RESUMEN
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.
Asunto(s)
Conducta Compulsiva/metabolismo , Locomoción/fisiología , NADPH Oxidasa 1/biosíntesis , NADPH Oxidasas/biosíntesis , Receptores de Dopamina D2/biosíntesis , Sinapsis/metabolismo , Animales , Células Cultivadas , Conducta Compulsiva/inducido químicamente , Conducta Compulsiva/psicología , Agonistas de Dopamina/farmacología , Agonistas de Dopamina/toxicidad , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 1/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Pirazolonas/farmacología , Piridonas/farmacología , Receptores de Dopamina D2/agonistas , Sinapsis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Trastorno del Espectro Autista/etiología , Conducta Animal/fisiología , NADPH Oxidasa 1/genética , Células de Purkinje/patología , Animales , Trastorno del Espectro Autista/inmunología , Cerebelo/embriología , Corteza Cerebral/embriología , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 1/metabolismo , Poli I-C/inmunología , Poli I-C/farmacología , EmbarazoRESUMEN
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.
Asunto(s)
Artritis Experimental/etiología , Colágeno/efectos adversos , Endotoxinas/efectos adversos , NADPH Oxidasa 1/fisiología , Animales , Células Cultivadas , Células Dendríticas , Progresión de la Enfermedad , Macrófagos , Masculino , Ratones Noqueados , Monocitos , NADPH Oxidasa 1/genética , NADPH Oxidasa 1/metabolismo , ARN Mensajero/metabolismo , Bazo/citología , Bazo/metabolismoRESUMEN
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.
Asunto(s)
Clioquinol/toxicidad , Proteínas Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Dopamina beta-Hidroxilasa/metabolismo , Chaperonas Moleculares/metabolismo , Neuronas/efectos de los fármacos , Norepinefrina/biosíntesis , Neuropatía Óptica Tóxica/etiología , Línea Celular Tumoral , Proteínas Transportadoras de Cobre/genética , Humanos , Chaperonas Moleculares/genética , Neuronas/enzimología , Oxidación-Reducción , Proteína-Lisina 6-Oxidasa/metabolismo , Vías Secretoras , Neuropatía Óptica Tóxica/enzimología , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Trastorno Depresivo/metabolismo , NADH NADPH Oxidorreductasas/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Trastorno Depresivo/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasas/deficiencia , Proteínas del Tejido Nervioso/genética , Oxidación-Reducción , Corteza Prefrontal , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
BACKGROUND & AIMS: Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. METHODS: Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. RESULTS: Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. CONCLUSIONS: NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.
Asunto(s)
Hígado Graso/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Resistencia a la Insulina , Cirrosis Hepática/tratamiento farmacológico , NADPH Oxidasas/metabolismo , NADP/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Biopsia , Dieta/métodos , Modelos Animales de Enfermedad , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/citología , Hígado/patología , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADP/administración & dosificación , NADPH Oxidasa 4 , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Proteína Fosfatasa 1/metabolismo , Pirazoles/metabolismo , Pirazolonas , Piridinas/metabolismo , Piridonas , Estrés Fisiológico/efectos de los fármacosRESUMEN
OBJECTIVE: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. APPROACH AND RESULTS: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. CONCLUSIONS: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.
Asunto(s)
Hipertensión Pulmonar/enzimología , NADH NADPH Oxidorreductasas/deficiencia , Arteria Pulmonar/enzimología , Actinas/metabolismo , Animales , Apoptosis , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar , Predisposición Genética a la Enfermedad , Hemodinámica , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertrofia , Hipertrofia Ventricular Derecha/enzimología , Hipertrofia Ventricular Derecha/etiología , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , Fenotipo , Potasio/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Interferencia de ARN , Ratas , TransfecciónRESUMEN
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.
Asunto(s)
Amebicidas/farmacología , Clioquinol/farmacología , Expresión Génica/efectos de los fármacos , Factores de Crecimiento Nervioso/genética , Neuroblastoma/genética , Neuropéptidos/genética , Proteínas Proto-Oncogénicas c-fos/genética , Amebicidas/efectos adversos , Clioquinol/efectos adversos , Humanos , Mielitis/inducido químicamente , Factores de Crecimiento Nervioso/metabolismo , Neuroblastoma/metabolismo , Neuropéptidos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neuritis Óptica/inducido químicamente , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Células Tumorales CultivadasRESUMEN
Clioquinol 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. We previously performed a global analysis of human neuroblastoma cells using DNA chips and demonstrated that clioquinol induced 1) DNA double-strand breaks and subsequent activation of ATM/p53 signaling; 2) the expression of VGF, the precursor of neuropeptides involved in pain reactions, by inducing c-Fos; 3) the expression of interleukin-8, which is reported to be involved in intestinal inflammation, optic neuropathy, and neuropathic pain, by down-regulating GATA-2 and GATA-3. We also demonstrated that clioquinol induced zinc influx and oxidation of the copper chaperone ATOX1, leading to the impairment of the functional maturation of a copper-dependent enzyme dopamine-ß-hydroxylase and the inhibition of noradrenaline biosynthesis. Thus, clioquinol-induced neurotoxicity in SMON seems to be mediated by multiple pathways.
Asunto(s)
Clioquinol , Enfermedades del Nervio Óptico , Humanos , Clioquinol/efectos adversos , Cobre , Médula Espinal , Japón , Proteínas Transportadoras de Cobre , Chaperonas MolecularesRESUMEN
BACKGROUND: Subacute myelo-optico-neuropathy (SMON) is a neurological disorder associated with the administration of clioquinol, particularly at very high doses. Although clioquinol has been used worldwide, there was an outbreak of SMON in the 1950s-1970s in which the majority of cases were in Japan, prompting speculation that the unique genetic background of the Japanese population may have contributed to the development of SMON. Recently, a possible association between loss-of-function polymorphisms in NQO1 and the development of SMON has been reported. In this study, we analyzed the relationship between NQO1 polymorphisms and SMON in Japan. METHODS: We analyzed 125 Japanese patients with SMON. NQO1 loss-of-function polymorphisms (rs1800566, rs10517, rs689452, and rs689456) were evaluated. The allele frequency distribution of each polymorphism was compared between the patients and the healthy Japanese individuals (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), as well as our in-house healthy controls. RESULTS: The frequencies of the loss-of-function NQO1 alleles in patients with SMON and the normal control group did not differ significantly. CONCLUSION: We conclude that known NQO1 polymorphisms are not associated with the development of SMON.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona) , Polimorfismo de Nucleótido Simple , Humanos , NAD(P)H Deshidrogenasa (Quinona)/genética , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adulto , Frecuencia de los Genes , Mutación con Pérdida de Función , JapónRESUMEN
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.
Asunto(s)
Células Estrelladas Hepáticas , Cirrosis Hepática , Oligopéptidos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Animales , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/inducido químicamente , Ratones , Masculino , Modelos Animales de Enfermedad , Tetracloruro de Carbono , Humanos , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Anfirregulina/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
The involvement of reactive oxygen species (ROS) in morphine-induced analgesia and tolerance has been suggested, yet how and where ROS take part in these processes remains largely unknown. Here, we report a novel role for the superoxide-generating enzyme NOX1/NADPH oxidase in the regulation of analgesia and acute analgesic tolerance. In mice lacking Nox1 (Nox1(-/Y)), the magnitude of the analgesia induced by morphine was significantly augmented. More importantly, analgesic tolerance induced by repeated administration of morphine was significantly suppressed compared with that in the littermates, wild-type Nox1(+/Y). In a membrane fraction obtained from the dorsal spinal cord, no difference was observed in morphine-induced [(35)S]GTPγS-binding between the genotypes, whereas morphine-stimulated GTPase activity was significantly attenuated in Nox1(-/Y). At 2 h after morphine administration, a significant decline in [(35)S]GTPγS-binding was observed in Nox1(+/Y) but not in Nox1(-/Y). No difference in the maximal binding and affinity of [(3)H]DAMGO was observed between the genotypes, but the translocation of protein kinase C isoforms to the membrane fraction following morphine administration was almost completely abolished in Nox1(-/Y). Finally, the phosphorylation of RGS9-2 and formation of a complex by Gαi2/RGS9-2 with 14-3-3 found in morphine-treated Nox1(+/Y) were significantly suppressed in Nox1(-/Y). Together, these results suggest that NOX1/NADPH oxidase attenuates the pharmacological effects of opioids by regulating GTPase activity and the phosphorylation of RGS9-2 by protein kinase C. NOX1/NADPH oxidase may thus be a novel target for the development of adjuvant therapy to retain the beneficial effects of morphine.
Asunto(s)
Tolerancia a Medicamentos/genética , Hiperalgesia/tratamiento farmacológico , Morfina/uso terapéutico , NADH NADPH Oxidorreductasas/metabolismo , Narcóticos/uso terapéutico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Encefalina Ala(2)-MeFe(4)-Gli(5)/metabolismo , GTP Fosfohidrolasas/metabolismo , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/farmacología , Hiperalgesia/genética , Masculino , Ratones , Ratones Noqueados , NADH NADPH Oxidorreductasas/deficiencia , NADPH Oxidasa 1 , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Proteína Quinasa C/metabolismo , Proteínas RGS/metabolismo , ARN Mensajero , Médula Espinal/citología , Isótopos de Azufre/metabolismo , Superóxidos/metabolismo , Tritio/metabolismoRESUMEN
The redox-sensitive transcription factors NF-κB and activator protein-1 (AP-1) are critical mediators of ANG II signaling. The promitogenic and promigratory factor interleukin (IL)-18 is an NF-κB- and AP-1-responsive gene. Therefore, we investigated whether ANG II-mediated smooth muscle cell (SMC) migration and proliferation involve IL-18. ANG II induced rat carotid artery SMC migration and proliferation and IL-18 and metalloproteinase (MMP)-9 expression via ANG II type 1 (AT(1)) receptor. ANG II-induced superoxide generation, NF-κB and AP-1 activation, and IL-18 and MMP-9 induction were all markedly attenuated by losartan, diphenyleneiodonium chloride (DPI), and Nox1 knockdown. Similar to ANG II, addition of IL-18 also induced superoxide generation, activated NF-κB and AP-1, and stimulated SMC migration and proliferation, in part via Nox1, and both ANG II and IL-18 induced NOX1 transcription in an AP-1-dependent manner. AT(1) physically associates with Nox1 in SMC, and ANG II enhanced this binding. Interestingly, exogenous IL-18 neither induced AT(1) binding to Nox1 nor enhanced the ANG II-induced increase in AT(1)/Nox1 binding. Importantly, IL-18 knockdown, or pretreatment with IL-18 neutralizing antibodies, or IL-18 binding protein, all attenuated the migratory and mitogenic effects of ANG II. Continuous infusion of ANG II for 7 days induced carotid artery hyperplasia in rats via AT(1) and was associated with increased AT(1)/Nox1 binding (despite lower AT(1) levels); increased DPI-inhibitable superoxide production; increased phospho-IKKß, JNK, p65, and c-Jun; and induction of IL-18 and MMP-9 in endothelium-denuded carotid arteries. These results indicate that IL-18 amplifies the ANG II-induced, redox-dependent inflammatory cascades by activating similar promitogenic and promigratory signal transduction pathways. The ANG II/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis.
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Angiotensina II/metabolismo , Movimiento Celular , Proliferación Celular , Interleucina-18/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Angiotensina II/administración & dosificación , Bloqueadores del Receptor Tipo 1 de Angiotensina II/administración & dosificación , Animales , Arterias Carótidas/enzimología , Arterias Carótidas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Genes Reporteros , Hiperplasia , Quinasa I-kappa B/metabolismo , Bombas de Infusión Implantables , Infusiones Subcutáneas , Interleucina-18/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Losartán/administración & dosificación , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Transducción de Señal , Superóxidos/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA/metabolismo , TransfecciónRESUMEN
UNLABELLED: Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1α mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of α-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27(kip1) , a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27(kip1) gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO. CONCLUSION: These findings indicate that reactive oxygen species derived from NOX1/NADPH oxidase oxidize and inactivate PTEN to positively regulate the Akt/FOXO4/p27(kip1) signaling pathway. NOX1 may thus promote proliferation of HSCs and accelerate the development of fibrosis following BDL-induced liver injury.
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Proliferación Celular , Células Estrelladas Hepáticas/patología , Cirrosis Hepática Experimental/etiología , NADH NADPH Oxidorreductasas/fisiología , NADP/fisiología , Animales , Tetracloruro de Carbono/toxicidad , Proteínas de Ciclo Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Factores de Transcripción Forkhead/fisiología , Ligadura , Cirrosis Hepática Experimental/patología , Ratones , NADPH Oxidasa 1 , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Subacute myelo-optico-neuropathy (SMON) is a severe neurological disorder associated with clioquinol administration, which frequently occurred in Japan during the 1950s and 1960s. The unique genetic background of the Japanese population is considered to be strongly involved in the development of this neurological disease. Recently, genetic variants of ABCC4 (OMIM: 605250) and ABCC11 (OMIM: 607040), which are particularly common in the Japanese population, were suggested as possible genetic susceptibility factors for the development of SMON. METHODS: We analyzed 125 Japanese SMON patients who provided consent for this study. Patient DNA was collected from peripheral blood, and genetic analysis was performed for ABCC4 rs3765534 (c.2268G>A, p.Glu857Lys) and ABCC11 rs17822931 (c.538G>A, p.Gly180Arg) polymorphisms using the Sanger sequencing method and/or TaqMan PCR method. The frequency distribution of each polymorphism was compared with that in healthy Japanese people recorded in two genomic databases (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), and each genotype was compared with the clinical features of patients. RESULTS: The frequencies of ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms in SMON patients and healthy Japanese people were not significantly different in the multifaceted analysis. CONCLUSION: We conclude that the ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms are not associated with the development of SMON.
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Clioquinol , Enfermedades del Sistema Nervioso Periférico , Transportadoras de Casetes de Unión a ATP , Humanos , Japón , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genéticaRESUMEN
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.
RESUMEN
OBJECTIVE: To examine the effect of an oxidized extracellular oxidation-reduction (redox) state (E(h)) on the expression of NADPH oxidases in vascular cells. METHODS AND RESULTS: The generation of reactive oxygen species by NADPH oxidase (Nox)-based NADPH oxidases activates redox-dependent signaling pathways and contributes to the development of "oxidative stress" in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans; however, the cellular mechanisms by which the extracellular redox state may cause disease are not known. Aortic segments and cultured aortic smooth muscle cells were exposed to E(h) between -150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized E(h) increased the expression of Nox1 and resulted in Nox1-dependent proliferation of smooth muscle cells. Oxidized E(h) rapidly induced epidermal growth factor receptor phosphorylation via shedding of epidermal growth factor-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2-dependent phosphorylation of the transcription factors activating transcription factor-1 and cAMP-response element-binding protein. Inhibition of epidermal growth factor receptor or extracellular signal-regulated kinase 1/2 activation, or addition of small interference RNA to activating transcription factor-1, prevented the increase in Nox1 expression. CONCLUSIONS: Our results identify a novel mechanism by which extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease.
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Receptores ErbB/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , NADH NADPH Oxidorreductasas/biosíntesis , Animales , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , NADPH Oxidasa 1 , Oxidación-Reducción , Estrés Oxidativo , Transducción de SeñalRESUMEN
INTRODUCTION: Estrogen-related receptor α (ERRα), one of orphan nuclear receptors with an unknown ligand, is expressed in various types of cancer. Increased ERRα levels are associated with a higher risk of recurrence and poor clinical outcome in breast cancer, suggesting that ERRα could be a negative prognostic factor. Recently, it has been suggested that vascular endothelial growth factor (VEGF) could be one of the transcriptional targets of ERRα in breast cancer. Here, we examined the expression of ERRα and the association of ERRα with VEGF in uterine cervical cancer cells and tissues. METHODS: We evaluated the expression of ERRα and VEGF by immunohistologic analysis using specimens from 40 patients with invasive cervical cancer. We also evaluated the VEGF promoter activity of ERRα in cervical cancer cell lines by transfection and luciferase assay. We overexpressed or knocked down ERRα and examined VEGF expression by real-time polymerase chain reaction. Finally, cell proliferation assay was performed to examine whether ERRα affects tumor growth in cervical cancer. RESULTS: Immunohistologic analysis demonstrated that ERRα expression in cervical cancer tissues was higher than that in noncancerous tissues and that there was a positive association between ERRα and VEGF expression in cancer tissues (P < 0.05). We showed that ERRα stimulated the VEGF promoter activity in cervical cancer cell lines. We further showed the overexpression and knockdown of ERRα-regulated VEGF expression level by real-time polymerase chain reaction. Moreover, we showed that ERRα and VEGF knockdown by small interfering RNA or an inverse agonist of ERRα, XCT 790, could suppress cell growth compared with control cells in cervical cancer. CONCLUSIONS: We have provided compelling evidence that ERRα affects VEGF expression and tumor growth in cervical cancer. These results justify further investigation into the use of ERRα as a therapeutic target for patients with uterine cervical cancer.
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Carcinoma/genética , Receptores de Estrógenos/genética , Receptores de Estrógenos/fisiología , Neoplasias del Cuello Uterino/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Carcinoma/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/fisiología , ARN Interferente Pequeño/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
NADPH oxidase is a superoxide (O(2)(-))-generating enzyme first identified in phagocytes that shows bactericidal activities. It has been reported that O(2)(-) is also produced in an NADPH-dependent manner in non-phagocytes. In the last decade, non-phagocyte-type NADPH oxidases have been identified, and the catalytic subunit NOX family has been found to be composed of five homologs, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. These NOX proteins have distinct features in dependency on other components for maximal enzymatic activity, tissue distribution, expressional regulation, and physiological functions. This review summarized the distinct characteristics of NOX family proteins, especially focusing on their functions and mechanisms of their expressional regulation.
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Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , NADPH Oxidasas/fisiología , Superóxidos/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , NADP/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/genéticaRESUMEN
The present review focuses primarily on the studies we made in recent years to improve the understanding of the molecular mechanisms of PGF2alpha-induced hypertrophy of Vascular Smooth Muscle Cells (VSMC). In this review, we will summarize the recent findings in the context of the PGF2alpha signaling pathway in three parts: PGF2alpha binding to its receptor, transactivation of EGF receptor, two independent signaling transduction pathways increasing the expression of NOX1 gene.