Melatonin nephroprotective action in Zucker diabetic fatty rats involves its inhibitory effect on NADPH oxidase

Excessive activity of NADPH oxidase (Nox) is considered to be of importance for the progress of diabetic nephropathy. The aim of the study was to elucidate the effect of melatonin, known for its nephroprotective properties, on Nox activity under diabetic conditions. The experiments were performed on three groups of animals: (i) untreated lean (?/+) Zucker diabetic fatty (ZDF) rats; (ii) untreated obese diabetic (fa/fa) ZDF rats; and (iii) ZDF fa/fa rats treated with melatonin (20 mg/L) in drinking water. Urinary albumin excretion was measured weekly. After 4 wk of the treatment, the following parameters were determined in kidney cortex: Nox activity, expression of subunits of the enzyme, their phosphorylation and subcellular distribution. Histological studies were also performed. Compared to ?/+ controls, ZDF fa/fa rats exhibited increased renal Nox activity, augmented expression of Nox4 and p47^phox subunits, elevated level of p47^phox phosphorylation, and enlarged phospho‐p47^phox and p67^phox content in membrane. Melatonin administration to ZDF fa/fa rats resulted in the improvement of renal functions, as manifested by considerable attenuation of albuminuria and some amelioration of structural abnormalities. The treatment turned out to nearly normalize Nox activity, which was accompanied by considerably lowered expression and diminished membrane distribution of regulatory subunits, that is, phospho‐p47^phox and p67^phox. Thus, it is concluded that: (i) melatonin beneficial action against diabetic nephropathy involves attenuation of the excessive activity of Nox; and (ii) the mechanism of melatonin inhibitory effect on Nox is based on the mitigation of expression and membrane translocation of its regulatory subunits.     

Gp91phox is the heme binding subunit of the superoxide-generating NADPH oxidase

The phagocyte NADPH oxidase flavocytochrome b₅₅₈ is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91^phox, and a nonglycosylated subunit, p22^phox. It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O₂), resulting in the generation of superoxide ion (O₂⁻). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91^phox, p22^phox, or both polypeptides to examine the relative role of each flavocytochrome b₅₅₈ subunit in heme binding and O₂⁻ formation. A similar membrane localization was observed when gp91^phox and p22^phox were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91^phox or both gp91^phox and p22^phox showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b₅₅₈. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22^phox. Furthermore, redox titration studies suggested that two heme groups were contained in gp91^phox expressed in COS7 membranes, with midpoint potentials of −264 and −233 mV that were very similar to those obtained for neutrophil flavocytochrome b₅₅₈. These results provide strong support for the hypothesis that gp91^phox is the sole heme binding subunit of flavocytochrome b₅₅₈. However, coexpression of gp91^phox and p22^phox in COS7 membranes was required to support O₂⁻ production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b₅₅₈.     

Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Superoxide anion (O₂⁻) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O₂⁻ is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O₂⁻ production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O₂⁻-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22^phox, gp91^phox, p47^phox, and p67^phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O₂⁻ production. Furthermore, immunodepletion of p67^phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67^phox. Further study into the regulation of this adventitial source of O₂⁻ is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.     

The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle

OBJECTIVE: NADPH oxidases are important sources of reactive oxygen species (ROS) in the vasculature. In phagocytic cells, the catalytic subunit of NADPH oxidase is a glycoprotein, gp91phox. However, vascular smooth muscle cells (VSMCs), which show prominent NADPH oxidase activity, lack gp91phox. Hence, we examined the role of Nox4, a gp91phox homologue, in superoxide production in mouse-cultured VSMCs. METHODS AND RESULTS: Incubation of VSMCs with NADPH increased ROS production whether detected by lucigenin-enhanced chemiluminescence or dichlorofluorescein. Superoxide production was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium and apocynin, but not by inhibitors of other potential sources of superoxide. In unstimulated VSMCs, phosphorothioate antisense oligonucleotides against Nox4 down-regulated mRNA expression of the subunit by 65% and attenuated superoxide production by 41% without affecting Nox1 expression. Interleukin-1beta (IL-beta) thrombin and platelet-derived growth factor (PDGF) also reduced Nox4 mRNA expression after 3 h without affecting Nox1 levels. Of these stimuli, only IL-beta reduced superoxide, but this effect was more rapid (< or =30 min) than its actions on Nox4. CONCLUSIONS: Under resting conditions, NADPH oxidase activity in VSMCs is largely dependent upon Nox4 expression. Proinflammatory mediators down-regulated Nox4 but did not affect Nox1 expression, so other factors must compensate to regulate superoxide production.     

Rac1 mediates sex difference in cardiac tumor necrosis factor-α expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia

The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91^phox-NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91^phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts.     

The Effects of Combined Treatment with an HMG-CoA Reductase Inhibitor and PPARγ Agonist on the Activation of Rat Pancreatic Stellate Cells

Background/Aims

Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) and peroxisome proliferator-activated receptor gamma (PPARγ) ligands can modulate cellular differentiation, proliferation, and apoptosis through various pathways. It has been shown that HMG-CoA reductase inhibitors and PPARγ agonists separately inhibit pancreatic stellate cell (PaSC) activation. We studied the effects of a combination of both types of drugs on activated PaSCs via platelet-derived growth factor (PDGF), which has not previously been reported. The present study was performed to elucidate the underlying mechanisms of these effects by focusing on the impact of the signaling associated with cell-cycle progression.

Methods

Primary cultures of rat PaSCs were exposed to simvastatin and troglitazone. Proliferation was quantified using the BrdU method, and cell-cycle analysis was performed using a fluorescent activated cell sorter. The protein expression levels of smooth muscle actin (SMA), extracellular signal-regulated kinase (ERK), and a cell cycle machinery protein (p27Kip1) were investigated using Western blot analysis.

Results

Simvastatin reversed the effects of PDGF on cell proliferation in a dose-dependent manner. The combination of a low concentration of simvastatin (1 mM) and troglitazone (10 mM) synergistically reversed the effects of PDGF on cell proliferation but had no effect on cell viability. The expression of a-SMA was markedly attenuated by combining the two drugs, which blocked the cell cycle beyond the G0/G1 phase by reducing the levels of phosphorylated ERK and reversed the expression of p27Kip1 interrupted by PDGF.

Conclusions

Simvastatin and troglitazone synergistically inhibited cell proliferation in activated PaSCs by blocking the cell cycle beyond the G0/G1 phase. This inhibition was due to the synergistic modulation of the ERK pathway and the cell cycle machinery protein p27Kip1.     

Induction of regulatory T cells by macrophages is dependent on production of reactive oxygen species

The phagocyte NAPDH–oxidase complex consists of several phagocyte oxidase (phox) proteins, generating reactive oxygen species (ROS) upon activation. ROS are involved in the defense against microorganisms and also in immune regulation. Defective ROS formation leads to chronic granulomatous disease (CGD) with increased incidence of autoimmunity and disturbed resolution of inflammation. Because regulatory T cells (Tregs) suppress autoimmune T-cell responses and are crucial in down-regulating immune responses, we hypothesized that ROS deficiency may lead to decreased Treg induction. Previously, we showed that in p47^phox-mutated mice, reconstitution of macrophages (Mph) with ROS-producing capacity was sufficient to protect the mice from arthritis. Now, we present evidence that Mph-derived ROS induce Tregs. In vitro, we showed that Mph ROS-dependently induce Treg, using an NADPH-oxidase inhibitor. This finding was confirmed genetically: rat or human CGD Mph with mutated p47^phox or gp91^phox displayed hampered Treg induction and T-cell suppression. However, basal Treg numbers in these subjects were comparable to those in controls, indicating a role for ROS in induction of peripheral Tregs. Induction of allogeneic delayed-type hypersensitivity with p47^phox-mutated Mph confirmed the importance of Mph-derived ROS in Treg induction in vivo. We conclude that NAPDH oxidase activity in Mph is important for the induction of Tregs to regulate T cell-mediated inflammation.     

Left ventricular remodeling after myocardial infarction in mice with targeted deletion of the NADPH oxidase subunit gp91PHOX

Background Oxidative stress is involved in progression of left ventricular hypertrophy and heart failure. Since NADPH oxidases are a major source of reactive oxygen species in the heart, we studied left ventricular remodeling after myocardial infarction in mice with targeted deletion of the NADPH oxidase subunit gp91^phox. Methods and results gp91^phox knockout (KO) and wild–type (WT) animals underwent coronary artery ligation. Mortality was significant higher in the gp91^phox KO mice. However, transthoracic echocardiography performed at days 1, 7, and 56 at mid–papillary levels revealed that progression of left ventricular remodeling was not influenced by the genotype. Moreover, systemic oxidative stress was not reduced in gp91^phox KO mice as indicated by a significant increase in lipid peroxides potentially mediated by an increase of the NADPH subunit nox–1 in gp91^phox KO mice. Conclusion Targeted deletion of the NADPH subunit gp91^phox does not affect left ventricular remodeling following myocardial infarction and does not decrease the production of oxidative stress. However, the final role of the different NADPH subunits in the heart under pathophysiologic conditions remains to be determined.     

Activation of natriuretic peptide receptor-C attenuates the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats: implication of Gialpha protein

We have recently shown that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of Giα proteins, which was attributed to the enhanced oxidative stress. Since C-ANP_4-23 that specifically interacts with natriuretic peptide C (NPR-C) receptor has been shown to decrease the expression of Giα protein in VSMC, the present study was undertaken to examine if C-ANP_4-23 can also decrease the enhanced expression of Giα protein in VSMC from SHR and whether it is attributed to its ability to attenuate the enhanced oxidative stress. Aortic VSMC from 12-week-old SHR and their age-matched Wistar-Kyoto (WKY) rats were used for the present studies. VSMC from SHR exhibited enhanced expression of Giα-2 and Giα-3 proteins, different subunits of NADPH oxidase such as Nox⁴ and p^47phox proteins but not of p^22phox, enhanced production of superoxide anion as well as NADPH oxidase activity as compared to age-matched WKY rats. Treatment of VSMC from SHR with C-ANP_4-23 decreased towards control levels the enhanced expression of Giα proteins, enhanced superoxide anion production and enhanced NADPH oxidase activity as well as the enhanced expression of Nox⁴ and p^47phox. However, C-ANP_4-23-induced attenuation of the enhanced level of O₂⁻ and NADPH oxidase activity occurs at 4 h before the decrease in the enhanced expression of p^47phox that occurs at 16 h of C-ANP_4-23 treatment. The decreased expression of NADPH oxidase in SHR was also associated with further decrease in O₂⁻ and NADPH oxidase activity. Furthermore, treatment of VSMC from SHR with pertussis toxin (PT) decreased the enhanced levels of superoxide anion as well as NADPH oxidase activity; however, the enhanced levels of different subunits of NADPH oxidase were not attenuated by PT treatment. These results suggest that C-ANP_4-23 decreases the enhanced oxidative stress in SHR by attenuating the enhanced expression of Giα proteins and also the enhanced levels of NADPH oxidase.     

NADPH oxidase inhibition prevents beta cell dysfunction induced by prolonged elevation of oleate in rodents

Aims/hypothesis

The activation of NADPH oxidase has been implicated in NEFA-induced beta cell dysfunction. However, the causal role of this activation in vivo remains unclear. Here, using rodents, we investigated whether pharmacological or genetic inhibition of NADPH oxidase could prevent NEFA-induced beta cell dysfunction in vivo.

Methods

Normal rats were infused for 48 h with saline or oleate with or without the NADPH oxidase inhibitor apocynin. In addition, NADPH oxidase subunit p47^phox-null mice and wild-type littermate controls were infused with saline or oleate for 48 h. This was followed by measurement of NADPH oxidase activity, reactive oxygen species (ROS) and superoxide imaging and assessment of beta cell function in isolated islets and hyperglycaemic clamps.

Results

Oleate infusion in rats increased NADPH oxidase activity, consistent with increased total but not mitochondrial superoxide in islets and impaired beta cell function in isolated islets and during hyperglycaemic clamps. Co-infusion of apocynin with oleate normalised NADPH oxidase activity and total superoxide levels and prevented beta cell dysfunction. Similarly, 48 h NEFA elevation in wild-type mice increased total but not mitochondrial superoxide and impaired beta cell function in isolated islets. p47^phox-null mice were protected against these effects when subjected to 48 h oleate infusion. Finally, oleate increased the levels of total ROS, in both models, whereas inhibition of NADPH oxidase prevented this increase, suggesting that NADPH oxidase is the main source of ROS in this model.

Conclusions/interpretation

These data show that NADPH-oxidase-derived cytosolic superoxide is increased in islets upon oleate infusion in vivo; and whole-body NADPH-oxidase inhibition decreases superoxide in concert with restoration of islet function.     

Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins

The phagocyte NADPH oxidase is dormant in resting cells but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defence. The catalytic core of this enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The oxidase activation requires the small GTPase Rac and the SH3 domain-containing proteins p47phox and p67phox; they normally exist in the cytoplasm and translocate upon cell stimulation to the membrane. The translocation depends on a stimulus-induced conformational change of p47phox, which leads to the SH3 domain-mediated interaction with p22phox, a binding required for the gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase that is likely involved in host defence at the colon, requires novel proteins homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively. Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of constitutively activating Nox1. The constitutive activation may be due to the property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate with p22phox, which is required for the Nox1 activation. We will also describe the mechanism underlying regulation of the third oxidase Nox3, which exits in fetal kidney and inner ears.     

Glucose,palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line

Aims/hypothesis Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47 ^phox component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). Methods Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47 ^phox production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. Results Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47 ^phox protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47 ^phox was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1β it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1β-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47 ^phox . Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1β plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. Conclusions/interpretation NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise. D. Morgan and H. R. Oliveira-Emilio contributed equally to this study.     

Apocynin restores endothelial dysfunction in streptozotocin diabetic rats through regulation of nitric oxide synthase and NADPH oxidase expressions

AimIncreased production of reactive oxygen species (ROS) in the diabetic vasculature results in the impairment of nitric oxide (NO)-mediated relaxations leading to impaired endothelium-dependent vasodilation. An important source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the inhibition of this enzyme is an active area of interest. This study aimed to investigate the effects of apocynin, an NADPH oxidase inhibitor, on endothelial dysfunction and on the expression of NO synthase (NOS) and NADPH oxidase in thoracic aorta of diabetic rats.MethodStreptozotocin (STZ)-diabetic rats received apocynin (16 mg/kg per day) for 4 weeks. Endothelium-dependent and -independent relaxations were determined in thoracic aortic rings. Western blotting and RT-PCR analysis were performed for NOSs and NADPH oxidase in the aortic tissue.ResultsAcetylcholine-induced relaxations and l-NAME-induced contractions were decreased in diabetic aorta. The decrease in acetylcholine and l-NAME responses were prevented by apocynin treatment without a significant change in plasma glucose levels. Endothelial NOS (eNOS) protein and mRNA expression exhibited significant decrease in diabetes, while protein and/or mRNA expressions of inducible NOS (iNOS) as well as p22^phox and gp91^phox subunits of NADPH oxidase were increased, and these alterations were markedly prevented by apocynin treatment.ConclusionNADPH oxidase expression is increased in diabetic rat aorta. NADPH oxidase-mediated oxidative stress is accompanied by the decreased eNOS and increased iNOS expressions, contributing to endothelial dysfunction. Apocynin effectively prevents the increased NADPH oxidase expression in diabetic aorta and restores the alterations in NOS expression, blocking the vicious cycle leading to diabetes-associated endothelial dysfunction.     

Oxidative stress in scleroderma: Maintenance of scleroderma fibroblast phenotype by the constitutive up‐regulation of reactive oxygen species generation through the NADPH oxidase complex pathway

Objective

To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc).

Methods

Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O₂⁻ and H₂O₂ released from fibroblasts were estimated by the superoxide dismutase (SOD)–inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti‐p47^phox–specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by ³H‐thymidine incorporation. Northern blot analysis was used to study α1 and α2 type I collagen gene expression.

Results

Unstimulated skin fibroblasts from SSc patients released more O₂⁻ and H₂O₂ in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47^phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin‐1β (IL‐1β), platelet‐derived growth factor type BB (PDGF‐BB), transforming growth factor β1 (TGFβ1), and H₂O₂. Treatment of normal and SSc fibroblasts with tumor necrosis factor α (TNFα), IL‐2, IL‐4, IL‐6, IL‐10, interferon‐α (IFNα), IFNγ, granulocyte–macrophage colony‐stimulating factor (GM‐CSP), G‐CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL‐1 receptor antagonist, or antibodies blocking the effect of TGFβ1, PDGF‐BB, and other agonists (IL‐4, IL‐6, TNFα, CTGF). In contrast, treatment of SSc fibroblasts with the membrane‐permeant antioxidant N‐acetyl‐L ‐cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down‐regulation of α1(I) and α2(I) collagen messenger RNA.

Conclusion

The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase–like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O₂⁻, H₂O₂, IL‐1β, TGFβ1, PDGF‐BB, IL‐4, IL‐6, TNFα, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

     

Phagocytosis-Coupled Activation of the Superoxide-Producing Phagocyte Oxidase, a Member of the NADPH Oxidase (Nox) Family

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in host defense by neutrophils and macrophages. When cells ingest invading microbes, this enzyme becomes activated to reduce molecular oxygen to superoxide, a precursor of microbicidal oxidants, in the phagosome. The catalytic core of the oxidase is membrane-bound cytochrome b558, which comprises gp91phox and p22phox. gp91phox belongs to the NADPH oxidase (Nox) family, which contains the entire electron-transporting apparatus from NADPH to molecular oxygen. In resting neutrophils, cytochrome b558 is mainly present in the membrane of the specific granule, an intracellular component, and is targeted to the phagosomal membrane during phagocytosis. Activation of gp91phox involves the integrated function of cytoplasmic proteins such as p47phox, p67phox, p40phox, and the small guanosine triphosphatase Rac; these proteins translocate to the phagosomal membrane to interact with cytochrome b558, leading to superoxide production. Here we describe a current molecular model for phagocytosis-coupled activation of the NADPH oxidase.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

Hematologically important mutations: The autosomal recessive forms of chronic granulomatous disease (second update)

Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22-phox, NCF2, encoding p67-phox, NCF1, encoding p47-phox, and NCF4, encoding p40-phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.     

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91^phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91^phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91^phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91^phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91^phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91^phox gene in human neutrophils, monocytes, and B lymphocytes.     

Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)-induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox-/- mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II-induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox-/- mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox-/- and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox-/- mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload-induced LVH in both gp91phox-/- and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.