Activated Nicotinamide Adenine Dinucleotide Phosphate Oxidase Research Articles

2,3′4,4′,5-Pentachlorobiphenyl induces hepatocellular carcinoma cell proliferation through pyruvate kinase M2-dependent glycolysis

Polychlorinated biphenyls (PCBs) are classic persistent organic pollutants (POPs) and are associated with the progression of many cancers, including liver cancer. The present study investigated the effect of 2,3’4,4′,5-pentachlorobiphenyl (PCB118) on hepatocellular carcinoma cell proliferation and its underlying mechanisms. The results indicated that PCB118 exposure promotes the proliferation and glycolysis of hepatocellular carcinoma SMMC-7721 cells. Moreover, PCB118 exposure increased the expression level of pyruvate kinase M2 (PKM2) and its nuclear translocation, whereas treatment with PKM2 shRNA suppressed the induction of cell proliferation and glycolysis by PCB118. PCB118 stimulated reactive oxygen species (ROS) production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Treatment with the antioxidants N-acetyl-L-cysteine (NAC) and superoxide dismutase (SOD) prevented PCB118-induced effects on PKM2, cell proliferation and glycolysis. Furthermore, we found that PCB118 activated NADPH oxidase through the aryl hydrocarbon receptor (AhR) in SMMC-7721 cells. Consistently, treatment with AhR shRNA suppressed PCB118-induced effects on PKM2, cell proliferation and glycolysis. Overall, these results indicated that PCB118 promotes HCC cell proliferation via PKM2-dependent upregulation of glycolysis, which is mediated by AhR/NADPH oxidase-induced ROS production.

Advanced oxidation protein products induce pre-osteoblast apoptosis through a nicotinamide adenine dinucleotide phosphate oxidase-dependent, mitogen-activated protein kinases-mediated intrinsic apoptosis pathway.

SummaryOsteoblast apoptosis contributes to age‐related bone loss. Advanced oxidation protein products (AOPPs) are recognized as the markers of oxidative stress and potent inducers of apoptosis. We have demonstrated that AOPP accumulation was correlated with age‐related bone loss. However, the effect of AOPPs on the osteoblast apoptosis still remains unknown. Exposure of osteoblastic MC3T3‐E1 cells to AOPPs caused the excessive generation of reactive oxygen species (ROS) by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Increased ROS induced phosphorylation of mitogen‐activated protein kinases (MAPKs), which subsequently triggered intrinsic apoptosis pathway by inducing mitochondrial dysfunction, endoplasmic reticulum stress, and Ca2+ overload and eventually leads to apoptosis. Chronic AOPP loading in aged Sprague‐Dawley rats induced osteoblast apoptosis and activated NADPH oxidase signaling cascade, in combination with accelerated bone loss and deteriorated bone microstructure. Our study suggests that AOPPs induce osteoblast apoptosis by the NADPH oxidase‐dependent, MAPK‐mediated intrinsic apoptosis pathway.

Production of Reactive Oxygen Species in the Diabetic Heart

Reactive oxygen species (ROS) are the main facilitators of cardiovascular complications in diabetes mellitus (DM), and the ROS level is increased in cultured cells exposed to high glucose concentrations or in diabetic animal models. Emerging evidence shows that mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are dominant mechanisms of ROS production in the diabetic heart. Hyperpolarization of the mitochondrial inner membrane potentials and impaired mitochondrial function promote ROS production in the mitochondria of the diabetic heart. Uncoupling proteins are upregulated and may reduce the ROS level by depolarizing the mitochondrial inner membrane potential. NADPH oxidase is another major site of ROS production and its contribution to DM-induced ROS increase has been elucidated not only in vascular smooth muscle cells and endothelial cells, but also in cardiomyocytes. Protein kinase C, angiotensin II, and advanced glycation endproducts (AGEs)/receptor for AGEs can activate NADPH oxidase. Increased intracellular calcium level mediated via the Na(+)-H(+) exchanger and subsequent activation of Ca(2+)/calmodulin-dependent protein kinase II may also activate NADPH oxidase. This review presents the current understanding of the mechanisms of ROS production, focusing especially on the roles of mitochondria and NADPH oxidase.

Breaking barriers: insight into the pathogenesis of neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (AMD) is a leading cause of central visual acuity loss in a growing segment of the population, those over the age of 60 years. Treatment has improved over the last decade, with the availability of agents that inhibit the bioactivity of vascular endothelial growth factor (VEGF), but it is still limited, because of tachyphylaxis and potential risk and toxicity of anti-VEGF agents. The authors have sought to understand the mechanisms of choroidal endothelial cell (CEC) activation and transmigration of the retinal pigment epithelium (RPE) and of RPE barrier dysfunction, events preceding vision-threatening neovascular AMD. The authors developed physiologically relevant human RPE and CEC coculture and transmigration models that have been important in helping to understand causes of events in human neovascular AMD. The authors can control for interactions between these cells and can separately assess activation of signaling pathways in each cell type relevant during CEC transmigration. Using these models, it was found that VEGF, particularly the cell-associated VEGF splice variant VEGF189, accounts for about 40% of CEC transmigration across the RPE. This percentage is in the range of similar reports following clinical inhibition of VEGF in neovascular AMD. RPE VEGF189 working through CEC VEGF receptor 2 activates the small guanosine triphosphatase (GTPase) of the Rho family, Rac1, in CECs, which in turn facilitates CEC transmigration. Conversely, inhibition of Rac1 activity prevents CEC transmigration. Once activated, Rac1 aggregates with subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in the generation of reactive oxygen species. Activated NADPH oxidase increases choroidal neovascularization in animal models of laser-induced injury. Rac1 is also downstream of the eotaxin-CCR3 pathway, another pathway important in human neovascular AMD. Studies also suggest that active Ras-related protein 1 (Rap1), another small GTPase, in RPE can strengthen the RPE barrier integrity and can resist CEC transmigration of the RPE, suggesting Rap1 activation may be another potential target for preventing neovascular AMD.

Nox1-Based NADPH Oxidase Is the Major Source of UVA-Induced Reactive Oxygen Species in Human Keratinocytes

UVA radiation is a major environmental stress on skin, causing acute and chronic photodamage. These responses are mediated by reactive oxygen species (ROS), although the cellular source of these ROS is unknown. We tested the hypotheses that UVA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required for ROS generation in human keratinocytes (HK) and that these ROS initiate rapid prostaglandin E2 (PGE2) synthesis. Treatment of HK with a non-toxic dose of UVA rapidly increased NADPH oxidase activity and intracellular ROS, which were partially blocked by an inhibitor of NADPH oxidase and by a mitochondria-selective antioxidant. Depleting the Nox1 isoform of the catalytic subunit of NADPH oxidase using small interfering RNA (siRNA) blocked the UVA-induced ROS increase, indicating that ROS produced by mitochondria or other sources are downstream from Nox1. Nox1 siRNA also blocked UVA-initiated PGE2 synthesis. The mechanism for activation of Nox1 is mediated by an increase in intracellular calcium. Ceramide, which has been proposed to mediate responses to UVA in HK, also activated NADPH oxidase. These results indicate that UVA activates Nox1-based NADPH oxidase to produce ROS that stimulate PGE2 synthesis, and that Nox1 may be an appropriate target for agents designed to block UVA-induced skin injury.

Hypoadiponectinemia as a Marker of Adipocyte Dysfunction—Part II: The Functional Significance of Low Adiponectin Secretion

Low adiponectin expression is common in obesity and is tightly linked to insulin resistance and fat mass expansion. Whereas normal adipocytes offer effective metabolic buffering through well-controlled release and uptake of free fatty acids on demand, adipocyte expansion induced by caloric excess and modulated by genetic, regional, and systemic factors elicits major unfavorable changes in fat cell phenotypes. Large, dysfunctional adipocytes show increased lipolysis and enhanced expression and secretion of proinflammatory and pro-oxidative cytokines. Low adiponectin secretion is a hallmark of impaired adipocyte function; its secretion is inhibited by cytokines such as tumor necrosis factor alpha, interleukin 6 and plasminogen activator inhibitor 1 and by high oxidative stress induced by increased fatty acids that activate nicotinamide adenine dinucleotide phosphate-oxidase. The ensuing hypoadiponectinemia may aggravate insulin resistance and facilitate the evolution of type 2 diabetes. Only massive weight loss allows true and sustained recovery of normal fat cell function as reflected by adiponectin secretion.

Bile Salt–Induced Apoptosis Involves NADPH Oxidase Isoform Activation

Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis. The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7). The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Czeta (PKCzeta). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase-dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCzeta, or NADPH oxidases. The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.

Investigation into the relationship between calyculin A-mediated potentiation of NADPH oxidase activity and inhibition of store-operated uptake of calcium by human neutrophils

The primary objective of the current study was to investigate possible relationships between calyculin A (CA)-mediated potentiation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and inhibition of store-operated uptake of Ca 2+ by chemoattractant-activated human neutrophils. Treatment of neutrophils with 100 nM CA, but not at lower concentrations (12.5–50 nM), prior to the addition of the N-formylated chemotactic tripeptide, N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) (1 μM), both potentiated and prolonged the activity of NADPH oxidase which was accompanied by exaggerated membrane depolarisation, delayed and attenuated membrane repolarisation, and inhibition of store-operated Ca 2+ influx. Inclusion of diphenylene iodonium chloride (DPI, 10 μM), an inhibitor of NADPH oxidase, antagonised the effects of CA on NADPH oxidase activity and the membrane repolarisation responses of FMLP-activated neutrophils, but failed to restore store-operated influx of Ca 2+. Similarly, CA also inhibited store-operated influx of Ca 2+ into FMLP-activated neutrophils from a patient with chronic granulomatous disease, a primary immunodeficiency disorder characterised by the absence of a functional NADPH oxidase. CA also inhibited the store-operated influx of Ca 2+ into control neutrophils treated with 1 μM thapsigargin, a selective inhibitor of the endomembrane Ca 2+-ATPase, which does not activate NADPH oxidase. Taken together, these observations demonstrate that augmentation of NADPH oxidase activity is not primarily involved in CA-mediated inhibition of the store-operated influx of Ca 2+ into activated human neutrophils.

Stable compounds of cigarette smoke induce endothelial superoxide anion production via NADPH oxidase activation.

Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (O2*). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial O2* production. Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in O2* production (20-fold, 3-fold, and 2-fold increase, respectively; P<0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequence-tat peptide but not by oxypurinol, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased O2* production 5-fold, which was prevented by prior inhibition of NADPH oxidase. These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial O2* production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity.

Autosomal recessive chronic granulomatous disease associated with 18q- syndrome and end-stage renal failure due to Henoch-Schönlein nephritis

Chronic granulomatous disease (CGD) is an inherited disorder in which phagocytes, including polymorphonuclear neutrophils, are unable to generate oxygen-derived microbicidal compounds, among them superoxide. Two main types of CGD are known, an X-linked form which is normally associated with the absence of cytochrome b558, a component of the membrane-associated reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which generates superoxide and an autosomal recessive form, in which cytochrome b558 is present, caused by the deficiency of a cytosolic factor required to activate NADPH oxidase. Patients with the X-linked type are highly susceptible to infections; those with the autosomal recessive form may be less severely affected. We report the unusual association of autosomal CGD with 18q--syndrome in a girl who developed terminal renal insufficiency caused by Henoch-Schönlein nephritis, and speculate on the possibility that the gene defect of autosomal recessive CGD may be located on chromosome 18.

Reduced nicotinamide adenine dinucleotide phosphate oxidase in adipocyte plasma membrane and its activation by insulin: Possible role in the hormone's effects on adenylate cyclase and the hexose monophosphate shunt

An oxidase activity utilizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and producing H 2O 2 was observed in intact adipocytes of rat, as well as in the isolated plasma membranes of these cells. A stoichiometry of 1 mol of H 2O 2 production per mole of NADPH disappearance was found with isolated plasma membranes. Activation of this enzyme (R) was produced by pretreatment of cells with insulin, dithiothreitol, or sulfhydryl inhibitors, e.g., p-chloromercuribenzoate or tosyl- l-lysine chloromethyl ketone. All of these agents also stimulated glucose oxidation via the hexose monophosphate shunt. Activation of R was also observed with biologically active derivatives of insulin, e.g., proinsulin or desalanine insulin, but not with an inactive derivative, desoctapeptide insulin. The enzyme could not be activated by exposing the cells to membrane perturbants, e.g., hypotonic conditions or Triton X-100 (0.01–0.1%). The enzyme activity in the plasma membrane had a pH optimum at 6.0 and, from the Lineweaver-Burke plot, V was determined at 230 nmol and K m for NADPH was at 5.8 × 10 −5, m. The activity remained unaltered in the presence of sodium azide or cyanide. Preincubation of adipocytes with insulin or SH reagents or direct addition of oxidants, e.g., H 2O 2, potassium ferricyanide, or phenazine methosulfate, to the membranes also caused inhibition of adenylate cyclase (AC). This enzyme activity could be restored in these preparations by adding thiols. It is suggested that inhibition of AC in whole cells in response to insulin may be caused by oxidation of its SH groups by the H 2O 2 generated from the activated NADPH oxidase. Reversal of this inhibition may involve cellular reducing equivalents. The evidence suggests that the plasma membrane enzymes, i.e., NADPH oxidase and adenylate cyclase, are controlled, in part, by the intracellular redox potential.