Kazumi Iwata

Nox1 Is Involved in Angiotensin II–Mediated Hypertension: A Study in Nox1-Deficient Mice

Background— Increased production of reactive oxygen species (ROSs) by angiotensin II (Ang II) is involved in the initiation and progression of cardiovascular diseases. NADPH oxidase is a major source of superoxide generated in vascular tissues. Although Nox1 has been identified in vascular smooth muscle cells as a new homolog of gp91phox (Nox2), a catalytic subunit of NADPH oxidase, the pathophysiological function of Nox1-derived ROSs has not been fully elucidated. To clarify the role of Nox1 in Ang II–mediated hypertension, we generated Nox1-deficient (−/Y) mice. Methods and Results— No difference in the baseline blood pressure was observed between Nox1+/Y and Nox1−/Y. Infusion of Ang II induced a significant increase in mean blood pressure, accompanied by augmented expression of Nox1 mRNA and superoxide production in the aorta of Nox1+/Y, whereas the elevation in blood pressure and production of superoxide were significantly blunted in Nox1−/Y. Conversely, the infusion of pressor as well as subpressor doses of Ang II did elicit marked hypertrophy in the thoracic aorta of Nox1−/Y similar to Nox1+/Y. Administration of a nitric oxide synthase inhibitor (L-NAME) to Nox1+/Y did not affect the Ang II–mediated increase in blood pressure, but it abolished the suppressed pressor response to Ang II in Nox1−/Y. Finally, endothelium-dependent relaxation and the level of cGMP in the isolated aorta were preserved in Nox1−/Y infused with Ang II. Conclusions— A pivotal role for ROSs derived from Nox1/NADPH oxidase was suggested in the pressor response to Ang II by reducing the bioavailability of nitric oxide.

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.

Potential role of the NADPH oxidase NOX1 in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice.

Although NADPH oxidase 1 (NOX1) has been shown to be highly expressed in the gastrointestinal tract, the physiological and pathophysiological roles of this enzyme are not yet fully understood. In the present study, we investigated the role of NOX1 in the pathogenesis of intestinal mucositis induced by the cancer chemotherapeutic agent 5-fluorouracil (5-FU) in mice. Intestinal mucositis was induced in Nox1 knockout (Nox1KO) and littermate wild-type (WT) mice via single, daily administration of 5-FU for 5 days. In WT mice, 5-FU caused severe intestinal mucositis characterized by a shortening of villus height, a disruption of crypts, a loss of body weight, and diarrhea. In Nox1KO mice, however, the severity of mucositis was significantly reduced, particularly with respect to crypt disruption. The numbers of apoptotic caspase-3- and caspase-8-activated cells in the intestinal crypt increased 24 h after the first 5-FU administration but were overall significantly lower in Nox1KO than in WT mice. Furthermore, the 5-FU-mediated upregulation of TNF-α, IL-1β, and NOX1 and the production of reactive oxygen species were significantly attenuated in Nox1KO mice compared with that in WT mice. These findings suggest that NOX1 plays an important role in the pathogenesis of 5-FU-induced intestinal mucositis. NOX1-derived ROS production following administration of 5-FU may promote the apoptotic response through upregulation of inflammatory cytokines.

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.

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation.

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.