NADPH oxidases: not just for leukocytes anymore!

In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b(558) of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.     

Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases

Reactive oxygen species (ROS) are important signal transduction molecules in ligand-induced signaling, regulation of cell growth, differentiation, apoptosis and motility. Recently NADPH oxidases (Nox) homologous to Nox2 (gp91phox) of phagocyte cytochrome b558 have been identified, which are an enzymatic source for ROS generation in epithelial cells. This study was undertaken to delineate the requirements for ROS generation by Nox4. Nox4, in contrast to other Nox proteins, produces large amounts of hydrogen peroxide constitutively. Known cytosolic oxidase proteins or the GTPase Rac are not required for this activity. Nox4 associates with the protein p22phox on internal membranes, where ROS generation occurs. Knockdown and gene transfection studies confirmed that Nox4 requires p22phox for ROS generation. Mutational analysis revealed structural requirements affecting expression of the p22phox protein and Nox activity. Mechanistic insight into ROS regulation is significant for understanding fundamental cell biology and pathophysiological conditions.     

Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.     

Stimulation of NO synthase activity in the immune-competent lepidopteran Estigmene acraea hemocyte line.

In contrast to the vertebrate immune system, nearly nothing is known about the immunological role of nitric oxide (NO) in invertebrates. This study provides evidence of the presence of a NO synthase (NOS) activity in an immune-competent, macrophage-like insect hemocyte line, previously established from larvae of the lepidopteran insect Estigmene acraea. As proven by photometric determination of nitroblue tetrazolium reduction after cell fixation, the E. acraea cells possess NADPH diaphorase (NADPHd) activity. This NADPH diaphorase activity was NADPH dependent, not inhibitable by superoxide dismutase, influenced by extracellular addition of L-arginine, and inhibited in a dose-dependent manner by the specific NOS inhibitor Nomega-monomethyl-L-arginine. Furthermore, the NADPH diaphorase activity was stimulated within 30 min by the addition of insect pathogenic bacteria (Bacillus thuringiensis var. kurstaki, Photorhabdus luminescens), bacterial lipopolysaccharide, and silica beads. In activated E. acraea cell suspensions strongly increased amounts of L-citrulline and enhanced levels of total nitrite/nitrate (as NO derivates) can be determined. This is the first report on stimulable NOS activity in insect hemocytes.     

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase

The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1μM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.     

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.     

Diaphorase P: a new fetal isozyme identified in human placenta

Human placenta contains a thermostable, cytosolic NADH-diaphorase which is different from the other diaphorases and which we designate as diaphorase P. It is specific for NADH and reduces artificial substrates such as dichlorophenol and tetrazolium derivatives, but not natural substrates such as methemoglobin, cytochrome b5 or lipoate. It is antigenically distinct from the ubiquitous red-cell type NADH-diaphorase (soluble cytochrome b5 reductase) specified by the DIA1 locus. Using electrophoretic and immunologic methods, it was possible to detect diaphorase P in various fetal tissues (brain, liver, kidney, muscle), whereas was not found in adult tissues with the exception of the brain. This enzyme, the physiological role of which remains unknown, appears to belong, therefore, to the category of fetal proteins. Its resurgance in primary liver cancer was demonstrated in three cases.     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.     

Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism

The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419-430 of Nox2. This region is required for binding Rac2 but not p47(phox) or p67(phox) cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419-430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.     

BMP-2 induces a profibrotic phenotype in adult renal progenitor cells through Nox4 activation

Adult renal progenitor cells (ARPCs) isolated from the human kidney may contribute to repair featuring acute kidney injury (AKI). Bone morphogenetic proteins (BMPs) regulate differentiation, modeling, and regeneration processes in several tissues. The aim of this study was to evaluate the biological actions of BMP-2 in ARPCs in vitro and in vivo. BMP-2 was expressed in ARPCs of normal adult human kidneys, and it was upregulated in vivo after delayed graft function (DGF) of renal transplantation, a condition of AKI. ARPCs expressed BMP receptors, suggesting their potential responsiveness to BMP-2. Incubation of ARPCs with this growth factor enhanced reactive oxygen species (ROS) production, NADPH oxidase activity, and Nox4 protein expression. In vivo, Nox4 was localized in BMP-2-expressing CD133+ cells at the tubular level after DGF. BMP-2 incubation induced α-smooth muscle actin (SMA), collagen I, and fibronectin protein expression in ARPCs. Moreover, α-SMA colocalized with CD133 in vivo after DGF. The oxidative stimulus (H(2)O(2)) induced α-SMA expression in ARPCs, while the antioxidant N-acetyl-cysteine inhibited BMP-2-induced α-SMA expression. Nox4 silencing abolished BMP-2-induced NADPH oxidase activation and myofibroblastic induction. We showed that 1) ARPCs express BMP-2, 2) this expression is increased in a model of AKI; 3) BMP-2 may induce the commitment of ARPCs toward a myofibroblastic phenotype in vitro and in vivo; and 4) this profibrotic effect is mediated by Nox4 activation. Our findings suggest a novel mechanism linking AKI with progressive renal damage.     

Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmembrane Nox4 topology revealed by topological determination by Ubiquitin Fusion Assay,a novel method to uncover membrane protein topology

The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available.To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins.Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.     

Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation

The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.     

IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells

Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.     

Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 microM) chemiluminescence detectable superoxide than BCA. Apocynin (10 microM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.