NADPH oxidase in human lung fibroblasts

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91-phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.     

Functional analysis of two-amino acid substitutions in gp91<Emphasis Type="Italic">phox</Emphasis> in a patient with X-linked flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>558</Subscript>-positive chronic granulomatous disease by means of transgenic PLB-985 cells

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b₅₅₈, which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91⁺ CGD. We have previously reported an X⁺ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X⁺ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism.Clara Bionda and Xing Jun Li contributed equally to this work     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

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^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^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.     

Proton conduction through full-length gp91phox requires histidine 115

Summary.  The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 ^phox , one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 ^phox has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 ^phox has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 ^phox . Stable CHO cell lines were established which expressed full-length gp91 ^phox in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 ^phox and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 ^phox . The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 ^phox . Therefore, histidine 115 is required for proton conduction by both full-length gp91 ^phox and the N-terminal 230-amino-acid fragment of gp91 ^phox . Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Bristol Institute for Transfusion Sciences, Bristol, United Kingdom. RID="*" ID="*" Correspondence and reprints: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.     

The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH₃ domains may become exposed follwoing phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a prton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncretain but could be related to some signal function of superoxide.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

NADPH oxidase subunit gp91phox: A proton pathway

Summary The generation of superoxide by the NADPH oxidase is an electrogenic process resulting in a rapid depolarisation of the membrane potential of the cell. The efflux of H⁺ ions through an arachidonate-activatable, Zn²⁺-inhibitable H⁺ pathway accompanies the efflux of electrons and provides the necessary charge compensation. Inhibition of H⁺ flux leads to inhibition of superoxide generation. The protein gp91^phox, a transmembrane component of the NADPH oxidase, was demonstrated to be capable of acting as the NADPH oxidase-associated H⁺ channel in a stable CHO cell line, CHO91. The N-terminal 230 amino acids contain all that is required for the protein to form an H⁺ channel and specifically histidine 115 is important to the ability of gp91^phox to conduct H⁺ ions. The recording of outward currents from CHO91 cells, in the whole-cell configuration, demonstrated that gp91^phox is also capable of functioning as a voltage-gated H⁺ conductance pathway. The similarity in properties between voltage-elicited outward currents, from both wild type and the mutations, and the arachidonate-activated H⁺ flux strongly suggests that these H⁺ pathways are one in the same. Among the recently identified homologues of gp91^phox only NOH-1S has so far been demonstrated to also act as an H⁺ conductance pathway.Abbreviation CGD chronic granulomatous disease     

Genetic deficiency of NADPH oxidase does not diminish,but rather enhances,LPS-induced acute inflammatory responses in vivo

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in the etiology of cell dysfunction and tissue damage in sepsis. However, there is limited and controversial evidence from in vivo studies that ROS mediate cell signaling processes that elicit acute inflammatory responses during sepsis. Because NADPH oxidase is one of the main cellular sources of ROS, we investigated the role of this enzyme in lipopolysaccharide (LPS)-induced acute inflammation in vivo, utilizing mice deficient in the gp91^phox or p47^phox subunits of NADPH oxidase. Age-and body weight-matched C57BL/6J wild-type (WT) and gp91^phox?/? and p47^phox?/? mice were injected ip with 50 μg LPS or saline vehicle and sacrificed at various time points up to 24 h. We found that LPS-induced acute inflammatory responses in serum and tissues were not significantly diminished in gp91^phox?/? and p47^phox?/? mice compared to WT mice. Rather, genetic deficiency of NADPH oxidase was associated with enhanced gene expression of inflammatory mediators and increased neutrophil recruitment to lung and heart. Furthermore, no protection from LPS-induced septic death was observed in either knockout strain. Our findings suggest that NADPH oxidase-mediated ROS production and cellular redox signaling do not promote, but instead limit, LPS-induced acute inflammatory responses in vivo.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O₂^–). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b₅₅₈ (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91⁰, X91^– or X91⁺) according to their cytochrome b₅₅₈ expression and NADPH oxidase activity. Nine patients had X91⁰ CGD, one had X91^– CGD and one had X91⁺ CGD. Six mutations in CYBB were novel. Of the four new X91⁰ CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91⁰ CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91^– CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91⁺ CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.     

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

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 NADPH oxidase 2 (NOX2)‐type 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/gp91^phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40^phox. p40^phox itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91^phox and p40^phox 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 colocalization of p40^phox with NOX2/gp91^phox 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.

     

Superoxide Anion Production and Expression of gp91phox and p47phox Are Increased in Glomeruli and Proximal Tubules of Cisplatin‐Treated Rats

The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91^phox and p47^phox, subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91^phox and p47^phox, is involved in the oxidative stress in cisplatin‐induced nephrotoxicity.     

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification,apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O₂^?-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91^phox and p22^phox) forming the catalytic core, three cytosolic proteins (p67^phox, p47^phox and p40^phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47^phox (aa 1–286), p67^phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.     

Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea

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.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi

NADPH oxidases (Nox) are membrane complexes that produce O₂^?. Researches in mammals, plants and fungi highlight the involvement of Nox‐generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91^phox/Nox2 is associated with p22^phox forming the flavocytochrome b₅₅₈ ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22^phox gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22^phox homologue as being mutated in the Podospora anserina mutant IDC⁵⁰⁹. Functional studies show that the fungal p22^phox, PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91^phox homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co‐localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.