The Phosphoinositide-Binding Protein p40phox Regulates NADPH Oxidase Activation Rather Than Assembly during FcγIIA Receptor-Induced Phagocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 678-678 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Chang-Il Suh ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Null Mouse ◽  
Target Sequence ◽  
Resting Cells ◽  
Nadph Oxidase Activity ◽  
Px Domain ◽  
Significant Difference ◽  
Nadph Oxidase Activation

Abstract Many critical features of the organization and regulation of the phagocyte NADPH oxidase, a complex multi-subunit enzyme that generates superoxide for microbial killing, remain poorly defined. The active enzyme includes a membrane-bound flavocytochrome b along with p47phox, p67phox, p40phox, and Rac-GTP that are present in the cytosol of resting cells. p67phox is linked by high affinity interactions with both p47phox and p40phox, which appear to translocate as a trimeric complex upon cellular activation. The p47phox subunit acts as an adaptor to promote translocation by docking at a proline-rich target sequence on the flavocytochrome, and p67phox is a Rac-GTP effector containing a domain that activates electron transport. In contrast, the function of p40phox, which is not required for high level oxidase activity in cell free systems, is poorly understood. Recently, our group showed that p40phox plays key role in the activation of superoxide production during phagocytosis of IgG-opsonized targets in COSphoxFcγR cells. This model cell line contains stable transgenes for the flavocytochrome, p47phox, p67phox, and the FcγIIA receptor, without or with an additional transgene for p40phox. p40phox-dependent coupling of FcγR-mediated phagocytosis to superoxide production required an intact p40phox PX domain, which binds to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide generated by class III PI3 kinases in phagosome membranes (Suh et al J Exp Med 203, 1915Suh et al J Exp Med 203, 2006). Furthermore, a newly developed p40phox-null mouse exhibits reduced neutrophil NADPH oxidase activity in response to selected agonists, including IgG-opsonized targets (Ellson et al J Exp Med 203, 1927Ellson et al J Exp Med 203, 2006). In the current study, we investigated whether p40phox is required for translocation of p67phox during phagocytosis. We generated COSphoxFcγR cells expressing YFP-tagged p67phox from a stable transgene instead of untagged p67phox. Following incubation with IgG-opsonized sheep red blood cells (IgG-RBC), p67phox was detected on phagosome membranes at both early stages of phagosome cup formation and after closure, independent of whether or not p40phox was also co-expressed. However, NADPH oxidase activity was not detected in IgG-RBC phagosomes in COSphoxFcγR-p67phox-YFP cells unless p40phox was present. PMA-activated superoxide production was independent of p40phox, and Western blotting indicated there was no significant difference in expression of the other oxidase subunits in COSphoxFcγR-p67phox-YFP cells without or with the p40phox transgene. Further studies in PLB-985 granulocytes expressing stable transgenes for either YFP-tagged p67phox or p40phox showed that the PI3K inhibitor wortmannin inhibited phagosome NADPH oxidase activity and translocation of p40phox, but localization of p67phox to phagosomes was unaffected. These results indicate that although p40phox positively regulates NADPH oxidase activation during phagocytosis, recruitment of p67phox to the phagosome is independent of p40phox. Taken together, these data suggest that the PX domain of p40phox acts as a PI3P-dependent switch to activate the membrane-assembled NADPH oxidase complex.

scholarly journals FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2386-2386
Author(s):  
Chaekyun Kim ◽  
Mary C. Dinauer
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Superoxide Production ◽  
Wild Type ◽  
Functional Responses ◽  
Nadph Oxidase Activity ◽  
Nadph Oxidase Activation

Abstract Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

Role for p40phox in Fcγ-Receptor-Induced NADPH Oxidase Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 653-653 ◽  
Author(s):  
Chang II Suh ◽  
Natalie Stull ◽  
Yoshito Fuji ◽  
Sergio Grinstein ◽  
Michael Yaffe ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Transient Expression ◽  
Oxidase Activity ◽  
Missense Mutations ◽  
Nadph Oxidase Activity ◽  
Px Domain ◽  
Flavocytochrome B ◽  
Derivatives Of ◽  
Nadph Oxidase Activation ◽  
Phosphatidylinositol 3

Abstract The phagocyte NADPH oxidase catalyzes the production of superoxide, the precursor to microbicidal oxidants that are essential for normal host defense. The active enzyme complex includes a membrane-bound flavocytochrome b along with p47−phox, p67phox, and Rac-GTP, which translocate from the cytosol to the membrane to activate electron transport through the flavocytochrome upon cellular stimulation with soluble or particulate agonists. Translocation of p67phox is mediated by p47phox, which can be isolated from the cytoplasm prior to oxidase activation as a complex along with a third protein, p40phox. Although p40phox also translocates to the membrane of activated cells, its role in regulation of the NADPH oxidase has been unclear. p40phox is not required for reconstitution of high-level NADPH oxidase activity in cell-free systems or in heterologous whole cell models based in COS7 or K562 cell lines. It has recently been recognized that p40phox binds phosphatidylinositol-3-phosphate (PI(3)P), a product of type III phosphatidylinositol 3-kinase, via an N-terminal PX domain, and that PI(3)P is generated in high levels in the phagosomal membrane, where it recruits proteins important for phagosomal maturation. To examine whether p40phox might play a role in NADPH oxidase activation during phagocytosis, we introduced a cDNA for the human FcγIIA receptor via retroviral-mediated gene transfer into COS7 cells expressing the phagocyte flavocytochrome b, p47phox, and p67phox from stable transgenes. As previously shown by a number of groups for parental COS7 cells, heterologous expression of FcγIIA enabled “COSphox” cells to ingest IgG-coated sheep red blood cells (IgG-SRBC). However, NADPH oxidase activity was not detected, using nitroblue tetrazolium as a probe to detect superoxide production, which is otherwise evident by the accumulation of intraphagosomal formazan deposits in macrophages and neutrophils undergoing phagocytosis. However, numerous NBT-positive (formazan-stained) phagosomes were seen in COSphoxFcγR cells upon transient or stable transfection of an expression plasmid for p40phox. NBT-positive phagosomes were detected within five minutes in a synchronized phagocytosis assay. Upon transient expression of derivatives of p40phox which had point missense mutations in the PX domain predicted to prevent binding of PI(3)P, only rare NBT-positive phagosomes were detected in COSphoxFcγR cells ingesting IgG-SRBC. Transient expression of p40phox derivatives with point mutations in the SH3 domain, which disrupts a potential binding site for p47phox, or in the PC motif, which should disrupt binding to p67phox, resulted in a ~2-fold reduction in the numbers of NBT-positive phagosomes. Introduction of both the SH3 and PC mutations into p40phox resulted in only rare NBT-positive phagosomes following transient transfection of the double mutant and subsequent phagocytosis of IgG-SRBC. Thus, this study identifies a role for p40phox in coupling NADPH oxidase activation to phagocytosis of IgG-coated particles. Results of experiments using mutant derivatives of p40phox suggest that its ability to interact with both PI(3)P and p47phox/p67phox are critical determinants in this process.

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

2008 ◽  
Vol 216 (3) ◽  
pp. 796-804 ◽  
Author(s):  
Rafael Herling Lambertucci ◽  
Sandro Massao Hirabara ◽  
Leonardo dos Reis Silveira ◽  
Adriana Cristina Levada‐Pires ◽  
Rui Curi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Electron Transport ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Electron Transport Chain ◽  
Muscle Cells ◽  
Superoxide Production ◽  
Mitochondrial Electron Transport Chain ◽  
Nadph Oxidase Activity ◽  
Transport Chain

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

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3309-3315 ◽  
Author(s):  
Juan D. Matute ◽  
Andres A. Arias ◽  
Nicola A. M. Wright ◽  
Iwona Wrobel ◽  
Christopher C. M. Waterhouse ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Oxidase Activity ◽  
Autosomal Recessive ◽  
Premature Stop Codon ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Recessive Mutations ◽  
Px Domain

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

Abstract 211: Inhibition of Heme Oxygenase-1 (HO-1) Induces Mild Hypertension in Pregnant Rats

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Eric M George ◽  
Frank Spradley ◽  
Joey P Granger
Keyword(s):  
Oxidative Stress ◽  
Blood Pressure ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Late Gestation ◽  
Heme Oxygenase 1 ◽  
Maternal Circulation ◽  
Pregnant Rats ◽  
Nadph Oxidase Activity ◽  
Significant Difference

In the preeclamptic patient, inadequate remodeling of the maternal vasculature exacerbates this effect, causing dramatically increased oxidative stress in the placenta, which has been shown to be an important component of the maternal hypertension. There is also increasing awareness that HO-1 may act as an important regulator of placental function during normal pregnancy and decreases in HO-1 activity have been implicated in the pathogenesis of preeclampsia. While previous work in pregnant mice demonstrated that pharmacological inhibition of HO-1 leads to elevations in blood pressure, the mechanisms involved in the hypertension are unclear. The purpose of this study was to test the hypothesis that HO inhibition in late gestation leads to increases in maternal blood pressure by altering angiogenic balance and increasing placental oxidative stress in pregnant rats. HO activity was inhibited with tin mesoporphyrin (SnMP) was administered on gestational day 14, and blood pressure was measured on gestational day 19 by indwelling carotid catheter before sacrifice. In response to SnMP treatment, maternal MAP was significantly increased (99±1 vs 113±2 mmHg, p<0.05, n=15 per group). Placental sFlt-1 (631±47 vs 648±26 pg/mg, p=0.76) levels in the placenta were not affected by HO inhibition. Additionally, there was no significant difference in free VEGF in the maternal circulation (287±22 vs 329±14 pg/ml, p=0.11). There was, however, a significant increase in placental NADPH oxidase activity in SnMP treated rats (2021±238 vs 3005±301 RLU/min/mg, p<0.05) as determined by NADPH dependent lucigenin luminescence. This is likely due to decreased production of bilirubin, which is known to inhibit NADPH oxidase activity, and suggests an important role for HO-1 as an antioxidant in the developing placenta.

Abstract WP274: Inhibition of Na+/H+ Exchanger Isoform 1 and/or Voltage Gated Proton Channel Improves Outcomes in an Embolic Stroke Model

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Weiguo Li ◽  
Rebecca Ward ◽  
Jingping Sun ◽  
Xinyue Guo ◽  
Adviye Ergul ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Infarct Size ◽  
Oxidase Activity ◽  
Embolic Stroke ◽  
Proton Channel ◽  
Experimental Stroke ◽  
Dependent Manner ◽  
Nadph Oxidase Activity ◽  
Voltage Gated ◽  
Significant Difference

While Na+/H+ exchanger isoform 1 inhibitor (NHEi) has been reported to be neuro- and cardio-protective in experimental stroke and myocardial infarction, respectively, clinical use of NHEi for cardioprotection has stalled due to increased cerebrovascular events. NHEi has been demonstrated to increase Hv1 activity and we recently showed that NHEi activates NADPH oxidase and results in amplified superoxide formation in a voltage gated proton channel Hv1-dependent manner. In the CNS, Hv1 is localized primarily to microglia and deletion of Hv1 is neuroprotective after permanent and transient middle cerebral artery occlusion (MCAO). In the current study, we hypothesized that beneficial effect of NHEi after MCAO will be greater in a rat model lacking Hv1 due to loss of a potentially deleterious increase in Hv1 activation and NADPH oxidase activity. The wild type (WT) and Hv1 knockout (KO) rats (n=4-6) were treated with vehicle or NHEi (KR-32568, 2 mg/kg, i.v.) at 30 min after embolic MCAO. The neurological deficiency, infarct size, HT index, and edema ratio were assessed 3 days after surgery (Table). Compare to WT rats, KO rats had smaller infarct, less edema, and better neurological outcomes as previously found in the suture model. NHEi decreased infarct size and edema in both strains. While there was no significant difference in HT between WT and KO rats, the HT was less in WT rats with NHEi. Functional outcomes were significantly improved with NHEi in WT group, while the KO groups had a trend for a better outcomes with NHEi. These data indicate that NHE inhibition in the acute stroke period is similarly effective in both WT and Hv1 KO animals in providing neurovascular protection. Our data do not support the hypothesis that a deleterious increase in Hv1 dependent NADPH oxidase activity limits the beneficial actions of NHEi in embolic stroke. Further studies are needed to explore the underlying mechanism of the interaction between NHEi and the Hv1 channel in ischemic stroke.

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