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1.  Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization ▿ †  
Molecular and Cellular Biology  2009;30(4):961-975.
Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H2O2 production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O2− generation. In contrast, Nox4 released H2O2 and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.
doi:10.1128/MCB.01393-09
PMCID: PMC2815567  PMID: 19995913
2.  Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain† 
Biochemistry  2010;49(11):2433-2442.
NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47phox and p67phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a Km for NADPH of 55 ± 10 μM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of ∼200 mol of H2O2 min−1 (mol of Nox4)−1. A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min−1, largely superoxide dismutase-independent), ferricyanide (470 min−1), and other electron acceptors (artificial dyes and cytochrome b5). Rates were similar to those observed for H2O2 production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.
doi:10.1021/bi9022285
PMCID: PMC2839512  PMID: 20163138
3.  Upregulation of phagocyte-like NADPH oxidase by cytokines in pancreatic beta-cells: Attenuation of oxidative and nitrosative stress by 2-bromopalmitate 
Biochemical pharmacology  2012;85(1):109-114.
Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.
doi:10.1016/j.bcp.2012.09.024
PMCID: PMC3529999  PMID: 23092759
Pancreatic β-cell; NADPH-oxidase; 2-Bromopalmitate; Cytokines; Oxidative stress; Nitrosative stress
4.  Nox regulation of smooth muscle contraction 
The catalytic subunit, gp91phox (a.k.a., Nox2) of the NADPH-oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology is being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.
doi:10.1016/j.freeradbiomed.2007.03.006
PMCID: PMC1989158  PMID: 17561091
5.  The Nicotinamide Adenine Dinucleotide Phosphate Oxidase Homologues NOX1 and NOX2/gp91phox Mediate Hepatic Fibrosis in Mice 
Hepatology (Baltimore, Md.)  2011;53(5):1730-1741.
NADPH oxidase (NOX) is a multicomponent enzyme that mediates electron transfer from NADPH to molecular oxygen, which leads to the production of superoxide. NOX2/gp91phox is a catalytic subunit of NOX expressed in phagocytic cells. Several homologues of NOX2, including NOX1, have been identified in non-phagocytic cells. We investigated the contributory role of NOX1 and NOX2 in hepatic fibrosis. Hepatic fibrosis was induced in wild-type (WT) mice, NOX1-knockout (NOX1KO) mice, and NOX2-knockout (NOX2KO) mice by either CCl4 injections or bile duct ligation (BDL). The functional contribution of NOX1 and NOX2 in endogenous liver cells, including hepatic stellate cells (HSCs), and bone marrow (BM) derived cells, including Kupffer cells (KCs), to hepatic reactive oxygen species (ROS) generation and hepatic fibrosis was assessed in vitro and in vivo using NOX1- or NOX2-BM chimeric mice. Hepatic NOX1 and NOX2 mRNA expression was increased in the two experimental mouse models of hepatic fibrosis. While NOX1 was expressed in HSCs but not in KCs, NOX2 was expressed in both HSCs and KCs. Hepatic fibrosis and ROS generation were attenuated in both NOX1KO and NOX2KO mice after CCl4 or BDL. Liver fibrosis in chimeric mice indicated that NOX1 mediates the profibrogenic effects in endogenous liver cells, while NOX2 mediates the profibrogenic effects in both endogenous liver cells and BM-derived cells. Multiple NOX1 and NOX2 components were upregulated in activated HSCs. Both NOX1- and NOX2-deficient HSCs had decreased ROS generation and failed to upregulate collagen α1(I) and TGF-β in response to angiotensin II.
Conclusion
Both NOX1 and NOX2 in endogenous liver cells, including HSCs, have an important role in hepatic fibrosis, while NOX2 in BM-derived cells has a lesser role.
doi:10.1002/hep.24281
PMCID: PMC3082608  PMID: 21384410
NADPH oxidase; oxidative stress; hepatic fibrosis; hepatic stellate cells
6.  Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase 
Background
Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.
Methods
Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (3H-naloxone) binding assays were performed in wild type and gp91phox-/- neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47phox to plasma membrane was assessed by western blot.
Results
Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of 3H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. 3H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91phox; in contrast, reduced 3H-naloxone binding was found in neutrophils deficient in gp91phox or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of 3H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91phox antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47phox to the membrane, leading to NOX2 inactivation.
Conclusions
Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.
doi:10.1186/1742-2094-9-32
PMCID: PMC3305409  PMID: 22340895
Neuroinflammation; Microglia; NADPH oxidase; Opioid receptor; Binding
7.  Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress 
PLoS ONE  2012;7(5):e38075.
It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47phox and p67phox, the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47phox and p67phox, which were prerequisites for NOX assembly and activation. Knocking down p47phox with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.
doi:10.1371/journal.pone.0038075
PMCID: PMC3360628  PMID: 22662267
8.  Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain† 
Biochemistry  2011;50(12):2013-2025.
Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of co-expressed, differentially tagged forms of Nox5 and occurred independently of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) were deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells or endogenously expressed in human aortic smooth muscle cells indicated molecular weights of about 350 kDa and 300 kDa, respectively, consistent with a tetramer as the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form Nox5S may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.
doi:10.1021/bi1020088
PMCID: PMC3073450  PMID: 21319793
9.  NADPH Oxidase Activation Is Required in Reactive Oxygen Species Generation and Cell Transformation Induced by Hexavalent Chromium 
Toxicological Sciences  2011;123(2):399-410.
Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2μM) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1–3 and NOX5) and subunits (p22phox, p47phox, p40phox, and p67phox). Cr(VI) also induced phosphorylation of p47phox and membrane translocation of p47phox and p67phox, further confirming NOX activation. Knockdown of p47phox with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to 3 months) to low doses of Cr(VI) (0.125, 0.25, and 0.5μM) also promoted ROS generation and the expression of NOX subunits, such as p47phox and p67phox, but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47phox or overexpression of SOD1, SOD2, or catalase (CAT) eliminated Cr(VI)-induced malignant transformation. Our results suggest that NOX plays an important role in Cr(VI)-induced ROS generation and carcinogenesis.
doi:10.1093/toxsci/kfr180
PMCID: PMC3179675  PMID: 21742780
hexavalent chromium; NADPH oxidase; ROS generation; carcinogenesis
10.  Regulation of NOXO1 Activity through Reversible Interactions with p22phox and NOXA1 
PLoS ONE  2010;5(5):e10478.
Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22phox for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22phox. Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22phox and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.
doi:10.1371/journal.pone.0010478
PMCID: PMC2864300  PMID: 20454568
11.  New p22-Phox Monoclonal Antibodies: Identification of a Conformational Probe for Cytochrome b558 
Journal of Innate Immunity  2009;1(6):556-569.
The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b558, formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b558 activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b558 conformational change, 6 monoclonal antibodies were produced against purified cytochrome b558. Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b558. These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox.
doi:10.1159/000231977
PMCID: PMC2919509  PMID: 20375611
Cytochrome b558; p22-phox conformation; Monoclonal antibody; Phagocyte NADPH oxidase; Nox
12.  Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes 
Background
The reactive oxygen-generating NADPH oxidases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits.
Results
Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested.
Conclusion
This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes.
doi:10.1186/1471-2148-7-178
PMCID: PMC2121648  PMID: 17900370
13.  Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction 
Basic Research in Cardiology  2011;106:527-538.
NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-011-0179-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00395-011-0179-7
PMCID: PMC3105229  PMID: 21528437
Endothelium; Vascular tone; NADPH oxidase; Hypertension; Reactive oxygen species
14.  Signaling Components of Redox Active Endosomes: The Redoxosomes 
Antioxidants & Redox Signaling  2009;11(6):1313-1333.
Abstract
Subcellular compartmentalization of reactive oxygen species (ROS) plays a critical role in transmitting cell signals in response to environmental stimuli. In this regard, signals at the plasma membrane have been shown to trigger NADPH oxidase-dependent ROS production within the endosomal compartment and this step can be required for redox-dependent signal transduction. Unique features of redox-active signaling endosomes can include NADPH oxidase complex components (Nox1, Noxo1, Noxa1, Nox2, p47phox, p67phox, and/or Rac1), ROS processing enzymes (SOD1 and/or peroxiredoxins), chloride channels capable of mediating superoxide transport and/or membrane gradients required for Nox activity, and novel redox-dependent sensors that control Nox activity. This review will discuss the cytokine and growth factor receptors that likely mediate signaling through redox-active endosomes, and the common mechanisms whereby they act. Additionally, the review will cover ligand-independent environmental injuries, such as hypoxia/reoxygenation injury, that also appear to facilitate cell signaling through NADPH oxidase at the level of the endosome. We suggest that redox-active endosomes encompass a subset of signaling endosomes that we have termed redoxosomes. Redoxosomes are uniquely equipped with redox-processing proteins capable of transmitting ROS signals from the endosome interior to redox-sensitive effectors on the endosomal surface. In this manner, redoxosomes can control redox-dependent effector functions through the spatial and temporal regulation of ROS as second messengers. Antioxid. Redox Signal. 11, 1313–1333.
doi:10.1089/ars.2008.2363
PMCID: PMC2842130  PMID: 19072143
15.  Direct interaction between Tks proteins and the N-terminal Proline Rich Region (PRR) of NoxA1 mediates Nox1-dependent ROS generation 
European journal of cell biology  2010;90(2-3):164-171.
NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date 7 members of this family have been reported, including Nox1–5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47phox-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior.
Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67phox, which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5.
Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.
doi:10.1016/j.ejcb.2010.05.007
PMCID: PMC3013238  PMID: 20609497
NADPH oxidase; Nox1; NoxA1; p67phox; Reactive Oxygen Species (ROS); Tks proteins; Invadopodia; Cancer
16.  Novel p47phox-related organizers regulate NADPH oxidase 1 (Nox1) activity and localization 
Science signaling  2009;2(88):ra54.
The mechanisms that determine localized formation of reactive oxygen species (ROS) via NADPH oxidases (Nox) in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 and Tks5 are functional members of a p47phox-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (vs. Nox2 and Nox4) activity in reconstituted cellular systems, and interact with the NoxA1 activator protein through an SH3-mediated interaction. Endogenous Tks4 is required for Rac GTPase-dependent ROS production by DLD1 colon cancer cells. Tks4 recruits Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent novel members of an organizer superfamily that link Nox to localized ROS formation.
doi:10.1126/scisignal.2000370
PMCID: PMC2850287  PMID: 19755710
17.  Hyperglycemia Enhances IGF-I–Stimulated Src Activation via Increasing Nox4-Derived Reactive Oxygen Species in a PKCζ-Dependent Manner in Vascular Smooth Muscle Cells 
Diabetes  2011;61(1):104-113.
IGF-I–stimulated sarcoma viral oncogene (Src) activation during hyperglycemia is required for propagating downstream signaling. The aim of the current study was to determine the mechanism by which hyperglycemia enhances IGF-I–stimulated Src activation and the role of NADPH oxidase 4 (Nox4) and protein kinase C ζ (PKCζ) in mediating this response in vascular smooth muscle cells (VSMCs). Nox4 expression was analyzed in VSMCs exposed to hyperglycemia. The role of Nox4-derived reactive oxygen species (ROS) in IGF-I–stimulated Src activation was investigated via knockdown of Nox4. Different isoforms of PKC were screened to investigate their role in hyperglycemia-induced Nox4. The oxidation of Src was shown to be a prerequisite for its activation in response to IGF-I during hyperglycemia. Hyperglycemia induced Nox4, but not Nox1, and p22 phagocyte oxidase (p22phox) expression and IGF-I stimulated Nox4/p22phox complex formation, leading to increased ROS generation. Knockdown of Nox4 prevented ROS generation and impaired the oxidation and activation of Src in response to IGF-I, whereas knockdown of Nox1 had no effect. PKCζ was shown to mediate the hyperglycemia-induced increase in Nox4 expression. The key observations in cultured VSMCs were confirmed in the diabetic mice. Nox4-derived ROS is responsible for the enhancing effect of hyperglycemia on IGF-I–stimulated Src activation, which in turn amplifies IGF-I–linked downstream signaling and biological actions.
doi:10.2337/db11-0990
PMCID: PMC3237650  PMID: 22148072
18.  Nox Activator 1 (NoxA1): A Potential Target for Modulation of Vascular Reactive Oxygen Species in Atherosclerotic Arteries 
Circulation  2010;121(4):549-559.
Background
Despite a concerted effort from many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homologue of p67phox, in VSMC NADPH oxidase function, and atherosclerosis.
Methods and Results
The presence of NoxA1 in mouse aortic VSMC was confirmed by RT-PCR and sequencing. NoxA1/p47phox interaction, following thrombin treatment, was observed by immunoprecipitation/Western analysis of lysates from p47phox−/− VSMC transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with AdNoxA1 significantly enhanced thrombin-induced ROS generation in wild-type but not in p47phox−/− and Nox1−/− VSMC. Thrombin-induced ROS production and VSMC proliferation were significantly reduced following downregulation of NoxA1 using shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of redox-sensitive protein kinases―Janus kinase 2, Akt and p38 mitogen-activated protein kinase―in VSMC. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMC and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE−/− mice compared with their age-matched wild-type mice. Further, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMC of human early carotid atherosclerotic lesions.
Conclusions
NoxA1 is the functional homologue of p67phox in VSMC and regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.
doi:10.1161/CIRCULATIONAHA.109.908319
PMCID: PMC2843418  PMID: 20083677
atherosclerosis; restenosis; neointimal hyperplasia; NADPH oxidase; thrombin; signal transduction
19.  Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through mitochondrial ROS–PKCε signaling axis in pulmonary artery smooth muscle cells 
Free radical biology & medicine  2008;45(9):1223-1231.
The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells including pulmonary artery smooth muscle cells (PASMCs) remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22phox, p47phox, and p67phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47phox protein to the plasma membrane in pulmonary, but not mesenteric arteries. The Nox inhibitor apocynin and p47phox gene deletion attenuated hypoxic increase in intracellular reactive oxygen species and Ca2+ concentration ([ROS]i and [Ca2+]i) as well as contraction in mouse PASMCs, and abolished hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCε translocation peptide inhibitor and PKCε gene deletion, but not the conventional PKC inhibitor GÖ6976, prevented hypoxic increase in Nox activity in pulmonary arteries and in [ROS]i in PASMCs. The PKC activator phorbol-12-myristate-13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H2O2 removal significantly inhibited hypoxic activation of Nox, whereas Gpx1 gene deletion had an opposite effect. Exogenous H2O2 increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS]i through mitochondrial ROS–PKCε signaling axis, providing a positive feedback mechanism to contribute to hypoxic increase in [ROS]i and [Ca2+]i as well as contraction in PASMCs.
doi:10.1016/j.freeradbiomed.2008.06.012
PMCID: PMC2586914  PMID: 18638544
hypoxia; NADPH oxidase; reactive oxygen species; intracellular calcium; mitochondria; protein kinase C; pulmonary artery
20.  NADPH Oxidases: Functions and Pathologies in the Vasculature 
Reactive oxygen species (ROS) are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of ROS in the vasculature: Nox1, Nox2, Nox4 and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1 and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2 and PDI. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these ROS, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.
doi:10.1161/ATVBAHA.108.181610
PMCID: PMC2841695  PMID: 19910640
NADPH oxidase; Reactive Oxygen Species; Blood Vessels; Hypertension; Atherosclerosis
21.  NADPH Oxidase and the Degeneration of Dopaminergic Neurons in Parkinsonian Mice 
Several lines of investigation have implicated oxidative stress in Parkinson's disease (PD) pathogenesis, but the mechanisms involved are still unclear. In this study, we characterized the involvement of NADPH oxidase (Nox), a multisubunit enzyme that catalyzes the reduction of oxygen, in the 6-hydroxydopamine- (6-OHDA-) induced PD mice model and compared for the first time the effects of this neurotoxin in mice lacking gp91phox−/−, the catalytic subunit of Nox2, and pharmacological inhibition of Nox with apocynin. Six-OHDA induced increased protein expression of p47phox, a Nox subunit, in striatum. gp91phox−/− mice appear to be completely protected from dopaminergic cell loss, whereas the apocynin treatment conferred only a limited neuroprotection. Wt mice treated with apocynin and gp91phox−/− mice both exhibited ameliorated apomorphine-induced rotational behavior. The microglial activation observed within the striatum and the substantia nigra pars compacta (SNpc) of 6-OHDA-injected Wt mice was prevented by apocynin treatment and was not detected in gp91phox−/− mice. Apocynin was not able to attenuate astrocyte activation in SN. The results support a role for Nox2 in the 6-OHDA-induced degeneration of dopaminergic neurons and glial cell activation in the nigrostriatal pathway and reveal that no comparable 6-OHDA effects were observed between apocynin-treated and gp91phox−/− mice groups.
doi:10.1155/2013/157857
PMCID: PMC3863573  PMID: 24379900
22.  Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration 
Background
Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death.
Methods
Primary co-cultures of cerebellar granule neurons and glia from rats were used to investigate the effect of NO (from iNOS, following lipopolysaccharide (LPS) and/or cytokine addition) or superoxide/hydrogen peroxide (from NOX, following phorbol 12-myristate 13-acetate (PMA), ATP analogue (BzATP), interleukin-1β (IL-1β) or arachidonic acid (AA) addition) on neuronal survival.
Results
Induction of glial iNOS caused little neuronal death. Similarly, activation of NOX alone resulted in little or no neuronal death. However, if NOX was activated (by PMA or BzATP) in the presence of iNOS (induced by LPS and interferon-γ) then substantial delayed neuronal death occurred over 48 hours, which was prevented by inhibitors of iNOS (1400W), NOX (apocynin) or a peroxynitrite decomposer (FeTPPS). Neurons and glia were also found to stain positive for nitrotyrosine (a putative marker of peroxynitrite) only when both iNOS and NOX were simultaneously active. If NOX was activated by weak stimulators (IL-1β, AA or the fibrillogenic prion peptide PrP106-126) in the presence of iNOS, it caused microglial proliferation and delayed neurodegeneration over 6 days, which was prevented by iNOS or NOX inhibitors, a peroxynitrite decomposer or a NMDA-receptor antagonist (MK-801).
Conclusion
These results suggest a dual-key mechanism, whereby glial iNOS or microglial NOX activation alone is relatively benign, but if activated simultaneously are synergistic in killing neurons, through generating peroxynitrite. This mechanism may mediate inflammatory neurodegeneration in response to cytokines, bacteria, ATP, arachidonate and pathological prions, in which case neurons may be protected by iNOS or NOX inhibitors, or scavengers of NO, superoxide or peroxynitrite.
doi:10.1186/1742-2094-2-20
PMCID: PMC1232863  PMID: 16156895
microglia; peroxynitrite; nitric oxide; prion protein; inflammation; cytokines
23.  The Involvement of the Tyrosine Kinase c-Src in the Regulation of Reactive Oxygen Species Generation Mediated by NADPH Oxidase-1 
Molecular Biology of the Cell  2008;19(7):2984-2994.
NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.
doi:10.1091/mbc.E08-02-0138
PMCID: PMC2441655  PMID: 18463161
24.  Dexamethasone inhibits the Nox-dependent ROS production via suppression of MKP-1-dependent MAPK pathways in activated microglia 
BMC Neuroscience  2011;12:49.
Background
Nox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear.
Results
The present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release.
Conclusion
In conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.
doi:10.1186/1471-2202-12-49
PMCID: PMC3121618  PMID: 21615929
microglia; Nox-2; MAPKs; ROS; dexamethasone
25.  Knock, knock: Who’s there? Nox1 
Circulation  2013;127(18):1850-1852.
It has become apparent that major sources of reactive oxygen species (ROS) in mammalian cells are the NADPH oxidases. These multimeric enzymes are composed of membrane bound catalytic subunits, or Nox enzymes and a small docking subunit termed p22phox. Depending on the Nox isoform, cytosolic regulatory subunits also play a role in their activation. There are 7 Nox isoforms. The first identified was Nox2, also termed gp91phox, which is present in phagocytic cells and is responsible for the oxidative burst. Unlike Nox2, the other Nox enzymes exhibit sustained production of ROS at somewhat lower levels. Their modes of activation differ, as do their cellular distribution. Nox 1-4 are expressed in vascular cells in rodents. Nox 5 has been identified in atherosclerotic lesions of humans but is not expressed in rodents. Stimulation of cells with angiotensin II, cytokines, catecholamines, high glucose, or mechanical stretch promote NADPH oxidase activation, in large part due to recruitment of cytosolic subunits to the membrane subunit, leading to formation of the functional enzyme complex that can transfer electrons molecular oxygen and formation of superoxide (O2 ·–).1 Superoxide serves as a progenitor for other ROS, including hydrogen peroxide (H2O2), peroxynitrite (OONO-), and hypochlorous acid (HOCl-). Enhanced activity the NADPH oxidases and increased expression of its subunits occur in many pathological conditions including hypertension, diabetes, atherosclerosis, cardiac hypertrophy and heart failure. Studies of genetically altered mice have shown that deletion of various NADPH oxidase subunits protect against these diseases, while their overexpression promotes pathology.2-4
doi:10.1161/CIRCULATIONAHA.113.002564
PMCID: PMC3909710  PMID: 23564667
diabetes mellitus; oxidant stress; NAD(P)H oxidase; arteriosclerosis; Editorial

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