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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as superoxide-generating multi-component enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2>Nox1>Nox3. Determinants of subcellular targeting include: 1) formation of Nox-p22phox heterodimeric complexes allowing plasma membrane translocation, 2) phospholipids-binding specificities of PX domain-containing organizer proteins (p47phox or Nox organizer 1 (Noxo1)), and 3) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox.

Abstract

Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing, and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as multicomponent superoxide-generating enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2 > Nox1 > Nox3. Determinants of subcellular targeting include: (a) formation of Nox-p22phox heterodimeric complexes allowing plasma membrane translocation, (b) phospholipids-binding specificities of PX domain-containing organizer proteins (p47phox or Nox organizer 1 (Noxo1 and p40phox), and (c) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox. Antioxid Redox Signal. 11, 2607–2619.

The NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to form reactive oxygen species (ROS), have increased in number during eukaryotic evolution1,2. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated3,4. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants5,6. The prototypical member of this family, NOX2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities7,8. Here, we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-β1 (TGF-β1) induces NOX4 expression in lung mesenchymal cells by a SMAD3-dependent mechanism. NOX4-dependent generation of hydrogen peroxide (H2O2) is required for TGF-β1-induced myofibroblast differentiation, extracellular matrix (ECM) production, and contractility. NOX4 is upregulated in lungs of mice subjected to non-infectious injury and in human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX4 abrogates fibrogenesis in two different murine models of lung injury. These studies support a novel function for NOX4 in tissue fibrogenesis and provide proof-of-concept for therapeutic targeting of NOX4 in recalcitrant fibrotic disorders.

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.

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.

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.

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.

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.

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.

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.

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.

The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a “respiratory burst” upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2 or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN-γ. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of Dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses.

NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b558, the redox core, is assembled with cytosolic p47phox, p67phox, p40phox, and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O2. Moreover, replacement of this loop by the Nox4-D-loop (D-loopNox4-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca2+ influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b558 was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loopNox4-Nox2 mutant may come from a change of responsiveness to intracellular Ca2+ level and to phosphorylation events during oxidase activation. Finally the D-loopNox4-Nox2 -PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.

SUMMARY

Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. Here we show that NOX–mediated damage can be inhibited by suppression of the voltage-gated proton channel, Hv1. Hv1 is required for full NOX activity since it compensates for loss of NOX–exported charge. We show that Hv1 is required for NOX–dependent ROS generation in brain microglia in situ and in vivo. Mouse and human brain microglia, but not neurons or astrocytes, express large Hv1-mediated currents. Mice lacking Hv1 were protected from NOX–mediated neuronal death and brain damage 24 hours after stroke. These results demonstrate that Hv1–dependent ROS production is responsible for a significant fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.

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