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.     

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.     

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.     

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91^phox (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3 × 10⁴ PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91^phox protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV–HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91^phox deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV–HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV–HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.     

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.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

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.     

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.     

Expression of functional mammal flavocytochrome b558 in yeast: Comparison with improved insect cell system

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b₅₅₈ (Cytb₅₅₈) which consists of a heterodimer of the gp91^phox and p22^phox subunits. The Cytb₅₅₈ is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb₅₅₈ (rCytb₅₅₈). We expressed the gp91^phox and p22^phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91^phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb₅₅₈ displayed comparable spectral properties to neutrophil Cytb₅₅₈. In contrast to rCytb₅₅₈ produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.     

Role for the first SH3 domain of p67 in activation of superoxide-producing NADPH oxidases

The membrane-bound NADPH oxidase in phagocytes, gp91^phox (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91^phox/Nox2 requires assembly with the cytosolic proteins p67^phox and p47^phox, each containing two SH3 domains. Although the C-terminal SH3 domain of p67^phox is responsible for binding to p47^phox, little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67^phox-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91^phox/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67^phox in cells, suggesting that SH3(N) primarily increases the affinity of p67^phox for the oxidase complex. On the other hand, p67^phox-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91^phox/Nox2. Thus p67^phox-SH3(N) specifically functions in gp91^phox/Nox2 activation probably via facilitating oxidase assembly.     

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     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O₂), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O₂⁻ molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91^phox) and p47^phox expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1β cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.     

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.     

NADPH oxidase is involved in protein kinase CKII down-regulation-mediated senescence through elevation of the level of reactive oxygen species in human colon cancer cells

We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIα small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22^phox siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII.     

Phosphorylation of p22phox on Threonine 147 Enhances NADPH Oxidase Activity by Promoting p47phox Binding

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22^phox, is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22^phox. We also explored the mechanism by which p22^phox phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22^phox in vitro by both protein kinase C-α and -δ. Moreover, this mutation blocked the p22^phox-p47^phox interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22^phox-p47^phox interaction in the membrane was restored. Maturation of gp91^phox was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47^phox still occurred. This study directly implicates threonine 147 of p22^phox as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity.     

Functional Reconstitution of Oxidase Activity in X-Linked Chronic Granulomatous Disease by Retrovirus-Mediated Gene Transfer

The feasibility of correction of the disease phenotype by gene gene transfer was investigated in cells of four patients with X-linked chronic granulomatous disease. These patients carry point mutations of the gp91-phoxgene, encoding for the large subunit of the catalytic core of the phagocytic cell NADPH oxidase. A retroviral vector expressing the gp91-phoxprotein was constructed and used to transduce lymphoblastoid cell lines established from the patients. Several transduced lymphoblastoid cell clones were investigated for mRNA and protein expression, and for functional reconstitution of oxidase activity. Although extensive quantitative variability was detected among different clones, functional reconstitution of O₂⁻production was obtained in most cases, with oxidase function within the same range as in B cell lines derived from normal individuals. The same vector was also used for transduction of hematopoietic precursors from bone marrow or peripheral blood either with or without enrichment for CD34⁺cells. A comprehensive analysis was performed on differentiated myeloid colonies, to evaluate the efficiency of transduction, the levels of gp91-phoxexpression, and the extent of functional reconstitution of oxidase activity. A high efficiency of transduction was obtained in most experiments, with 60–100% of colonies containing proviral DNA. Among the transduced colonies, an extensive variability in the levels of expression of the transduced gene and of functional restoration of NADPH oxidase activity was observed. These results represent a step toward the development of a gene therapy protocol for these patients.     

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.     

Cellular crosstalk between TNF-α, NADPH oxidase, PKCβ2, and C2GNT in human leukocytes

Increasing evidence suggests that chronic, sub-clinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have established the potential role of the inflammatory enzyme, core 2 β-1, 6-N-acetylglucosaminyltransferase (C2GNT) in diabetic retinopathy. The present study was designed to explore the NADPH oxidase signaling pathway in the tumor necrosis factor-alpha (TNF-α)-induced activity of C2GNT in leukocytes. Human leukocytes (U937 cells) and an Epstein-Barr-transformed lymphoblastoid cell line deficient in p47phox (F10007 cells) were used for the study. Cells were exposed to TNF-α for 24 h in the presence and absence of 1) NADPH oxidase inhibitors (apocynin and scrambled and unscrambled gp91ds-tat), 2) LY379196 (specific protein kinase C β1/2 (PKCβ1/2) inhibitor), and 3) the antioxidant tiron. Subsequent C2GNT and NADPH activity was measured and the adhesion of U937 and F10007 cells to endothelial cells was assessed. TNF-α-induced C2GNT activity (1813 ± 326 pmol/h/mg protein) (mean ± SEM) in human leukocytes was significantly reversed with apocynin (153 ± 82 pmol/h/mg protein), unscrambled gp91ds-tat (244 ± 122 pmol/h/mg protein) and tiron (756 ± 87 pmol/h/mg protein). We further supported this C2GNT-NADPH oxidase link using p47phox-deficient leukocytes. The deficiency in p47phox prevented TNF-α-induced NADPH oxidase and C2GNT activity and adherence to endothelial cells. The response to TNF-α was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. Our results demonstrate for the first time a novel signaling crosstalk between TNF-α, NADPH oxidase, PKCβ1/2 and C2GNT in leukocytes.     

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca^2 +-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca^2 + also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca^2 +.