HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP-binding-protein Ras on the redox signalling of the human neuroblastoma cell line, SK-N-BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK-HaRas) cells than in control cells. The treatment of cells with 4-(2-aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK-HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67(phox) and Rac to the plasma membrane. These effects depended on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas-mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67(phox) and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas-induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.     

Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes.

We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.     

Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes.     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

Adriamycin effects of transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbal myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions.

The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme.     

Inhibition by linoleic acid hydroperoxide of alveolar macrophage superoxide production: effects upon mitochondrial and plasma membrane potentials

Linoleic acid hydroperoxide (LOOH) is a naturally occurring product of lipid peroxidation. Incubation of rat alveolar macrophages with LOOH produced alterations of membrane properties and function at concentrations of LOOH as low as 0.1 microM. These included phorbol myristate acetate (PMA)-stimulated superoxide production, mitochondrial membrane potential, and plasma membrane potentials. These effects were clearly separated from gross loss of structural integrity as measured by lactate dehydrogenase release, in terms of both time of incubation and concentration of LOOH. PMA-stimulated superoxide production measured 15 min after addition of 10 microM LOOH was inhibited approximately 50%; however, addition of this concentration of the hydroperoxide after PMA stimulation was without effect. Superoxide production was also measured in a cell-free system produced by incubation of alveolar macrophages with sodium dodecyl sulfate. Prior incubation of alveolar macrophages with LOOH, H2O2, or t-butyl hydroperoxide, under conditions that significantly inhibited superoxide production by the intact cells, did not produce inhibition of the NADPH-dependent superoxide generating system in the cell-free preparation. These results suggest that the effect of LOOH was upon signal transduction involved in the stimulation of superoxide production rather than on the NADPH oxidase itself. Measurements of membrane potential changes were made using the lipophilic ions, 3,3'-dipentyloxacarbocyanine (DiOC5(3] and bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V). On the basis of their charge, DiOC5(3) fluorescence primarily reports mitochondrial potential and oxonol V absorbance reports plasma membrane potential. With 10 microM LOOH, depolarization of the plasma and mitochondrial membranes appeared to occur within seconds. As prior depolarization depresses superoxide production, these hydroperoxide-induced changes in membrane potential may be responsible for decreased PMA-stimulated superoxide production.     

The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects.

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, O2-. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were diassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate O2- upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.     

Superoxide generation and tyrosine kinase.

NADPH oxidase is a multi-subunit enzyme complex responsible for superoxide generation in many cells, for example, B-lymphocytes and osteoclasts. NADPH oxidase is localized on the cell surface and generates superoxide extracellularly. After synthesis, components of this oxidase are transported to the cell membrane where the functional NADPH oxidase complex is assembled. The mechanism by which the membrane-bound components are transported to the cell surface of osteoclasts remains unclear. In this study, we examined the role of tyrosine kinase activity in the transport of NADPH oxidase components. When B-lymphocytes and osteoclasts were treated with herbimycin A, a specific inhibitor of tyrosine kinase, superoxide production was significantly decreased. The amount of p91, the catalytic subunit of NADPH oxidase, was decreased in the cellular membrane of herbimycin A treated cells compared to untreated controls. Similar results were obtained for the movement of a regulatory subunit of the NADPH oxidase complex, p47, in B-lymphocytes. Thus, inhibition of tyrosine kinase decreases superoxide production by disrupting the translocation of the NADPH oxidase complex.     

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.     

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.     

Swell activated chloride channel function in human neutrophils

Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I_e has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I_e must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.     

Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages.

Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium-free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester- and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.     

Superoxide prevents nitric oxide-mediated suppression of helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide adenine dinucleotide phosphate oxidase knockout mice

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2-) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2- production. In contrast, macrophages from p47phox -/- (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2- in this system. To examine the NO-O2- interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.     

HIV‐1 Nef induces p47phox phosphorylation leading to a rapid superoxide anion release from the U937 human monoblastic cell line

The Nef protein of the human immunodeficiency virus type 1 (HIV‐1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV‐1 Nef in human macrophages cell line modulates in bi‐phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef‐induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef‐mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3‐binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef‐induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47^phox to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef‐mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3‐kinase (PI3K) inhibited both the Nef‐induced p47^phox phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K. J. Cell. Biochem. 106: 812–822, 2009. © 2009 Wiley‐Liss, Inc.     

The oxidative burst protects plants against pathogen attack: Mechanism and role as an emergency signal for plant bio-defence — a review

Various aspects, mechanisms and functions of the oxidative burst with generation of O₂⁻ superoxide anions in plant cells, which is stimulated by active defence-inducing agents such as fungal infection or elicitor treatment, were reviewed mainly on the basis of experimental evidence obtained in a system of Solanaceae plants and Phytophthora spp. The oxidative burst may be due to an O₂⁻-generating NADPH oxidase in the plasma membrane, which is activated with combinations of cytosolic proteins, Ca²⁺, calmodulin and protein kinase, following stimulation by elicitor molecules. The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defence.     

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Cytosolic phospholipase A2 (cPLA2)-generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.     

High glucose downregulates the number of caveolae in monocytes through oxidative stress from NADPH oxidase: implications for atherosclerosis

Atherosclerosis, an inflammatory disease, is closely associated with hyperglycemia, major sign of diabetes mellitus. Caveolae are vesicular invaginations of the plasma membrane that mediate the intracellular transport of lipids such as cholesterol. We evaluated the relationship between the expression of caveolin-1 and the number of caveolae in macrophages under conditions of high glucose concentration. Increased superoxide production, induction of inducible nitric oxide synthase (iNOS), and decreased caveolin-1 were observed in a concentration-dependent manner in THP-1 derived macrophages with high glucose concentrations. Mannitol, used as an osmotic control, showed no effects. Furthermore, co-localization of the NADPH oxidase component, p47(phox), and caveolin was confirmed by confocal microscopy. An atomic force microscopy (AFM) study showed that high glucose concentrations reduced the number and size of the caveolae. The percentage of cells with fragmented DNA was increased in cells grown in hyperglycemic media. Taken together, high glucose concentrations suppress the levels of caveolin-1 expression and reduce the number of caveolae. This might be due to the actions of superoxide via the activation of NADPH oxidase by translocation of its component and uncoupling of induced iNOS in macrophages. Furthermore, the apoptosis of macrophages might occur with high glucose concentrations, leading to the spreading of lipids from macrophages into intracellular spaces in the vessel wall.     

Electron and proton transport by NADPH oxidases

The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside-out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.     

Consequences of the electrogenic function of the phagocytic NADPH oxidase

NADPH oxidase of phagocytic cells transfers a single electron from intracellular NADPH to extracellular O2, producing superoxide (O.-2), the precursor to several other reactive oxygen species. The finding that a genetic defect of the enzyme causes chronic granulomatous disease (CGD), characterized by recurrent severe bacterial infections, linked O.-2 generation to destruction of potentially pathogenic micro-organisms. In this review, we focus on the consequences of the electrogenic functioning of NADPH oxidase. We show that enzyme activity depends on the possibilities for compensating charge movements. In resting neutrophils K+ conductance dominates, but upon activation the plasma membrane rapidly depolarizes beyond the opening threshold of voltage-gated H+ channels and H+ efflux becomes the major charge compensating factor. K+ release is likely to contribute to the killing of certain bacteria but complete elimination only occurs if O.-2 production can proceed at full capacity. Finally, the reversed membrane potential of activated neutrophils inhibits Ca2+ entry, thereby preventing overloading the cells with Ca2+. Absence of this limiting mechanism in CGD cells may contribute to the pathogenesis of the disease.