A possible biochemical link between NADPH oxidase (Nox) 1 redox-signalling and ERp72

Emerging evidence indicates that Nox (NADPH oxidase) 1-generated ROS (reactive oxygen species) play critical regulatory roles in various cellular processes, yet little is known of direct targets for the oxidase. In the present study we show that one of the proteins selectively oxidized in response to Nox1-generated ROS was ERp72 (endoplasmic reticulum protein 72 kDa) with TRX (thioredoxin) homology domains. Oxidation of ERp72 by Nox1 resulted in an inhibition of its reductase activity. EGF treatment of cells stimulated the Nox1 activity and the activated Nox1 subsequently mediated EGF-induced suppression of the ERp72 reductase activity. Co-immunoprecipitation, GST (glutathione transferase) pulldown assays and mutational analysis, indicated that Nox1 associates with ERp72, which involves its N-terminus encompassing a Ca(2+)-binding site and the first TRX-like motif. Furthermore, confocal microscopy showed co-localization between Nox1 and ERp72 at the plasma membrane. These results suggest that Nox1 functionally associates with ERp72, regulating redox-sensitive signalling pathways in a cellular context.     

Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.     

Backbone 1H, 15N, and 13C resonance assignments for the NOXO1β PX domain

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47 ^phox . NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.     

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.     

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase,Nox4, in Mesangial Cells

Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.     

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln⁵⁸ had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.     

Differential upregulation of Nox homologues of NADPH oxidase by tumor necrosis factor-alpha in human aortic smooth muscle and embryonic kidney cells

NADPH oxidases are important sources of vascular superoxide, which has been linked to the pathogenesis of atherosclerosis. Previously we demonstrated that the Nox4 subunit of NADPH oxidase is a critical catalytic component for superoxide production in quiescent vascular smooth muscle cells. In this study we sought to determine the role of Nox4 in superoxide production in human aortic smooth muscle cells (AoSMC) and embryonic kidney (HEK293) cells under proinflammatory conditions. Incubation with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) for 12 h increased superoxide production in both cell types, whereas angiotensin II, platelet-derived growth factor or interleukin-1beta had little effects. Superoxide production was completely abolished by the NADPH oxidase inhibitors diphenyline iodonium and apocynin, but not by inhibitors of xanthine oxidase, nitric oxide synthase or mitochondrial electron transport. TNF-alpha upregulated the expression of Nox4 in AoSMC at both message and protein levels, while Nox1 and Nox2 were unchanged. In contrast, upregulation of Nox2 appeared to mediate the enhanced superoxide production by TNF-alpha in HEK293 cells. We suggest that Nox4 may be involved in increased superoxide generation in vascular smooth muscle cells under proinflammatory conditions.     

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 the topology and functional domains of PAQR10

PAQR10 (progestin and adipoQ receptor 10) is a Golgi-localized protein that is able to enhance the retention and activation of Ras proteins in the Golgi apparatus, subsequently leading to a sustained ERK (extracellular-signal-regulated kinase) signalling. However, little is known about the topology and functional domains of PAQR10. In the present study, we extensively dissected and analysed the structure of PAQR10. The topology analysis reveals that PAQR10 is an integral membrane protein with its N-terminus facing the cytosol. Multiple domains, including the membrane-proximal region at the N-terminus, the membrane-proximal region at the C-terminus and the three loops facing the cytosol, were found to be required for PAQR10 to reside in the Golgi apparatus, to stimulate ERK phosphorylation and to tether Ras to the Golgi apparatus. Furthermore, when PAQR10 was artificially forced to be expressed in the endoplasmic reticulum, it could neither mobilize Ras to the Golgi apparatus nor increase ERK phosphorylation. Finally, the PAQR10 mutants that lost Golgi localization failed to promote differentiation of PC12 cells. Collectively, the results of the present study indicate that Golgi localization is indispensable for PAQR10 to implement its regulatory functions in the Ras signalling cascade.     

Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.     

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.     

Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase

The extracellular matrix (ECM) facilitates pancreatic cancer cells survival, which is of central importance for pancreatic adenocarcinoma that is highly fibrotic. Here, we show that reactive oxygen species (ROS) mediate the prosurvival effect of ECM in human pancreatic cancer cells. Fibronectin and laminin stimulated ROS production and NADPH oxidase activation in pancreatic cancer cells. Both pharmacological and molecular approaches show that fibronectin stimulated ROS production through activation of NADPH oxidase and NADPH oxidase-independent pathways and that 5-lipoxygenase (5-LO) mediates both these pathways. Analyses of the mechanisms of ROS production by ECM proteins and growth factors indicate that activation of NADPH oxidase (Nox4) is a common mechanism employed both by ECM proteins and growth factors to increase ROS in pancreatic cancer cells. We also found that Nox4 is present in human pancreatic adenocarcinoma tissues and that these tissues display membrane NADPH oxidase activity. ECM proteins and growth factors activate NADPH oxidase through different mechanisms; in contrast to ECM proteins, growth factors activate NADPH oxidase through 5-LO-independent mechanisms. Inhibition of 5-LO or NADPH oxidase with pharmacological inhibitors of these enzymes and with Nox4 or 5-LO antisense oligonucleotides markedly stimulated apoptosis in cancer cells cultured on fibronectin. Our results indicate that ROS generation via 5-LO and downstream NADPH oxidase mediates the prosurvival effect of ECM in pancreatic cancer cells. These mechanisms may play an important role in pancreatic cancer resistance to treatments and thus represent novel therapeutic targets.     

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.     

A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition

To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE). PE is required for the proper conformation and active transport function of LacY. The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa. Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport. These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures.     

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.     

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.     

Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1–10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.