Reactive Oxygen Species and NRF2 Signaling, Friends or Foes in Cancer?

The imbalance between reactive oxygen species (ROS) production and clearance causes oxidative stress and ROS, which play a central role in regulating cell and tissue physiology and pathology. Contingent upon concentration, ROS influence cancer development in contradictory ways, either stimulating cancer survival and growth or causing cell death. Cells developed evolutionarily conserved programs to sense and adapt redox the fluctuations to regulate ROS as either signaling molecules or toxic insults. The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2)-KEAP1 system is the master regulator of cellular redox and metabolic homeostasis. NRF2 has Janus-like roles in carcinogenesis and cancer development. Short-term NRF2 activation suppresses tissue injury, inflammation, and cancer initiation. However, cancer cells often exhibit constitutive NRF2 activation due to genetic mutations or oncogenic signaling, conferring advantages for cancer cells' survival and growth. Emerging evidence suggests that NRF2 hyperactivation, as an adaptive cancer phenotype under stressful tumor environments, regulates all hallmarks of cancer. In this review, we summarized the source of ROS, regulation of ROS signaling, and cellular sensors for ROS and oxygen (O2), we reviewed recent progress on the regulation of ROS generation and NRF2 signaling with a focus on the new functions of NRF2 in cancer development that reach beyond what we originally envisioned, including regulation of cancer metabolism, autophagy, macropinocytosis, unfolded protein response, proteostasis, and circadian rhythm, which, together with anti-oxidant and drug detoxification enzymes, contributes to cancer development, metastasis, and anticancer therapy resistance.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling.

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.

Bortezomib is an effective enhancer for chemical probe-dependent superoxide detection.

Various chemical probes for the detection of reactive oxygen species have been developed to examine oxidative stress associated with different pathologies. L-012, a luminol-based chemiluminescent probe, is widely used to detect extracellular superoxide because of its high sensitivity. We herein demonstrated that the co-application of the peptide boronic acid proteasome inhibitor, bortezomib, with L-012 significantly increased its luminescence without affecting the background. More than a 5-fold increase was detected in the total luminescence of L-012 in both NADPH oxidase-expressing cells and the xanthine oxidase-dependent cell-free superoxide generation system, but not in their background. Therefore, bortezomib increased the signal-to-background ratio and improved the detection of low levels of superoxide. The application of MLN2238, another peptide boronic acid proteasome inhibitor, also enhanced the luminescence of L-012. In contrast, carfilzomib, an epoxyketone proteasome inhibitor, did not increase luminescence, suggesting that the effects of bortezomib depend on the chemical structure of the peptide boronic acid, but not on its pharmacological effects. Bortezomib-induced enhancements appeared to be specific to the detection of superoxide because the detection of H2O2 by Amplex Red/HRP was not affected by the application of bortezomib. In the quantitative detection of the superoxide-specific oxidative product 2-hydroxyethidium (2-OH-E+), the application of bortezomib resulted in a 2-fold increase in the level of 2-OH-E+. Therefore, bortezomib sensitizes the detection of superoxide in both cell-based and cell-free systems, highlighting a novel feature of compounds containing the peptide boronic acid as powerful enhancers for the detection of superoxide.

NOX1/NADPH oxidase is involved in the LPS-induced exacerbation of collagen-induced arthritis.

We investigate as yet an unidentified role of NOX1, a non-phagocytic isoform of the superoxide-generating NADPH oxidase, in immune responses using Nox1-knockout mice (Nox1-KO). The transcripts of NOX1 was expressed in lymphoid tissues, including the spleen, thymus, bone marrow, and inguinal lymphoid nodes. When antibody production after ovalbumin (OVA) immunization was examined, no significant differences were observed in serum anti-OVA IgG levels between wild-type mice (WT) and Nox1-KO. In the experimental asthma, the infiltration of eosinophils and the Th2 cytokine response after the induction of asthma with OVA were similar between the two genotypes. However, the severity and incidence of experimental collagen-induced arthritis (CIA) following the administration of a low dose of endotoxin (LPS) were significantly lower in Nox1-KO. While neither serum levels of autoantibodies nor in vitro cytokine responses were affected by Nox1 deficiency, NOX1 mRNA levels in the spleen significantly increased after the LPS challenge. Among the spleen cells, remarkable LPS-induced upregulation of NOX1 was demonstrated in both CD11b+ monocytes/macrophages and CD11c+ dendritic cells, suggesting that LPS-inducible NOX1 in monocytes/macrophages/dendritic cells may modulate the development of experimental CIA. Therapeutic targeting of NOX1 may therefore control the onset and/or severity of arthritis which is exacerbated by bacterial infection.

1,3-Diradicals Embedded in Curved Paraphenylene Units: Singlet versus Triplet State and In-Plane Aromaticity.

Curved π-conjugated molecules and open-shell structures have attracted much attention from the perspective of fundamental chemistry, as well as materials science. In this study, the chemistry of 1,3-diradicals (DRs) embedded in curved cycloparaphenylene (CPPs) structures, DR-(n+3)CPPs (n = 0-5), was investigated to understand the effects of the curvature and system size on the spin-spin interactions and singlet versus triplet state, as well as their unique characteristics such as in-plane aromaticity. A triplet ground state was predicted for the larger 1,3-diradicals, such as the seven- and eight-paraphenylene-unit-containing diradicals DR-7CPP (n = 4) and DR-8CPP (n = 5), by quantum chemical calculations. The smaller-sized diradicals DR-(n+3)CPPs (n = 0-3) were found to possess singlet ground states. Thus, the ground-state spin multiplicity is controlled by the size of the paraphenylene cycle. The size effect on the ground-state spin multiplicity was confirmed by the experimental generation of DR-6CPP in the photochemical denitrogenation of its azo-containing precursor (AZ-6CPP). Intriguingly, a unique type of in-plane aromaticity emerged in the smaller-sized singlet states such as S-DR-4CPP (n = 1), as proven by nucleus-independent chemical shift calculations (NICS) and an analysis of the anisotropy of the induced current density (ACID), which demonstrate that homoconjugation between the 1,3-diradical moiety arises because of the curved and distorted bonding system.

NOX1/NADPH Oxidase Promotes Synaptic Facilitation Induced by Repeated D2 Receptor Stimulation: Involvement in Behavioral Repetition.

Repetitive behavior is a widely observed neuropsychiatric symptom. Abnormal dopaminergic signaling in the striatum is one of the factors associated with behavioral repetition; however, the molecular mechanisms underlying the induction of repetitive behavior remain unclear. Here, we demonstrated that the NOX1 isoform of the superoxide-producing enzyme NADPH oxidase regulated repetitive behavior in mice by facilitating excitatory synaptic inputs in the central striatum (CS). In male C57Bl/6J mice, repeated stimulation of D2 receptors induced abnormal behavioral repetition and perseverative behavior. Nox1 deficiency or acute pharmacological inhibition of NOX1 significantly shortened repeated D2 receptor stimulation-induced repetitive behavior without affecting motor responses to a single D2 receptor stimulation. Among brain regions, Nox1 showed enriched expression in the striatum, and repeated dopamine D2 receptor stimulation further increased Nox1 expression levels in the CS, but not in the dorsal striatum. Electrophysiological analyses revealed that repeated D2 receptor stimulation facilitated excitatory inputs in the CS indirect pathway medium spiny neurons (iMSNs), and this effect was suppressed by the genetic deletion or pharmacological inhibition of NOX1. Nox1 deficiency potentiated protein tyrosine phosphatase activity and attenuated the accumulation of activated Src kinase, which is required for the synaptic potentiation in CS iMSNs. Inhibition of NOX1 or ß-arrestin in the CS was sufficient to ameliorate repetitive behavior. Striatal-specific Nox1 knockdown also ameliorated repetitive and perseverative behavior. Collectively, these results indicate that NOX1 acts as an enhancer of synaptic facilitation in CS iMSNs and plays a key role in the molecular link between abnormal dopamine signaling and behavioral repetition and perseveration.SIGNIFICANCE STATEMENT Behavioral repetition is a form of compulsivity, which is one of the core symptoms of psychiatric disorders, such as obsessive-compulsive disorder. Perseveration is also a hallmark of such disorders. Both clinical and animal studies suggest important roles of abnormal dopaminergic signaling and striatal hyperactivity in compulsivity; however, the precise molecular link between them remains unclear. Here, we demonstrated the contribution of NOX1 to behavioral repetition induced by repeated stimulation of D2 receptors. Repeated stimulation of D2 receptors upregulated Nox1 mRNA in a striatal subregion-specific manner. The upregulated NOX1 promoted striatal synaptic facilitation in iMSNs by enhancing phosphorylation signaling. These results provide a novel mechanism for D2 receptor-mediated excitatory synaptic facilitation and indicate the therapeutic potential of NOX1 inhibition in compulsivity.

Clioquinol inhibits dopamine-ß-hydroxylase secretion and noradrenaline synthesis by affecting the redox status of ATOX1 and copper transport in human neuroblastoma SH-SY5Y cells.

Clioquinol (5-chloro-7-indo-8-quinolinol), a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. In the present study, a reporter assay revealed that clioquinol (20-50 µM) activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, suggesting zinc influx due to its ionophore effects. On the other hand, clioquinol (20-50 µM) significantly increased the cellular level of copper within 24 h. Clioquinol (50 µM) induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-ß-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was altered by clioquinol (20-50 µM). Noradrenaline levels were reduced by clioquinol (20-50 µM). Disruption of the ATOX1 gene suppressed the secretion of DBH. This study suggested that the disturbance of cellular copper transport by the inactivation of ATOX1 is one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

NOX1/NADPH oxidase affects the development of autism-like behaviors in a maternal immune activation model.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic and environmental factors. Among the environmental factors, maternal infection is known as one of the principal risk factors for ASD. On the other hand, postmortem studies suggested the relationship of oxidative stress with ASD etiology. However, the role of oxidative stress in the development of ASD remains unclear. Here, we report the involvement of NOX1/NADPH oxidase, an enzyme generating reactive oxygen species (ROS), in behavioral and anatomical abnormalities in a maternal immune activation (MIA) model. In the MIA model of gestational polyinosinic-polycytidylic acid (poly(I:C)) exposure, increased serum levels of IL-6 were observed in both wild-type (WT) and Nox1-deficient mice (Nox1KO). Following the comparable induction of MIA in the two genotypes, impairment of social preference and defects in motor coordination were observed in WT offspring but not in offspring deficient in Nox1. MIA up-regulated NOX1 mRNA in the cerebral cortex and cerebellum of the fetus but not in the adult offspring. Although the development of cortical neurons was unaffected by MIA in either genotype, the dropout of Purkinje cells in lobule VII of MIA-affected offspring was significantly ameliorated in Nox1KO. Taken together, these results suggested that NOX1/NADPH oxidase plays an essential role in some behavioral phenotypes observed in ASD, possibly by promoting the loss of Purkinje cells in the cerebellum.

Correction to: Cycloartane triterpenoid (23R, 24E)-23-acetoxymangiferonic acid inhibited proliferation and migration in B16-F10 melanoma via MITF downregulation caused by inhibition of both ß-catenin and c-Raf-MEK1-ERK signaling axis.

The article Cycloartane triterpenoid (23R, 24E)-23-acetoxymangiferonic acid inhibited proliferation and migration in B16-F10 melanoma via MITF downregulation caused by inhibition of both ß-catenin.

NOX1/NADPH oxidase in bone marrow-derived cells modulates intestinal barrier function.

The involvement of reactive oxygen species (ROS) has been suggested in the development of inflammatory bowel disease (IBD). An impaired intestinal barrier function is common in IBD patients. Here, we report the central role of NOX1/NADPH oxidase, a major source of ROS in nonphagocytic cells, in intestinal barrier dysfunction. By in vivo imaging using L-012 as a probe, a time-dependent increase in ROS was demonstrated in the abdomen of wild-type mice (WT) administered lipopolysaccharide (LPS: 6 mg/kg i.p.), but it was almost completely abolished in mice deficient in Nox1 (Nox1-KO) or the inducible nitric oxide synthase gene (iNOS-KO). By ex vivo imaging, increased ROS production was mainly shown in the ileum, where enhanced immunostaining of NOX1 was observed on the apical side of the epithelium. On the other hand, a punctate staining pattern of 3-nitrotyrosine, a marker of peroxynitrite production, was demonstrated in the lamina propria. When LPS-induced intestinal hyperpermeability was assessed by the oral administration of fluorescein isothiocyanate-conjugated dextran (FD-4), it was significantly suppressed in Nox1-KO as well as iNOS-KO. When Nox1-KO adoptively transferred with WT bone marrow were treated with LPS, the serum level of FD-4 was significantly elevated, whereas it remained unchanged in WT receiving bone marrow derived from Nox1-KO. Concomitantly, the activation of matrix metalloproteinase-9 induced by LPS was alleviated not only in intestinal tissue but also in peritoneal macrophages of Nox1-KO. Up-regulation of iNOS by LPS was significantly inhibited in macrophages deficient in Nox1, illustrating a functional hierarchy in NOX1/iNOS signaling. Together, these findings suggest that NOX1 in bone marrow-derived cells, but not epithelial cells, perturbs intestinal barrier integrity during endotoxemia.

Curve Effect on Singlet Diradical Contribution in Kekulé-type Diradicals: A Sensitive Probe for Quinoidal Structure in Curved π-Conjugated Molecules.

Curved (non-planar) aromatic compounds have attracted significant research attention in the fields of basic chemistry and materials science. The contribution of the quinoidal structure in the curved π-conjugated structures has been proposed to be the key for materials functions. In this study, the curve effect on the quinoidal contribution was investigated in Kekulé-type singlet diradicals (S-DR1-4) as a sensitive probe for quinoidal structures in curved π-conjugated molecules. The quinoidal contribution in S-DR1-4 was found to increase with increasing the curvature of the curved structure, which was quantitatively analyzed using NBO analysis and the natural orbital occupation numbers computed by the CASSCF method. The curve effect on the singlet-triplet energy gap was examined by the CASPT2 method. The singlet-triplet energy gaps for the highly π-conjugated diradicals were determined for the first time using the CASPT2 method. Substantial quinoidal contribution was found in the curved structures of the delocalized singlet diradicals S-DR1-4, in contrast to its absence in the corresponding triplet states T-DR1-4.

NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.

The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependent decline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.

Cycloartane triterpenoid (23R, 24E)-23-acetoxymangiferonic acid inhibited proliferation and migration in B16-F10 melanoma via MITF downregulation caused by inhibition of both ß-catenin and c-Raf-MEK1-ERK signaling axis.

We recently reported that (23R, 24E)-23-acetoxymangiferonic acid (23R-AMA), a cycloartane triterpenoid isolated by activity-guided separation from a methanol extract of Garcinia sp. bark, inhibited melanin production via inhibition of tyrosinase (TYR) expression in the B16-F10 melanoma cell line. Since 23R-AMA also inhibited microphthalmia-associated transcription factor (MITF) expression, an upstream factor of TYR, these features of 23R-AMA were thought to be appropriate for development of whitening cosmetics. However, 23R-AMA exhibited growth inhibition other than inhibition of melanin production in B16-F10 cells. Therefore, we investigated biological activities of 23R-AMA in detail, focused on its application as an anti-melanoma compound. In this study, we demonstrated that 23R-AMA inhibited cell proliferation and basic FGF (bFGF)-induced migration in B16-F10 cells. Furthermore, 23R-AMA promoted ser45/thr41 phosphorylation of ß-catenin and suppressed its intranuclear accumulation, which was suggested to be related to inhibition of MITF expression. The transcriptional activity of MITF is known to be regulated by phosphorylation via activated ERK. Further investigation revealed that 23R-AMA inhibited phosphorylation of c-Raf, MEK-1, and ERK, and also that of upstream molecules including FAK and c-Src. These results suggested that 23R-AMA inhibited growth and migration of B16-F10 melanoma by regulating both MITF expression and its activity. The activities of 23R-AMA reported in this study are new aspects of cycloartane triterpenoids.

[The involvement of NOX1/NADPH oxidase in the development of non-alcoholic steatohepatitis].

Reactive oxygen species (ROS) are known to play a critical role in the development of non-alcoholic steatohepatitis (NASH). To clarify the source of ROS, we examined the expression of superoxide-generating NADPH oxidase isoforms in the liver of high-fat and high-cholesterol (HFC) diet-fed mice. The mRNA expression of NOX1 was significantly elevated in mice on HFC diet for 8 weeks. Increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 in HFC diet-fed wild-type mice (WT) were significantly ameliorated in mice deficient in Nox1 (Nox1-KO). Increased nitrotyrosine adduct formation, a marker of peroxynitrite-induced injury, was observed in hepatic sinusoids of WT, which was significantly suppressed in NOX1-KO. NOX1 mRNA was mainly expressed in liver sinusoidal endothelial cells (LSECs), and it was significantly up-regulated in primary cultured LSECs treated with palmitic acid (PA). The production of nitric oxide by LSECs and LSECs-dependent relaxation of hepatic stellate cells were significantly attenuated by PA treatment. In contrast, these effects of PA were not observed in cells isolated from Nox1-KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impair hepatic microcirculation through reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the development of NASH.

Clioquinol increases the expression of interleukin-8 by down-regulating GATA-2 and GATA-3.

Clioquinol was used in the mid-1900s as an amebicide to treat indigestion and diarrhea. However, it was withdrawn from the market in Japan because it was linked to subacute myelo-optic neuropathy (SMON). The pathogenesis of SMON has not yet been elucidated in detail. As reported previously, we performed a global analysis on human neuroblastoma cells using DNA chips. The global analysis and quantitative PCR demonstrated that the mRNA level of interleukin-8 (IL-8) was significantly increased when SH-SY5Y neuroblastoma cells were treated with clioquinol. An enzyme-linked immunosorbent assay also demonstrated that clioquinol induced the secretion of IL-8 into culture media. Promoter analyses on SH-SY5Y cells revealed that a region responsive to clioquinol exists between -152 and -144 of the human IL-8 gene, which contains a consensus GATA-binding site sequence. The introduction of mutations at this site or the activator protein (AP)-1 site sequence at -126/-120 significantly reduced clioquinol-induced transcriptional activation. Among the GATA transcription factors expressed in SH-SY5Y cells, GATA-2 and GATA-3 protein levels were significantly decreased by the addition of clioquinol. Electrophoresis mobility shift assays using a probe corresponding to -159/-113 of the human IL-8 gene revealed two major shifted bands, one of which was increased and the other was decreased by clioquinol. The introduction of mutations showed that the former corresponded to binding to the AP-1 site, and the latter to binding to the GATA site. Supershift analyses revealed that the binding of c-Jun and c-Fos was increased, whereas that of GATA-3 was decreased by clioquinol. Genome editing against GATA-2 or GATA-3, not GATA-4 significantly enhanced clioquinol-induced IL-8 mRNA expression. On the other hand, the stable expression of GATA-2 or GATA-3 attenuated clioquinol-induced IL-8 mRNA expression and IL-8 secretion. These results suggest that the clioquinol-induced suppression of GATA-2 and GATA-3 expression mediates the up-regulation of IL-8.

Nicotine and methyl vinyl ketone, major components of cigarette smoke extracts, increase protective amyloid-ß peptides in cells harboring amyloid-ß precursor protein.

The increased ratio of longer amyloid-ß (Aß1-42)/shorter amyloid-ß (Aß1-40) peptides, generated from amyloid precursor protein (APP), is known to promote the development of Alzheimer's disease (AD). To investigate the role of smoking in Aß production, we determined the production of Aß species in the presence of nicotine or methyl vinyl ketone (MVK), major components of cigarette smoke extracts, in Flp-In™ T-REx™-293 (T-REx293) cells harboring a single copy of human APP. While treatment with nicotine or MVK did not affect the amount of APP, the levels of Aß1-40 in the culture media were significantly increased. On the other hand, the levels of Aß1-42 were unaltered by nicotine or MVK treatment. The Aß1-42/Aß1-40 ratio was therefore attenuated by cigarette smoke extracts. Similar results were obtained in T-REx293 cells harboring APP of Swedish- or London-type mutation linked to familial AD. T-REx293 cells expressed the nicotinic acetylcholine receptor (nAchR) and tubocurarine, an nAChR antagonist, completely blocked the effects of nicotine. Treatment with nicotine significantly elevated cellular levels of ß-secretase that cleaves APP prior to Aß generation. Taken together, a protective role of nicotine against AD pathology was suggested by enhanced extracellular Aß1-40 production, which may suppress Aß fibrillogenesis.

Up-regulation of NOX1/NADPH oxidase following drug-induced myocardial injury promotes cardiac dysfunction and fibrosis.

Cardiac fibrosis is a common feature in failing heart and therapeutic strategy to halt the progression of fibrosis is highly needed. We here report on NOX1, a non-phagocytic isoform of superoxide-producing NADPH oxidase, which promotes cardiac fibrosis in a drug-induced myocardial injury model. A single-dose administration of doxorubicin (DOX) elicited cardiac dysfunction accompanied by increased production of reactive oxygen species and marked elevation of NOX1 mRNA in the heart. In mice deficient in Nox1 (Nox1-/Y), cardiac functions were well retained and overall survival was significantly improved. However, increased level of serum creatine kinase was equivalent to that of wild-type mice (Nox1+/Y). At 4 days after DOX treatment, severe cardiac fibrosis accompanied by increased hydroxyproline content and activation of matrix metalloproteinase-9 was demonstrated in Nox1+/Y, but it was significantly attenuated in Nox1-/Y. When H9c2 cardiomyocytes were exposed to their homogenate, a dose-dependent increase in NOX1 mRNA was observed. Up-regulation of NOX1 mRNA in H9c2 co-incubated with their homogenate was abolished in the presence of TAK242, a TLR4 inhibitor. When isolated cardiac fibroblasts were exposed to H9c2 homogenates, increased proliferation and up-regulation of collagen 3a1 mRNA were demonstrated. These changes were significantly attenuated in cardiac fibroblasts exposed to homogenates from H9c2 harboring disrupted Nox1. These findings suggest that up-regulation of NOX1 following cellular damage promotes cardiac dysfunction and fibrosis by aggravating the pro-fibrotic response of cardiac fibroblasts. Modulation of the NOX1/NADPH oxidase signaling pathway may be a novel therapeutic strategy for preventing heart failure after myocardial injury.

The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.

The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.

Depressive-Like Behaviors Are Regulated by NOX1/NADPH Oxidase by Redox Modification of NMDA Receptor 1.

Involvement of reactive oxygen species (ROS) has been suggested in the development of psychiatric disorders. NOX1 is a nonphagocytic form of NADPH oxidase whose expression in the nervous system is negligible compared with other NOX isoforms. However, NOX1-derived ROS increase inflammatory pain and tolerance to opioid analgesia. To clarify the role of NOX1 in the brain, we examined depressive-like behaviors in mice deficient in Nox1 (Nox1-/Y). Depressive-like behaviors induced by chronic social defeat stress or administration of corticosterone (CORT) were significantly ameliorated in Nox1-/Y Generation of ROS was significantly elevated in the prefrontal cortex (PFC) of mice administrated with CORT, while NOX1 mRNA was upregulated only in the ventral tegmental area (VTA) among brain areas responsible for emotional behaviors. Delivery of miRNA against NOX1 to VTA restored CORT-induced depressive-like behaviors in wild-type (WT) littermates. Administration of CORT to WT, but not to Nox1-/Y, significantly reduced transcript levels of brain-derived neurotrophic factor (bdnf), with a concomitant increase in DNA methylation of the promoter regions in bdnf Delivery of miRNA against NOX1 to VTA restored the level of BDNF mRNA in WT PFC. Redox proteome analyses demonstrated that NMDA receptor 1 (NR1) was among the molecules redox regulated by NOX1. In cultured cortical neurons, hydrogen peroxide significantly suppressed NMDA-induced upregulation of BDNF transcripts in NR1-expressing cells but not in cells harboring mutant NR1 (C744A). Together, these findings suggest a key role of NOX1 in depressive-like behaviors through NR1-mediated epigenetic modification of bdnf in the mesoprefrontal projection.SIGNIFICANCE STATEMENT NADPH oxidase is a source of reactive oxygen species (ROS) that have been implicated in the pathogenesis of various neurological disorders. We presently showed the involvement of a nonphagocytic type of NADPH oxidase, NOX1, in major depressive disorders, including behavioral, biochemical, and anatomical changes in mice. The oxidation of NR1 by NOX1-derived ROS was demonstrated in prefrontal cortex (PFC), which may be causally linked to the downregulation of BDNF, promoting depressive-like behaviors. Given that NOX1 is upregulated only in VTA but not in PFC, mesocortical projections appear to play a crucial role in NOX1-dependent depressive-like behaviors. Our study is the first to present the potential molecular mechanism underlying the development of major depression through the NOX1-induced oxidation of NR1 and epigenetic modification of bdnf.

NOX1/NADPH Oxidase Expressed in Colonic Macrophages Contributes to the Pathogenesis of Colonic Inflammation in Trinitrobenzene Sulfonic Acid-Induced Murine Colitis.

NOX1/NADPH oxidase, a nonphagocytic isoform of reactive oxygen species-producing enzymes, is highly expressed in the colon, but the physiologic and pathophysiologic roles of this isoform are not fully understood. The present study investigated the role of NOX1 in the development of colonic inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Intrarectal injection of TNBS caused severe colitis accompanied by body weight loss, diarrhea, and increased myeloperoxidase (MPO) activity in wild-type (WT) mice. In contrast, the severity of colitis was significantly attenuated in NOX1-deficient (NOX1KO) mice (the inhibitions of macroscopic damage score, body weight loss, diarrhea score, and MPO activity were 73.1%, 36.8%, 83.3%, and 98.4%, respectively). TNBS-induced upregulation of inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß), chemokines (CXCL1 and CXLC2), and inducible nitric oxide synthase (iNOS) was also significantly less in NOX1KO than in WT mice (the inhibitions were 100.8%, 89.0%, 63.5%, 96.7%, and 97.1%, respectively). Expression of NOX1 mRNA was detected not only in the lamina propria but also in peritoneal macrophages isolated from WT mice. Increased expression of TNF-α, IL-1ß, and iNOS in peritoneal macrophages exposed to lipopolysaccharide was significantly attenuated in macrophages isolated from NOX1KO mice (68.1%, 67.0%, and 79.3% inhibition, respectively). These findings suggest that NOX1/NADPH oxidase plays an important role in the pathogenesis of TNBS-induced colonic inflammation via upregulation of inflammatory cytokines, chemokines, and iNOS. NOX1 in colonic macrophages may become a potential target in pharmacologic intervention for inflammatory bowel disease.