Carfilzomib shows therapeutic potential for reduction of liver fibrosis by targeting hepatic stellate cell activation.

Because hepatic stellate cells (HSCs) play a major role in fibrosis, we focused on HSCs as a potential target for the treatment of liver fibrosis. In this study, we attempted to identify drug candidates to inactivate HSCs and found that several proteasome inhibitors (PIs) reduced HSC viability. Our data showed that a second-generation PI, carfilzomib (CZM), suppressed the expression of fibrotic markers in primary murine HSCs at low concentrations of 5 or 10 nM. Since CZM was not toxic to HSCs up to a concentration of 12.5 nM, we examined its antifibrotic effects further. CZM achieved a clear reduction in liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis without worsening of liver injury. Mechanistically, RNA sequence analysis of primary HSCs revealed that CZM inhibits mitosis in HSCs. In the CCl4-injured liver, amphiregulin, which is known to activate mitogenic signaling pathways and fibrogenic activity and is upregulated in murine and human metabolic dysfunction-associated steatohepatitis (MASH), was downregulated by CZM administration, leading to inhibition of mitosis in HSCs. Thus, CZM and next-generation PIs in development could be potential therapeutic agents for the treatment of liver fibrosis via inactivation of HSCs without liver injury.

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.

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.

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.

All-Electrical Ca2+-Independent Signal Transduction Mediates Attractive Sodium Taste in Taste Buds.

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.

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.

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.

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.

A protective role of Nox1/NADPH oxidase in a mouse model with hypoxia-induced bradycardia.

Although it has been reported that endotoxin-induced expression of Nox1 in the heart contributes to apoptosis in cardiomyocytes, functional role of Nox1 at the physiological expression level has not been elucidated. The aim of this study was to clarify the role of Nox1 under a hypoxic condition using wild-type (WT, Nox1(+/Y)) and Nox1-deficient (Nox1(-/Y)) mice. ECG recordings from anesthetized mice revealed that Nox1(-/Y) mice were more sensitive to hypoxia, resulting in bradycardia, compared to WT mice. Atrial and ventricular electrocardiograms recorded from Langendorff-perfused hearts revealed that hypoxic perfusion more rapidly decreased heart rate in Nox1(-/Y) hearts compared with WT hearts. Sinus node recovery times measured under a hypoxic condition were prolonged more markedly in the Nox1(-/Y) hearts. Sinoatrial node dysfunction of Nox1(-/Y) hearts during hypoxia was ameriolated by the pre-treatment with the Ca(2+) channel blocker nifedipine or the K(+) channel opener pinacidil. Spontaneous action potentials were recorded from enzymatically-isolated sinoatrial node (SAN) cells under a hypoxic condition. There was no significant difference in the elapsed times from the commencement of hypoxia to asystole between WT and Nox1(-/Y) SAN cells. These findings suggest that Nox1 may have a protective effect against hypoxia-induced SAN dysfunction.

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney.

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/ß, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated ß-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16(INK4A) and p21(Cip1), were equivalent between the genotypes, increased levels of p27(Kip1) and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27(Kip1) signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.

Clioquinol increases the expression of VGF, a neuropeptide precursor, through induction of c-Fos expression.

Clioquinol was used extensively in the mid-1900s as an amebicide to treat indigestion and diarrhea. It was eventually withdrawn from the market because it was linked to subacute myelo-optic neuropathy (SMON) in Japan. However, 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 VGF (nonacronymic), the precursor of neuropeptides involved in pain reactions, was significantly increased when SH-SY5Y and IMR-32 neuroblastoma cells were treated with clioquinol. Promoter analyses in SH-SY5Y cells revealed that a region responsive to clioquinol exists between -1381 and -1349 of the human VGF gene, which contains an activator protein (AP)-1 site-like sequence. The introduction of mutations at this site significantly reduced clioquinol-induced transcriptional activation. Clioquinol induced the expression of the AP-1 family transcription factors, c-Jun and c-Fos. Electrophoresis mobility shift assays demonstrated that c-Jun and c-Fos could bind to the AP-1 site at -1374/-1368 in SH-SY5Y cells treated with clioquinol. RNA interference against c-Fos significantly suppressed clioquinol-induced VGF mRNA expression. These results suggest that the clioquinol-induced expression of c-Fos mediates the induction of VGF expression.