027166

  18. NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

    PubMed Central

    Kuwabara, Wilson Mitsuo Tatagiba; Zhang, Liling; Schuiki, Irmgard; Curi, Rui; Volchuk, Allen; Alba-Loureiro, Tatiana Carolina

    2015-01-01

    Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells. PMID:25668518

  19. Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species.

    PubMed

    Alili, Lirija; Sack, Maren; Puschmann, Katharina; Brenneisen, Peter

    2013-12-01

    Tumor-stroma interaction is a prerequisite for tumor progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumor-associated fibroblasts to myofibroblasts by growth factors, for example transforming growth factor beta (TGFβ). In this study, the question was addressed of whether fibroblast associated NAD(P)H oxidase, known to be activated by TGFβ1, is involved in the fibroblast-to-myofibroblast switch. The up regulation of alpha smooth muscle actin (αSMA), a biomarker for myofibroblasts, is mediated by a TGFβ1-dependent increase in the intracellular level of reactive oxygen species (ROS). The source of these ROS was identified to be NAD(P)H oxidase, showing two activity peaks over time with an early and late activity peak after treatment with the growth factor. The late (second) activity peak of NAD(P)H oxidase was identified to be responsible for the downstream signaling resulting in reactive oxygen species triggered activation of the stress kinase p38 and expression of αSMA. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-myofibroblast switch and may be important for chemoprevention. PMID:24299025

  20. NADPH oxidase mediates TNF-α-evoked in vitro brain barrier dysfunction: roles of apoptosis and time.

    PubMed

    Abdullah, Zuraidah; Bayraktutan, Ulvi

    2014-07-01

    The pro-inflammatory cytokine TNF-α severely perturbs the integrity of the blood-brain barrier (BBB). This study explored the specific roles of NADPH oxidase and associated downstream effectors by using human brain microvascular endothelial cells (HBMECs) and human astrocytes (HAs), the key components of BBB, alone or in co-cultures to mimic human BBB. Exposure to TNF-α (6h) impaired BBB integrity as evidenced by marked decreases in transendothelial electrical resistance and concurrent increases in paracellular flux which appeared to subside with time (24h). Increased barrier dysfunction concurred with increases in endothelial NADPH oxidase activity, O2(-) production, actin stress fibre formation, MMP-2/9 activities and concomitant decreases in antioxidant (CuZn-SOD and catalase) and tight junction (claudin-5 and occludin) protein expressions. Conversely, TNF-α did not affect astrocytic MMP activities and antioxidant enzyme expressions. Unlike BBB damage, rates of HBMEC and HA apoptosis increased by time. Suppression of NADPH oxidase by apocynin or diphenyleneiodonium prevented TNF-α-evoked morphological changes and apoptosis, attenuated endothelial MMP activity and helped retain usual tight junction protein expression and barrier function. In conclusion, HBMECs constitute the main source of oxidative stress and basement-membrane degrading endopeptidases in inflammatory conditions associated with excessive release of TNF-α where targeting NADPH oxidase may prove extremely beneficial in maintaining proper barrier activity through prevention of cytoskeletal and tight junction reorganisations. PMID:24907586

  1. Loss of functional NADPH oxidase-2 protects against alcohol-induced bone resorption in female p47phox-/- mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In bone, oxidant signaling through NADPH oxidase (NOX)-derived reactive oxygen species (ROS) is an important stimulus for osteoclast differentiation and activity. We have previously demonstrated that chronic alcohol abuse produces bone loss through NOX-dependent mechanisms. In the current study, s...

  2. NOX family NADPH oxidases: not just in mammals.

    PubMed

    Bedard, Karen; Lardy, Bernard; Krause, Karl-Heinz

    2007-09-01

    NOX family NADPH oxidases are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). NOX enzymes in mammalian organisms have received most attention. However, NOX enzymes are widely distributed in different kingdoms of life. While they are not found in prokaryotes and most unicellular eukaryotes, they are present in fungi, plants, and animals. The identity of the ancestral NOX is not known, but most likely it: (i) possessed the basic NOX structure consisting of 6 transmembrane domains (containing two assymmetrical hemes) and a long cytoplasmic C-terminal (containing the FAD and NADPH binding sites); and (ii) emerged before the divergence of life into fungi, plants, and animals. During evolution, acquisition of a Ca(2+)-binding EF hand domain by an ancestral NOX, led to NOX5-like isoforms. DUOX isoforms presumably developed from a NOX5-like isoform through the additional acquisition of a peroxidase homology domain. The expression pattern of NOX enzymes is specific to each kingdom of life. Fungi express only ancestral-type isoforms, and plants only NOX5-like isoforms. NOX expression patterns in animals are complex and ancestral NOXes, NOX5-like isoforms and DUOXes are generally found. But there are exceptions; for example rodents lack NOX5 and Caenorhabditis elegans expresses only DUOXes. Biological functions of NOX enzymes include, among others, host defense, post-translational modification of proteins, and regulation cell growth and differentiation. In summary, the invention of NOX enzymes early in the development of life was a success story: there is no evidence of multicellular life without NOX enzymes. PMID:17400358

  3. Polymorphisms in NADPH oxidase CYBA gene modify the risk of ESRD in patients with chronic glomerulonephritis.

    PubMed

    Zhou, Hui; Chen, Min; Zhu, Ying; Wang, Bing; Liu, Xiao-Ning; Zuo, Zhi; Tang, Feng-Ying

    2016-03-01

    End-stage renal disease (ESRD) was defined as start of renal replacement therapy or death due to kidney disease. However, death due to acute kidney injury was not included. It typically occurs when chronic renal failure progresses to a point where the kidneys are permanently functioning at less than 10% of their capacity. Oxidative stress (OS) plays a crucial role in ESRD. Nicotinamide adenine dinucleotide phosphate (NADPH) is one of the most important enzymes during oxidative stress. Cytochrome b light chain (CYBA), encoded by a polymorphic gene, which is a critical component of the nicotinamide adenine dinucleotide (NADH)/NADPH oxidase system and plays an important role in electron transport and superoxide anion production, is located on chromosome band 16q24 and has six exons spanning almost 7.76kb of genomic DNA. CYBA gene polymorphisms can influence the activity of NADPH oxidase. To evaluate the association between CYBA gene polymorphisms and ESRD, we genotyped five CYBA polymorphisms using TaqMan allelic discrimination assay on DNA samples from 306 healthy controls and 332 patients with ESRD. Our results suggested that rs1049255 polymorphism of CYBA modified the risk of ESRD (p ?=??0.019; OR ?=??0.625; 95%CI ?=??0.424-0.921). GG genotype and G allele might be a protective factor against the risk of ESRD, especially in patients with chronic glomerulonephritis. PMID:26627442

  4. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    PubMed Central

    Hidalgo, María A.; Carretta, María D.; Teuber, Stefanie E.; Zárate, Cristian; Cárcamo, Leonardo; Concha, Ilona I.; Burgos, Rafael A.

    2015-01-01

    N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils. PMID:26634216

  5. Over-inhibition of NADPH oxidase reduce the wound healing in liver of finfish.

    PubMed

    Wu, Yu-Sheng; Huang, Shih-Ling; Nan, Fan-Hua; Chang, Ching-Sheng; Hsiao, Chien-Mei; Lai, Kam-Chiu; Chen, Shiu-Nan

    2014-09-01

    Wound healing is a complex process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells in a response that occurs after surgical procedure or traumatic injury. The present study aims to investigate the ROS producing from the injury that involved in the wound healing using the ZFL (zebrafish liver cell) and tilapia partial hepatectomy model. In the ZFL, we observed that while over-inhibition of NADPH activity leading to reduce the wound healing moreover, experiment of the oxidative stress by the extracellular hydrogen peroxide exactly presented to increase the PCNA, BrdU and Ki-67 histopathological repair response of tilapia liver follow partial hepatectomy. We conclude that over inhibition of the NADPH oxidase by DPI may reduce the cell even the tissue in the progress of healing after the injury. PMID:25017371

  6. Regulation of cyclooxygenase-2 and cytosolic phospholipase A2 gene expression by lipopolysaccharide through the RNA-binding protein HuR: involvement of NADPH oxidase, reactive oxygen species and mitogen-activated protein kinases

    PubMed Central

    Lin, Wei-Ning; Lin, Chih-Chung; Cheng, Hsin-Yi; Yang, Chuen-Mao

    2011-01-01

    BACKGROUND AND PURPOSE Lipopolysaccharide (LPS)-induced expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) has been implicated in several respiratory diseases. HuR is known to enhance the expression of genes by binding to 3′-untranslated region (3′-UTR) of mRNA and stabilizing mRNA. However, the exact mechanisms by which HuR affects the stability of mRNA and modulates LPS-induced COX-2 and cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) are not known. EXPERIMENTAL APPROACH The expression of prostaglandin E2 (PGE2) was measured by ELISA, and pro-inflammatory proteins were determined by use of a promoter assay, PCR or Western blot analysis. Overexpression of siRNAs to knock down the target components was used to manipulate the expression of HuR. Release of reactive oxygen species (ROS) was detected by fluorescence dye. The activation of signalling components was assessed by comparing phosphorylation levels, localization of protein kinases or coimmunoprecipitation assay. KEY RESULTS LPS induced COX-2 and cPLA2 expression via post-translational regulation of mRNA stabilization, which were attenuated by transfection with HuR siRNA in HTSMCs. In addition, LPS-stimulated NADPH oxidase activation and ROS generation were attenuated by the NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin (APO). Generation of ROS induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and JNK1/2, which was attenuated by DPI and APO and the ROS scavenger N-acetylcysteine. CONCLUSIONS AND IMPLICATIONS These results suggested that in HTSMCs, LPS-induced COX-2 and cPLA2 expression is mediated through NADPH oxidase/ROS-dependent MAPKs associated with HuR accumulation in the cytoplasm. Activated MAPKs may regulate the nucleocytoplasmic shuttling of HuR, and thus induce the cytoplasmic accumulation of HuR. PMID:21391979

  7. NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives

    PubMed Central

    McCann, Sarah K.; Roulston, Carli L.

    2013-01-01

    Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation. PMID:24961415

  8. NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

    PubMed

    Villmow, Marten; Klöckner, Udo; Heymes, Christophe; Gekle, Michael; Rueckschloss, Uwe

    2015-09-01

    Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes. PMID:26173391

  9. Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis.

    PubMed

    Suzuki, Shugo; Shiraga, Kazuhide; Sato, Shinya; Punfa, Wanisa; Naiki-Ito, Aya; Yamashita, Yoriko; Shirai, Tomoyuki; Takahashi, Satoru

    2013-12-01

    Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer. PMID:24118288

  10. Defensive Mutualism Rescues NADPH Oxidase Inactivation in Gut Infection.

    PubMed

    Pircalabioru, Gratiela; Aviello, Gabriella; Kubica, Malgorzata; Zhdanov, Alexander; Paclet, Marie-Helene; Brennan, Lorraine; Hertzberger, Rosanne; Papkovsky, Dmitri; Bourke, Billy; Knaus, Ulla G

    2016-05-11

    NOX/DUOX family of NADPH oxidases are expressed in diverse tissues and are the primary enzymes for the generation of reactive oxygen species (ROS). The intestinal epithelium expresses NOX1, NOX4, and DUOX2, whose functions are not well understood. To address this, we generated mice with complete or epithelium-restricted deficiency in the obligatory NOX dimerization partner Cyba (p22(phox)). We discovered that NOX1 regulates DUOX2 expression in the intestinal epithelium, which magnified the epithelial ROS-deficiency. Unexpectedly, epithelial deficiency of Cyba resulted in protection from C. rodentium and L. monocytogenes infection. Microbiota analysis linked epithelial Cyba deficiency to an enrichment of H2O2-producing bacterial strains in the gut. In particular, elevated levels of lactobacilli physically displaced and attenuated C. rodentium virulence by H2O2-mediated suppression of the virulence-associated LEE pathogenicity island. This transmissible compensatory adaptation relied on environmental factors, an important consideration for prevention and therapy of enteric disease. PMID:27173933

  11. Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src, NADPH Oxidase, PI3K, and Akt in Human Colon Cancer Cells

    PubMed Central

    Lien, Gi-Shih; Wu, Ming-Shun; Bien, Mauo-Ying; Chen, Chien-Hsin; Lin, Chien-Huang; Chen, Bing-Chang

    2014-01-01

    Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation. PMID:25122478

  12. The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

    PubMed Central

    Cross, A R; Parkinson, J F; Jones, O T

    1984-01-01

    An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence. PMID:6497852

  13. NADPH oxidase 4 contributes to connective tissue growth factor expression through Smad3-dependent signaling pathway.

    PubMed

    Liu, Xin-Hua; Zhang, Qiu-Yan; Pan, Li-Long; Liu, Si-Yu; Xu, Peng; Luo, Xiao-Ling; Zou, Si-Li; Xin, Hong; Qu, Le-Feng; Zhu, Yi-Zhun

    2016-05-01

    Transforming growth factor-β (TGF-β)/Smad signaling has been implicated in connective tissue growth factor (CTGF) expression in vascular smooth muscle cells (VSMC). Reactive oxygen species (ROS) are involved in activation of TGF-β/Smad signaling. However, detailed mechanisms underlying the process remain unclear. In present study, we demonstrated TGF-β1 strongly induced CTGF expression, Smad3 activation, NADPH oxidase 4 (Nox4) expression and increased ROS production in primary rat VSMC in vitro. NADPH oxidases inhibitor diphenylene iodonium (DPI) eliminated TGF-β1-induced CTGF expression and ROS generation. In addition, small-interfering RNA (siRNA) silencing of Smad3 or Nox4 significantly suppressed TGF-β1-mediated CTGF expression in VSMC. Furthermore, Nox4 silencing or inhibition eliminated TGF-β1-induced Smad3 activation and interaction between Nox4 and Smad3. In vivo studies further identified a positive correlation of Nox4 levels with Smad3 activation and CTGF expression in atherosclerotic arteries of patients and animal models. These data established that a novel mechanistic link of Nox4-dependent activation of Smad3 to increased TGF-β1-induced CTGF in the process of vascular remodeling, which suggested a new potential pathway for therapeutic interventions. PMID:26945889

  14. NADPH Oxidase: a Target for the Modulation of the Excessive Oxidase Damage Induced by Overtraining in Rat Neutrophils

    PubMed Central

    Dong, Jingmei; Chen, Peijie; Wang, Ru; Yu, Dongzhen; Zhang, Yajun; Xiao, Weihua

    2011-01-01

    Objective: The purpose of this study is to demonstrate that NADPH oxidase mediating the ROS production is the major pathway for ROS generation in neutrophils during exercise. NADPH oxidase, as a target can modulate oxidative damage induced by overtraining, which can be value to the prevention of exercise-induced immunosuppression. Methods: Thirty male Wistar rats were randomly divided into three groups: a negative control group (C, n = 10), an overtraining group (E, n = 10) and an overtraining + DPI intervention group (D, n =10). Groups E and D were trained on a standard treadmill with progressive load for 11 weeks. After 36-40 h from the last training, eight rats were randomly selected from each group, and blood was sampled from the orbital vein. ELISAs were used to measure serum cytokine levels and lipid peroxidation in blood plasma. Flow cytometry with Annexin V / PI double staining was used to measure neutrophil apoptosis and necrosis. DNA damage in lymphocytes was tested using single cell gel electrophoresis (SCGE). The co-localization between gp91phox and p47phox of the NADPH-oxidase was detected using immunocytochemistry and confocal microscopy. Results: 1) Compared with group C, the concentrations of IL-1β, IL-8, and TNF-α were significantly increased and MCP-1, and CINC were significantly decreased in blood plasma from group E (P < 0.01 and P < 0.05, respectively). Concentrations of IL-1β and MCP-1 were decreased (P < 0.05), and IL-8 and TNF-α were significantly increased (P <0.05) in blood plasma from group D. MDA and MPO were elevated in plasma from groups E and D (P < 0.01 and P < 0.05, respectively). 2) Compared with group C, the percentage of neutrophils apoptosis were significantly elevated (P < 0.01) in both groups E and D, and the percentage of cell death was raised in group E (P < 0.05). No significant change was observed in group D. 3) Compared with group C, the number of comet cells, an indicator of DNA damage, was significantly increased (P < 0.01), and the width and tail length of comet cells were notably increased in group E, while no significant increase was observed in group D. 4) The p47phox protein translocated to the cell membrane and co-localized with the gp91phox subunit of NADPH oxidase in neutrophils activated by overtraining. Conclusion: 1) Excessive exercise led to an increased secretion of inflammatory cytokines and chemokines in peripheral blood, and it may have induced tissue inflammation 2) Overtraining can activate the NADPH oxidase-mediated overproduction of ROS, leading to increased lipid peroxidation. 3) NADPHoxidase in neutrophils as a target, was responsible for ROS, oxidative damage to phagocytes and lymphocytes and changes to inflammatory cytokines and immune regulatory factors all affect cellular immune functions and may be causative factors for exercise-induced immunosuppression. PMID:21814483

  15. Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

    PubMed Central

    Yu, XiaoTong; Wang, PengYan; Shi, ZhengMing; Dong, Kun; Feng, Ping; Wang, HongXia; Wang, XueJiang

    2015-01-01

    Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC. PMID:26658815

  16. NADPH Oxidase-Derived ROS Induced by Chronic Intermittent Hypoxia Mediates Hypersensitivity of Lung Vagal C Fibers in Rats

    PubMed Central

    Yang, Chang-Huan; Zhuang, Wei-Ling; Shen, Yan-Jhih; Lai, Ching Jung; Kou, Yu Ru

    2016-01-01

    Obstructive sleep apnea (OSA), manifested by exposure to chronic intermittent hypoxia (CIH) and excess production of reactive oxygen species (ROS) in the airways, is associated with hyperreactive airway diseases. ROS, particularly when created by NADPH oxidase, are known to sensitize lung vagal C fibers (LVCFs), which may contribute to airway hypersensitivity pathogenesis. We investigated whether CIH augments the reflex and afferent responses of LVCFs to chemical stimulants and the roles of ROS and NADPH oxidase in such airway hypersensitivity. Rats were exposed to room air (RA) or CIH with/without daily treatment with MnTMPyP (a superoxide anion scavenger), apocynin (an NADPH oxidase inhibitor), or vehicle. At 16 h after their last exposure, intravenous capsaicin, adenosine, or α,β-methylene-ATP evoked an augmented apneic response in anesthetized rats with 14-days CIH exposure, compared to anesthetized rats with 14-days RA exposure. The augmented apneic responses to these LVCF stimulants were abolished by bilateral vagotomy or perivagal capsaicin treatment, which block LVCFs neural conduction and were significantly suppressed by treatment with MnTMPyP or apocynin, but not vehicle. Electrophysiological studies revealed that 14-days CIH exposure potentiated the responses of LVCFs to these stimulants. This effect was inhibited by treatment with MnTMPyP or apocynin treatment and was not seen in rats who received 7-days of CIH exposure. Biochemical analysis indicated that 14-days CIH exposure increased both lung lipid peroxidation, which is indicative of oxidative stress, and expression of the p47phox subunit in the membrane fraction of lung tissue, which is an index of NADPH oxidase activation. The former was prevented by treatment with either MnTMPyP or apocynin, while the later was prevented by treatment with apocynin only. These results suggest that 14-days CIH exposure sensitizes LVCFs in rats, leading to an exaggerated reflex and afferent responses to stimulants and that this sensitization is mediated via ROS generated by NADPH oxidase.

  17. Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord.

    PubMed

    Zhao, Wei-Cheng; Zhang, Bin; Liao, Mei-Juan; Zhang, Wen-Xuan; He, Wan-You; Wang, Han-Bing; Yang, Cheng-Xiang

    2014-02-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main enzymes that produce oxidative stress, which plays an important role in painful diabetic neuropathy. Curcumin has been reported to exert an antinociceptive effect in a rat model of diabetic neuropathy by suppressing oxidative stress in the spinal cord. However, it remains unknown whether the mechanism by which curcumin ameliorates diabetic neuropathy can be attributed to spinal NADPH oxidases. This study was designed to determine the effect of curcumin on diabetic neuropathy and to investigate its precise mechanism in relation to NADPH oxidase-mediating oxidative stress in the spinal cord. Diabetic neuropathy was induced in Sprague-Dawley rats by intraperitoneal injection with 1% streptozotocin (STZ; 60 mg/kg). After the onset of diabetic neuropathy, a subset of the diabetic rats received daily intragastric administrations of curcumin (200mg/kg) or intraperitoneal injections of apocynin (2.5mg/kg) for 14 consecutive days, whereas other diabetic rats received equivalent volumes of normal saline (NS). STZ resulted in diabetic neuropathy with hyperglycemia and a lower paw withdrawal threshold (PWT), accompanied by elevations in the expression of the NADPH oxidase subunits p47(phox) and gp91(phox) and in the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and a reduction in superoxide dismutase (SOD) activity (P<0.05) in the spinal cord. Both curcumin and apocynin ameliorated diabetic neuropathy. In conclusion, curcumin attenuated neuropathic pain in diabetic rats, at least partly by inhibiting NADPH oxidase-mediating oxidative stress in the spinal cord. PMID:24370596

  18. NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

    PubMed Central

    Deliyanti, Devy; Rana, Indrajeetsinh; Miller, Antonia G.; Agrotis, Alex; Armani, Roksana; Szyndralewiez, Cédric; Wingler, Kirstin; Touyz, Rhian M.; Cooper, Mark E.; Jandeleit-Dahm, Karin A.; Schmidt, Harald H.H.W.

    2014-01-01

    Abstract Aims: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Results: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Innovation: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Conclusions: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies. Antioxid. Redox Signal. 20, 2726–2740. PMID:24053718

  19. Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

    PubMed Central

    2011-01-01

    Background Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target. Methods NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation. Results Nigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis. Conclusions A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives. PMID:21975039

  20. Temporal changes in the expression of mRNA of NADPH oxidase subunits in renal epithelial cells exposed to oxalate or calcium oxalate crystals

    PubMed Central

    Khan, Saeed R.; Khan, Aslam; Byer, Karen J.

    2011-01-01

    Background. Exposure of renal epithelial cells to oxalate (Ox) or calcium oxalate (CaOx) crystals leads to the production of reactive oxygen species and cell injury. We have hypothesized that Ox and CaOx crystals activate NADPH oxidase through upregulation of its various subunits. Methods. Human renal epithelial-derived cell line, HK-2, was exposed to 100 μmol Ox or 66.7 μg/cm2 CaOx monohydrate crystals for 6, 12, 24 or 48 h. After exposure, the cells and media were processed to determine activation of NADPH oxidase, production of superoxide and 8-isoprostane (8IP), and release of lactate dehydrogenase (LDH). RT-PCR was performed to determine mRNA expression of NADPH subunits p22phox, p40phox, p47phox, p67phox and gp91phox as well as Rac-GTPase. Results. Exposure to Ox and CaOx crystals resulted in increase in LDH release, production of 8-IP, NADPH oxidase activity and production of superoxide. Exposure to CaOx crystals resulted in significantly higher NADPH oxidase activity, production of superoxide and LDH release than Ox exposure. Exposure to Ox and CaOx crystals altered the expression of various subunits of NADPH oxidase. More consistent were increases in the expression of membrane-bound p22phox and cytosolic p47phox. Significant and strong correlations were seen between NADPH oxidase activity, the expression of p22phox and p47phox, production of superoxide and release of LDH when cells were exposed to CaOx crystals. The expressions of neither p22phox nor p47phox were significantly correlated with increased NADPH oxidase activity after the Ox exposure. Conclusions. As hypothesized, exposure to Ox or CaOx crystals leads to significant increases in the expression of p22phox and p47phox, leading to activation of NADPH oxidase. Increased NADPH oxidase activity is associated with increased superoxide production and lipid peroxidation. Different pathways appear to be involved in the stimulation of renal epithelial cells by exposure to Ox and CaOx crystals. PMID:21079197

  1. NADPH oxidases: an overview from structure to innate immunity-associated pathologies

    PubMed Central

    Panday, Arvind; Sahoo, Malaya K; Osorio, Diana; Batra, Sanjay

    2015-01-01

    Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), play important roles in immunity, cell growth, and cell signaling. In excess, however, ROS are lethal to cells, and the overproduction of these molecules leads to a myriad of devastating diseases. The key producers of ROS in many cells are the NOX family of NADPH oxidases, of which there are seven members, with various tissue distributions and activation mechanisms. NADPH oxidase is a multisubunit enzyme comprising membrane and cytosolic components, which actively communicate during the host responses to a wide variety of stimuli, including viral and bacterial infections. This enzymatic complex has been implicated in many functions ranging from host defense to cellular signaling and the regulation of gene expression. NOX deficiency might lead to immunosuppression, while the intracellular accumulation of ROS results in the inhibition of viral propagation and apoptosis. However, excess ROS production causes cellular stress, leading to various lethal diseases, including autoimmune diseases and cancer. During the later stages of injury, NOX promotes tissue repair through the induction of angiogenesis and cell proliferation. Therefore, a complete understanding of the function of NOX is important to direct the role of this enzyme towards host defense and tissue repair or increase resistance to stress in a timely and disease-specific manner. PMID:25263488

  2. NADPH oxidase mediates glucolipotoxicity-induced beta cell dysfunction--clinical implications.

    PubMed

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2010-03-01

    An impairment of glucose-stimulated insulin secretion--reflecting decreased glucokinase expression--and a moderate decrease in beta cell mass attributable to increased apoptosis, constitute the key features of beta cell failure in type 2 diabetes. Oxidative stress, provoked by prolonged exposure to excessive levels of glucose and/or fatty acids (glucolipotoxicity), appears to be a key mediator of these defects. Oxidant-provoked JNK activation induces nuclear export of the PDX-1 transcription factor, required for expression of glucokinase and other beta cell proteins. Conversely, increases in cAMP induced by incretin hormones promote the nuclear importation of PDX-1, counteracting the diabetogenic impact of oxidant stress; this may explain the utility of measures that slow dietary carbohydrate absorption for diabetes prevention. The ability of oxidative stress to boost apoptosis in beta cells is poorly understood, but may also entail JNK activation. Recent work establishes a phagocyte-type NADPH oxidase as the chief source of glucotoxicity-mediated oxidative stress in beta cells. Since bilirubin is now known to function physiologically as an inhibitor of NADPH oxidase, and phycocyanobilin (PCB) derived from spirulina likewise can inhibit this enzyme complex, supplemental PCB may have utility in the prevention and control of diabetes, and Gilbert syndrome, associated with chronically elevated free bilirubin, may be associated with decreased diabetes risk. PMID:19576699

  3. The Anorexigenic Effect of Serotonin Is Mediated by the Generation of NADPH Oxidase-Dependent ROS

    PubMed Central

    Wang, Li-Na; Yang, Jing; Zeng, Qing-Jie; Cheng, Xiao; Zhang, Zhi-Qi; Wang, Song-Bo; Gao, Ping; Zhu, Xiao-Tong; Xi, Qian-Yun; Zhang, Yong-Liang; Jiang, Qing-Yan

    2013-01-01

    Serotonin (5-HT) is a central inhibitor of food intake in mammals. Thus far, the intracellular mechanisms for the effect of serotonin on appetite regulation remain unclear. It has been recently demonstrated that reactive oxygen species (ROS) in the hypothalamus are a crucial integrative target for the regulation of food intake. To investigate the role of ROS in the serotonin-induced anorexigenic effects, conscious mice were treated with 5-HT alone or combination with Trolox (a ROS scavenger) or Apocynin (an NADPH oxidase inhibitor) by acute intracerebroventricular injection. Both Trolox and Apocynin reversed the anorexigenic action of 5-HT and the 5-HT-induced hypothalamic ROS elevation. The mRNA and protein expression levels of pro-opiomelanocortin (POMC) were dramatically increased after ICV injection with 5-HT. The anorexigenic action of 5-HT was accompanied by markedly elevated hypothalamic MDA levels and GSH-Px activity, while the SOD activity was decreased. Moreover, 5-HT significantly increased the mRNA expression of UCP-2 but reduced the levels of UCP-3. Both Trolox and Apocynin could block the 5-HT-induced changes in UCP-2 and UCP-3 gene expression. Our study demonstrates for the first time that the anorexigenic effect of 5-HT is mediated by the generation of ROS in the hypothalamus through an NADPH oxidase-dependent pathway. PMID:23326391

  4. NOX, NOX Who is There? The Contribution of NADPH Oxidase One to Beta Cell Dysfunction

    PubMed Central

    Taylor-Fishwick, David A.

    2013-01-01

    Predictions of diabetes prevalence over the next decades warrant the aggressive discovery of new approaches to stop or reverse loss of functional beta cell mass. Beta cells are recognized to have a relatively high sensitivity to reactive oxygen species (ROS) and become dysfunctional under oxidative stress conditions. New discoveries have identified NADPH oxidases in beta cells as contributors to elevated cellular ROS. Reviewed are recent reports that evidence a role for NADPH oxidase-1 (NOX-1) in beta cell dysfunction. NOX-1 is stimulated by inflammatory cytokines that are elevated in diabetes. First, regulation of cytokine-stimulated NOX-1 expression has been linked to inflammatory lipid mediators derived from 12-lipoxygenase activity. For the first time in beta cells these data integrate distinct pathways associated with beta cell dysfunction. Second, regulation of NOX-1 in beta cells involves feed-forward control linked to elevated ROS and Src-kinase activation. This potentially results in unbridled ROS generation and identifies candidate targets for pharmacologic intervention. Third, consideration is provided of new, first-in-class, selective inhibitors of NOX-1. These compounds could have an important role in assessing a disruption of NOX-1/ROS signaling as a new approach to preserve and protect beta cell mass in diabetes. PMID:23565109

  5. Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

    PubMed Central

    Hirano, Kazufumi; Chen, Woei Shin; Chueng, Adeline L.W.; Dunne, Angela A.; Seredenina, Tamara; Filippova, Aleksandra; Ramachandran, Sumitra; Bridges, Angela; Chaudry, Laiq; Pettman, Gary; Allan, Craig; Duncan, Sarah; Lee, Kiew Ching; Lim, Jean; Ma, May Thu; Ong, Agnes B.; Ye, Nicole Y.; Nasir, Shabina; Mulyanidewi, Sri; Aw, Chiu Cheong; Oon, Pamela P.; Liao, Shihua; Li, Dizheng; Johns, Douglas G.; Miller, Neil D.; Davies, Ceri H.; Browne, Edward R.; Matsuoka, Yasuji; Chen, Deborah W.; Jaquet, Vincent

    2015-01-01

    Abstract Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358–374. PMID:26135714

  6. NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

    PubMed Central

    Peck, Ammon B.; Khan, Saeed R.

    2013-01-01

    A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

  7. NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

    PubMed Central

    Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  8. NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

    PubMed

    Brothers, Kimberly M; Gratacap, Remi L; Barker, Sarah E; Newman, Zachary R; Norum, Ashley; Wheeler, Robert T

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  9. Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis

    PubMed Central

    2013-01-01

    Background Non-steroidal anti-inflammatory drugs (NSAIDs) aspirin, naproxen, nimesulide, and piroxicam lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. Conclusions NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID. PMID:23718778

  10. A pepducin designed to modulate P2Y2R function interacts with FPR2 in human neutrophils and transfers ATP to an NADPH-oxidase-activating ligand through a receptor cross-talk mechanism.

    PubMed

    Gabl, Michael; Holdfeldt, André; Winther, Malene; Oprea, Tudor; Bylund, Johan; Dahlgren, Claes; Forsman, Huamei

    2016-06-01

    Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin. PMID:26996596

  11. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells

    PubMed Central

    2014-01-01

    Background Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Methods Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. Results In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. Conclusions In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria. PMID:25012725

  12. FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress.

    PubMed

    Pereira, Sandra; Park, Edward; Mori, Yusaku; Haber, C Andrew; Han, Ping; Uchida, Toyoyoshi; Stavar, Laura; Oprescu, Andrei I; Koulajian, Khajag; Ivovic, Alexander; Yu, Zhiwen; Li, Deling; Bowman, Thomas A; Dewald, Jay; El-Benna, Jamel; Brindley, David N; Gutierrez-Juarez, Roger; Lam, Tony K T; Najjar, Sonia M; McKay, Robert A; Bhanot, Sanjay; Fantus, I George; Giacca, Adria

    2014-07-01

    Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKβ- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKβ and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKβ/JNK → impaired hepatic insulin signaling. PMID:24824652

  13. A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity.

    PubMed

    Xu, Juan; Xie, Jie; Yan, Chengfei; Zou, Xiaoqin; Ren, Dongtao; Zhang, Shuqun

    2014-01-01

    Plant recognition of pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin-derived flg22 triggers rapid activation of mitogen-activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). Arabidopsis has at least four PAMP/pathogen-responsive MAPKs: MPK3, MPK6, MPK4 and MPK11. It was speculated that these MAPKs may function downstream of ROS in plant immunity because of their activation by exogenously added H2 O2 . MPK3/MPK6 or their orthologs in other plant species have also been reported to be involved in the ROS burst from the plant respiratory burst oxidase homolog (Rboh) of the human neutrophil gp91phox. However, detailed genetic analysis is lacking. Using a chemical genetic approach, we generated a conditional loss-of-function mpk3 mpk6 double mutant. Consistent with results obtained using a conditionally rescued mpk3 mpk6 double mutant generated previously, the results obtained using the new conditional loss-of-function mpk3 mpk6 double mutant demonstrate that the flg22-triggered ROS burst is independent of MPK3/MPK6. In Arabidopsis mutants lacking a functional AtRbohD, the flg22-induced ROS burst was completely blocked. However, activation of MPK3/MPK6 was not affected. Based on these results, we conclude that the rapid ROS burst and MPK3/MPK6 activation are two independent early signaling events in plant immunity, downstream of FLS2. We also found that MPK4 negatively affects the flg22-induced ROS burst. In addition, salicylic acid pre-treatment enhances the AtRbohD-mediated ROS burst, which is again independent of MPK3/MPK6 based on analysis of the mpk3 mpk6 double mutant. The establishment of an mpk3 mpk6 double mutant system using a chemical genetic approach provides a powerful tool to investigate the function of MPK3/MPK6 in the plant defense signaling pathway. PMID:24245741

  14. NOX2 NADPH OXIDASE PROMOTES PATHOLOGIC CARDIAC REMODELING ASSOCIATED WITH DOXORUBICIN CHEMOTHERAPY

    PubMed Central

    Zhao, Youyou; McLaughlin, Declan; Robinson, Emma; Harvey, Adam P.; Hookham, Michelle B.; Shah, Ajay M.; McDermott, Barbara J.; Grieve, David J.

    2010-01-01

    Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2−/−) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2−/− versus WT mice (fractional shortening: 29.5±1.4 vs 25.7±1.0 %; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2−/− versus WT mice (ejection fraction: 52.6±2.5 vs 28.5±2.3 %, LVdPdtmin: −8379±416 vs −5198±527 mmHg s−1, EDPVR: 0.051±0.009 vs 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2−/− mice exhibited less myocardial atrophy, cardiomyocyte apoptosis and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, TGF-β3, connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist, losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically-derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy. PMID:20884632

  15. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

    SciTech Connect

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to ethanol-induced MMP-12 expression. • p38 MAPK/NF-κB signaling pathway modulates ethanol-induced Nox2 expression.

  16. The senescence-accelerated mouse prone-8 (SAM-P8) oxidative stress is associated with upregulation of renal NADPH oxidase system.

    PubMed

    Baltans, Ana; Solesio, Maria E; Zalba, Guillermo; Galindo, Mara F; Fortuo, Ana; Jordn, Joaqun

    2013-12-01

    Herein, we investigate whether the NADPH oxidase might be playing a key role in the degree of oxidative stress in the senescence-accelerated mouse prone-8 (SAM-P8). To this end, the activity and expression of the NADPH oxidase, the ratio of glutathione and glutathione disulfides (GSH/GSSG), and the levels of malonyl dialdehyde (MDA) and nitrotyrosine (NT) were determined in renal tissue from SAM-P8 mice at the age of 1 and 6 months. The senescence-accelerated-resistant mouse (SAM-R1) was used as control. At the age of 1 month, NADPH oxidase activity and Nox2 protein expression were higher in SAM-P8 than in SAM-R1 mice. However, we found no differences in the GSH/GSSG ratio, MDA, NT, and Nox4 levels between both groups of animals. At the age of 6 months, SAM-R1 mice in comparison to SAM-P8 mice showed an increase in NADPH oxidase activity, which is associated with higher levels of NT and increased Nox4 and Nox2 expression levels. Furthermore, we found oxidative stress hallmarks including depletion in GSH/GSSG ratio and increase in MDA levels in the kidney of SAM-P8 mice. Finally, NADPH oxidase activity positively correlated with Nox2 expression in all the animals (r?=?0.382, P?NADPH oxidase activity might be an early hallmark to predict future oxidative stress in renal tissue during the aging process that takes place in SAM-P8 mice. PMID:23839498

  17. Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans.

    PubMed

    van der Hoeven, Ransome; Cruz, Melissa R; Chávez, Violeta; Garsin, Danielle A

    2015-01-01

    Dual oxidases (DUOX) are enzymes that contain an NADPH oxidase domain that produces hydrogen peroxide (H2O2) and a peroxidase domain that can utilize H2O2 to carry out a variety of reactions. The model organism Caenorhabditis elegans produces the DUOX, BLI-3, which has roles in both cuticle development and in protection against infection. In previous work, we demonstrated that while certain peroxidases were protective against the human bacterial pathogen Enterococcus faecalis, the peroxidase domain of BLI-3 was not, leading to the postulate that the NADPH oxidase domain is the basis for BLI-3's protective effects. In this work, we show that a strain carrying a mutation in the NADPH oxidase domain of BLI-3, bli-3(im10), is more susceptible to E. faecalis and the human fungal pathogen Candida albicans. Additionally, less H2O2 is produced in response to pathogen using both an established Amplex Red assay and a strain of C. albicans, WT-OXYellow, which acts as a biosensor of reactive oxygen species (ROS). Finally, a C. elegans line containing a BLI-3::mCherry transgene was generated. Previous work suggested that BLI-3 is produced in the hypodermis and the intestine. Expression of the transgene was observed in both these tissues, and additionally in the pharynx. The amount and pattern of localization of BLI-3 did not change in response to pathogen exposure. PMID:25909649

  18. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    NASA Astrophysics Data System (ADS)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  19. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    PubMed Central

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Federman Gross, Aya; Rafalowski, Meirav; Pick, Edgar

    2015-01-01

    The superoxide (O·−2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O·−2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components. PMID:25699251

  20. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 (phox.).

    PubMed

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Federman Gross, Aya; Rafalowski, Meirav; Pick, Edgar

    2015-01-01

    The superoxide (O(·-) 2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b 558 (a heterodimer of Nox2 and p22 (phox) ), and four cytosolic components, p47 (phox) , p67 (phox) , p40 (phox) , and Rac. The catalytic component, responsible for O(·-) 2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67 (phox) . Using a peptide-protein binding assay, we found that Nox2 peptides containing a (369)CysGlyCys(371) triad (CGC) bound p67 (phox) with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67 (phox) only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67 (phox) via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67 (phox) , in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67 (phox) to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components. PMID:25699251

  1. Role of the NADPH Oxidases DUOX and NOX4 in Thyroid Oxidative Stress.

    PubMed

    Carvalho, Denise P; Dupuy, Corinne

    2013-09-01

    Somatic mutations are present at high levels in the rat thyroid gland, indicating that the thyrocyte is under oxidative stress, a state in which cellular oxidant levels are high. The most important class of free radicals, or reactive metabolites, is reactive oxygen species (ROS), such as superoxide anion (O2 (-)), hydroxyl radical (OH) and hydrogen peroxide (H2O2). The main source of ROS in every cell type seems to be mitochondrial respiration; however, recent data support the idea that NADPH:O(2) oxidoreductase flavoproteins or simply NADPH oxidases (NOX) are enzymes specialized in controlled ROS generation at the subcellular level. Several decades ago, high concentrations of H2O2 were detected at the apical surface of thyrocytes, where thyroid hormone biosynthesis takes place. Only in the last decade has the enzymatic source of H2O2 involved in thyroid hormone biosynthesis been well characterized. The cloning of two thyroid genes encoding NADPH oxidases dual oxidases 1 and 2 (DUOX1 and DUOX2) revealed that DUOX2 mutations lead to hereditary hypothyroidism in humans. Recent reports have also described the presence of NOX4 in the thyroid gland and have suggested a pathophysiological role of this member of the NOX family. In the present review, we describe the participation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, particularly the involvement of these enzymes in the regulation of thyroid oxidative stress. PMID:24847449

  2. Alcohol-induced bone loss is blocked in p47phox -/- mice lacking functional nadph oxidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a resu...

  3. Bacillus Calmette-Guerin Infection in NADPH Oxidase Deficiency: Defective Mycobacterial Sequestration and Granuloma Formation

    PubMed Central

    Deffert, Christine; Schäppi, Michela G.; Pache, Jean-Claude; Cachat, Julien; Vesin, Dominique; Bisig, Ruth; Ma Mulone, Xiaojuan; Kelkka, Tiina; Holmdahl, Rikard

    2014-01-01

    Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (∼50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-α, IFN-γ, IL-17 and IL-12) early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and impaired granuloma formation. PMID:25188296

  4. NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice

    PubMed Central

    Murray, Thomas V.A.; Dong, Xuebin; Sawyer, Greta J.; Caldwell, Anna; Halket, John; Sherwood, Roy; Quaglia, Alberto; Dew, Tracy; Anilkumar, Narayana; Burr, Simon; Mistry, Rajesh K.; Martin, Daniel; Schröder, Katrin; Brandes, Ralf P.; Hughes, Robin D.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins. PMID:26472193

  5. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues.

    PubMed

    Jiménez-Quesada, María J; Traverso, José Á; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen-pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed. PMID:27066025

  6. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues

    PubMed Central

    Jiménez-Quesada, María J.; Traverso, José Á.; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen–pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed. PMID:27066025

  7. The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

    SciTech Connect

    Riganti, Chiara

    2008-05-01

    We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

  8. Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1β-exposed Caco-2 cells.

    PubMed

    Tesoriere, L; Attanzio, A; Allegra, M; Gentile, C; Livrea, M A

    2014-02-01

    Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 μM), and IL-1β prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level. PMID:23931157

  9. NADPH oxidase-dependent H2O2 production is required for salt-induced antioxidant defense in Arabidopsis thaliana.

    PubMed

    Ben Rejeb, Kilani; Benzarti, Maâli; Debez, Ahmed; Bailly, Christophe; Savouré, Arnould; Abdelly, Chedly

    2015-02-01

    The involvement of hydrogen peroxide (H2O2) generated by nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) in the antioxidant defense system was assessed in salt-challenged Arabidopsis thaliana seedlings. In the wild-type, short-term salt exposure led to a transient and significant increase of H2O2 concentration, followed by a marked increase in catalase (CAT, EC 1.11.16), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities. Pre-treatment with either a chemical trap for H2O2 (dimethylthiourea) or two widely used NADPH oxidase inhibitors (imidazol and diphenylene iodonium) significantly decreased the above-mentioned enzyme activities under salinity. Double mutant atrbohd/f plants failed to induce the antioxidant response under the culture conditions. Under long-term salinity, the wild-type was more salt-tolerant than the mutant based on the plant biomass production. The better performance of the wild-type was related to a significantly higher photosynthetic activity, a more efficient K(+) selective uptake, and to the plants' ability to deal with the salt-induced oxidative stress as compared to atrbohd/f. Altogether, these data suggest that the early H2O2 generation by NADPH oxidase under salt stress could be the beginning of a reaction cascade that triggers the antioxidant response in A. thaliana in order to overcome the subsequent reactive oxygen species (ROS) production, thereby mitigating the salt stress-derived injuries. PMID:25462961

  10. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

    NASA Technical Reports Server (NTRS)

    McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

    2003-01-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  11. Endothelin-1 impairs coronary arteriolar dilation: Role of p38 kinase-mediated superoxide production from NADPH oxidase.

    PubMed

    Thengchaisri, Naris; Hein, Travis W; Ren, Yi; Kuo, Lih

    2015-09-01

    Elevated levels of endothelin-1 (ET-1), a potent vasoactive peptide, are implicated as a risk factor for cardiovascular diseases by exerting vasoconstriction. The aim of this study was to address whether ET-1, at sub-vasomotor concentrations, elicits adverse effects on coronary microvascular function. Porcine coronary arterioles (50-100μm) were isolated, cannulated and pressurized without flow for in vitro study. Diameter changes were recorded using a videomicrometer. Arterioles developed basal tone (60±3μm) and dilated to the endothelium-dependent nitric oxide (NO)-mediated vasodilators serotonin (1nmol/L to 0.1μmol/L) and adenosine (1nmol/L to 10μmol/L). Treating the vessels with a clinically relevant sub-vasomotor concentration of ET-1 (10pmol/L, 60min) significantly attenuated arteriolar dilations to adenosine and serotonin but not to endothelium-independent vasodilator sodium nitroprusside. The arteriolar wall contains ETA receptors and the adverse effect of ET-1 was prevented by ETA receptor antagonist BQ123, the superoxide scavenger Tempol, the NADPH oxidase inhibitors apocynin and VAS2870, the NOX2-based NADPH oxidase inhibitor gp91 ds-tat, or the p38 kinase inhibitor SB203580. However, ETB receptor antagonist BQ788, H2O2 scavenger catalase, scrambled gp91 ds-tat, or inhibitors of xanthine oxidase (allopurinol), PKC (Gö 6983), Rho kinase (Y27632), and c-Jun N-terminal kinase (SP600125) did not protect the vessel. Immunohistochemical staining showed that ET-1 elicited Tempol-, apocynin- and SB203580-sensitive superoxide productions in the arteriolar wall. Our results indicate that exposure of coronary arterioles to a pathophysiological, sub-vasomotor concentration of ET-1 leads to vascular dysfunction by impairing endothelium-dependent NO-mediated dilation via p38 kinase-mediated production of superoxide from NADPH oxidase following ETA receptor activation. PMID:26211713

  12. Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase.

    PubMed

    Inoguchi, Toyoshi; Sonta, Toshiyo; Tsubouchi, Hirotaka; Etoh, Takashi; Kakimoto, Maiko; Sonoda, Noriyuki; Sato, Naoichi; Sekiguchi, Naotaka; Kobayashi, Kunihisa; Sumimoto, Hideki; Utsumi, Hideo; Nawata, Hajime

    2003-08-01

    Hyperglycemia seems to be an important causative factor in the development of micro- and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKC-dependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes. PMID:12874436

  13. The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ pump

    PubMed Central

    Tazzeo, T; Worek, F; Janssen, LJ

    2009-01-01

    Background and purpose: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. Experimental approach: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca2+ pump activity was assessed by Ca2+ uptake into smooth muscle microsomes. Key results: Contractions to acetylcholine were markedly enhanced by DPI (10−4 M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC50∼8 × 10−6 M and 6 × 10−7 M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca2+ pump. Direct measurement of microsomal Ca2+ uptake revealed that DPI modestly inhibits the internal Ca2+ pump. Conclusions and implications: DPI inhibits cholinesterase activity and the internal Ca2+ pump in tracheal smooth muscle. PMID:19788497

  14. A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase.

    PubMed

    Joo, Jung Hee; Huh, Jeong-Eun; Lee, Jee Hyun; Park, Doo Ri; Lee, Yoonji; Lee, Seul Gee; Choi, Sun; Lee, Hwa Jeong; Song, Seong-Won; Jeong, Yongmi; Goo, Ja-Il; Choi, Yongseok; Baek, Hye Kyung; Yi, Sun Shin; Park, Soo Jin; Lee, Ji Eun; Ku, Sae Kwang; Lee, Won Jae; Lee, Kee-In; Lee, Soo Young; Bae, Yun Soo

    2016-01-01

    Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis. PMID:26975635

  15. A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase

    PubMed Central

    Joo, Jung Hee; Huh, Jeong-Eun; Lee, Jee Hyun; Park, Doo Ri; Lee, Yoonji; Lee, Seul Gee; Choi, Sun; Lee, Hwa Jeong; Song, Seong-Won; Jeong, Yongmi; Goo, Ja-Il; Choi, Yongseok; Baek, Hye Kyung; Yi, Sun Shin; Park, Soo Jin; Lee, Ji Eun; Ku, Sae Kwang; Lee, Won Jae; Lee, Kee-In; Lee, Soo Young; Bae, Yun Soo

    2016-01-01

    Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis. PMID:26975635

  16. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  17. NADPH oxidase-dependent redox signaling in TGF-β-mediated fibrotic responses☆

    PubMed Central

    Jiang, Fan; Liu, Guei-Sheung; Dusting, Gregory J.; Chan, Elsa C.

    2014-01-01

    Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-β has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-β would have undesired systemic side effects due to the multiple physiological functions of TGF-β. Further characterization of the downstream signaling pathway(s) involved in TGF-β-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-β-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-β/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders. PMID:24494202

  18. Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

    PubMed Central

    Gorin, Yves; Wauquier, Fabien

    2015-01-01

    Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-β. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted. PMID:25824546

  19. Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways.

    PubMed

    Cui, Yanji; Park, Jee-Yun; Wu, Jinji; Lee, Ji Hyung; Yang, Yoon-Sil; Kang, Moon-Seok; Jung, Sung-Cherl; Park, Joo Min; Yoo, Eun-Sook; Kim, Seong-Ho; Ahn Jo, Sangmee; Suk, Kyoungho; Eun, Su-Yong

    2015-05-01

    Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 g/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91 (phox) , which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways. PMID:25954126

  20. Prostaglandin E2 Suppresses Bacterial Killing in Alveolar Macrophages by Inhibiting NADPH Oxidase

    PubMed Central

    Serezani, Carlos H.; Chung, Jooho; Ballinger, Megan N.; Moore, Bethany B.; Aronoff, David M.; Peters-Golden, Marc

    2007-01-01

    Prostaglandin E2 (PGE2) is a potent lipid mediator that effects changes in cell functions through ligation of four distinct G protein–coupled E prostanoid (EP) receptors (EP1–EP4). PGE2 inhibits bacterial killing and reactive oxygen intermediate (ROI) production by alveolar macrophages (AMs), although little is known about the operative molecular mechanisms. The aims of this study were to evaluate the molecular mechanisms and the specific EP receptors through which PGE2 inhibits killing of Klebsiella pneumoniae by AMs. The treatment of AMs with PGE2 suppressed the killing of K. pneumoniae, and this effect was blocked by an adenylyl cyclase inhibitor and mimicked by agonists for the stimulatory G protein (Gs)-coupled EP2 and EP4 receptors. Conversely, microbicidal activity was augmented by pretreatment with the cyclooxygenase inhibitor, indomethacin, and antagonists of EP2 and EP4. Similar results were found when ROI production was examined. PGE2 inhibition of killing and ROI generation was associated with its activation of the cAMP effectors, protein kinase A and exchange protein directly activated by cAMP-1, as well as attenuation of the phosphorylation and translocation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component, p47phox, to the phagosomal membrane. We conclude that PGE2 suppresses the microbicidal activity of AMs through the Gs-coupled EP2/EP4 receptors, with increased cAMP inhibiting the assembly and activation of p47phox. PMID:17585108

  1. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

    PubMed Central

    Altenhöfer, Sebastian; Radermacher, Kim A.; Kleikers, Pamela W.M.; Wingler, Kirstin

    2015-01-01

    Abstract Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406–427. PMID:24383718

  2. Influenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances susceptibility to secondary methicillin-resistant Staphylococcus aureus infection.

    PubMed

    Sun, Keer; Metzger, Dennis W

    2014-04-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. In this study, we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, even though MRSA superinfection had no significant effect on viral burdens. Compared with mice infected with bacteria alone, postinfluenza MRSA-infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for reactive oxygen species production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91(phox)-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to wild-type animals, gp91(phox-/-) mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91(phox+/-) mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Taken together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection. PMID:24563256

  3. NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus

    PubMed Central

    Ryder, Lauren S.; Dagdas, Yasin F.; Mentlak, Thomas A.; Kershaw, Michael J.; Thornton, Christopher R.; Schuster, Martin; Chen, Jisheng; Wang, Zonghua; Talbot, Nicholas J.

    2013-01-01

    The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics. PMID:23382235

  4. Fetal–maternal interface impedance parallels local NADPH oxidase related superoxide production

    PubMed Central

    Guedes-Martins, L.; Silva, E.; Gaio, A.R.; Saraiva, J.; Soares, A.I.; Afonso, J.; Macedo, F.; Almeida, H.

    2015-01-01

    Blood flow assessment employing Doppler techniques is a useful procedure in pregnancy evaluation, as it may predict pregnancy disorders coursing with increased uterine vascular impedance, as pre-eclampsia. While the local causes are unknown, emphasis has been put on reactive oxygen species (ROS) excessive production. As NADPH oxidase (NOX) is a ROS generator, it is hypothesized that combining Doppler assessment with NOX activity might provide useful knowledge on placental bed disorders underlying mechanisms. A prospective longitudinal study was performed in 19 normal course, singleton pregnancies. Fetal aortic isthmus (AoI) and maternal uterine arteries (UtA) pulsatility index (PI) were recorded at two time points: 20–22 and 40–41 weeks, just before elective Cesarean section. In addition, placenta and placental bed biopsies were performed immediately after fetal extraction. NOX activity was evaluated using a dihydroethidium-based fluorescence method and associations to PI values were studied with Spearman correlations. A clustering of pregnancies coursing with higher and lower PI values was shown, which correlated strongly with placental bed NOX activity, but less consistently with placental tissue. The study provides evidence favoring that placental bed NOX activity parallels UtA PI enhancement and suggests that an excess in oxidation underlies the development of pregnancy disorders coursing with enhanced UtA impedance. PMID:25912167

  5. NADPH Oxidase 4 Mediates TGFβ1-induced CCN2 in Gingival Fibroblasts.

    PubMed

    Yang, W H; Deng, Y T; Hsieh, Y P; Wu, K J; Kuo, M Y P

    2015-07-01

    Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFβ in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFβ. We previously showed that Src, JNK, and Smad3 mediate TGFβ1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFβ. In this study, we demonstrated that TGFβ1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFβ1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFβ1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFβ1-induced NOX4 expression in HGFs. PMID:25858818

  6. Methamphetamine alters occludin expression via NADPH oxidase-induced oxidative insult and intact caveolae

    PubMed Central

    Park, Minseon; Hennig, Bernhard; Toborek, Michal

    2012-01-01

    Abstract Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. This study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells. PMID:21435178

  7. The link between angiotensin II-mediated anxiety and mood disorders with NADPH oxidase-induced oxidative stress

    PubMed Central

    Liu, Feng; Havens, Jennifer; Yu, Qi; Wang, Gang; Davisson, Robin L.; Pickel, Virginia M.; Iadecola, Costantino

    2012-01-01

    The renin-angiotensin system (RAS) and its active peptide angiotensin II (AngII) have major involvements not only in hypertension but also in mood and anxiety disorders. Substantial evidence supports the notion that AngII acts as a neuromodulator in the brain. In this review, we provide an overview of the link between the RAS and anxiety or mood disorders, and focus on recent advances in the understanding of AngII-linked, NADPH oxidase-derived oxidative stress in the central nervous system, which may underlie pathogenesis of mood and anxiety disorders. PMID:22461954

  8. Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase.

    PubMed

    Youn, Ji-Youn; Zhang, Jun; Zhang, Yixuan; Chen, Houzao; Liu, Depei; Ping, Peipei; Weiss, James N; Cai, Hua

    2013-09-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways. PMID:23643589

  9. Role of NADPH oxidase in the regulation of autophagy in cardiomyocytes.

    PubMed

    Sciarretta, Sebastiano; Yee, Derek; Ammann, Paul; Nagarajan, Narayani; Volpe, Massimo; Frati, Giacomo; Sadoshima, Junichi

    2015-04-01

    In the past several years, it has been demonstrated that the reactive oxygen species (ROS) may act as intracellular signalling molecules to activate or inhibit specific signalling pathways and regulate physiological cellular functions. It is now well-established that ROS regulate autophagy, an intracellular degradation process. However, the signalling mechanisms through which ROS modulate autophagy in a regulated manner have only been minimally clarified. NADPH oxidase (Nox) enzymes are membrane-bound enzymatic complexes responsible for the dedicated generation of ROS. Different isoforms of Nox exist with different functions. Recent studies demonstrated that Nox-derived ROS can promote autophagy, with Nox2 and Nox4 representing the isoforms of Nox implicated thus far. Nox2- and Nox4-dependent autophagy plays an important role in the elimination of pathogens by phagocytes and in the regulation of vascular- and cancer-cell survival. Interestingly, we recently found that Nox is also important for autophagy regulation in cardiomyocytes. We found that Nox4, but not Nox2, promotes the activation of autophagy and survival in cardiomyocytes in response to nutrient deprivation and ischaemia through activation of the PERK (protein kinase RNA-like endoplasmic reticulum kinase) signalling pathway. In the present paper, we discuss the importance of Nox family proteins and ROS in the regulation of autophagy, with a particular focus on the role of Nox4 in the regulation of autophagy in the heart. PMID:25515000

  10. Oxidative Stress in Atrial Fibrillation: An Emerging Role of NADPH Oxidase

    PubMed Central

    Youn, Ji-Youn; Zhang, Jun; Zhang, Yixuan; Chen, Houzao; Liu, Depei; Ping, Peipei; Weiss, James N.; Cai, Hua

    2013-01-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as Statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and Statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways. PMID:23643589

  11. Role of NADPH oxidases in the redox biology of liver fibrosis

    PubMed Central

    Crosas-Molist, Eva; Fabregat, Isabel

    2015-01-01

    Liver fibrosis is the pathological consequence of chronic liver diseases, where an excessive deposition of extracellular matrix (ECM) proteins occurs, concomitantly with the processes of repair and regeneration. It is characterized by increased production of matrix proteins, in particular collagens, and decreased matrix remodelling. The principal source of ECM accumulation is myofibroblasts (MFB). Most fibrogenic MFB are endogenous to the liver, coming from hepatic stellate cells (HSC) and portal fibroblasts. Dysregulated inflammatory responses have been associated with most (if not all) hepatotoxic insults and chronic oxidative stress play a role during the initial liver inflammatory phase and its progression to fibrosis. Redox-regulated processes are responsible for activation of HSC to MFB, as well as maintenance of the MFB function. Increased oxidative stress also induces hepatocyte apoptosis, which contributes to increase the liver injury and to transdifferentiate HSC to MFB, favouring the fibrogenic process. Mitochondria and other redox-active enzymes can generate superoxide and hydrogen peroxide as a by-product in liver cells. Moreover, accumulating evidence indicates that NADPH oxidases (NOXs), which play a critical role in the inflammatory response, may contribute to reactive oxygen species (ROS) production during liver fibrosis, being important players in HSC activation and hepatocyte apoptosis. Based on the knowledge of the pathogenic role of ROS, different strategies to prevent or reverse the oxidative damage have been developed to be used as therapeutic tools in liver fibrosis. This review will update all these concepts, highlighting the relevance of redox biology in chronic fibrogenic liver pathologies. PMID:26204504

  12. NOX4 NADPH Oxidase-Dependent Mitochondrial Oxidative Stress in Aging-Associated Cardiovascular Disease

    PubMed Central

    Vendrov, Aleksandr E.; Vendrov, Kimberly C.; Smith, Alberto; Yuan, Jinling; Sumida, Arihiro; Robidoux, Jacques; Madamanchi, Nageswara R.

    2015-01-01

    Abstract Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe−/− mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe−/− and Apoe−/−/p47phox−/− mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox−/− mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe−/− mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD. Antioxid. Redox Signal. 23, 1389–1409. PMID:26054376

  13. Matrix compliance regulates Rac1b localization, NADPH oxidase assembly, and epithelialmesenchymal transition

    PubMed Central

    Lee, KangAe; Chen, Qike K.; Lui, Cecillia; Cichon, Magdalena A.; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Epithelialmesenchymal transition (EMT) is a form of epithelial plasticity implicated in fibrosis and tumor metastasis. Here we show that the mechanical rigidity of the microenvironment plays a pivotal role in the promotion of EMT by controlling the subcellular localization and downstream signaling of Rac GTPases. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against EMT, whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Rac1b, a highly activated splice variant of Rac1 found in tumors, localizes to the plasma membrane in cells cultured on stiff substrata or in collagen-rich regions of human breast tumors. At the membrane, Rac1b forms a complex with NADPH oxidase and promotes the production of reactive oxygen species, expression of Snail, and activation of the EMT program. In contrast, soft microenvironments inhibit the membrane localization of Rac1b and subsequent redox changes. These results reveal a novel mechanotransduction pathway in the regulation of epithelial plasticity via EMT. PMID:22918955

  14. Neuroprotection of taurine against reactive oxygen species is associated with inhibiting NADPH oxidases.

    PubMed

    Han, Zhou; Gao, Li-Yan; Lin, Yu-Hui; Chang, Lei; Wu, Hai-Yin; Luo, Chun-Xia; Zhu, Dong-Ya

    2016-04-15

    It is well established that taurine shows potent protection against glutamate-induced injury to neurons in stroke. The neuroprotection may result from multiple mechanisms. Increasing evidences suggest that NADPH oxidases (Nox), the primary source of superoxide induced by N-methyl-d-aspartate (NMDA) receptor activation, are involved in the process of oxidative stress. We found that 100μM NMDA induced oxidative stress by increasing the reactive oxygen species level, which contributed to the cell death, in vitro. Neuron cultures pretreated with 25mM taurine showed lower percentage of death cells and declined reactive oxygen species level. Moreover, taurine attenuated Nox2/Nox4 protein expression and enzyme activity and declined intracellular calcium intensity during NMDA-induced neuron injury. Additionally, taurine also showed neuroprotection against H2O2-induced injury, accompanying with Nox inhibition. So, we suppose that protection of taurine against reactive oxygen species during NMDA-induced neuron injury is associated with Nox inhibition, probably in a calcium-dependent manner. PMID:26945820

  15. Nox Family NADPH Oxidases in Mechano-Transduction: Mechanisms and Consequences

    PubMed Central

    Weissmann, Norbert; Schröder, Katrin

    2014-01-01

    Abstract Significance: The majority of cells in a multi-cellular organism are continuously exposed to ever-changing physical forces. Mechano-transduction links these events to appropriate reactions of the cells involving stimulation of signaling cascades, reorganization of the cytoskeleton and alteration of gene expression. Recent Advances: Mechano-transduction alters the cellular redox balance and the formation of reactive oxygen species (ROS). Nicotine amide adenine dinucleotide reduced form (NADPH) oxidases of the Nox family are prominent ROS generators and thus, contribute to this stress-induced ROS formation. Critical Issues: Different types and patterns of mechano-stress lead to Nox-dependent ROS formation and Nox-mediated ROS formation contributes to cellular responses and adaptation to physical forces. Thereby, Nox enzymes can mediate vascular protection during physiological mechano-stress. Despite this, over-activation and induction of Nox enzymes and a subsequent substantial increase in ROS formation also promotes oxidative stress in pathological situations like disturbed blood flow or extensive stretch. Future Directions: Individual protein targets of Nox-mediated redox-signaling will be identified to better understand the specificity of Nox-dependent ROS signaling in mechano-transduction. Nox-inhibitors will be tested to reduce cellular activation in response to mechano-stimuli. Antioxid. Redox Signal. 20, 887–898. PMID:23682993

  16. Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase

    PubMed Central

    Kim, Gab Seok; Jung, Joo Eun; Narasimhan, Purnima; Sakata, Hiroyuki; Yoshioka, Hideyuki; Song, Yun Seon; Okami, Nobuya; Chan, Pak H

    2012-01-01

    Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α′ and dephosphorylation of CK2β against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2′-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury. PMID:22146192

  17. Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase.

    PubMed

    Kim, Gab Seok; Jung, Joo Eun; Narasimhan, Purnima; Sakata, Hiroyuki; Yoshioka, Hideyuki; Song, Yun Seon; Okami, Nobuya; Chan, Pak H

    2012-04-01

    Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α' and dephosphorylation of CK2β against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2'-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury. PMID:22146192

  18. Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase.

    TOXLINE Toxicology Bibliographic Information

    Kim GS; Jung JE; Narasimhan P; Sakata H; Yoshioka H; Song YS; Okami N; Chan PH

    2012-04-01

    Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α' and dephosphorylation of CK2β against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2'-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury.

  19. Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

    PubMed

    Arai, Yasuyuki; Nishinaka, Yoko; Arai, Toshiyuki; Morita, Makiko; Mizugishi, Kiyomi; Adachi, Souichi; Takaori-Kondo, Akifumi; Watanabe, Tomohiro; Yamashita, Kouhei

    2014-01-10

    Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia. PMID:24326071

  20. New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

    PubMed Central

    Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

    2014-01-01

    NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by ∆nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in ∆nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and ∆nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of ∆nox2, lacking the NADPH oxidase 2 gene, ∆nor1, and transcription factor deletion mutant ∆ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi. PMID:24407906

  1. New insights into the roles of NADPH oxidases in sexual development and ascospore germination in Sordaria macrospora.

    PubMed

    Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domnguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kck, Ulrich

    2014-03-01

    NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of nox2, lacking the NADPH oxidase 2 gene, nor1, and transcription factor deletion mutant ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein ?-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi. PMID:24407906

  2. Activation of NADPH oxidase by transforming growth factor-β in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-κB-dependent mechanism

    PubMed Central

    Murillo, Miguel M.; Carmona-Cuenca, Irene; del Castillo, Gaelle; Ortiz, Conrad; Roncero, César; Sánchez, Aránzazu; Fernández, Margarita; Fabregat, Isabel

    2007-01-01

    The TGF-β (transforming growth factor-β) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor α-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-α and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-β in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-β, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TβR-I (TGF-β receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-β up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TβR-I-dependent manner. TGF-β mediates activation of the nuclear factor-κB pathway, which is inhibited by DPI and is required for up-regulation of TGF-α and HB-EGF. In contrast, EGFR activation is not required for TGF-β-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-β in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-β-treated cells. PMID:17407446

  3. Expression of NADPH oxidase homologues and accessory genes in human cancer cell lines, tumours and adjacent normal tissues

    PubMed Central

    JUHASZ, AGNES; GE, YUN; MARKEL, SUSAN; CHIU, ALICE; MATSUMOTO, LINDA; VAN BALGOOY, JOSEPHUS; ROY, KRISHNENDU; DOROSHOW, JAMES H

    2010-01-01

    The family of NADPH oxidase (NOX) genes produces reactive oxygen species (ROS) pivotal for both cell signalling and host defense. To investigate whether NOX and NOX accessory gene expression might be a factor common to specific human tumour types, this study measured the expression levels of NOX genes 1–5, dual oxidase 1 and 2, as well as those of NOX accessory genes NoxO1, NoxA1, p47phox, p67phox and p22phox in human cancer cell lines and in tumour and adjacent normal tissue pairs by quantitative, real-time RT-PCR. The results demonstrate tumour-specific patterns of NOX gene expression that will inform further studies of the role of NOX activity in tumour cell invasion, growth factor response and proliferative potential. PMID:19431059

  4. Protection against LPS-induced acute lung injury by a mechanism-based inhibitor of NADPH oxidase (type 2)

    PubMed Central

    Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Feinstein, Sheldon I.

    2014-01-01

    The phospholipase A2 activity of peroxiredoxin 6 is inhibited by the transition state analog, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33). This activity is required for the activation of NADPH oxidase, type 2. The present study evaluated the effect of MJ33 on manifestations of acute lung injury. Mice were injected intratracheally (IT) with LPS from Escherichia coli 0111:B4 (LPS, 1 or 5 mg/kg), either concurrently with LPS or 2 h later, and evaluated for lung injury 24 h later. MJ33 inhibited reactive oxygen species (ROS) generation by lungs when measured at 24 h after LPS. LPS at either a low or high dose significantly increased lung infiltration with inflammatory cells, secretion of proinflammatory cytokines (IL-6, TNF-α, and the chemokine macrophage inflammatory protein-2), expression of lung vascular cell adhesion molecule, lung permeability (protein in bronchoalveolar lavage fluid, leakage of FITC-dextran, lung wet-to-dry weight ratio), tissue lipid peroxidation (thiobarbituric acid reactive substances, 8-isoprostanes), tissue protein oxidation (protein carbonyls), and activation of NF-κB. MJ33, given either concurrently or 2 h subsequent to LPS, significantly reduced all of these measured parameters. Previous studies of toxicity showed a high margin of safety for MJ33 in the intact mouse. Thus we have identified MJ33 as a potent, nontoxic, and specific mechanism-based inhibitor of NADPH oxidase type 2-mediated ROS generation that protects mice against lung injury associated with inflammation. PMID:24487388

  5. Expression dynamics of NADPH oxidases during early zebrafish development.

    PubMed

    Weaver, Cory J; Leung, Yuk Fai; Suter, Daniel M

    2016-07-01

    Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX-derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130-2141, 2016. © 2015 Wiley Periodicals, Inc. PMID:26662995

  6. The Arabidopsis NADPH oxidases RbohD and RbohF display differential expression patterns and contributions during plant immunity.

    PubMed

    Morales, Jorge; Kadota, Yasuhiro; Zipfel, Cyril; Molina, Antonio; Torres, Miguel-Angel

    2016-04-01

    Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity. PMID:26798024

  7. NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology

    PubMed Central

    Schiavone, S; Jaquet, V; Sorce, S; Dubois-Dauphin, M; Hultqvist, M; Bäckdahl, L; Holmdahl, R; Colaianna, M; Cuomo, V; Trabace, L; Krause, K-H

    2012-01-01

    Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47phox was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47phox were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations. PMID:22832955

  8. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana.

    PubMed

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi; Shirasu, Ken; Yoshioka, Hirofumi

    2015-09-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  9. NADPH Oxidase Inhibitor Apocynin Attenuates PCB153-Induced Thyroid Injury in Rats

    PubMed Central

    Abliz, Ablikim; Chen, Chen; Deng, Wenhong; Sun, Rongze

    2016-01-01

    PCBs, widespread endocrine disruptors, cause the disturbance of thyroid hormone (TH) homeostasis in humans and animals. However, the exact mechanism of thyroid dysfunction caused by PCBs is still unknown. In order to clarify the hypotheses that NADPH oxidase (NOX) and subsequent NF-κB pathway may play roles in thyroid dysfunction, sixty Sprague-Dawley rats were randomly divided into four groups: control group, PCB153 treated (PCB) group, received apocynin with PCB153 treatment (APO + PCB) group, and drug control (APO) group. Serum thyroid hormone levels were evaluated. The morphological change of thyroid tissue was analyzed under the light and transmission electron microscopy. NOX2, 8-OHdG, and NF-κB expression in the thyroid tissue was evaluated by immune-histochemical staining. Oxidative stress and inflammatory cytokines were detected. The following results were reduced after apocynin treatment: (1) serum thyroid hormone, (2) thyroid pathological injuries, (3) thyroid MDA, (4) thyroid ultrastructural change, (5) serum inflammatory cytokines, and (6) thyroid expression of NOX2, 8-OHdG, and NF-κB. These results suggested that NOX inhibition attenuates thyroid dysfunction induced by PCB in rats, presumably because of its role in preventing ROS generation and inhibiting the activation of NF-κB pathway. Our findings may provide new therapeutic targets for PCBs induced thyroid dysfunction. PMID:27047545

  10. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein*

    PubMed Central

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S.; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M.; Brandes, Ralf P.

    2016-01-01

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. PMID:26861875

  11. NADPH oxidases-do they play a role in TRPC regulation under hypoxia?

    PubMed

    Malczyk, Monika; Veith, Christine; Schermuly, Ralph T; Gudermann, Thomas; Dietrich, Alexander; Sommer, Natascha; Weissmann, Norbert; Pak, Oleg

    2016-01-01

    In the lung, acute alveolar hypoxia causes hypoxic pulmonary vasoconstriction (HPV) to maintain ventilation perfusion matching and thus optimal oxygenation of blood. In contrast, global chronic hypoxia triggers a pathological thickening of pulmonary arterial walls, called pulmonary vascular remodelling, leading to persistence of pulmonary hypertension (PH). Moreover, ischaemia or hypoxia can lead to a damage of pulmonary endothelial cells with subsequent oedema formation. Alterations in reactive oxygen species (ROS) have been suggested as a crucial mediator of such responses. Among the various sources of cellular ROS production, NADPH oxidases (NOXs) have been found to contribute to these physiological and pathophysiological signalling processes. NOXs are the only known examples that generate ROS as the primary function of the enzyme system. However, the downstream targets of NOX-derived ROS signalling in hypoxia are still not known. Canonical transient receptor potential (TRPC) channels recently have been recognised as directly or indirectly ROS-activated channels and have been shown to be essential for hypoxia-dependent vascular regulatory processes in the lung. Against this background, we here summarise the current knowledge on NOX-mediated TRPC channel signalling during hypoxia in the pulmonary circulation. PMID:26424109

  12. The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

    PubMed

    Prior, Kim-Kristin; Wittig, Ilka; Leisegang, Matthias S; Groenendyk, Jody; Weissmann, Norbert; Michalak, Marek; Jansen-Dürr, Pidder; Shah, Ajay M; Brandes, Ralf P

    2016-03-25

    Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. PMID:26861875

  13. Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

    PubMed Central

    Seil, Michèle; Fontanils, Unai; Etxebarria, Irantzu Gorrono; Pochet, Stéphanie; Garcia-Marcos, Mikel; Marino, Aida

    2008-01-01

    ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X7 knockout mice or in cells pretreated with a P2X7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species. PMID:18581262

  14. NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

    PubMed Central

    Hofstetter, Amelia R.; De La Cruz, Juan A.; Cao, Weiping; Patel, Jenish; Belser, Jessica A.; McCoy, James; Liepkalns, Justine S.; Amoah, Samuel; Cheng, Guangjie; Ranjan, Priya; Diebold, Becky A.; Shieh, Wun-Ju; Zaki, Sherif; Katz, Jacqueline M.; Sambhara, Suryaprakash; Lambeth, J. David; Gangappa, Shivaprakash

    2016-01-01

    The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection. PMID:26910342

  15. Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress.

    PubMed

    Williams, Robert; Lemaire, Paul; Lewis, Philip; McDonald, Fiona B; Lucking, Eric; Hogan, Sean; Sheehan, David; Healy, Vincent; O'Halloran, Ken D

    2015-01-01

    Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling. PMID:25688214

  16. Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells.

    PubMed

    Verzola, Daniela; Famà, Annamaria; Villaggio, Barbara; Di Rocco, Maia; Simonato, Alchiede; D'Amato, Elena; Gianiorio, Fabio; Garibotto, Giacomo

    2012-11-01

    Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 ± 23%, p67 (phox) +108 ± 22% and gp91 (phox) +75 ± 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-L-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI. PMID:22403019

  17. Caveolin-1 is a Negative Regulator of NADPH Oxidase-Derived Reactive Oxygen Species

    PubMed Central

    Chen, Feng; Barman, Scott; Yu, Yanfang; Haigh, Steven; Wang, Yusi; Dou, Huijuan; Bagi, Zsolt; Han, Weihong; Su, Yunchao; Fulton, David J.R.

    2014-01-01

    Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Nox), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PA) and that Cav-1 expression is reduced in isolated PA from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1-5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In additional to post-translational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 though inhibition of the NF-kB pathway. Lastly, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS. PMID:24835767

  18. Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

    PubMed

    Chignalia, Andreia Z; Oliveira, Maria Aparecida; Debbas, Victor; Dull, Randal O; Laurindo, Francisco R M; Touyz, Rhian M; Carvalho, Maria Helena C; Fortes, Zuleica B; Tostes, Rita C

    2015-07-01

    The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk. PMID:25700020

  19. Inhibition of NADPH oxidase prevents acute lung injury in obese rats following severe trauma.

    PubMed

    Xiang, Lusha; Lu, Silu; Mittwede, Peter N; Clemmer, John S; Hester, Robert L

    2014-03-01

    Lung capillary filtration coefficient (Kf) and impacts of oxidative stress have not been determined in the setting of severe trauma, especially in obese patients who exhibit increased lung injury. We hypothesized that severe trauma leads to a greater increase in lung Kf in obesity due to exacerbated production of and/or vulnerability to oxidative stress. Severe trauma was induced in lean and obese Zucker rats by muscle injury, fibula fracture, and bone component injection to both hindlimbs, with or without 24-h treatments of apocynin, a NADPH oxidase (NOX) inhibitor. Lung wet/dry weight ratios, lung vascular Kf, lung neutrophil counts, lung NOX and myeloperoxidase (MPO) activity, and plasma IL-6 levels were measured 24 h after trauma. In an additional study, lungs were isolated from nontrauma lean and obese rats to determine the acute effect of phenazime methosulfate, a superoxide donor, on pulmonary vascular Kf. After trauma, compared with lean rats, obese rats exhibited greater increases in lung capillary Kf, neutrophil accumulation, NOX and MPO activity, and plasma IL-6. The lung wet/dry weight ratio was increased in obese rats but not in lean rats. Apocynin treatment decreased lung Kf, neutrophil counts, NOX and MPO activities, wet/dry weight ratio, and plasma IL-6 in obese rats. Phenazime methosulfate treatment resulted in a greater increase in lung Kf in nontrauma obese rats compared with nontrauma lean rats. These results suggest that obese rats are susceptible to lung injury following severe trauma due to increased production of and responsiveness to pulmonary oxidative stress. PMID:24414071

  20. C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells.

    PubMed

    Manea, Simona-Adriana; Todirita, Andra; Raicu, Monica; Manea, Adrian

    2014-07-01

    In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress. PMID:24797079

  1. C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells

    PubMed Central

    Manea, Simona-Adriana; Todirita, Andra; Raicu, Monica; Manea, Adrian

    2014-01-01

    In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress. PMID:24797079

  2. Endothelial NADPH oxidase 4 protects ApoE-/- mice from atherosclerotic lesions.

    PubMed

    Craige, Siobhan M; Kant, Shashi; Reif, Michaella; Chen, Kai; Pei, Yongmei; Angoff, Rebecca; Sugamura, Koichi; Fitzgibbons, Timothy; Keaney, John F

    2015-12-01

    Vascular reactive oxygen species (ROS) are known to be involved in atherosclerosis development and progression. NADPH oxidase 4 (Nox4) is a constitutively active ROS-producing enzyme that is highly expressed in the vascular endothelium. Nox4 is unique in its biology and has been implicated in vascular repair, however, the role of Nox4 in atherosclerosis is unknown. Therefore, to determine the effect of endothelial Nox4 on development of atherosclerosis, Apoe E-/- mice +/- endothelial Nox4 (ApoE-/- + EC Nox4) were fed a high cholesterol/high fat (Western) diet for 24 weeks. Significantly fewer atherosclerotic lesions were observed in the ApoE-/- + EC Nox4 mice as compared to the ApoE-/- littermates, which was most striking in the abdominal region of the aorta. In addition, markers of T cell populations were markedly different between the groups; T regulatory cell marker (FoxP3) was increased whereas T effector cell marker (T-bet) was decreased in aorta from ApoE-/- + EC Nox4 mice compared to ApoE-/- alone. We also observed decreased monokine induced by gamma interferon (MIG; CXCL9), a cytokine known to recruit and activate T cells, in plasma and tissue from ApoE-/- + EC Nox4 mice. To further investigate the link between endothelial Nox4 and MIG expression, we utilized cultured endothelial cells from our EC Nox4 transgenic mice and human cells with adenoviral overexpression of Nox4. In these cultured cells, upregulation of Nox4 attenuated endothelial cell MIG expression in response to interferon-gamma. Together these data suggest that endothelial Nox4 expression reduces MIG production and promotes a T cell distribution that favors repair over inflammation, leading to protection from atherosclerosis. PMID:26169727

  3. Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation

    PubMed Central

    Montiel, Jesús; Arthikala, Manoj-Kumar; Cárdenas, Luis; Quinto, Carmen

    2016-01-01

    Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant’s response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis. PMID:27213330

  4. Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation.

    PubMed

    Montiel, Jesús; Arthikala, Manoj-Kumar; Cárdenas, Luis; Quinto, Carmen

    2016-01-01

    Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant's response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis. PMID:27213330

  5. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  6. NADPH oxidase 4 is a critical mediator in Ataxia telangiectasia disease

    PubMed Central

    Weyemi, Urbain; Redon, Christophe E.; Aziz, Towqir; Choudhuri, Rohini; Maeda, Daisuke; Parekh, Palak R.; Bonner, Michael Y.; Arbiser, Jack L.; Bonner, William M.

    2015-01-01

    Ataxia telangiectasia (A-T), a rare autosomal recessive disorder characterized by progressive cerebellar degeneration and a greatly increased incidence of cancer among other symptoms, is caused by a defective or missing ataxia telangiectasia mutated (ATM) gene. The ATM protein has roles in DNA repair and in the regulation of reactive oxygen species (ROS). Here, we provide, to our knowledge, the first evidence that NADPH oxidase 4 (NOX4) is involved in manifesting A-T disease. We showed that NOX4 expression levels are higher in A-T cells, and that ATM inhibition leads to increased NOX4 expression in normal cells. A-T cells exhibit elevated levels of oxidative DNA damage, DNA double-strand breaks and replicative senescence, all of which are partially abrogated by down-regulation of NOX4 with siRNA. Sections of degenerating cerebelli from A-T patients revealed elevated NOX4 levels. ATM-null mice exhibit A-T disease but they die from cancer before the neurological symptoms are manifested. Injecting Atm-null mice with fulvene-5, a specific inhibitor of NOX4 and NADPH oxidase 2 (NOX2), decreased their elevated cancer incidence to that of the controls. We conclude that, in A-T disease in humans and mice, NOX4 may be critical mediator and targeting it will open up new avenues for therapeutic intervention in neurodegeneration. PMID:25646414

  7. NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia

    PubMed Central

    Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Seo, Ju-Ha; Kim, Hahn Young; Shin, Chan Young; Han, Jung-Soo; Han, Seol-Heui; Kim, Yoon-Seong

    2014-01-01

    Abstract Aims: Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Results: Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. Innovation and Conclusion: We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD. Antioxid. Redox Signal. 21, 533–550. PMID:24294978

  8. Selective inhibition of NADPH oxidase reverses the over contraction of diabetic rat aorta?

    PubMed Central

    Rehman, Atif ur; Dugic, Elma; Benham, Chris; Lione, Lisa; Mackenzie, Louise S.

    2013-01-01

    Abnormal vascular responsiveness in diabetes has been attributed to a number of changes in contractile pathways, affected in part by the overproduction of reactive oxygen species (ROS). It has been reported that NADPH oxidase (NOX) is increased in diabetic (streptozotocin treated; STZ) rat arteries; however the pharmacological agents used to inhibit NOX activity are known to be unsuitable for in vitro studies and have a high level of non-selectivity. Here we have used the highly selective NOX inhibitor VAS2870 in diabetic rat aorta and compared its effects with apocynin, SOD, and allopurinol on phenylephrine and U46619 induced contraction. Male Wistar rats were injected intraperitoneally with 65mg/kg STZ and development of diabetes was confirmed by testing blood glucose levels. Rats were killed by CO2 asphyxiation, and the thoracic aorta removed and mounted in an organ bath under a tension of 1g. Diabetic rat aortas exhibit a greatly increased response to phenylephrine, which was reduced to a level consistent with control rat aorta by 10?5M VAS2870 and 150U/ml SOD. Incubation with VAS2870 led to an increase in normal rat aorta contraction, but led to a significant reduction in phenylephrine and U46619 induced tone in diabetic rat aorta, which indicates that ROS in diabetic rats directly contributes to these contractile responses. Apocynin and allopurinol had no effect on contraction in diabetic or normal rat aorta. This data is the first to show that selective inhibition of NOX reduces diabetic arterial contraction in direct comparison with inhibition of other known contributors of ROS. PMID:25460721

  9. Selective inhibition of NADPH oxidase reverses the over contraction of diabetic rat aorta.

    PubMed

    Rehman, Atif Ur; Dugic, Elma; Benham, Chris; Lione, Lisa; Mackenzie, Louise S

    2013-12-11

    Abnormal vascular responsiveness in diabetes has been attributed to a number of changes in contractile pathways, affected in part by the overproduction of reactive oxygen species (ROS). It has been reported that NADPH oxidase (NOX) is increased in diabetic (streptozotocin treated; STZ) rat arteries; however the pharmacological agents used to inhibit NOX activity are known to be unsuitable for in vitro studies and have a high level of non-selectivity. Here we have used the highly selective NOX inhibitor VAS2870 in diabetic rat aorta and compared its effects with apocynin, SOD, and allopurinol on phenylephrine and U46619 induced contraction. Male Wistar rats were injected intraperitoneally with 65mg/kg STZ and development of diabetes was confirmed by testing blood glucose levels. Rats were killed by CO2 asphyxiation, and the thoracic aorta removed and mounted in an organ bath under a tension of 1g. Diabetic rat aortas exhibit a greatly increased response to phenylephrine, which was reduced to a level consistent with control rat aorta by 10(-5)M VAS2870 and 150U/ml SOD. Incubation with VAS2870 led to an increase in normal rat aorta contraction, but led to a significant reduction in phenylephrine and U46619 induced tone in diabetic rat aorta, which indicates that ROS in diabetic rats directly contributes to these contractile responses. Apocynin and allopurinol had no effect on contraction in diabetic or normal rat aorta. This data is the first to show that selective inhibition of NOX reduces diabetic arterial contraction in direct comparison with inhibition of other known contributors of ROS. PMID:25460721

  10. Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice

    PubMed Central

    Bhandarkar, Sulochana S.; Jaconi, Marisa; Fried, Levi E.; Bonner, Michael Y.; Lefkove, Benjamin; Govindarajan, Baskaran; Perry, Betsy N.; Parhar, Ravi; Mackelfresh, Jamie; Sohn, Allie; Stouffs, Michael; Knaus, Ulla; Yancopoulos, George; Reiss, Yvonne; Benest, Andrew V.; Augustin, Hellmut G.; Arbiser, Jack L.

    2009-01-01

    Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function. PMID:19620773

  11. Nox1 NADPH oxidase is necessary for late but not early myocardial ischaemic preconditioning

    PubMed Central

    Jiang, Shuxia; Streeter, Jennifer; Schickling, Brandon M.; Zimmerman, Kathy; Weiss, Robert M.; Miller, Francis J.

    2014-01-01

    Aims Ischaemic preconditioning (IPC) is an adaptive mechanism that renders the myocardium resistant to injury from subsequent hypoxia. Although reactive oxygen species (ROS) contribute to both the early and late phases of IPC, their enzymatic source and associated signalling events have not yet been understood completely. Our objective was to investigate the role of the Nox1 NADPH oxidase in cardioprotection provided by IPC. Methods and results Wild-type (WT) and Nox1-deficient mice were treated with three cycles of brief coronary occlusion and reperfusion, followed by prolonged occlusion either immediately (early IPC) or after 24 h (late IPC). Nox1 deficiency had no impact on the cardioprotection afforded by early IPC. In contrast, deficiency of Nox1 during late IPC resulted in a larger infarct size, cardiac remodelling, and increased myocardial apoptosis compared with WT hearts. Furthermore, expression of Nox1 in WT hearts increased in response to late IPC. Deficiency of Nox1 abrogated late IPC-mediated activation of cardiac nuclear factor-κB (NF-κB) and induction of tumour necrosis factor-α (TNF-α) in the heart and circulation. Finally, knockdown of Nox1 in cultured cardiomyocytes prevented TNF-α induction of NF-κB and the protective effect of IPC on hypoxia-induced apoptosis. Conclusions Our data identify a critical role for Nox1 in late IPC and define a previously unrecognized link between TNF-α and NF-κB in mediating tolerance to myocardial injury. These findings have clinical significance considering the emergence of Nox1 inhibitors for the treatment of cardiovascular disease. PMID:24501329

  12. Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

    PubMed

    Yeligar, Samantha M; Harris, Frank L; Hart, C Michael; Brown, Lou Ann S

    2014-03-01

    Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 μM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia. PMID:24441868

  13. Role of mitochondria and NADPH oxidase derived reactive oxygen species in hyperoxaluria induced nephrolithiasis: therapeutic intervention with combinatorial therapy of N-acetyl cysteine and Apocynin.

    PubMed

    Sharma, Minu; Kaur, Tanzeer; Singla, S K

    2016-03-01

    The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidase, are known to play an imperative role in the pathogenesis of hyperoxaluria-induced nephrolithiasis. The present study was designed to investigate the protective effect of a combinatorial therapy based on the attenuation of oxidative stress with antioxidant (N-acetyl cysteine), and NADPH oxidase inhibitor (apocynin), that might be required to effectively eliminate hyperoxaluric manifestations. Hyperoxaluria was induced in male Wistar rats by administering 0.4% ethylene glycol with 1% ammonium chloride in drinking water for 9days. Hyperoxaluria accentuated renal oxidative stress in terms of increased ROS production and lipid peroxidation. Mitochondrial dysfunction, a central deleterious event in renal stone crystallization, was evident by decreased activities of electron transport chain complex I, II and IV, augmented mitochondrial ROS, reduced GSH/GSSG ratio, which resulted in the mitochondrial permeability transition pore (mPTP) opening as indicated by increased mitochondrial swelling in hyperoxaluric rats. Furthermore, NADPH oxidase activity was significantly increased, with raised expression of NOX1, NOX2, NOX4, p38MAPK and MnSOD, in the renal tissue of hyperoxaluric rats compared to control. However, combinatorial therapy with N-acetyl cysteine (50mg/kg/day) and apocynin (200mg/kg/day), intraperitoneally, significantly improved renal functions in hyperoxaluric rats and considerably ameliorated mitochondrial dysfunction. NAC with apocynin was also found to be effective in reducing the redundant activity of NADPH oxidase in renal tissue of hyperoxaluric rats. Hence, our investigation provides novel mechanistic insights that combinatorial approaches using targeted modulators of ROS offer therapeutic benefits in hyperoxaluria-induced nephrolithiasis. PMID:26779823

  14. Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells*

    PubMed Central

    Shah, Anu; Xia, Ling; Goldberg, Howard; Lee, Ken W.; Quaggin, Susan E.; Fantus, I. George

    2013-01-01

    Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN. PMID:23329835

  15. Mild exposure of RIN-5F β-cells to human islet amyloid polypeptide aggregates upregulates antioxidant enzymes via NADPH oxidase-RAGE: An hormetic stimulus☆

    PubMed Central

    Borchi, Elisabetta; Bargelli, Valentina; Guidotti, Valentina; Berti, Andrea; Stefani, Massimo; Nediani, Chiara; Rigacci, Stefania

    2013-01-01

    The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP), a hallmark of type 2 diabetes, contributes to pancreatic β-cell impairment, where oxidative stress plays a key role. A contribution of NADPH oxidase to reactive oxygen species (ROS) generation after cell exposure to micromolar concentrations of hIAPP aggregates has been suggested. However, little is known about β-cells exposure to lower amounts of hIAPP aggregates, similar to those found in human pancreas. Thus, we aimed to investigate the events resulting from RIN-5F cells exposure to nanomolar concentrations of toxic hIAPP aggregates. We found an early and transient rise of NADPH oxidase activity resulting from increased Nox1 expression following the engagement of receptor for advanced glycation end-products (RAGE) by hIAPP aggregates. Unexpectedly, NADPH oxidase activation was not accompanied by a significant ROS increase and the lipoperoxidation level was significantly reduced. Indeed, cell exposure to hIAPP aggregates affected the antioxidant defences, inducing a significant increase of the expression and activity of catalase and glutathione peroxidase. We conclude that exposure of pancreatic β-cells to nanomolar concentrations of hIAPP aggregates for a short time induces an hormetic response via the RAGE-Nox1 axis; the latter stimulates the enzymatic antioxidant defences that preserve the cells against oxidative stress damage. PMID:24416718

  16. Nitroglycerine-Induced Nitrate Tolerance Compromises Propofol Protection of the Endothelial Cells against TNF-α: The Role of PKC-β2 and NADPH Oxidase

    PubMed Central

    Su, Wating; Liu, Huimin; Xia, Zhong-yuan; Yang, Qing-jun; Qiao, Xin; Du, Yun; Zhang, Liangqing; Xia, Zhengyuan

    2013-01-01

    Continuous treatment with organic nitrates causes nitrate tolerance and endothelial dysfunction, which is involved with protein kinase C (PKC) signal pathway and NADPH oxidase activation. We determined whether chronic administration with nitroglycerine compromises the protective effects of propofol against tumor necrosis factor (TNF-) induced toxicity in endothelial cells by PKC-β2 dependent NADPH oxidase activation. Primary cultured human umbilical vein endothelial cells were either treated or untreated with TNF-α (40 ng/mL) alone or in the presence of the specific PKC-β2 inhibitor CGP53353 (1 μM)), nitroglycerine (10 μM), propofol (100 μM), propofol plus nitroglycerin, or CGP53353 plus nitroglycerine, respectively, for 24 hours. TNF-α increased the levels of superoxide, Nox (nitrate and nitrite), malondialdehyde, and nitrotyrosine production, accompanied by increased protein expression of p-PKC-β2, gP91phox, and endothelial cell apoptosis, whereas all these changes were further enhanced by nitroglycerine. CGP53353 and propofol, respectively, reduced TNF-α induced oxidative stress and cell toxicity. CGP53353 completely prevented TNF-α induced oxidative stress and cell toxicity in the presence or absence of nitroglycerine, while the protective effects of propofol were neutralized by nitroglycerine. It is concluded that nitroglycerine comprises the protective effects of propofol against TNF-α stimulation in endothelial cells, primarily through PKC-β2 dependent NADPH oxidase activation. PMID:24396568

  17. Topological organization of NADPH-oxidase in haematopoietic stem cell membrane: preliminary study by fluorescence near-field optical microscopy.

    PubMed

    Frassanito, M C; Piccoli, C; Capozzi, V; Boffoli, D; Tabilio, A; Capitanio, N

    2008-03-01

    The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O(2) sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane 'rafts'-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms. PMID:18331504

  18. Extracellular ATP Induces the Accumulation of Superoxide via NADPH Oxidases in Arabidopsis1

    PubMed Central

    Song, Charlotte J.; Steinebrunner, Iris; Wang, Xuanzhi; Stout, Stephen C.; Roux, Stanley J.

    2006-01-01

    Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2−) in a biphasic, dose-dependent manner, with a threshold at 500 nm ATP. This effect did not require ATP hydrolysis for it was mimicked by ATPγS. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2−, indicating that NADPH oxidases are responsible for the induced O2− accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2− production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2− accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 μm, well above the level needed to induce O2− accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2− production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses. PMID:16428598

  19. NADPH oxidase is implicated in the pathogenesis of oxidative phosphorylation dysfunction in mice fed a high-fat diet.

    PubMed

    García-Ruiz, Inmaculada; Solís-Muñoz, Pablo; Fernández-Moreira, Daniel; Grau, Montserrat; Muñoz-Yagüe, Teresa; Solís-Herruzo, José A

    2016-01-01

    The aim of this study was to evaluate the role of NADPH oxidase (NADPHox) in the pathogenesis of oxidative phosphorylation (OXPHOS) dysfunction as found in mice fed a high-fat diet (HFD). C57BL/6J mice were distributed in four groups: WT/SCD: six wild-type (WT) mice fed a standard chow diet (SCD); WT/HFD, six WT mice fed a HFD; NOX2(-/-)/SCD, six NADPHox-deficient mice on a SCD; (4) NOX2(-/-)/HFD, six NADPHox-deficient mice on a HFD. After 32 weeks, we studied the liver for: histology; OXPHOS complex activity; fully assembled OXPHOS complexes and their subunits; gene expression of OXPHOS subunits; oxidative and nitrosative stress; and oxidative DNA damage. In the liver of WT/HFD mice, we found a significant decreased in the activity of all OXPHOS complexes, in fully assembled complexes, in the amount of OXPHOS subunits, and in gene expression of mitochondrial DNA-encoded subunits. 8-hydroxy-2'-deoxyguanosine was only increased in mitochondrial DNA. The liver of NOX(-/-)/HFD mice showed mild steatosis but no non-alcoholic steatohepatitis (NASH) lesions were found. OXPHOS activity, OXPHOS subunits, and assembly of subunits into OXPHOS complexes were normal in these mice. We conclude that this study shows that NADPH deficiency protects mice from developing OXPHOS dysfunction and NASH caused by a HFD. PMID:27173483

  20. NADPH oxidase is implicated in the pathogenesis of oxidative phosphorylation dysfunction in mice fed a high-fat diet

    PubMed Central

    García-Ruiz, Inmaculada; Solís-Muñoz, Pablo; Fernández-Moreira, Daniel; Grau, Montserrat; Muñoz-Yagüe, Teresa; Solís-Herruzo, José A.

    2016-01-01

    The aim of this study was to evaluate the role of NADPH oxidase (NADPHox) in the pathogenesis of oxidative phosphorylation (OXPHOS) dysfunction as found in mice fed a high-fat diet (HFD). C57BL/6J mice were distributed in four groups: WT/SCD: six wild-type (WT) mice fed a standard chow diet (SCD); WT/HFD, six WT mice fed a HFD; NOX2−/−/SCD, six NADPHox-deficient mice on a SCD; (4) NOX2−/−/HFD, six NADPHox-deficient mice on a HFD. After 32 weeks, we studied the liver for: histology; OXPHOS complex activity; fully assembled OXPHOS complexes and their subunits; gene expression of OXPHOS subunits; oxidative and nitrosative stress; and oxidative DNA damage. In the liver of WT/HFD mice, we found a significant decreased in the activity of all OXPHOS complexes, in fully assembled complexes, in the amount of OXPHOS subunits, and in gene expression of mitochondrial DNA-encoded subunits. 8-hydroxy-2′-deoxyguanosine was only increased in mitochondrial DNA. The liver of NOX−/−/HFD mice showed mild steatosis but no non-alcoholic steatohepatitis (NASH) lesions were found. OXPHOS activity, OXPHOS subunits, and assembly of subunits into OXPHOS complexes were normal in these mice. We conclude that this study shows that NADPH deficiency protects mice from developing OXPHOS dysfunction and NASH caused by a HFD. PMID:27173483

  1. Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.

    PubMed Central

    Xing, T; Higgins, V J; Blumwald, E

    1997-01-01

    The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants. PMID:9061955

  2. Renal denervation attenuates NADPH oxidase-mediated oxidative stress and hypertension in rats with hydronephrosis.

    PubMed

    Peleli, Maria; Al-Mashhadi, Ammar; Yang, Ting; Larsson, Erik; Wåhlin, Nils; Jensen, Boye L; G Persson, A Erik; Carlström, Mattias

    2016-01-01

    Hydronephrosis is associated with the development of salt-sensitive hypertension. Studies have suggested that increased sympathetic nerve activity and oxidative stress play important roles in hypertension and the modulation of salt sensitivity. The present study primarily aimed to examine the role of renal sympathetic nerve activity in the development of hypertension in rats with hydronephrosis. In addition, we aimed to investigate if NADPH oxidase (NOX) function could be affected by renal denervation. Partial unilateral ureteral obstruction (PUUO) was created in 3-wk-old rats to induce hydronephrosis. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high-, and low-salt diets. The renal excretion pattern, NOX activity, and expression as well as components of the renin-angiotensin-aldosterone system were characterized after treatment with the normal salt diet. On the normal salt diet, rats in the PUUO group had elevated blood pressure compared with control rats (115 ± 3 vs. 87 ± 1 mmHg, P < 0.05) and displayed increased urine production and lower urine osmolality. The blood pressure change in response to salt loading (salt sensitivity) was more pronounced in the PUUO group compared with the control group (15 ± 2 vs. 5 ± 1 mmHg, P < 0.05). Renal denervation in PUUO rats attenuated both hypertension (97 ± 3 mmHg) and salt sensitivity (5 ± 1 mmHg, P < 0.05) and normalized the renal excretion pattern, whereas the degree of renal fibrosis and inflammation was not changed. NOX activity and expression as well as renin and ANG II type 1A receptor expression were increased in the renal cortex from PUUO rats and normalized by denervation. Plasma Na(+) and K(+) levels were elevated in PUUO rats and normalized after renal denervation. Finally, denervation in PUUO rats was also associated with reduced NOX expression, superoxide production, and fibrosis in the heart. In conclusion, renal denervation attenuates hypertension and restores the renal excretion pattern, which is associated with reduced renal NOX and components of the renin-angiotensin-aldosterone system. This study emphasizes a link between renal nerves, the development of hypertension, and modulation of NOX function. PMID:26538440

  3. Role of NADPH oxidases and reactive oxygen species in regulation of bone turnover and the skeletal toxicity of alcohol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies with genetically modified mice and dietary antioxidants have suggested an important role for superoxide derived from NADPH oxidase (NOX) enzymes and other reactive oxygen species (ROS) such as hydrogen peroxide in regulation of normal bone turnover during development and also in the r...

  4. Angiotensin II-dependent chronic hypertension and cardiac hypertrophy are unaffected by gp91phox-containing NADPH oxidase.

    PubMed

    Touyz, Rhian M; Mercure, Chantel; He, Ying; Javeshghani, Danesh; Yao, Guoying; Callera, Glaucia E; Yogi, Alvaro; Lochard, Nadheige; Reudelhuber, Timothy L

    2005-04-01

    The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II-dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91-), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91-). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P<0.05) in the heart and aorta of TTRhRen/gp91- and gp91-mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P<0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated. PMID:15753233

  5. Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: Implication of NADPH oxidase

    PubMed Central

    Rouhanizadeh, Mahsa; Hwang, Juliana; Clempus, Roza E.; Marcu, Laura; Lassègue, Bernard; Sevanian, Alex; Hsiai, Tzung K.

    2007-01-01

    Modified low-density lipoprotein (LDL) induces reactive oxygen species (ROS) production by vascular cells. It is unknown if specific oxidized components in these LDL particles such as oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) can stimulate ROS production. Bovine aortic endothelial cells (BAEC) were incubated with ox-PAPC (50 μg/ml). At 4 h, ox-PAPC significantly enhanced the rate of O2−• production. Pretreatment of BAEC in glucose-free Dulbecco’s modified Eagle’s medium plus 10 mM 2-deoxyglucose (2-DOG), the latter being an antimetabolite that blocks NADPH production by the pentose shunt, significantly reduced the rate of O2−• production. The intensity of NAD(P)H autofluorescence decreased by 28 ± 12% in BAEC incubated with ox-PAPC compared to untreated cells, with a further decrease in the presence of 2-DOG. Ox-PAPC also increased Nox4 mRNA expression by 2.4-fold ± 0.1 while pretreatment of BAEC with the small interfering RNA (siNox4) attenuated Nox4 RNA expression. Ox-PAPC further reduced the level of glutathione while pretreatment with apocynin (100 μM) restored the GSH level (control = 22.54 ± 0.23, GSH = 18.06 ± 0.98, apocynin = 22.55 ± 0.60, ox-PAPC + apocynin = 21.17 ± 0.36 nmol/106 cells). Treatment with ox-PAPC also increased MMP-2 mRNA expression accompanied by a 1.5-fold increase in MMP-2 activity. Ox-PAPC induced vascular endothelial O2−• production that appears to be mediated largely by NADPH oxidase activity. PMID:16274886

  6. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease

    PubMed Central

    Egan, Martin J.; Wang, Zheng-Yi; Jones, Mark A.; Smirnoff, Nicholas; Talbot, Nicholas J.

    2007-01-01

    One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Here, we report that Magnaporthe grisea, the causal agent of rice blast disease, undergoes an oxidative burst of its own during plant infection, which is associated with its development of specialized infection structures called appressoria. Scavenging of these oxygen radicals significantly delayed the development of appressoria and altered their morphology. We targeted two superoxide-generating NADPH oxidase-encoding genes, Nox1 and Nox2, and demonstrated genetically, that each is independently required for pathogenicity of M. grisea. ?nox1 and ?nox2 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. The initiation of rice blast disease therefore requires production of superoxide by the invading pathogen. PMID:17600089

  7. NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

    PubMed

    Sahoo, Sanghamitra; Meijles, Daniel N; Pagano, Patrick J

    2016-03-01

    Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of 'longevity genes' and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population. PMID:26814203

  8. Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models.

    PubMed

    Sirokmány, Gábor; Donkó, Ágnes; Geiszt, Miklós

    2016-04-01

    Nox/Duox NADPH oxidases are now considered the primary, regulated sources of reactive oxygen species (ROS). These enzymes are expressed in diverse cells and tissues, and their products are essential in several physiological settings. Knockout mouse models are instrumental in identifying the physiological functions of Nox/Duox enzymes as well as in exploring the impact of their pharmacological targeting on disease progression. The currently available data from experiments on knockout animals suggest that the lack of non-phagocytic Nox/Duox enzymes often modifies the course and phenotype in many disease models. Nevertheless, as illustrated by studies on Nox4-deficient animals, the absence of Nox-derived ROS can also lead to aggravated disease manifestation, reinforcing the need for a more balanced view on the role of ROS in health and disease. PMID:26861575

  9. In Silico Sequence Analysis Reveals New Characteristics of Fungal NADPH Oxidase Genes

    PubMed Central

    Détry, Nicolas; Choi, Jaeyoung; Kuo, Hsiao-Che; Asiegbu, Fred O.

    2014-01-01

    NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes. PMID:25346600

  10. Biochemistry, Physiology and Pathophysiology of NADPH Oxidases in the Cardiovascular System

    PubMed Central

    Lassègue, Bernard; San Martín, Alejandra; Griendling, Kathy K.

    2012-01-01

    The NADPH oxidase (Nox) enzymes are critical mediators of cardiovascular physiology and pathophysiology. These proteins are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels and proteins that regulate the cytoskeleton. Nox enzymes have been implicated in many different cardiovascular pathologies: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4 and 5), their roles in cardiovascular cell biology, and their contributions to disease development. PMID:22581922

  11. Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases

    PubMed Central

    Nauseef, William M.

    2013-01-01

    BACKGROUND The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for reassuring oxidants released extracellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. SCOPE OF REVIEW Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O2· and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. MAJOR CONCLUSIONS Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. Several reliable methods for measurement of extracellular O2· and H2O2 by NOX proteins are available. Chemiluminescent probes for both extracellular and intracellular O2· and H2O2 detection have shortcomings that limit their use Options for quantitation of intracellular O2· and H2O2 are very limited However, non-redox sensitive probes and genetically encoded reporters promise to provide spatial and temporal detection of O2· and H2O2 GENERAL SIGNIFICANCE The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. PMID:23660153

  12. NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

    PubMed

    Ameziane-El-Hassani, Rabii; Talbot, Monique; de Souza Dos Santos, Maria Carolina; Al Ghuzlan, Abir; Hartl, Dana; Bidart, Jean-Michel; De Deken, Xavier; Miot, Françoise; Diallo, Ibrahima; de Vathaire, Florent; Schlumberger, Martin; Dupuy, Corinne

    2015-04-21

    Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect. PMID:25848056

  13. NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation

    PubMed Central

    Ameziane-El-Hassani, Rabii; Talbot, Monique; de Souza Dos Santos, Maria Carolina; Al Ghuzlan, Abir; Hartl, Dana; Bidart, Jean-Michel; De Deken, Xavier; Miot, Françoise; Diallo, Ibrahima; de Vathaire, Florent; Schlumberger, Martin; Dupuy, Corinne

    2015-01-01

    Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect. PMID:25848056

  14. NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS

    PubMed Central

    Cifuentes-Pagano, Eugenia; Csanyi, Gabor; Pagano, Patrick J.

    2015-01-01

    NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns. PMID:22585059

  15. Similar and distinct roles of NADPH oxidase components in the tangerine pathotype of Alternaria alternata.

    PubMed

    Yang, Siwy Ling; Chung, Kuang-Ren

    2013-08-01

    The fungal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, which has been implicated in the production of low-level reactive oxygen species (ROS), contains mainly NoxA, NoxB (gp91(phox) homologues) and NoxR (p67(phox) homologue). Here, we report the developmental and pathological functions of NoxB and NoxR in the tangerine pathotype of Alternaria alternata. Loss-of-function genetics revealed that all three Nox components are required for the accumulation of cellular hydrogen peroxide (H₂O₂). Alternaria alternata strains lacking NoxA, NoxB or NoxR also displayed an increased sensitivity to H₂O₂ and many ROS-generating oxidants. These phenotypes are highly similar to those previously seen for the Δyap1 mutant lacking a YAP1 transcriptional regulator and for the Δhog1 mutant lacking a HOG1 mitogen-activated protein (MAP) kinase, implicating a possible link among them. A fungal strain carrying a NoxA NoxB or NoxA NoxR double mutation was more sensitive to the test compounds than the strain mutated at a single gene, implicating a synergistic function among Nox components. The ΔnoxB mutant strain failed to produce any conidia; both ΔnoxA and ΔnoxR mutant strains showed a severe reduction in sporulation. Mutant strains carrying defective NoxB had higher chitin content than the wild-type and were insensitive to calcofluor white, Congo red and the fungicides vinclozolin and fludioxonil. Virulence assays revealed that all three Nox components are required for the elaboration of the penetration process. The inability to penetrate the citrus host, observed for Δnox mutants, could be overcome by wounding and by reacquiring a dominant Nox gene. The A. alternata NoxR did not influence the expression of NoxB, but negatively regulated NoxA. Importantly, the expression of both YAP1 and HOG1 genes, whose products are involved in resistance to ROS, was down-regulated in fungi carrying defective NoxA, NoxB or NoxR. Our results highlight the requirement of Nox in ROS resistance and provide insights into its critical role in regulating both YAP1 and HOG1 in A. alternata. PMID:23527595

  16. NADPH Oxidase-Dependent Reactive Oxygen Species Mediate Amplified TLR4 Signaling and Sepsis-Induced Mortality in Nrf2-deficient Mice

    PubMed Central

    Kong, Xiaoni; Thimmulappa, Rajesh; Kombairaju, Ponvijay; Biswal, Shyam

    2010-01-01

    Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and lipopolysaccharide (LPS) shock model; however, the underlying mechanisms are unclear. In the present study, compared to wild-type (Nrf2+/+) macrophages, we observed greater PKC-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2−/−) macrophages that was modulated by glutathione (GSH) levels. To address the NADPH oxidase-mediated hyper-inflammatory response and sepsis-induced lung injury and mortality in Nrf2−/− mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91phox (Nrf2−/−//Gp91phox−/−). Compared to Nrf2+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MYD88 and TRIF in Nrf2−/− macrophages that was diminished by ablation of gp91phox. Similarly, phosphorylation of IκB and IRF3 as well as cytokine expression was markedly higher in Nrf2−/− macrophages, while it was similar in Nrf2+/+ and Nrf2−/−//Gp91phox−/−. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2−/− mice that was significantly reduced by ablation of gp91phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2−/− mice, while Nrf2−/−//Gp91phox−/− showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis. PMID:20511556

  17. Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes.

    PubMed Central

    Wallach, T M; Segal, A W

    1996-01-01

    Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1. PMID:8947464

  18. Persistent activation of microglia and NADPH drive hippocampal dysfunction in experimental multiple sclerosis.

    PubMed

    Di Filippo, Massimiliano; de Iure, Antonio; Giamp, Carmela; Chiasserini, Davide; Tozzi, Alessandro; Orvietani, Pier Luigi; Ghiglieri, Veronica; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Mancini, Andrea; Costa, Cinzia; Sarchielli, Paola; Fusco, Francesca Romana; Calabresi, Paolo

    2016-01-01

    Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression. PMID:26887636

  19. Persistent activation of microglia and NADPH drive hippocampal dysfunction in experimental multiple sclerosis

    PubMed Central

    Di Filippo, Massimiliano; de Iure, Antonio; Giampà, Carmela; Chiasserini, Davide; Tozzi, Alessandro; Orvietani, Pier Luigi; Ghiglieri, Veronica; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Mancini, Andrea; Costa, Cinzia; Sarchielli, Paola; Fusco, Francesca Romana; Calabresi, Paolo

    2016-01-01

    Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression. PMID:26887636

  20. The plasma membrane NADPH oxidase OsRbohA plays a crucial role in developmental regulation and drought-stress response in rice.

    PubMed

    Wang, Xiang; Zhang, Mao-Mao; Wang, Ya-Jing; Gao, Yin-Tao; Li, Ri; Wang, Gang-Feng; Li, Wen-Qiang; Liu, Wen-Ting; Chen, Kun-Ming

    2016-04-01

    Plasma membrane NADPH oxidases are major producers of reactive oxygen species (ROS) in plant cells under normal growth and stress conditions. In the present study the total activity of rice NADPH oxidases and the transcription of OsRbohA, which encodes an Oryza sativa plasma membrane NADPH oxidase, were stimulated by drought. OsRbohA was expressed in all tissues examined throughout development. Its mRNA was upregulated by a number of factors, including heat, drought, salt, oxidative stress and methyl jasmonate treatment. Compared with wild-type (WT), the OsRbohA-knockout mutant osrbohA exhibited upregulated expression of other respiratory burst oxidase homolog genes and multiple abnormal agronomic traits, including reduced biomass, low germination rate and decreased pollen viability and seed fertility. However, OsRbohA-overexpressing transgenic plants showed no differences in these traits compared with WT. Although osrbohA leaves and roots produced more ROS than WT, the mutant had lesser intracellular ROS. In contrast, OsRbohA-overexpressing transgenic plants exhibited higher ROS production at the intracellular level and in tissues. Ablation of OsRbohA impaired the tolerance of plants to various water stresses, whereas its overexpression enhanced the tolerance. In addition, a number of genes related to energy supply, substrate transport, stress response and transcriptional regulation were differentially expressed in osrbohA plants even under normal growth conditions, suggesting that OsRbohA has fundamental and broad functions in rice. These results indicate that OsRbohA-mediated processes are governed by complex signaling pathways that function during the developmental regulation and drought-stress response in rice. PMID:26400148

  1. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    SciTech Connect

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-12-19

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91{sup phox} was dose-dependent. Meanwhile, the cytoplasmic subunit p47{sup phox} was translocated to the cell membrane and localized with p22{sup phox} and gp91{sup phox} to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  2. Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension

    PubMed Central

    Du, Jianhai; Welak, Scott; Guan, Tongju; Eis, Annie; Shi, Yang; Konduri, Girija G.

    2012-01-01

    Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91phox and p47phox, NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis. PMID:22245997

  3. AP-1-dependent transcriptional regulation of NADPH oxidase in human aortic smooth muscle cells: role of p22phox subunit.

    TOXLINE Toxicology Bibliographic Information

    Manea A; Manea SA; Gafencu AV; Raicu M; Simionescu M

    2008-05-01

    OBJECTIVE: NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions.METHODS AND RESULTS: Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-alpha. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF-alpha-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67(phox), p47(phox), p22(phox)). Inhibitors of AP-1 significantly diminished the Ang II or TNF-alpha-stimulated p22(phox) promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22(phox) promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1-binding sites in the p22(phox) gene promoter.CONCLUSIONS: In SMCs exposed to Ang II or TNF-alpha, inhibition of AP-1-related pathways reduces NADPHox expression and the O(2)(-) production. The physical interaction of AP-1 with p22(phox) gene promoter facilitates NADPHox regulation.

  4. Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease.

    PubMed Central

    Dinauer, M C; Pierce, E A; Erickson, R W; Muhlebach, T J; Messner, H; Orkin, S H; Seger, R A; Curnutte, J T

    1991-01-01

    Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O2-) and other microbial oxidants due to mutations in any one of four components of the O2(-)-generating complex, NADPH oxidase. We report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b [where phox indicates phagocyte oxidase (used to designate protein components of the phagocyte NADPH oxidase)] results in a nonfunctional oxidase and failure of neutrophils to produce O2- in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. We postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of a nonfunctional cytochrome b. A single-base substitution (C----A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro----Gln substitution at residue 156. The same mutation was also identified in all clones sequenced from patient genomic DNA, demonstrating homozygosity for the mutant allele. An antipeptide antibody against p22 residues 153-164 was found to bind only to permeabilized neutrophils, indicating that the mutation occurs in a cytoplasmic domain. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function. Images PMID:1763037

  5. Amyloid β25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

    PubMed

    Part, Kristin; Künnis-Beres, Kai; Poska, Helen; Land, Tiit; Shimmo, Ruth; Zetterström Fernaeus, Sandra

    2015-12-10

    Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aβ) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aβ through ROS and metals are mainly studied in neuronal cells using full-length Aβ1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aβ25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aβ25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aβ25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aβ25-35 induced ROS. PMID:26505916

  6. Anandamide Protects HT22 Cells Exposed to Hydrogen Peroxide by Inhibiting CB1 Receptor-Mediated Type 2 NADPH Oxidase

    PubMed Central

    Jia, Ji; Wu, Mingchun; Zhang, Lei; Zhang, Xiajing; Zhai, Qian; Jiang, Tao; Xiong, Lize

    2014-01-01

    Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells. PMID:25136404

  7. Role of neuronal NADPH oxidase 1 in the peri-infarct regions after stroke.

    PubMed

    Choi, Dong-Hee; Kim, Ji-Hye; Lee, Kyoung-Hee; Kim, Hahn-Young; Kim, Yoon-Seong; Choi, Wahn Soo; Lee, Jongmin

    2015-01-01

    The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia-reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke. PMID:25617620

  8. Role of Neuronal NADPH Oxidase 1 in the Peri-Infarct Regions after Stroke

    PubMed Central

    Choi, Dong-Hee; Kim, Ji-Hye; Lee, Kyoung-Hee; Kim, Hahn-Young; Kim, Yoon-Seong; Choi, Wahn Soo; Lee, Jongmin

    2015-01-01

    The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia—reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2′-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke. PMID:25617620

  9. The Role of the NADPH Oxidase NOX2 in Prion Pathogenesis

    PubMed Central

    Sorce, Silvia; Nuvolone, Mario; Keller, Annika; Falsig, Jeppe; Varol, Ahmet; Schwarz, Petra; Bieri, Monika; Budka, Herbert; Aguzzi, Adriano

    2014-01-01

    Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis. PMID:25502554

  10. NADPH oxidase enzymes in skin fibrosis: molecular targets and therapeutic agents.

    PubMed

    Babalola, Olubukola; Mamalis, Andrew; Lev-Tov, Hadar; Jagdeo, Jared

    2014-05-01

    Fibrosis is characterized by the excessive deposition of extracellular matrix components eventually resulting in organ dysfunction and failure. In dermatology, fibrosis is the hallmark component of many skin diseases, including systemic sclerosis, graft-versus-host disease, hypertrophic scars, keloids, nephrogenic systemic fibrosis, porphyria cutanea tarda, restrictive dermopathy and other conditions. Fibrotic skin disorders may be debilitating and impair quality of life. There are few FDA-approved anti-fibrotic drugs; thus, research in this area is crucial in addressing this deficiency. Recent investigations elucidating the pathogenesis of skin fibrosis have implicated endogenous reactive oxygen species produced by the multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzyme complex. In this review, we discuss Nox enzymes and their role in skin fibrosis. An overview of the Nox enzyme family is presented and their role in the pathogenesis of skin fibrosis is discussed. The mechanisms by which Nox enzymes influence specific fibrotic skin disorders are also reviewed. Finally, we describe the therapeutic approaches to ameliorate skin fibrosis by directly targeting Nox enzymes with the use of statins, p47phox subunit modulators, or GKT137831, a competitive inhibitor of Nox enzymes. Nox enzymes can also be targeted indirectly via scavenging ROS with antioxidants. We believe that Nox modulators are worthy of further investigation and have the potential to transform the management of skin fibrosis by dermatologists. PMID:24155025

  11. Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding.

    PubMed

    Ueyama, Takehiko; Nakakita, Junya; Nakamura, Takashi; Kobayashi, Takeshi; Kobayashi, Toshihiro; Son, Jeonghyun; Sakuma, Megumi; Sakaguchi, Hirofumi; Leto, Thomas L; Saito, Naoaki

    2011-11-25

    During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms. PMID:21956105

  12. Salviaolate Protects Rat Brain from Ischemia-Reperfusion Injury through Inhibition of NADPH Oxidase.

    PubMed

    Lou, Zheng; Ren, Kai-Di; Tan, Bin; Peng, Jing-Jie; Ren, Xian; Yang, Zhong-Bao; Liu, Bin; Yang, Jie; Ma, Qi-Lin; Luo, Xiu-Ju; Peng, Jun

    2015-10-01

    Salviaolate is a group of depside salts isolated from Danshen (a traditional Chinese herbal medicine), with ≥ 85 % of magnesium lithospermate B. This study aims to investigate whether salviaolate is able to protect the rat brain from ischemia/reperfusion injury and the underlying mechanisms. Rats were subjected to 2 h of cerebral ischemia and 24 h of reperfusion to establish an ischemia/reperfusion injury model. The neuroprotective effects of salviaolate at different dosages were evaluated. A dosage (25 mg/kg) was chosen to explore the neuroprotective mechanisms of salviaolate. Neurological function, infarct volume, cellular apoptosis, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H2O2 content were measured. In a nerve cell model of hypoxia/reoxygenation injury, magnesium lithospermate B was applied. Cellular apoptosis, lactate dehydrogenase, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H2O2 content were examined. Ischemia/reperfusion treatment significantly increased the neurological deficit score, infarct volume, and cellular apoptosis accompanied by the elevated nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 content in the rat brains. Administration of salviaolate reduced ischemia/reperfusion-induced cerebral injury in a dose-dependent manner concomitant with a decrease in nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 production. Magnesium lithospermate B (20 mg/kg) and edaravone (6 mg/kg, the positive control) achieved the same beneficial effects as salviaolate did. In the cell experiments, the injury (indicated by apoptosis ratio and lactate dehydrogenase release), nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 content were dramatically increased following hypoxia/reoxygenation, which were attenuated in the presence of magnesium lithospermate B (10(-5) M), VAS2870 (nicotinamide adenine dinucleotide phosphate-oxidase inhibitor), or edaravone (10(-5) M). The results suggest that salviaolate is able to protect the brain from ischemia/reperfusion oxidative injury, which is related to the inhibition of nicotinamide adenine dinucleotide phosphate-oxidase and a reduction of reactive oxygen species production. PMID:26252829

  13. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  14. The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis.

    PubMed

    Libik-Konieczny, Marta; Kozieradzka-Kiszkurno, Małgorzata; Desel, Christine; Michalec-Warzecha, Żaneta; Miszalski, Zbigniew; Konieczny, Robert

    2015-03-01

    This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2 (•-)) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3'-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2 (•-) was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2 (•-) is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth. PMID:25172434

  15. Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques

    PubMed Central

    Quesada, I.M.; Lucero, A.; Amaya, C.; Meijles, D.N.; Cifuentes, M.E.; Pagano, P.J.; Castro, C.

    2016-01-01

    Background A variety of NADPH oxidase (Nox) isoforms including Noxs 1, 2, 4 and 5 catalyze the formation of reactive oxygen species (ROS) in the vascular wall. The Nox2 isoform complex has arguably received the greatest attention in the progression of atherogenesis in animal models. Thus, in the current study we postulated that specific Nox2 oxidase inhibition could reverse or attenuate atherosclerosis in mice fed a high-fat diet. Methods We evaluated the effect of isoform-selective Nox2 assembly inhibitor on the progression and vascularization of atheromatous plaques. Apolipoprotein E-deficient mice (ApoE−/−) were fed a high fat diet for two months and treated over 15 days with Nox2ds-tat or control sequence (scrambled); 10 mg/ kg/day, i.p. Mice were sacrificed and superoxide production in arterial tissue was detected by cytochrome C reduction assay and dihydroethidium staining. Plaque development was evaluated and the angiogenic markers VEGF, HIF1-α and visfatin were quantified by real time qRT-PCR. MMP-9 protein release and gelatinolytic activity was determined as a marker for vascularization. Results Nox2ds-tat inhibited Nox-derived superoxide determined by cytochrome C in carotid arteries ( 2.3±0.1vs1.7±0.1O2•-nmol/min∗mg protein; P < 0.01) and caused a significant regression in atherosclerotic plaques in aorta (66 ± 6 μm2 vs 37 ± 1 μm2; scrmb vs. Nox2ds-tat; P < 0.001). Increased VEGF, HIF-1α, MMP-9 and visfatin expression in arterial tissue in response to high-fat diet were significantly attenuated by Nox2ds-tat which in turn impaired both MMP-9 protein expression and activity. Conclusion Given these results, it is quite evident that selective Nox inhibitors can reverse vascular pathology arising with atherosclerosis. PMID:26298737

  16. NAD(P)H oxidase-dependent intracellular and extracellular O2·- production in coronary arterial myocytes from CD38 knockout mice

    PubMed Central

    Xu, Ming; Zhang, Yang; Xia, Min; Li, Xiao-Xue; Ritter, Joseph K; Zhang, Fan; Li, Pin-Lan

    2011-01-01

    Activation of NAD(P)H oxidase has been reported to produce superoxide (O2 ·-) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use NAD(P)H oxidase product, NAD+ or NADP+ to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), which mediates intracellular Ca2+ signaling. The present study was designed to test a hypothesis that CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2 ·-in mouse coronary arterial myocytes (CAMs). By fluorescent microscopic imaging, we simultaneously monitored extra- and intracellular O2 ·-production in wild-type (CD38+/+) and CD38 knockout (CD38-/-) CAMs in response to oxotremorine (OXO), a muscarinic type 1 (M1) receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2 ·-production in CAMs. Consistently, the OXO-induced intracellular O2 ·-production was markedly inhibited by CD38 shRNA or CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2 ·- production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2 ·-production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2+ concentration and restored intracellular O2 ·-production in CD38-/- CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that CD38/cADPR pathway importantly controls Nox4-mediated intracellular O2 ·-production and that CD38-dependent intracellular O2 ·-production is augmented via an autocrine manner of CD38-independent Nox1-derived extracellular O2 ·-production in CAMs. PMID:22100343

  17. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model.

    PubMed

    Harraz, Maged M; Marden, Jennifer J; Zhou, Weihong; Zhang, Yulong; Williams, Aislinn; Sharov, Victor S; Nelson, Kathryn; Luo, Meihui; Paulson, Henry; Schöneich, Christian; Engelhardt, John F

    2008-02-01

    Neurodegeneration in familial amyotrophic lateral sclerosis (ALS) is associated with enhanced redox stress caused by dominant mutations in superoxide dismutase-1 (SOD1). SOD1 is a cytosolic enzyme that facilitates the conversion of superoxide (O(2)(*-)) to H(2)O(2). Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase-dependent (Nox-dependent) O(2)(*-) production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H(2)O(2) uncoupled SOD1 binding in a reversible fashion, producing a self-regulating redox sensor for Nox-derived O(2)(*-) production. This process of redox-sensitive uncoupling of SOD1 from Rac1 was defective in SOD1 ALS mutants, leading to enhanced Rac1/Nox activation in transgenic mouse tissues and cell lines expressing ALS SOD1 mutants. Glial cell toxicity associated with expression of SOD1 mutants in culture was significantly attenuated by treatment with the Nox inhibitor apocynin. Treatment of ALS mice with apocynin also significantly increased their average life span. This redox sensor mechanism may explain the gain-of-function seen with certain SOD1 mutations associated with ALS and defines new therapeutic targets. PMID:18219391

  18. Oxidative stress, redox signalling and endothelial dysfunction in ageing-related neurodegenerative diseases: a role of NADPH oxidase 2

    PubMed Central

    Cahill-Smith, Sarah; Li, Jian-Mei

    2014-01-01

    Chronic oxidative stress and oxidative damage of the cerebral microvasculature and brain cells has become one of the most convincing theories in neurodegenerative pathology. Controlled oxidative metabolism and redox signalling in the central nervous system are crucial for maintaining brain function; however, excessive production of reactive oxygen species and enhanced redox signalling damage neurons. While several enzymes and metabolic processes can generate intracellular reactive oxygen species in the brain, recently an O2−-generating enzyme, NADPH oxidase 2 (Nox2), has emerged as a major source of oxidative stress in ageing-related vascular endothelial dysfunction and neurodegenerative diseases. The currently available inhibitors of Nox2 are not specific, and general antioxidant therapy is not effective in the clinic; therefore, insights into the mechanism of Nox2 activation and its signalling pathways are needed for the discovery of novel drug targets to prevent or treat these neurodegenerative diseases. This review summarizes the recent developments in understanding the mechanisms of Nox2 activation and redox-sensitive signalling pathways and biomarkers involved in the pathophysiology of the most common neurodegenerative diseases, such as ageing-related mild cognitive impairment, Alzheimer’s disease and Parkinson’s disease. PMID:25279404

  19. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    PubMed

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular junction. Our data suggest that the location of P2Y receptors in lipid rafts speeds up the modulatory effect of ATP. Uncovered mechanisms may contribute to motor dysfunctions and neuromuscular diseases associated with oxidative stress. PMID:25463521

  20. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana[OPEN

    PubMed Central

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi

    2015-01-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  1. Superoxide deficiency attenuates promotion of hepatocarcinogenesis by cytotoxicity in NADPH oxidase knockout mice.

    PubMed

    Parzefall, Wolfram; Freiler, Constanze; Lorenz, Olga; Koudelka, Helga; Riegler, Teresa; Nejabat, Marzieh; Kainzbauer, Eveline; Grasl-Kraupp, Bettina; Schulte-Hermann, Rolf

    2015-08-01

    Long-term exposure to carcinogens combined with chronic hepatitis contributes greatly to the worldwide high incidence of hepatocellular carcinoma (HCC). It is still unclear to which extent the release of pro-inflammatory reactive oxygen or nitrogen species contributes to the development of this malignancy. Here, we aim to elucidate the role of superoxide in a model of chemical hepatocarcinogenesis. p47(phox) knockout mice (KO), lacking superoxide formation by phagocytic NADPH oxidase (phox), and wild-type animals (WT) were subjected to two different initiation-promotion protocols: (1) single dose of diethylnitrosamine (DEN) at 6 weeks of age followed by phenobarbital (PB) via diet, or ethanol (EtOH) in drinking water; (2) DEN at neonatal age followed by three cytotoxic doses of DEN at intervals of 6-7 weeks. The appearance of tumors and prestages was quantified. There was no obvious difference in the capacity of DEN to initiate hepatocarcinogenesis in KO and WT mice. PB promoted tumor development in both genotypes without significant difference. EtOH induced steatosis significantly less in KO than in WT liver, but had no effect on tumor formation in either genotype. However, hepatocarcinogenesis by three cytotoxic DEN doses after neonatal initiation was attenuated significantly in KO. Macrophages/monocytes identified by the specific antigen F4/80 were more abundant in KO than in WT liver, possibly reflecting a compensatory response. We conclude that phox-derived superoxide is not essential but is supportive for the promotion of hepatocarcinogenesis by cytotoxic doses of DEN. The production of superoxide may therefore contribute to the promotion of hepatocarcinogenesis by cytotoxic/pro-inflammatory stimuli. PMID:25182417

  2. Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases.

    PubMed

    Yeligar, Samantha M; Harris, Frank L; Hart, C Michael; Brown, Lou Ann S

    2012-04-15

    Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function. PMID:22412195

  3. Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK.

    PubMed

    Wang, Xi-Jin; Zhang, Shi; Yan, Zhi-Qiang; Zhao, Yan-Xin; Zhou, Hai-Yan; Wang, Ying; Lu, Guo-Qiang; Zhang, Jing-Dong

    2011-05-01

    CD200-CD200R signaling holds microglia in a quiescent state. Parkinson disease (PD) neurodegeneration may be associated with impairment of CD200-CD200R-mediated microglia silencing in the substantia nigra (SN). In this study, an anti-CD200R blocking antibody (ACDR) selectively and significantly enhanced the susceptibility of dopaminergic neurons to neurotoxicity induced by rotenone (Rot) and iron (Ir) in mesencephalic neuron/glia cultures. Microglia were shown to mediate dopaminergic neurotoxicity induced by ACDR/Rot (combination of ACDR and Rot) and ACDR/Ir (combination of ACDR and Ir). ACDR significantly enhanced the microglial activation induced by Rot and Ir in neuron/glia cultures. NADPH oxidase-mediated superoxide generation was a key contributor to dopaminergic neurotoxicity induced by ACDR/Rot and ACDR/Ir. p38 MAPK contributed to NADPH oxidase activation induced by ACDR/Rot and ACDR/Ir. Interestingly, there were a decrease in CD200 expression (mRNA and protein) and an enhancement of microglial response (MHCII mRNA and ICAM-1 protein) in the rat SN with aging. ICAM-1 expression was significantly inversely correlated with CD200 expression. These results strongly indicate the participation of SN CD200-CD200R dysfunction in the etiopathogenesis of PD and provide a new insight into the molecular mechanisms underlying the involvement of aging in PD and help to elucidate the mechanisms of the combined involvement of immune/inflammatory factors, environmental substances, and aging in PD. PMID:21295135

  4. beta-aminobutyric acid primes an NADPH oxidase-dependent reactive oxygen species production during grapevine-triggered immunity.

    PubMed

    Dubreuil-Maurizi, Carole; Trouvelot, Sophie; Frettinger, Patrick; Pugin, Alain; Wendehenne, David; Poinssot, Benoît

    2010-08-01

    The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase-dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem. PMID:20615112

  5. Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

    PubMed

    Ben Rejeb, Kilani; Lefebvre-De Vos, Delphine; Le Disquet, Isabel; Leprince, Anne-Sophie; Bordenave, Marianne; Maldiney, Régis; Jdey, Asma; Abdelly, Chedly; Savouré, Arnould

    2015-12-01

    Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant. PMID:26180024

  6. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    SciTech Connect

    Eum, Sung Yong Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-10-15

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  7. Monocytic AML cells inactivate antileukemic lymphocytes: role of NADPH oxidase/gp91phox expression and the PARP-1/PAR pathway of apoptosis

    PubMed Central

    Aurelius, Johan; Thorén, Fredrik B.; Akhiani, Ali A.; Brune, Mats; Palmqvist, Lars; Hansson, Markus; Martner, Anna

    2012-01-01

    Dysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91phox. Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1−dependent apoptosis in adjacent NK cells, CD4+ T cells, and CD8+ T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91phox, did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91phox mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10−11) or FAB-M2 cells (P < 10−9). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity. PMID:22550344

  8. Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides

    PubMed Central

    Gonzalez, Angel; Hung, Chiung-Yu; Cole, Garry T.

    2011-01-01

    Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2-/- knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2-/- mice died earlier than the WT strain. Immunization of the NOX2-/- and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2-/- mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis. PMID:21896326

  9. Exploring the protective role of apocynin, a specific NADPH oxidase inhibitor, in cisplatin-induced cardiotoxicity in rats.

    PubMed

    El-Sawalhi, Maha M; Ahmed, Lamiaa A

    2014-01-25

    Despite the clinical reports, few studies have focused on reducing the cardiotoxicity of cisplatin. In the present study, cardiotoxicity was examined after a single ip injection of cisplatin (7mg/kg) in rats. Apocynin was given in drinking water (600mg/L) for five successive days before and after cisplatin injection. At the end of the experiment, hemodynamic parameters were recorded, animals were sacrificed and serum creatine kinase-MB activity was determined. The whole ventricle was isolated for estimation of tumor necrosis factor-alpha (TNF-α) content, NADPH oxidase, myeloperoxidase and caspase-3 activities in addition to nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB) gene expressions. Furthermore, oxidative stress markers and antioxidant enzymes were measured in postmitochondrial and mitochondrial fractions. Mitochondrial membrane potential, nuclear DNA fragmentation and cardiomyocyte cross-sectional area were also evaluated. Apocynin was effective against cisplatin-induced decrement in heart rate and blood pressure. Moreover, pretreatment with apocynin notably ameliorated the state of oxidative stress, mitigated inflammation and preserved mitochondrial membrane potential. Apocynin provided also a significant cardioprotection as revealed by alleviating the overexpression of Nrf2, HO-1 and NF-κB, the elevation of caspase-3 activity, the prominent nuclear DNA fragmentation and the decreased cardiomyocyte cross-sectional area. This study highlights the potential role of apocynin in inhibiting cisplatin-induced hemodynamic changes, postmitochondrial and mitochondrial damage as indicated by improvement in the state of oxidative stress, inflammation and apoptosis. PMID:24291008

  10. Involvement of NADPH oxidases and non-muscle myosin light chain in senescence of endothelial progenitor cells in hyperlipidemia.

    PubMed

    Li, Ting-Bo; Zhang, Jie-Jie; Liu, Bin; Liu, Wei-Qi; Wu, Yan; Xiong, Xiao-Ming; Luo, Xiu-Ju; Ma, Qi-Lin; Peng, Jun

    2016-03-01

    NADPH oxidase (NOX)-derived reactive oxygen species (ROS) is involved in endothelial dysfunction of hyperlipidemia, and non-muscle myosin regulatory light chain (nmMLC20) is reported to have a transcriptional function in regulation of gene expression. The purposes of this study are to determine whether NOX-derived ROS can promote endothelial progenitor cell (EPC) senescence and whether nmMLC20 can regulate NOX expression through a phosphorylation-dependent manner. The rats were subjected to 8weeks of high-fat diet feeding to establish a hyperlipidemic model, which showed an increase in plasma lipids and the accelerated senescence and reduced number of circulating EPCs, accompanied by an increase in myosin light chain kinase (MLCK) and NOX activities, p-nmMLC20 level, NOX (NOX2, NOX4) expression, and H2O2 content. Next, EPCs isolated from normal rats were incubated with ox-LDL (100?g/mL) for 24h to establish a senescent model in vitro. Consistent with our in vivo findings, ox-LDL treatment increased the senescence of EPCs concomitant with an increase in MLCK and NOX activities, p-nmMLC20 level (in total or nuclear proteins), NOX expression, and H2O2 content; these phenomena were reversed by MLCK inhibitor. NOX inhibitor achieved similar results to that of MLCK inhibitor except that there is no effect on MLCK activity and p-nmMLC20 level. Furthermore, knockdown of nmMLC20, NOX2, or NOX4 led to a down-regulation in NOX and a reduction in ox-LDL-induced EPC senescence. These results suggest that NOX-derived ROS promotes the senescence of circulating EPCs in hyperlipidemia and nmMLC20 may play a transcriptional role in the upregulation of NOX through a phosphorylation-dependent manner. PMID:26685858

  11. Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases

    PubMed Central

    Wang, Qingshan; Chu, Chun-Hsien; Oyarzabal, Esteban; Jiang, Lulu; Chen, Shih-Heng; Wilson, Belinda; Qian, Li; Hong, Jau-Shyong

    2014-01-01

    Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson’s disease (PD). The inhibition of NOX2-generated superoxide has become an effective strategy for developing disease-modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long-acting NOX2 inhibitor. However, due to its non-specificity for NOX2 and high cytotoxicity at standard doses (μM), DPI has been precluded from human studies. In this study, using ultra-low doses of DPI, we aimed to: 1) investigate whether these problems could be circumvented and 2) determine whether ultra-low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10−14 and 10−13 M) displays no toxicity in primary midbrain neuron-glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)-induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein-containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post-treatment study. When subpicomolar DPI was added to neuron-glia cultures pretreated with lipopolysaccharide (LPS), 1-methyl-4-phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI’s unique combination of high specificity towards NOX2, low cytotoxicity and potent neuroprotective efficacy in post-treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials. PMID:25043383

  12. The Crystal Structure of the Plant Small GTPase OsRac1 Reveals Its Mode of Binding to NADPH Oxidase*

    PubMed Central

    Kosami, Ken-ichi; Ohki, Izuru; Nagano, Minoru; Furuita, Kyoko; Sugiki, Toshihiko; Kawano, Yoji; Kawasaki, Tsutomu; Fujiwara, Toshimichi; Nakagawa, Atsushi; Shimamoto, Ko; Kojima, Chojiro

    2014-01-01

    Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr39 and Asp45 substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins. PMID:25128531

  13. The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

    PubMed

    Kosami, Ken-ichi; Ohki, Izuru; Nagano, Minoru; Furuita, Kyoko; Sugiki, Toshihiko; Kawano, Yoji; Kawasaki, Tsutomu; Fujiwara, Toshimichi; Nakagawa, Atsushi; Shimamoto, Ko; Kojima, Chojiro

    2014-10-10

    Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins. PMID:25128531

  14. Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms in human aortic smooth muscle cells.

    PubMed

    Manea, Adrian; Manea, Simona A; Florea, Irina C; Luca, Catalina M; Raicu, Monica

    2012-05-01

    NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs) underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood. Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of various proinflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5' deletion mutants to identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiation site was detected by rapid amplification of the 5'-cDNA ends. In silico analysis indicated the existence of typical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhanced chemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition and the silencing of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 reduced significantly the interferon γ-induced Ca(2+)-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellular Ca(2+), an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators of Nox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals in excess, further contributing to SMCs dysfunction in atherosclerosis. PMID:22348975

  15. NADPH Oxidase/ROS-Dependent VCAM-1 Induction on TNF-α-Challenged Human Cardiac Fibroblasts Enhances Monocyte Adhesion

    PubMed Central

    Lin, Chih-Chung; Yang, Chien-Chung; Wang, Chen-Yu; Tseng, Hui-Ching; Pan, Chih-Shuo; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    The inflammation-dependent adhesion molecule expressions are characterized in cardiovascular diseases and myocardial tissue infiltrations. Several pro-inflammatory cytokines are elevated in the acute myocardial injury and infarction. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is raised in the injury tissues and inflammatory regions and involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. In fibroblasts, TNF-α-triggered expression of vascular cell adhesion molecule (VCAM)-1 aggravated the heart inflammation. However, the mechanisms underlying TNF-α-mediated VCAM-1 expression in cardiac fibroblasts remain unclear. Here, the primary cultured human cardiac fibroblasts (HCFs) were used to investigate the effects of TNF-α on VCAM-1 expression. The molecular evidence, including protein, mRNA, and promoter analyses, indicated that TNF-α-induced VCAM-1 gene expression is mediated through the TNFR-dependent manner. Activation of TNF-α/TNFR system triggered PKCα-dependent NADPH oxidase (Nox)/reactive oxygen species (ROS) signal linking to MAPK cascades, and then led to activation of the transcription factor, AP-1. Moreover, the results of mRNA and promoter assay demonstrated that c-Jun/AP-1 phosphorylated by TNF-α turns on VCAM-1 gene expression. Subsequently, up-regulated VCAM-1 on the cell surface of TNF-α-challenged HCFs increased the number of monocytes adhering to these cells. These results indicated that in HCFs, activation of AP-1 by PKCα-dependent Nox/ROS/MAPKs cascades is required for TNF-α-induced VCAM-1 expression. To clarify the mechanisms of TNF-α-induced VCAM-1 expression in HCFs may provide therapeutic strategies for heart injury and inflammatory diseases. PMID:26858641

  16. Alzheimer's disease: a gas model. The NADPH oxidase-Nitric Oxide system as an antibubble biomachinery.

    PubMed

    Denis, Pierre A

    2013-12-01

    Alzheimer's disease (AD) is a neurodegenerative disease of unknown origin. The pathological lesions that define AD would be linked to the insidious accumulation of nitrogen, having invaded the brain interstitial fluid (ISF) from the blood via the physiological cycling pool of vascular glucose transporters (GLUT-1). According to this hypothesis, the nitrogen nanobubbles, being chemically inert and actually indestructible for human beings, can not escape from the ISF anymore. They would exert a huge and deleterious pressure against cellular components, especially in microglia and in astrocytes. They could enhance the existing cell oxygen anisotropy, which might enhance the natural bubble nucleation of O2-2O2 in cells or in mitochondria. Indeed, with the help of a new symbolic representation for gas nuclei in chemical reactions, the NADPH oxidase-NO system is identified for the first time, as an antibubble biomachinery, able to break O2-2O2 bubbles up as it releases superoxide O2-. Superoxide is considered as a quantum bubble, which collapses through the reactivity of the gaseous NO radical. Their combination in soluble peroxinitrite provides the change from one state of matter to another, avoiding any risk of a bubble enlargement, and finally avoiding the risk of enzyme crowding or of a bulk pressure variation. However, a bubble is expected to entrap Nitric Oxide (NO), which leads theoretically to a decrease in its bioavailability, and is expected to trigger a guanylyl-cyclase-mediated inflammatory cascade, that could explain the inflammation in AD. In vitro, any increase in the hydrostatic pressure has already been linked to the microtubule disorganization. The amyloid deposits, also known as senile plaques, would behave as a sponge toward ISF nitrogen; Aβ is considered as a foam-stabilizing agent. By taking the shape of cerebral amyloid angiopathy, the amyloid could confine the nitrogen leak from the blood, and progressively insulate the Blood-Brain Barrier against the pollutant. All these theoretical features finally lead to the death of the neurons. The comprehensive statement of the theoretical pro-inflammatory action of inert gases is a real upheaval for the whole medicine. PMID:24119763

  17. Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease

    SciTech Connect

    Dinauer, M.C. ); Pierce, E.A. ); Erickson, R.W.; Curnutte, J.T. ); Muhlbeach, T.J.; Seger, R.A. ); Messner, H. ); Orkin, S.H. Howard Hughes Medical Inst., Boston, MA )

    1991-12-15

    Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O{sub 2}{sup {minus}}) and other microbial oxidants due to mutations in any one of four components of the O{sub 2}{sup {minus}}-generating complex, NADPH oxidase. The authors report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b results in a nonfunctional oxidase and failure of neutrophils to produce O{sub 2}{sup {minus}} in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. They postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of nonfunctional cytochrome b. A single-base substitution (C {yields} A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro {yields} Gln substitution at residue 156. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function.

  18. A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs.

    PubMed Central

    Keller, T; Damude, H G; Werner, D; Doerner, P; Dixon, R A; Lamb, C

    1998-01-01

    Rapid generation of O2- and H2O2, which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA (for respiratory burst oxidase homolog A) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91phox. This domain contains two Ca2+ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA, which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91phox, does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca2+ and G protein stimulation of O2- and H2O2 production at the cell surface. PMID:9490748

  19. Transient congenital hypothyroidism caused by compound heterozygous mutations affecting the NADPH-oxidase domain of DUOX2.

    PubMed

    Yoshizawa-Ogasawara, Atsuko; Abe, Kiyomi; Ogikubo, Sayaka; Narumi, Satoshi; Hasegawa, Tomonobu; Satoh, Mari

    2016-03-01

    Here, we describe three cases of loss-of-function mutations in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) domain of dual oxidase 2 (DUOX2) occurring along with concurrent missense mutations in thyroid peroxidase (TPO), leading to transient congenital hypothyroidism (CH). Three Japanese boys with nonconsanguineous parents were diagnosed with CH during their neonatal screenings. All patients presented with moderate-to-severe neonatal hypothyroidism and were diagnosed with transient CH after re-evaluation of thyroid function. Two siblings were compound heterozygous for p.[R1110Q]+[Y1180X] in DUOX2; one of them was also heterozygous for p.[R361L] in TPO. The third patient was compound heterozygous for p.[L1160del]+[R1334W] in DUOX2 and heterozygous for p.[P883S] in TPO. This is the first report of a de novo L1160del mutation affecting the DUOX2 gene and of the novel mutations Y1180X in DUOX2 and R361L in TPO. R1110Q and L1160del were found to reduce H2O2 production (5%-9%, p<0.01), while Y1180X, which introduces a premature stop codon, did not confer detectable H2O2 production (-0.7%±0.6%, p<0.01). Moreover, R1334W, a missense mutation possibly affecting electron transfer, led to reduced H2O2 production (24%±0.9%, p<0.01) in vitro, and R1110Q and R1334W resulted in reduced protein expression. Y1180X was detected in a 120 kDa truncated form, whereas L1160del expression was maintained. Further, R361L, a novel missense mutation in TPO, caused partial reduction in peroxidase activity (20.6%±0.8%, p=0.01), whereas P883S, a missense variant, increased it (133.7%±2.8%, p=0.02). The protein expression levels in the case of R361L and P883S were maintained. In conclusion, we provide clinical and in vitro demonstrations of different functional defects and phenotypic heterogeneity in the same thyroid hormonogenesis pathway. PMID:26565538

  20. Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass

    PubMed Central

    Sun, Jinlong; Ming, Leiguo; Shang, Fengqing; Shen, Lijuan; Chen, Jihua; Jin, Yan

    2015-01-01

    Because of the reduced potential for osteogenesis in aging bone marrow stromal cells, the balance of bone metabolism becomes disrupted, leading to various bone diseases. An increase in reactive oxygen species has been determined to be one of the key factors that accelerates the aging process in BMSCs. In these cells, increased expression of NADPH oxidases is the major source of ROS. In the current study, we suppressed the expression of NOX using apocynin, an effective antioxidant and free radical scavenger, and the results showed that aging BMSCs exhibited an enhanced potential for osteogenesis. The expression of potential key targets influencing this reversal was evaluated using qRT-PCR, and the expression of p53 was shown to be reduced with the suppression of NOX. We speculate that this may be one of the major reasons for the reversal of the aging process. We also examined the effect of apocynin in vivo, and the results showed that in SAMP6 mice, bone mineral density and total bone volume were increased after 3 months of apocynin treatment. In conclusion, our results demonstrate that in aging BMSCs, suppression of NADPH oxidase by apocynin partially reverses the aging process and enhances osteogenic potential. PMID:26686764

  1. Arctigenin reduces blood pressure by modulation of nitric oxide synthase and NADPH oxidase expression in spontaneously hypertensive rats.

    PubMed

    Liu, Ying; Wang, Guoyuan; Yang, Mingguang; Chen, Haining; zhao, Yan; Yang, Shucai; Sun, Changhao

    2015-12-25

    Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation. PMID:26585490

  2. Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

    SciTech Connect

    Tateishi, Yoshihisa Sasabe, Eri; Ueta, Eisaku; Yamamoto, Tetsuya

    2008-02-08

    Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by {gamma}-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

  3. Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis.

    PubMed

    Manea, Adrian; Manea, Simona-Adriana; Gan, Ana Maria; Constantin, Alina; Fenyo, Ioana Madalina; Raicu, Monica; Muresian, Horia; Simionescu, Maya

    2015-05-22

    Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 μg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis. PMID:25871798

  4. Molecular Mechanisms of the Crosstalk Between Mitochondria and NADPH Oxidase Through Reactive Oxygen Species—Studies in White Blood Cells and in Animal Models

    PubMed Central

    Kröller-Schön, Swenja; Steven, Sebastian; Kossmann, Sabine; Scholz, Alexander; Daub, Steffen; Oelze, Matthias; Xia, Ning; Hausding, Michael; Mikhed, Yuliya; Zinßius, Elena; Mader, Michael; Stamm, Paul; Treiber, Nicolai; Scharffetter-Kochanek, Karin; Li, Huige; Schulz, Eberhard; Wenzel, Philip; Münzel, Thomas

    2014-01-01

    Abstract Aims: Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. Results: NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD+/−) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD+/− mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. Innovation: These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. Conclusions: Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension. Antioxid. Redox Signal. 20, 247–266. PMID:23845067

  5. Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.

    PubMed

    Lin, Chih-Chung; Yang, Chien-Chung; Cho, Rou-Ling; Wang, Chen-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47 (phox) , and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment. PMID:27065868

  6. Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    PubMed Central

    Lin, Chih-Chung; Yang, Chien-Chung; Cho, Rou-Ling; Wang, Chen-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment. PMID:27065868

  7. Propofol Protects Against H2O2-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca(2+)-Dependent NADPH Oxidase.

    PubMed

    Chen, Xiao-Hui; Zhou, Xue; Yang, Xiao-Yu; Zhou, Zhi-Bin; Lu, Di-Han; Tang, Ying; Ling, Ze-Min; Zhou, Li-Hua; Feng, Xia

    2016-05-01

    Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H2O2)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H2O2 exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H2O2-induced decrease in cell viability, prevented H2O2-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91(phox) (NOX2) as well as the NOX activity following H2O2 exposure in PC12 cells. In addition, blocking of L-type Ca(2+) channels with nimodipine reduced H2O2-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H2O2. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca(2+)-dependent NADPH oxidase. PMID:26162968

  8. Curcuminoids Modulate the PKCδ/NADPH Oxidase/Reactive Oxygen Species Signaling Pathway and Suppress Matrix Invasion during Monocyte-Macrophage Differentiation.

    PubMed

    Huang, Shao-Lan; Chen, Pei-Yi; Wu, Ming-Jiuan; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-10-14

    Monocyte recruitment and invasion play critical roles in the initiation and progression of atherosclerosis. The reduction in monocyte adhesion and infiltration is thought to exert antiatherosclerotic effects. Curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are the major active components of curcuminoids and exhibit several biological activities, including anti-inflammatory, anticarcinogenic, and hypocholesterolemic activities. The aim of this study was to investigate the antiatherogenic effects and mechanisms of curcuminoids during monocyte to macrophage differentiation. The results showed that curcumin, DMC, and BDMC (20 μM) suppressed matrix invasion from 100.0 ± 5.0% to 24.8 ± 1.4%, 26.6 ± 2.9%, and 33.7 ± 1.7%, respectively, during PMA-induced THP-1 differentiation. We found that curcuminoids significantly reduced PMA-induced CD11b and MMP-9 expression by THP-1 cells. Production of reactive oxygen species (ROS) induced by PMA (126.7 ± 2.1%) was markedly attenuated by curcumin, DMC, and BDMC to 99.5 ± 7.8%, 87.8 ± 8.2%, and 89.8 ± 7.6%, respectively, resulting in the down-regulation of CD11b and MMP-9 expression. We demonstrated that curcuminoids inhibited NADPH oxidase through the down-regulation of NOX2 expression and the reduction of p47phox membrane translocation. Moreover, we found involvement of PKCδ in the PMA-induced NOX2, CD11b, and MMP-9 mRNA expression. Curcumin, DMC, and BDMC decreased the active form of PKCδ protein stimulated by PMA in THP-1 cells. Overall, our results reveal that curcuminoids suppress matrix invasion through the inhibition of the PKCδ/NADPH oxidase/ROS signaling pathway during monocyte-macrophage differentiation. PMID:26414495

  9. Glucose oxidase activity of actinomycetes.

    PubMed

    St Vlahov, S

    1978-01-01

    The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established. PMID:76424

  10. Genipin induces cyclooxygenase-2 expression via NADPH oxidase, MAPKs, AP-1, and NF-κB in RAW 264.7 cells.

    PubMed

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Chung, Young Chul; Kim, Hee Suk; Park, Youn-Joon; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-02-01

    Genipin is a compound found in gardenia fruit extract with diverse pharmacological activities. However, the mechanism underlying genipin-induced cyclooxygenase-2 (COX-2) expression remains unknown. In this study, we investigated the effects of genipin on COX-2 expression and determined that exposure to genipin dose-dependently enhanced the production of prostaglandin E2 (PGE2), a major COX-2 metabolite, in RAW 264.7 cells. These effects were mediated by genipin-induced activation of the COX-2 promoter, as well as AP-1 and NF-κB luciferase constructs. Phosphatidylinositol-3-kinase/Akt and MAPKs were also significantly activated by genipin, and Akt and MAPKs inhibitors (PD98059, SB20358, SP600125, and LY294002) inhibited genipin-induced COX-2 expression. Moreover, genipin increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2 and NOX3. Inhibition of NADPH with diphenyleneiodonium attenuated ROS production, COX-2 expression and NF-κB and AP-1 activation. These results suggest that the molecular mechanism mediating ROS-dependent COX-2 up-regulation and PGE2 production by genipin involves activation of Akt, MAPKs and AP-1/NF-κB. PMID:24296130

  11. High Expression Levels of NADPH Oxidase 3 in the Cerebrum of Ten-Week-Old Stroke-Prone Spontaneously Hypertensive Rats.

    PubMed

    Michihara, Akihiro; Oda, Asaki; Mido, Mayuko

    2016-01-01

    We previously demonstrated that the high levels of oxidative stress in the brains of ten-week-old stroke-prone hypertensive rats (SHRSP) were attributable to intrinsic, not extrinsic factors (Biol. Pharm. Bull., 33, 2010, Michihara et al.). The aim of the present study was to determine whether increases in the enzymes producing reactive oxygen species (ROS), reductions in the enzymes and proteins removing ROS, or increases in an enzyme and transporter removing antioxidants promoted oxidative stress in the SHRSP cerebrum. No significant decreases were observed in the mRNA levels of enzymes that remove ROS between SHRSP and normotensive Wistar Kyoto rats. The activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and the protein and mRNA levels of NOX3, an enzyme that produces ROS, were significantly increased in the SHRSP cerebrum. These results suggested that the high expression levels of NOX3 increased oxidative stress in the SHRSP cerebrum. PMID:26830484

  12. Allelic variations in the CYBA gene of NADPH oxidase and risk of kidney complications in patients with type 1 diabetes.

    PubMed

    Patente, Thiago A; Mohammedi, Kamel; Bellili-Muoz, Nama; Driss, Fathi; Sanchez, Manuel; Fumeron, Frdric; Roussel, Ronan; Hadjadj, Samy; Corra-Giannella, Maria Lcia; Marre, Michel; Velho, Gilberto

    2015-09-01

    Oxidative stress plays a pivotal role in the pathophysiology of diabetic nephropathy, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is an important source of reactive oxygen species in hyperglycemic conditions in the kidney. Plasma concentration of advanced oxidation protein products (AOPP), a marker of oxidative stress, is increased in patients with diabetic nephropathy. We investigated associations of variants in the CYBA gene, encoding the regulatory subunit p22(phox) of NADPH oxidase, with diabetic nephropathy and plasma AOPP and myeloperoxidase (MPO) concentrations in type 1 diabetic patients. Seven SNPs in the CYBA region were analyzed in 1357 Caucasian subjects with type 1 diabetes from the SURGENE (n=340), GENEDIAB (n=444), and GENESIS (n=573) cohorts. Duration of follow-up was 10, 9, and 6 years, respectively. Cox proportional hazards and logistic regression analyses were used to estimate hazard ratios (HR) or odds ratios (OR) for incidence and prevalence of diabetic nephropathy. The major G-allele of rs9932581 was associated with the incidence of renal events defined as new cases of microalbuminuria or the progression to a more severe stage of nephropathy during follow-up (HR 1.59, 95% CI 1.17-2.18, P=0.003) in SURGENE. The same allele was associated with established/advanced nephropathy (OR 1.52, 95% CI 1.22-1.92, P=0.0001) and with the incidence of end-stage renal disease (ESRD) (HR 2.01, 95% CI 1.30-3.24, P=0.001) in GENEDIAB/GENESIS pooled studies. The risk allele was also associated with higher plasma AOPP concentration in subsets of SURGENE and GENEDIAB, with higher plasma MPO concentration in a subset of GENEDIAB, and with lower estimated glomerular filtration rate (eGFR) in the three cohorts. In conclusion, a functional variant in the promoter of the CYBA gene was associated with lower eGFR and with prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. These results are consistent with a role for NADPH oxidase in the pathophysiology of kidney complications of diabetes. PMID:25862415

  13. Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A Role for Caspases but Not for SHP-1, Src Kinases, NADPH Oxidase or Reactive Oxygen

    PubMed Central

    Mao, Hui; Kano, Gen; Hudson, Sherry A.; Brummet, Mary; Zimmermann, Nives; Zhu, Zhou; Bochner, Bruce S.

    2013-01-01

    Background Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ. Methods Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used. Results Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process. Conclusions These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans. PMID:23840825

  14. Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells

    SciTech Connect

    Manea, Adrian; Tanase, Laurentia I.; Raicu, Monica; Simionescu, Maya

    2010-06-11

    Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

  15. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases.

    PubMed

    Zielonka, Jacek; Cheng, Gang; Zielonka, Monika; Ganesh, Thota; Sun, Aiming; Joseph, Joy; Michalski, Radosław; O'Brien, William J; Lambeth, J David; Kalyanaraman, Balaraman

    2014-06-01

    Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action. PMID:24764302

  16. Phycobiliprotein C-phycocyanin from Spirulina platensis is powerfully responsible for reducing oxidative stress and NADPH oxidase expression induced by an atherogenic diet in hamsters.

    PubMed

    Riss, Jérome; Décordé, Kelly; Sutra, Thibault; Delage, Martine; Baccou, Jean-Claude; Jouy, Nicolas; Brune, Jean-Pierre; Oréal, Henri; Cristol, Jean-Paul; Rouanet, Jean-Max

    2007-09-19

    The effects of spirulina and its chromophore phycocyanin, both without bound Se or selenium-enriched, were studied on plasma cholesterol, early atherosclerosis, cardiac production of superoxide anions, and NAD(P)H oxidase expression in hamsters. Forty hamsters were divided into 5 groups of 8 and fed an atherogenic diet for 12 weeks. They received by gavage either 7.14 mL/(kg day) phycocyanin (PC), Se-rich phycocyanin (SePC), spirulina (SP) or Se-rich spirulina (SeSP) in water, or water as control. SeSP and SePC supplied 0.4 microg of Se per 100 g body weight. Plasma cholesterol and non-HDL cholesterol concentrations were lower in group consuming SePC. HDL-cholesterol was never affected. SePC significantly increased plasma antioxidant capacity by 42% compared with controls. A sparing effect in liver glutathione peroxidase (87% on average) and superoxide dismutase (56% on average) activity was observed for all the groups compared to controls. Aortic fatty streak area was significantly reduced in the experimental groups, especially by PC (82%) and SePC (85%). Cardiac production of superoxide anion significantly decreased by approximately 46-76% in the four experimental groups and especially in SePC group (76%). The expression of p22phox subunit of NAD(P)H oxidase decreased by 34% after consumption of SePC. The results indicate that chronic consumption of Se-rich spirulina phycocyanin powerfully prevents the development of atherosclerosis. The underlying mechanism is related mainly to inhibiting pro-oxidant factors and at a lesser extent improving the serum lipid profile. PMID:17696484

  17. Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes

    SciTech Connect

    Shvedova, A.A. Kisin, E.R.; Murray, A.R.; Kommineni, C.; Castranova, V.; Fadeel, B.; Kagan, V.E.

    2008-09-01

    Single-walled carbon nanotubes (SWCNT) have been introduced into a large number of new technologies and consumer products. The combination of their exceptional features with very broad applications raised concerns regarding their potential health effects. The prime target for SWCNT toxicity is believed to be the lung where exposure may occur through inhalation, particularly in occupational settings. Our previous work has demonstrated that SWCNT cause robust inflammatory responses in rodents with very early termination of the acute phase and rapid onset of chronic fibrosis. Timely elimination of polymorphonuclear neutrophils (PMNs) through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. Thus, we hypothesized that NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. To experimentally address the hypothesis, we employed NADPH oxidase-deficient mice which lack the gp91{sup phox} subunit of the enzymatic complex. We found that NADPH oxidase null mice responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-{beta}, and significantly lower levels of collagen deposition, as compared to C57BL/6 control mice. These results demonstrate a role for NADPH oxidase-derived reactive oxygen species in determining course of pulmonary response to SWCNT.

  18. Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons.

    PubMed

    Case, Adam J; Li, Shumin; Basu, Urmi; Tian, Jun; Zimmerman, Matthew C

    2013-07-01

    Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling. PMID:23624625

  19. Persistence of the bacterial pathogen Granulibacter bethesdensis in Chronic Granulomatous Disease monocytes and macrophages lacking a functional NADPH oxidase1

    PubMed Central

    Chu, Jessica; Song, Helen H.; Zarember, Kol A.; Mills, Teresa A.; Gallin, John I.

    2013-01-01

    Granulibacter bethesdensis is a Gram-negative pathogen in patients with Chronic Granulomatous Disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to non-oxidative killing mechanisms. While G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with interferon-γ (IFN-γ) to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 days) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, likely relating to the persistence of this microorganism in infected CGD patients. PMID:23956436

  20. Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity

    PubMed Central

    Sosna, Justyna; Döring, Claudia; Klapper, Wolfram; Küppers, Ralf; Böttcher, Sebastian; Adam, Dieter; Siebert, Reiner; Schütze, Stefan

    2013-01-01

    The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL. PMID:24376854

  1. Phagocyte-like NADPH oxidase (Nox2) promotes activation of p38MAPK in pancreatic β-cells under glucotoxic conditions: Evidence for a requisite role of Ras-related C3 botulinum toxin substrate 1 (Rac1).

    PubMed

    Sidarala, Vaibhav; Veluthakal, Rajakrishnan; Syeda, Khadija; Vlaar, Cornelis; Newsholme, Philip; Kowluru, Anjaneyulu

    2015-06-15

    It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet β-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20mM; 24h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated β-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the β-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes. PMID:25881746

  2. Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

    PubMed

    Mangum, Lee C; Borazjani, Abdolsamad; Stokes, John V; Matthews, Anberitha T; Lee, Jung Hwa; Chambers, Janice E; Ross, Matthew K

    2015-04-20

    Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox) levels and enhanced p47(phox) membrane localization compared to that in vehicle-treated cells. p47(phox) is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47(phox). In addition, ATK also blocked OC-induced p47(phox) serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation. PMID:25633958

  3. NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2

    PubMed Central

    Muise, Aleixo M; Xu, Wei; Guo, Cong-Hui; Walters, Thomas D; Wolters, Victorien M; Fattouh, Ramzi; Lam, Grace Y; Hu, Pingzhao; Murchie, Ryan; Sherlock, Mary; Gana, Juan Cristóbal; Russell, Richard K; Glogauer, Michael; Duerr, Richard H; Cho, Judy H; Lees, Charlie W; Satsangi, Jack; Wilson, David C; Paterson, Andrew D; Griffiths, Anne M; Silverberg, Mark S; Brumell, John H

    2013-01-01

    Objective The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that is histologically similar to Crohn's colitis, suggesting a common aetiology for both diseases. The aim of this study is to determine if polymorphisms in NOX2 NADPH oxidase complex genes that do not cause CGD are associated with the development of inflammatory bowel disease (IBD). Methods Direct sequencing and candidate gene approaches were used to identify susceptibility loci in NADPH oxidase complex genes. Functional studies were carried out on identified variants. Novel findings were replicated in independent cohorts. Results Sequence analysis identified a novel missense variant in the neutrophil cytosolic factor 2 (NCF2) gene that is associated with very early onset IBD (VEO-IBD) and subsequently found in 4% of patients with VEO-IBD compared with 0.2% of controls (p=1.3×10−5, OR 23.8 (95% CI 3.9 to 142.5); Fisher exact test). This variant reduced binding of the NCF2 gene product p67phox to RAC2. This study found a novel genetic association of RAC2 with Crohn's disease (CD) and replicated the previously reported association of NCF4 with ileal CD. Conclusion These studies suggest that the rare novel p67phox variant results in partial inhibition of oxidase function and are associated with CD in a subgroup of patients with VEO-IBD; and suggest that components of the NADPH oxidase complex are associated with CD. PMID:21900546

  4. NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium.

    PubMed

    Felmy, Boas; Songhet, Pascal; Slack, Emma Marie Caroline; Müller, Andreas J; Kremer, Marcus; Van Maele, Laurye; Cayet, Delphine; Heikenwalder, Mathias; Sirard, Jean-Claude; Hardt, Wolf-Dietrich

    2013-01-01

    Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface. PMID:24143212

  5. NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent, TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

    PubMed Central

    Slack, Emma Marie Caroline; Müller, Andreas J.; Kremer, Marcus; Van Maele, Laurye; Cayet, Delphine; Heikenwalder, Mathias; Sirard, Jean-Claude; Hardt, Wolf-Dietrich

    2013-01-01

    Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tmavir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb−/− mice are efficiently infected by S.Tmavir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb−/−Myd88−/− mice indicated that the S.Tmavir-inflicted disease in Cybb−/− mice hinges on CD11c+CX3CR1+ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tmavir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface. PMID:24143212

  6. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  7. NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

    PubMed Central

    Wang, Dashou; Youngson, Charlotte; Wong, Veronica; Yeger, Herman; Dinauer, Mary C.; de Miera, Eleazar Vega-Saenz; Rudy, Bernardo; Cutz, Ernest

    1996-01-01

    Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms. PMID:8917565

  8. NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson's disease.

    PubMed

    Pal, Rituraj; Bajaj, Lakshya; Sharma, Jaiprakash; Palmieri, Michela; Di Ronza, Alberto; Lotfi, Parisa; Chaudhury, Arindam; Neilson, Joel; Sardiello, Marco; Rodney, George G

    2016-01-01

    Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson's disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD. PMID:26960433

  9. NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

    PubMed Central

    Pal, Rituraj; Bajaj, Lakshya; Sharma, Jaiprakash; Palmieri, Michela; Di Ronza, Alberto; Lotfi, Parisa; Chaudhury, Arindam; Neilson, Joel; Sardiello, Marco; Rodney, George G.

    2016-01-01

    Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD. PMID:26960433

  10. Effects of rosuvastatin on ADMA, rhokinase, NADPH oxidase, caveolin-1, hsp 90 and NFkB levels in a rat model of myocardial ischaemia–reperfusion

    PubMed Central

    Burma, Oktay; Uysal, Ayhan; Onat, Elif; Sahna, Engin; Ilhan, Necip; Erol, Deniz; Ozcan, Mete

    2014-01-01

    Summary Aim Endothelial dysfunction, oxidative stress and inflammation are among the most important mechanisms of ischaemia–reperfusion (I/R) injury. Besides their cholesterol-lowering effects, statins are known to provide protection against myocardial dysfunction and vascular endothelial injury via nitric oxide-dependent mechanisms. The aim of this study was to investigate the effects of rosuvastatin on certain intermediates involved in the generation of nitric oxide (asymmetrical dimethyl arginin, ADMA, caveolin-1 and hsp 90), oxidative stress (rhokinase, NADPH oxidase) and inflammation (NFkB), using an in vivo model of myocardial infarction in the rat. Methods Adult male Sprague Dawley rats were divided into three groups (control, I/R and I/R after 15 days of rosuvastatin administration). Reperfusion was applied for 120 min following left anterior descending coronary artery ischaemia for 30 min. Caveolin-1, hsp 90 and NFkB levels were evaluated with the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and ADMA, rhokinase and NADPH oxidase levels were evaluated with ELISA. Results While NFkB and hsp 90 levels were higher in the I/R group, their levels were significantly lower in the rosuvastatin group. While ADMA and NADPH oxidase levels significantly increased with I/R, they were lower in the rosuvastatin-treated group, but not statistically significant. Rhokinase levels were significantly lower in the rosuvastatin group. Caveolin-1 levels were not different between the groups. Conclusion Our results suggest that ADMA, rhokinase, NADPH oxidase, hsp 90 and NFkB could facilitate I/R injury, and rosuvastatin significantly reduced levels of these parameters. These results indicate that rosuvastatin may have a protective role in I/R injury via mechanisms targeting inflammation, endothelial dysfunction and oxidative stress. PMID:25629537

  11. Super oxide from NADPH oxidase as second messenger for expression of osteopontin and monocyte chemoattractant-1 in renal epithelial cells exposed to calcium oxalate crystals

    PubMed Central

    Umekawa, Tohru; Uemura, Hirotsugu; Khan, Saeed R.

    2013-01-01

    Objective Calcium oxalate monohydrate crystals (COM) stimulate OPN and MCP-1 expression in renal tubular cells. Reactive oxygen species (ROS) play an important but not yet fully defined role in expression of inflammatory genes such as MCP-1 and OPN. Recently, it has been proposed that ROS serve as intracellular signals. NADPH oxidase is the major source of superoxide in many tissues including kidney. We tested the hypothesis that COM exposure of a renal epithelial cell line, NRK 52E, will upregulate NADPH oxidase subunit p47phox, enhance superoxide production and increase MCP-1 and OPN mRNA levels. The latter will be attenuated by diphenileneiodium chloride (DPI), an inhibitor for NADPH Oxidase. Materials and Methods Confluent cultures of NRK52E cells were exposed to COM (66.7μg/cm2) with or without pre treatment with DPI (10×10−6M) under serum free conditions. The conditioned medium was collected and total cellular RNA isolated from the cells, and subjected to enzyme-linked immunosorbent assay (ELISA) and real time PCR. Production of ROS was estimated by dihydroethidium (DHE) staining using fluorescence microscope. Immunohistochemistry and real time PCR were used to analyze p47phox in NRK52E cells. Results COM treated NRK52E cells showed enhanced expression of p47phox and production of superoxide. COM-induced production of MCP-1 and OPN was significantly reduced following treatment with DPI. Conclusions While the generation of a large amount of ROS may play a major role in tissue injury or death, the regulated generation of low concentration of ROS, possibly by NADPH oxidase, may represent a second messenger system for generation of COM induced MCP-1 and OPN in the renal tubules. PMID:19220254

  12. Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.

    PubMed

    Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A

    2013-12-01

    Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress. PMID:23816524

  13. Activation of Polyphenol Oxidase of Chloroplasts 1

    PubMed Central

    Tolbert, N. E.

    1973-01-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or —18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density. Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles × mg−1 chlorophyll × hr−1. Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes. Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 μg of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 μg of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold. Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 μmoles × mg−1 chlorophyll × hr−1. Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency. PMID:16658308

  14. Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba

    PubMed Central

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

  15. Diapocynin, a dimer of the NADPH oxidase inhibitor apocynin, reduces ROS production and prevents force loss in eccentrically contracting dystrophic muscle.

    PubMed

    Ismail, Hesham M; Scapozza, Leonardo; Ruegg, Urs T; Dorchies, Olivier M

    2014-01-01

    Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches. PMID:25329652

  16. Diapocynin, a Dimer of the NADPH Oxidase Inhibitor Apocynin, Reduces ROS Production and Prevents Force Loss in Eccentrically Contracting Dystrophic Muscle

    PubMed Central

    Ismail, Hesham M.; Scapozza, Leonardo; Ruegg, Urs T.; Dorchies, Olivier M.

    2014-01-01

    Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches. PMID:25329652

  17. NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection

    PubMed Central

    Macedo, Juan P.; Utsch, Lara; Tafuri, Wagner L.; Campagnole-Santos, Maria José; Alves, Rosana O.; Alves-Filho, José C. F.; Romanha, Alvaro J.; Cunha, Fernando Queiroz; Teixeira, Mauro M.; Radi, Rafael; Vieira, Leda Q.

    2012-01-01

    •NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91phox−/− or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with •NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi. PMID:22348160

  18. Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.

    PubMed

    Antony, Smitha; Wu, Yongzhong; Hewitt, Stephen M; Anver, Miriam R; Butcher, Donna; Jiang, Guojian; Meitzler, Jennifer L; Liu, Han; Juhasz, Agnes; Lu, Jiamo; Roy, Krishnendu K; Doroshow, James H

    2013-12-01

    Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation. PMID:23851018

  19. NAD(P)H Oxidase Nox-4 Mediates 7-Ketocholesterol-Induced Endoplasmic Reticulum Stress and Apoptosis in Human Aortic Smooth Muscle Cells

    PubMed Central

    Pedruzzi, Eric; Guichard, Cécile; Ollivier, Véronique; Driss, Fathi; Fay, Michèle; Prunet, Céline; Marie, Jean-Claude; Pouzet, Cécile; Samadi, Mohammad; Elbim, Carole; O'Dowd, Yvonne; Bens, Marcelle; Vandewalle, Alain; Gougerot-Pocidalo, Marie-Anne; Lizard, Gérard; Ogier-Denis, Eric

    2004-01-01

    The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca2+ oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH2-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis. PMID:15572675

  20. Non-muscle myosin light chain promotes endothelial progenitor cells senescence and dysfunction in pulmonary hypertensive rats through up-regulation of NADPH oxidase.

    PubMed

    Liu, Bin; Li, Tao; Peng, Jing-Jie; Zhang, Jie-Jie; Liu, Wei-Qi; Luo, Xiu-Ju; Ma, Qi-Lin; Gong, Zhi-Cheng; Peng, Jun

    2016-03-15

    Non-muscle myosin regulatory light chain (nmMLC20) is reported to exert transcriptional function in regulation of gene expression, and NADPH oxidase (NOX)-derived reactive oxygen species contribute to vascular remodeling of pulmonary artery hypertension (PAH). This study aims to determine if nmMLC20 can promote endothelial progenitor cells (EPCs) senescence and dysfunction through up-regulation of NOX in PAH rats. The rats were exposed to10% hypoxia for 3 weeks to establish a PAH model, which showed an increase in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, and the accelerated senescence and impaired functions in EPCs, accompanied by an increase in Rho-kinase (ROCK) and NOX activities, p-nmMLC20 level, NOX expression and H2O2 content; these phenomena were reversed by fasudil, a selective inhibitor of ROCK. Next, normal EPCs were cultured under hypoxia to induce senescence in vitro. Consistent with the in vivo findings, hypoxia increased the senescence and dysfunction of EPCs concomitant with an increase in ROCK and NOX activities, p-nmMLC20 level, NOX expression and H2O2 content; these phenomena were reversed by fasudil. Knockdown of nmMLC20 showed similar results to that of fasudil except no effect on ROCK activity. Based on these observations, we conclude that nmMLC20 could promote the senescence and dysfunctions of EPCs in PAH through up-regulation of NOX in a phosphorylation-dependent manner. PMID:26872992

  1. The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli.

    PubMed

    Miller, Gad; Schlauch, Karen; Tam, Rachel; Cortes, Diego; Torres, Miguel A; Shulaev, Vladimir; Dangl, Jeffery L; Mittler, Ron

    2009-01-01

    Cell-to-cell communication and long-distance signaling play a key role in the response of plants to pests, mechanical wounding, and extreme environmental conditions. Here, we report on a rapid systemic signal in Arabidopsis thaliana that traveled at a rate of 8.4 centimeters per minute and was dependent on the respiratory burst oxidase homolog D (RbohD) gene. Signal propagation was accompanied by the accumulation of reactive oxygen species (ROS) in the extracellular spaces between cells and was inhibited by the suppression of ROS accumulation at locations distant from the initiation site. The rapid systemic signal was triggered by wounding, heat, cold, high-intensity light, and salinity stresses. Our results reveal the profound role that ROS play in mediating rapid, long-distance, cell-to-cell propagating signals in plants. PMID:19690331

  2. Sanguisorbae radix protects against 6-hydroxydopamine-induced neurotoxicity by regulating NADPH oxidase and NF-E2-related factor-2/heme oxygenase-1 expressions.

    PubMed

    Oh, Hyein; Hur, Jinyoung; Park, Gunhyuk; Kim, Hyo Geun; Kim, Young Ock; Oh, Myung Sook

    2013-07-01

    6-Hydroxydopamine (6-OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS-induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase-1 (HO-1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6-OHDA-induced cell toxicity by regulating ROS in SH-SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6-OHDA-induced cell damage dose dependently in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X ratio and decreased cytochrome C release and caspase-3 activity. SRE also abolished 6-OHDA-induced ROS by inhibiting NOX expression and by inducing HO-1 expression via NF-E2-related factor-2 activation. Taken together, these results demonstrate that SRE has protective effects against 6-OHDA-induced cell death by regulating ROS in SH-SY5Y cells. PMID:22933385

  3. The antiangiogenic agent SU5416 down-regulates phorbol ester-mediated induction of cyclooxygenase 2 expression by inhibiting nicotinamide adenine dinucleotide phosphate oxidase activity.

    PubMed

    Saha, Debabrata; Sekhar, Konjeti R; Cao, Carolyn; Morrow, Jason D; Choy, Hak; Freeman, Michael L

    2003-10-15

    Increased expression of cyclooxygenase (COX) 2 and the production of PGs appear to provide a survival advantage to transformed cells through the inhibition of apoptosis, increased attachment to extracellular matrix, increased invasiveness and the stimulation of angiogenesis. The purpose of this study was to determine whether an angiogenic antagonist, SU5416, could inhibit endogenous and phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 expression. SU5416 (5 micro M) inhibited endogenous as well as PMA-mediated induction of COX-2 expression when analyzed by immunoblot and Northern blot analysis. However, COX-1 expression remained unchanged under similar conditions. PMA is a potent inducer of reactive oxygen species that can play an important role during the induction of COX-2 expression. Our results demonstrated that PMA-mediated induction of COX-2 expression was found to be dependent on NADPH oxidase activity. An inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked the PMA-mediated induction of COX-2 expression. The oxidase complex exhibited a temporal pattern of activation after exposure to PMA in which maximum activation was observed at 30 min after the addition of PMA. Activation of NADPH oxidase was also inhibited by SU5416, whereas an inhibitor of epidermal growth factor receptor signaling was unable to prevent the PMA-mediated induction of NADPH oxidase activity. When we blocked the PMA-mediated production of reactive oxygen species by blocking NADPH oxidase with SU5416, COX-2 expression and PGE(2) synthesis were also inhibited. Our results suggest that inhibition of NADPH oxidase activity, blocking of COX-2 expression, and PGE(2) synthesis may represent novel targets for SU5416. PMID:14583492

  4. Effect of NADPH oxidase inhibition on the expression of kidney injury molecule and calcium oxalate crystal deposition in hydroxy-L-proline-induced hyperoxaluria in the male Sprague–Dawley rats

    PubMed Central

    Zuo, Jian; Khan, Aslam; Glenton, Patricia A.; Khan, Saeed R.

    2011-01-01

    Background. Renal calcium oxalate (CaOx) crystal deposition is associated with epithelial injury and movement of inflammatory cells into the interstitium. We have proposed that oxalate (Ox)- and CaOx crystal-induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Methods. Present study was undertaken to determine the effect of NADPH oxidase inhibitor apocynin on the expression of kidney injury molecule-1 (KIM-1) and renal CaOx crystal deposition in rats with hyperoxaluria. We also investigated the urinary excretion of KIM-1, osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1) and renal expression of OPN and ED-1. Male Sprague–Dawley rats were fed a diet containing 5% hydroxyl-L-proline (HLP) and 4 mmol apocynin to drink for 28 days. Urine was collected on Days 7, 14, 21 and 28. After that, rats were sacrificed and their kidneys processed for various microscopic and molecular investigations. Results. HLP consumption produced heavy deposits of CaOx crystals. Renal expression of KIM-1 and OPN and urinary excretion of KIM-1, OPN, H2O2 and MCP-1 was significantly increased. ED-1-positive cells migrated into renal interstitium. Apocynin treatment caused significant reduction of crystal deposits, injured and dilated tubules; renal expression of KIM-1, OPN and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H2O2. Apocynin had no effect on the urinary excretion of Ox. Conclusions. This is the first study of urinary excretion and renal expression of KIM-1 in association with renal CaOx crystal deposition, experimental or clinical. The results indicate that NADPH oxidase inhibition leads to reduction in KIM-1 expression and urinary excretion as well as renal CaOx crystal deposition. KIM-1 is an important marker of renal epithelial injury. The results provide further support to our proposal that renal epithelial injury is critical for crystal retention and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase. PMID:21378157

  5. The regulation of methyl jasmonate on hyphal branching and GA biosynthesis in Ganoderma lucidum partly via ROS generated by NADPH oxidase.

    PubMed

    Shi, Liang; Gong, Li; Zhang, Xiangyang; Ren, Ang; Gao, Tan; Zhao, Mingwen

    2015-08-01

    Ganoderma lucidum is one of the best known medicinal basidiomycetes because it produces many pharmacologically active compounds, and methyl jasmonate (MeJA) was previously reported to induce the biosynthesis of ganoderic acids (GA) in G. lucidum. In this study, we found that MeJA not only increased the amount of GA but also increased the distance between hyphal branches by approximately 1.2-fold. Further analysis showed that MeJA could increase the intracellular ROS (reactive oxygen species) content by approximately 2.2-2.7-fold. Furthermore, the hyphal branching and GA biosynthesis regulated by MeJA treatment could be abolished by ROS scavengers to a level similar to or lower than that of the control group. These results indicated that the regulation of hyphal branching and GA biosynthesis by MeJA might occur via a ROS signaling pathway. Further analysis revealed that NADPH oxidase (NOX) plays an important role in MeJA-regulated ROS generation. Importantly, our results highlight that NOX functions in signaling cross-talk between ROS and MeJA. In addition, these findings provide an excellent opportunity to identify potential pathways linking ROS networks to MeJA signaling in fungi and suggest that plants and fungi share a conserved signaling-crosstalk mechanism. PMID:25512263

  6. NADPH Oxidase 1 Controls the Persistence of Directed Cell Migration by a Rho-Dependent Switch of α2/α3 Integrins▿ †

    PubMed Central

    Sadok, Amine; Pierres, Anne; Dahan, Laetitia; Prévôt, Charles; Lehmann, Maxime; Kovacic, Hervé

    2009-01-01

    NADPH oxidase 1 (Nox1) is expressed mainly in colon epithelial cells and produces superoxide ions as a primary function. We showed that Nox1 knockdown inhibits directional persistence of migration on collagen I. This paper dissects the mechanism by which Nox1 affects the direction of colonic epithelial cell migration in a two-dimensional model. Transient activation of Nox1 during cell spreading on collagen 1 temporarily inactivated RhoA and led to efficient exportation of α2β1 integrin to the cell surface, which supported persistent directed migration. Nox1 knockdown led to a loss of directional migration which takes place through a RhoA-dependent α2/α3 integrin switch. Transient RhoA overactivation upon Nox1 inhibition led to transient cytoskeletal reorganization and increased cell-matrix contact associated with a stable increase in α3 integrin cell surface expression. Blocking of α3 integrin completely reversed the loss of directional persistence of migration. In this model, Nox1 would represent a switch between random and directional migration through RhoA-dependent integrin cell surface expression modulation. PMID:19451223

  7. NADPH oxidase 3-associated oxidative stress and caspase 3-dependent apoptosis in the cochleae of D-galactose-induced aged rats

    PubMed Central

    DU, ZHENGDE; LI, SHUO; LIU, LIN; YANG, QIONG; ZHANG, HONGWEI; GAO, CHUNSHENG

    2015-01-01

    Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis. PMID:26498835

  8. Hepatitis C Virus NS5A Protein Triggers Oxidative Stress by Inducing NADPH Oxidases 1 and 4 and Cytochrome P450 2E1

    PubMed Central

    Smirnova, Olga A.; Ivanova, Olga N.; Bartosch, Birke; Valuev-Elliston, Vladimir T.; Mukhtarov, Furkat; Kochetkov, Sergey N.; Ivanov, Alexander V.

    2016-01-01

    Replication of hepatitis C virus (HCV) is associated with the induction of oxidative stress, which is thought to play a major role in various liver pathologies associated with chronic hepatitis C. NS5A protein of the virus is one of the two key viral proteins that are known to trigger production of reactive oxygen species (ROS). To date it has been considered that NS5A induces oxidative stress by altering calcium homeostasis. Herein we show that NS5A-induced oxidative stress was only moderately inhibited by the intracellular calcium chelator BAPTA-AM and not at all inhibited by the drug that blocks the Ca2+ flux from ER to mitochondria. Furthermore, ROS production was not accompanied by induction of ER oxidoreductins (Ero1), H2O2-producing enzymes that are implicated in the regulation of calcium fluxes. Instead, we found that NS5A contributes to ROS production by activating expression of NADPH oxidases 1 and 4 as well as cytochrome P450 2E1. These effects were mediated by domain I of NS5A protein. NOX1 and NOX4 induction was mediated by enhanced production of transforming growth factor β1 (TGFβ1). Thus, our data show that NS5A protein induces oxidative stress by several multistep mechanisms. PMID:27200149

  9. The NADPH oxidase NOX2 as a novel biomarker for suicidality: evidence from human post mortem brain samples.

    PubMed

    Schiavone, S; Neri, M; Mhillaj, E; Morgese, M G; Cantatore, S; Bove, M; Riezzo, I; Tucci, P; Pomara, C; Turillazzi, E; Cuomo, V; Trabace, L

    2016-01-01

    Recent evidence points towards a role of oxidative stress in suicidality. However, few studies were carried out on the sources of reactive oxygen species (ROS) in subjects with suicidal behaviour. We have previously demonstrated that the NADPH oxidase NOX2-derived oxidative stress has a major role in the development of neuropathological alterations observed in an animal model of psychosis. Here, we investigated the possible increase in NOX2 in post mortem brain samples of subjects who died by asphyctic suicide (AS) compared with controls (CTRL) and subjects who died by non-suicidal asphyxia (NSA). We found that NOX2 expression was significantly higher in the cortex of AS subjects than in the other two experimental groups. NOX2 immunostaining was mainly detected in GABAergic neurons, with a minor presence of NOX2-positive-stained cells in glutamatergic and dopaminergic neurons, as well as astrocytes and microglia. A sustained increase in the expression of 8-hydroxy-2'-deoxyguanosine, an indirect marker of oxidative stress, was also detected in the cortex of AS subjects, compared with CTRL and NSA subjects. A significant elevation in cortical interleukin-6 immunoreactivity in AS subjects suggested an involvement of cytokine-associated molecular pathways in NOX2 elevations. Our results suggest that the increase in NOX2-derived oxidative stress in the brain might be involved in the neuropathological pathways leading to suicidal behaviour. These results may open innovative insights in the identification of new pathogenetic and necroscopic biomarkers, predictive for suicidality and potentially useful for suicide prevention. PMID:27187235

  10. Puerarin Improves Diabetic Aorta Injury by Inhibiting NADPH Oxidase-Derived Oxidative Stress in STZ-Induced Diabetic Rats.

    PubMed

    Li, Wenping; Zhao, Wenwen; Wu, Qin; Lu, Yuanfu; Shi, Jingshan; Chen, Xiuping

    2016-01-01

    Objective. Puerarin is a natural flavonoid isolated from the TCM lobed kudzuvine root. This study investigated the effect and mechanisms of puerarin on diabetic aorta in rats. Methods. Streptozotocin- (STZ-) induced diabetic rats were administered with puerarin for 3 weeks. Levels of serum insulin (INS), PGE2, endothelin (ET), glycated hemoglobin (GHb), H2O2, and nitric oxide (NO) in rats were measured by ELISA and colorimetric assay kits. The aortas were stained with H&E. Moreover, the mRNA expression of ICAM-1, LOX-1, NADPH oxidase 2 (NOX2), and NOX4 and the protein expression of ICAM-1, LOX-1, NF-κB p65, E-selectin, NOX2, and NOX4 in aorta tissues were measured by real-time PCR and Western blot, respectively. The localization of ICAM-1, NF-κB p65, NOX2, and NOX4 in the aorta tissues was also determined through immunohistochemistry. Results. Puerarin treatment exerted no effect on fasting blood glucose levels but significantly reduced the serum levels of INS, GHb, PGE2, ET, H2O2, and NO. In addition, puerarin improved the pathological alterations and inhibited the expression of ICAM-1, LOX-1, NOX2, and NOX4 at both mRNA and protein levels. Puerarin also significantly reduced the number of cells showing positive staining for ICAM-1, NOX2, NOX4, and NF-κB p65. Conclusion. Puerarin demonstrated protective effect on the STZ-induced diabetic rat aorta. The protective mechanisms may include regulation of NF-κB and inhibition of NOX2 and NOX4 followed by inhibition of cell adhesion molecule expression. PMID:26881260

  11. Puerarin Improves Diabetic Aorta Injury by Inhibiting NADPH Oxidase-Derived Oxidative Stress in STZ-Induced Diabetic Rats

    PubMed Central

    Li, Wenping; Zhao, Wenwen; Wu, Qin; Lu, Yuanfu; Shi, Jingshan; Chen, Xiuping

    2016-01-01

    Objective. Puerarin is a natural flavonoid isolated from the TCM lobed kudzuvine root. This study investigated the effect and mechanisms of puerarin on diabetic aorta in rats. Methods. Streptozotocin- (STZ-) induced diabetic rats were administered with puerarin for 3 weeks. Levels of serum insulin (INS), PGE2, endothelin (ET), glycated hemoglobin (GHb), H2O2, and nitric oxide (NO) in rats were measured by ELISA and colorimetric assay kits. The aortas were stained with H&E. Moreover, the mRNA expression of ICAM-1, LOX-1, NADPH oxidase 2 (NOX2), and NOX4 and the protein expression of ICAM-1, LOX-1, NF-κB p65, E-selectin, NOX2, and NOX4 in aorta tissues were measured by real-time PCR and Western blot, respectively. The localization of ICAM-1, NF-κB p65, NOX2, and NOX4 in the aorta tissues was also determined through immunohistochemistry. Results. Puerarin treatment exerted no effect on fasting blood glucose levels but significantly reduced the serum levels of INS, GHb, PGE2, ET, H2O2, and NO. In addition, puerarin improved the pathological alterations and inhibited the expression of ICAM-1, LOX-1, NOX2, and NOX4 at both mRNA and protein levels. Puerarin also significantly reduced the number of cells showing positive staining for ICAM-1, NOX2, NOX4, and NF-κB p65. Conclusion. Puerarin demonstrated protective effect on the STZ-induced diabetic rat aorta. The protective mechanisms may include regulation of NF-κB and inhibition of NOX2 and NOX4 followed by inhibition of cell adhesion molecule expression. PMID:26881260

  12. Apocynin protects against ethanol-induced gastric ulcer in rats by attenuating the upregulation of NADPH oxidases 1 and 4.

    PubMed

    El-Naga, Reem N

    2015-12-01

    Gastric ulcer is a common gastrointestinal disorder affecting many people all over the world. Absolute ethanol (5 ml/kg) was used to induce gastric ulceration in rats. Apocynin (50 mg/kg) was given orally one hour before the administration of absolute ethanol. Omeprazole (20 mg/kg) was used as a standard. Interestingly, apocynin pre-treatment provided 93.5% gastroprotection against ethanol-induced ulceration. Biochemically, gastric mucin content was significantly increased with apocynin pre-treatment. This finding was further supported by alcian blue staining of stomach sections obtained from the different treated groups. Also, gastric juice volume and acidity were significantly reduced. Apocynin significantly ameliorated ethanol-induced oxidative stress by replenishing reduced glutathione and superoxide dismutase levels as well as reducing elevated malondialdehyde levels in gastric tissues. Besides, ethanol-induced pro-inflammatory response was significantly decreased by apocynin pre-treatment via reducing elevated levels of pro-inflammatory markers; interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2 and inducible nitric oxide synthase. Additionally, caspase-3 tissue level was significantly reduced in apocynin pre-treated group. Interestingly, NADPH oxidase-1 (NOX-1) and NOX-4 up-regulation was shown to be partially involved in the pathogenesis of ethanol-induced gastric ulceration and was significantly reversed by apocynin pre-treatment. Gastroprotective properties of apocynin were confirmed by histopathological examination. It is worth mentioning that apocynin was superior in all aspects except gastric mucin content parameter where it was signif