Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67(phox) and p47(phox)

The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A(2) (cPLA(2)) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA(2) regulation of NADPH oxidase activation by evaluating the effects of cPLA(2) on translocation and phosphorylation of p67(phox) and p47(phox). We report that translocation and phosphorylation of p67(phox), as well as p47(phox), occur upon activation of human monocytes and that decreased cPLA(2) protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA(2) mRNA, blocked the stimulation-induced translocation of p47(phox) and p67(phox) from the cytosol to the membrane fraction. Inhibition of translocation of both p47(phox) and p67(phox) by cPLA(2) AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA(2) enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA(2)-deficient human monocytes. These results represent the first report that cPLA(2) activity or AA is required for p67(phox) and p47(phox) translocation in human monocytes. Although cPLA(2) was required for translocation of p47(phox) and p67(phox), it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA(2) regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47(phox) and p67(phox). These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.     

Essential requirement of cytosolic phospholipase A(2) for stimulation of NADPH oxidase-associated diaphorase activity in granulocyte-like cells.

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.     

Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.     

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.     

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.     

Glycated albumin activates NADPH oxidase in rat mesangial cells through up-regulation of p47phox

Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-β (TGF-β) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-β expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-β expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-β and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy.     

Cytosolic phospholipase A2alpha is targeted to the p47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A2alpha (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, F?rster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.     

Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 microM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67(phox) localized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.     

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.     

Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells.

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.     

Amyloid-beta peptide induces temporal membrane biphasic changes in astrocytes through cytosolic phospholipase A2

Oligomeric amyloid-beta peptide (Abeta) is known to induce cytotoxic effects and to damage cell functions in Alzheimer's disease. However, mechanisms underlying the effects of Abeta on cell membranes have yet to be fully elucidated. In this study, Abeta 1-42 (Abeta(42)) was shown to cause a temporal biphasic change in membranes of astrocytic DITNC cells using fluorescence microscopy of Laurdan. Abeta(42) made astrocyte membranes became more molecularly-disordered within the first 30 min to 1 h, but gradually changed to more molecularly-ordered after 3 h. However, Abeta(42) caused artificial membranes of vesicles made of rat whole brain lipid extract to become more disordered only. The trend for more molecularly-ordered membranes in astrocytes induced by Abeta(42) was abrogated by either an NADPH oxidase inhibitor, apocynin, or an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), but not by an inhibitor of calcium-independent PLA(2) (iPLA(2)). Apocynin also suppressed the increased production of superoxide anions (O(2)(-)) and phosphorylation of cPLA(2) induced by Abeta(42). In addition, hydrolyzed products of cPLA(2), arachidonic acid (AA), but not lysophosphatidylcholine (LPC) caused astrocyte membranes to become more molecularly-ordered. These results suggest (1) a direct interaction of Abeta(42) with cell membranes making them more molecularly-disordered, and (2) Abeta(42) also indirectly makes membranes become more molecularly-ordered by triggering the signaling pathway involving NADPH oxidase and cPLA(2) in astrocytes.     

Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase

The superoxide-producing phagocyte NADPH oxidase can be activated by arachidonic acid (AA) or by phosphorylation of p47(phox) under cell-free conditions. The molecular mechanism underlying the activation, however, has remained largely unknown. Here we demonstrate that AA, at high concentrations (50-100 micrometer), induces direct interaction between the oxidase factors p47(phox) and p22(phox) in parallel with superoxide production. The interaction, being required for the oxidase activation, is mediated via the Src homology 3 (SH3) domains of p47(phox) (p47-(SH3)(2)), which are intramolecularly masked in a resting state. We also show that AA disrupts complexation of p47-(SH3)(2) with its intramolecular target fragment (amino acids 286-340) without affecting association of p47-(SH3)(2) with p22(phox), indicating that the disruption plays a crucial role in the induced interaction with p22(phox). Phosphorylation of p47(phox) by protein kinase C partially replaces the effects of AA; treatment of the SH3 target fragment with PKC in vitro results in a completely impaired interaction with p47-(SH3)(2), and the same treatment of the full-length p47(phox) leads to both interaction with p22(phox) and oxidase activation without AA, but to a lesser extent. Furthermore, phosphorylated p47(phox) effectively binds to p22(phox) and activates the oxidase in the presence of AA at low concentrations (1-5 micrometer), where an unphosphorylated protein only slightly supports superoxide production. Thus AA, at high concentrations, fully induces the interaction of p47(phox) with p22(phox) by itself, whereas, at low concentrations, AA synergizes with phosphorylation of p47(phox) to facilitate the interaction, thereby activating the NADPH oxidase.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.     

The role of cytosolic phospholipase A2-alfa in regulation of phagocytic functions

Phospholipase A2(s) (PLA2(s)) are a family of enzymes that is present in a variety of mammalian and nonmammalian sources. Phagocytic cells contain cytosolic PLA2 (cPLA2) as well as several types of secreted PLA2, all of which have the potential to produce proinflammatory lipid mediators. The role of the predominant form of cPLA2 present in neutrophils is cPLA2alpha was studied by many groups. By modulating its expression in a variety of phagocytes it was found that it plays a major role in formation of eicosanoids. In addition, it was reported that cPLA2alpha also regulates the NADPH oxidase activation. The specificity of its effect on the NADPH oxidase is evident by results demonstrating that the differentiation process as well as other phagocytic functions are normal in cPLA2alpha-deficient PLB cell model. The novel dual subcellular localization of cPLA2alpha in different compartments, in the plasma membranes and in the nucleus, provides a molecular mechanism for the participation of cPLA2alpha in different processes (stimulation of NADPH oxidase and formation of eicosanoids) in the same cells.     

A critical role of protein kinase C delta activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47(phox) and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity

The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67(phox). Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67(phox) and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67(phox) with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67(phox) and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67(phox) targets Vav1-mediated Rac activation.