Masakazu Ibi

Reactive Oxygen Species Derived from NOX1/NADPH Oxidase Enhance Inflammatory Pain

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1−/Y), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1+/Y, pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1−/Y. Concomitantly, PMA-induced translocation of PKCε was markedly perturbed in Nox1−/Y or Nox1+/Y DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCε, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCε but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCε in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

NOX1/nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase promotes proliferation of stellate cells and aggravates liver fibrosis induced by bile duct ligation.

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 p27kip1, 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 p27kip1 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.

NOX1/NADPH oxidase is involved in endotoxin-induced cardiomyocyte apoptosis

The functional significance of NOX1/NADPH oxidase in the heart has not been explored due to its low expression relative to other NOX homologs identified so far. We aimed to clarify the role of NOX1/NADPH oxidase in the septic heart by utilizing mice deficient in the Nox1 gene (Nox1(-/Y)). Sepsis was induced by intraperitoneal administration of lipopolysaccharides (LPS: 25mg/kg) or cecal ligation and puncture (CLP) surgery. A marked elevation of NOX1 mRNA was demonstrated in cardiac tissue, which was accompanied by increased production of reactive oxygen species (ROS). In Nox1(-/Y) treated with LPS, cardiac dysfunction and survival were significantly improved compared with wild-type mice (Nox1(+/Y)) treated with LPS. Concomitantly, LPS-induced cardiomyocyte apoptosis and activation of caspase-3 were alleviated in Nox1(-/Y). The level of phosphorylated Akt in cardiac tissue was significantly lowered in Nox1(+/Y) but not in Nox1(-/Y) treated with LPS or that underwent CLP surgery. Increased oxidation of cysteine residues of Akt and enhanced interaction of Akt with protein phosphatase 2A (PP2A), a major phosphatase implicated in the dephosphorylation of Akt, were demonstrated in LPS-treated Nox1(+/Y). These responses to LPS were significantly attenuated in Nox1(-/Y). Taken together, ROS derived from NOX1/NADPH oxidase play a pivotal role in endotoxin-induced cardiomyocyte apoptosis by increasing oxidation of Akt and subsequent dephosphorylation by PP2A. Marked up-regulation of NOX1 may affect the risk of mortality under systemic inflammatory conditions.

Involvement of NOX1/NADPH Oxidase in Morphine-Induced Analgesia and Tolerance

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 [35S]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 [35S]GTPγS-binding was observed in Nox1+/Y but not in Nox1−/Y. No difference in the maximal binding and affinity of [3H]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.

Superoxide dismutase activity in organotypic midbrain–striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity

We have previously demonstrated that dopaminergic neurons in midbrain–striatum slice co‐cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain–striatum co‐cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2′‐(hydroxynitrosohydrazono)bis‐ethanamine (NOC‐18) and 3‐morpholinosydnonimine (SIN‐1). The cytotoxicity of NMDA (30 µm) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor Nω‐nitro‐l‐arginine (100 µm), whereas the toxicity in co‐cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co‐cultures than those in single cultures. Single cultures and co‐cultures did not show appreciable differences in the number or distribution of NOS‐containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn‐SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co‐cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.

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/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-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.

Myocyte enhancer factor 2B is involved in the inducible expression of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F2α and platelet‐derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1 gene. RT‐PCR and 5′‐rapid amplification of cDNA ends‐based analyses revealed a novel 5′‐terminal exon of the rat NOX1 gene located approximately 28 kb upstream of the exon containing the start codon. Both PGF2α and PDGF enhanced the transcriptional activity of the − 3.6 kb 5′‐flanking region of the NOX1 gene in A7r5 cells, a rat vascular smooth muscle cell line. A PGF2α‐response element was located between −146 and −125 in the 5′‐flanking region containing a consensus binding site for myocyte enhancer factor 2 (MEF2), to which binding of MEF2 was augmented by PGF2α. Gene silencing of MEF2B by RNA interference significantly suppressed the expression of NOX1, while silencing of activating transcription factor (ATF)‐1, previously implicated in up‐regulation of NOX1, abolished the PGF2α‐ or PDGF‐induced expression of MEF2B. These results indicate that superoxide production in vascular smooth muscle cells is regulated by the ATF‐1–MEF2B cascade by induction of the expression of the NOX1 gene.

Deficiency of NOX1/Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Oxidase Leads to Pulmonary Vascular Remodeling

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 &agr;-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-1&agr; and HIF-2&agr;, 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.

Sp3 transcription factor is crucial for transcriptional activation of the human NOX4 gene

NOX is the catalytic subunit of NADPH oxidase, the superoxide‐generating enzyme. Among several isoforms of NOX, NOX4 is abundantly expressed in various tissues. To clarify the mechanisms of constitutive and ubiquitous expression of NOX4, the promoter activities of the human NOX4 gene were analyzed by reporter assays. The 5’‐flanking and non‐coding regions of the human NOX4 gene are known to contain multiple GC bases. Among them, three GC‐boxes containing putative Sp/Klf‐binding sites, which were not found in rodent genes, were suggested to be essential for the basal expression of the NOX4 gene in SH‐SY5Y and HEK293 cells. Electrophoresis mobility shift assays demonstrated that Sp1 and Sp3 could bind to GC‐boxes at positions −239/−227 and +69/+81 in these cells. Chromatin immunoprecipitation assays showed that Sp1 and Sp3 could also bind to GC‐boxes at positions −239/−227 and +69/+81 in vivo. The promoter activity of the NOX4 gene was reduced in SH‐SY5Y and HEK293 cells by transfection of an anti‐Sp3 short hairpin RNA‐expression plasmid. Taken together, these results suggest that Sp3 plays a key role in the expression of NOX4 in various cell lineages in humans.

Clioquinol induces DNA double-strand breaks, activation of ATM, and subsequent activation of p53 signaling

Clioquinol, a Cu²⁺/Zn²⁺/Fe²⁺ chelator/ionophor, was used extensively in the mid 1900s as an amebicide for treating indigestion and diarrhea. It was eventually withdrawn from the market because of a link to subacute myelo-optic neuropathy (SMON) in Japan. The pathogenesis of SMON, however, is not fully understood. To clarify the molecular mechanisms of clioquinol-induced neurotoxicity, a global analysis using DNA chips was carried out on human neuroblastoma cells. The global analysis and quantitative PCR demonstrated that mRNA levels of p21(Cip1), an inhibitor of cyclins D and E, and of GADD45α, a growth arrest and DNA damage-inducible protein, were significantly increased by clioquinol treatment in SH-SY5Y and IMR-32 neuroblastoma cells. Activation of p53 by clioquinol was suggested, since clioquinol induced phosphorylation of p53 at Ser15 to enhance its stabilization. The phosphorylation of p53 was inhibited by KU-55933, an inhibitor of ataxia-telangiectasia mutated kinase (ATM), but not by NU7026, an inhibitor of DNA-dependent protein kinase (DNA-PK). Clioquinol in fact induced phosphorylation of ATM and histone H2AX, a marker of DNA double-strand breaks (DSBs). These results suggest that clioquinol-induced neurotoxicity is mediated by DSBs and subsequent activation of ATM/p53 signaling.