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.

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 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.

ROS-generating oxidases Nox1 and Nox4 contribute to oncogenic Ras-induced premature senescence.

Activated oncogenes induce premature cellular senescence, a permanent state of proliferative arrest in primary rodent and human fibroblasts. Recent studies suggest that generation of reactive oxygen species (ROS) is involved in oncogenic Ras-induced premature senescence. However, the signaling mechanism controlling this oxidant-mediated irreversible growth arrest is not fully understood. Here, we show that through the Ras/MEK pathway, Ras oncogene up-regulated the expression of superoxide-generating oxidases, Nox1 in rat REF52 cells and Nox4 in primary human lung TIG-3 cells, leading to an increase in intracellular level of ROS. Ablation of Nox1 and Nox4 by small interfering RNAs (siRNAs) blocked the RasV12 senescent phenotype including ß-galactosidase activity, growth arrest and accumulation of tumor suppressors such as p53 and p16Ink4a. This suggests that Nox-generated ROS transduce senescence signals by activating the p53 and p16Ink4a pathway. Furthermore, Nox1 and Nox4 siRNAs inhibited both Ras-induced DNA damage response and p38MAPK activation, whereas overexpression of Nox1 and Nox4 alone was able to induce senescence. The involvement of Nox1 in Ras-induced senescence was also confirmed with embryonic fibroblasts derived from Nox1 knockout mice. Together, these findings suggest that Nox1- and Nox4-generated ROS play an important role in Ras-induced premature senescence, which may involve DNA damage response and p38MAPK signaling pathways.

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.

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

Clioquinol, a Cu²âº/Zn²âº/Fe²âº 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.

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-1ß, 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.

Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme.

NADPH oxidase is a superoxide (O(2) (•-))-generating enzyme first identified in phagocytes, essential for their bactericidal activities. Later, in non-phagocytes, production of O(2) (•-) was also demonstrated in an NADPH-dependent manner. In the last decade, several non-phagocyte-type NADPH oxidases have been identified. The catalytic subunit of these oxidases, NOX, constitutes the NOX family. There are five homologs in the family, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. Transgenic or gene-disrupted mice of the NOX family have also been established. NOX/DUOX proteins possess distinct features in the dependency on other components for their enzymatic activities, tissue distributions, and physiological functions. This review summarized the characteristics of the NOX family proteins, especially focused on their functions clarified through studies using gene-modified mice.

A crucial role for Nox 1 in redox-dependent regulation of Wnt-ß-catenin signaling.

Canonical Wnt signaling critically regulates cell fate and proliferation in developmental stages and adult tissues. Redox regulation through nucleoredoxin (NRX) has recently been identified in canonical Wnt signaling. However, the source of reactive oxygen species (ROS) affecting the redox state of NRX remains elusive. Our principal aim in this study was to investigate whether superoxide-generating NADPH oxidase1 (Nox1) is involved in NRX-regulated Wnt signaling in intestinal and colon epithelial cells. Here, we demonstrate that Wnt treatment of mouse intestinal cells induces production of ROS through Nox1. This Nox1 action is regulated by Rac1 GTPase through Wnt-induced activation of the Rac1 guanine nucleotide exchange factor Vav2 by Src-mediated tyrosine phosphorylation. Nox1-generated ROS oxidize and inactivate NRX, thereby releasing the NRX-dependent suppression of Wnt-ß-catenin signaling through dissociation of NRX from Dvl. Nox1 small-interference RNA inhibits cell response to Wnt, including stabilization of ß-catenin, expression of cyclin D1 and c-Myc via the TCF transcription factor, and accelerated cell proliferation. Nox1 mediates Wnt-induced cell growth in colon cancer cells with the normal Wnt pathway, but not in APC-deficient colon cancer cells, which are constitutively active in Wnt signaling. Together, these results suggest the mediating role of Nox1 in redox-dependent regulation of canonical Wnt-ß-catenin signaling and provide further insight into the regulatory mechanism of the Wnt pathway.

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 [(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.

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

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.

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.

NADPH oxidase isoforms and anti-hypertensive effects of atorvastatin demonstrated in two animal models.

Beneficial effects of statins on cardiovascular diseases have been attributed to decreased generation of reactive oxygen species (ROS). We tested the hypothesis that atorvastatin protects against the development of hypertension by reducing levels of NADPH oxidase-derived ROS in two hypertensive animal models. Atorvastatin was given to mice chronically infused with angiotensin (Ang) II or to apolipoprotein E (ApoE)-deficient mice fed a high-fat diet. Increased mean blood pressure (MBP) demonstrated in both animal models was significantly suppressed by atorvastatin with reduced ROS production in the aorta. Treatment with atorvastatin did not alter the mRNA level of NOX1, a catalytic subunit of NADPH oxidase, but decreased the levels of other NOX isoforms, NOX2 and NOX4, in the ApoE-deficient mice fed a high-fat diet. In the Ang II-infused model treated with statin, only the NOX4 mRNA level was reduced. Membrane translocation of Rac1 was significantly reduced in the Ang II-infused mice treated with atorvastatin. Finally, atorvastatin administered to Ang II-infused mice lacking the Nox1 gene elicited an additional decline in MBP compared to Nox1-deficient mice treated with vehicle. Together, these findings suggest that reduced expression and activity of the isoforms of NADPH oxidase, involving NOX1, NOX2, and possibly NOX4, mediate the anti-hypertensive effect of atorvastatin.

Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy.

Megakaryocytes (MKs) undergo an endomitotic cell cycle, leading to polyploidy. We examined the expression of the flavoproteins and oxidative stress-promoting enzymes, NADPH oxidases (Nox's), in MKs because of their known role in promoting the cell cycle. Although the expression of Nox isoforms varies between cell types, they are induced at the mRNA level by mitogenic stimuli. Western blotting or reverse transcription-polymerase chain reaction of purified mouse MKs isolated from thrombopoietin (TPO)-treated bone marrow (BM) cultures indicated high expression of Nox1, a weak expression of Nox4, and no significant expression of Nox2. Immunofluorescence of freshly isolated MKs confirmed strong expression of Nox1 in one-third of MKs, whereas Nox1 staining was detected in nearly all MKs in TPO-stimulated BM cultures. Treatment of mouse BM cultures with Nox inhibitors resulted in accumulation of MKs with low DNA content levels and significant reduction of higher ploidy MKs. Purified, Nox-inhibited MKs showed a notable decrease in the level of the G(1) phase cyclin E, a cyclin associated with MK polyploidy, and its up-regulation restored most of the effect of Nox inhibitors. Hence, this study shows the expression of Nox isoforms in MKs and highlights a potential role of flavoproteins in promoting polyploidization in this lineage.

Receptor activator of nuclear factor-kappaB ligand-induced mouse osteoclast differentiation is associated with switching between NADPH oxidase homologues.

Reactive oxygen species (ROS) have been suggested to regulate receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated osteoclast differentiation. Stimulation of wild-type mouse bone marrow monocyte/macrophage lineage (BMM) cells by RANKL down-regulated NADPH oxidase 2 (Nox2) mRNA expression by half. RANKL reciprocally increased Nox1 mRNA levels and newly induced Nox4 transcript expression. BMM cells from Nox1 knockout (Nox1(-/-)) as well as Nox2(-/-) mice generated ROS in response to RANKL and differentiated into osteoclasts in the same way as wild-type BMM cells, which was assessed by the appearance of tartrate-resistant acid phosphatase-positive, multinucleated cells having the ability to form resorption pits and by the expression of osteoclast marker genes. A small interfering RNA (siRNA) targeting Nox1 or Nox2 failed to inhibit the RANKL-stimulated ROS generation and osteoclast formation in wild-type cells, whereas Nox1 and Nox2 siRNAs significantly suppressed the ROS generation and osteoclast formation in Nox2(-/-) and Nox1(-/-) cells, respectively. We also confirmed that Nox4 siRNA did not affect the RANKL-dependent events in Nox2(-/-) cells, whereas p22(phox) siRNA suppressed the events in both wild-type and Nox1(-/-) cells. Collectively, our results suggest that there may be a flexible compensatory mechanism between Nox1 and Nox2 for RANKL-stimulated ROS generation to facilitate osteoclast differentiation.

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 PKCepsilon was markedly perturbed in Nox1(-/Y) or Nox1(+/Y) DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCepsilon, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCepsilon but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCepsilon in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

The AP-1 site is essential for the promoter activity of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme: Possible involvement of the ERK1/2-JunB pathway.

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. The expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F(2alpha), and platelet-derived growth factor (PDGF). It was reported previously that the inducible expression of NOX1 is governed by the activating transcription factor-1 (ATF-1)-myocyte enhancer factor 2B (MEF2B) cascade downstream of phosphoinositide 3 (PI3) kinase. It was also reported that extracellular signal-regulated kinase (ERK) 1/2 is involved in the expression of NOX1. To further clarify the factors involved in NOX1 induction downstream of ERK1/2, the promoter region of the NOX1 gene was analyzed. A consensus activator protein-1 (AP-1) site was found at -98/-92 in the 5'-flanking region of the rat NOX1 gene. The introduction of mutations at this site abolished PGF(2alpha)-induced transcriptional activation in a luciferase assay. Electrophoresis mobility shift assays demonstrated that PGF(2alpha) and PDGF augmented the binding of JunB to this sequence. PD98059, an inhibitor of MAPK/ERK kinase, suppressed the expression of JunB induced by PGF(2alpha) or PDGF. These results suggest that the ERK1/2-JunB pathway is a key regulator of the inducible expression of the NOX1 gene in vascular smooth muscle cells.

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) F(2alpha) 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 PGF(2alpha) 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 PGF(2alpha)-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 PGF(2alpha). 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 PGF(2alpha)- 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.

The activity of aldose reductase is elevated in diabetic mouse heart.

The importance of aldose reductase (AR) has been implicated in the pathogenesis of diabetic complications, although the alterations in the expression and activity of AR during hyperglycemia in the heart have not been well characterized. We investigated the expression and enzyme activity of AR in a murine diabetic model. Three weeks after the induction of hyperglycemia with streptozotocin, the level of AR mRNA was significantly reduced in the cardiac ventricles of BDF-1 mice. In contrast, the activity of AR was significantly elevated in the heart without any significant change in the protein level. In these mice, the level of cardiac thiobarbituric acid-reactive substances was unaltered, whereas the level of reduced glutathione (GSH) was significantly increased. Daily administration of insulin for 3 weeks completely normalized the level of AR mRNA and the enzyme activity. On the other hand, daily administration of an antioxidant, N-acetylcysteine significantly reduced the level of AR mRNA in the heart with a concomitant elevation in the enzyme activity. These results suggest that the activity of AR in the heart is affected by GSH dynamics. Augmented AR activity at the early stage of hyperglycemia may perturb glycolysis and affect cardiac performance.

Aldose reductase inhibitors improve myocardial reperfusion injury in mice by a dual mechanism.

Aldose reductase (AR) has been implicated in the pathogenesis of diabetic complications, although the clinical efficacy of AR inhibitors has not been clearly proven. To clarify the pathophysiological role of AR in the heart, we investigated effects of AR inhibitors applied either during the pre-ischemic phase, or during the post-ischemic reperfusion phase on ischemia-reperfusion injury in isolated heart from transgenic mice overexpressing human AR. On reperfusion following global ischemia, transgenic mouse hearts exhibited lower left developed pressure, increased release of creatine kinase, and lower ATP content compared with their littermates. When inhibitors of AR were applied during the pre-ischemic phase, they significantly improved deranged cardiac function, creatine kinase release, and ATP content. On the other hand, inhibition of AR during the post-ischemic reperfusion phase did not affect cardiac performance and ATP content, but it significantly attenuated creatine kinase release and the level of thiobarbiturate-reactive substances in transgenic mouse hearts. These results suggest a dual role of AR in ischemia-reperfusion injury. Inhibition of AR during ischemia preserved generation of ATP via glycolysis, whereas inhibition during the reperfusion phase reduced myocardial injury by attenuating oxidative stress elicited by ischemic insult and reoxygenation.