Nox Family Of NADPH Oxidases Research Articles

Progressive Alterations of Mitochondrial Function in the Kidney and Heart During the Development of Salt‐Induced Hypertension

IntroductionHypertension is a multi‐factorial disease and a leading cause of morbidity and mortality globally. Nearly half of these individuals are sensitive to dietary salt intake with the kidney and oxidative stress playing a key role in this disease leading to eventual failure of both the kidney and heart. Although oxidative stress and mitochondrial dysfunction in the kidney and heart have been studied in various models of hypertension, progressive alterations of mitochondrial function and development of oxidative stress in these two organs have not been studies in parallel during the development of hypertension. The goal of the present study was to investigate progressive alterations in mitochondrial energy metabolism in both the kidney and heart concurrently in the Dahl salt‐sensitive (SS) rats during the development of hypertension when rats were switched from a 0.4% NaCl (LS) to a 4% NaCl (HS) diet.MethodMitochondria were isolated from the ventricles of the heart and outer medulla (OM) and cortex of the kidney of SS rats fed a LS diet since weaning and studied at days 3, 7, 14 & 21 of a HS diet feeding. Oxygen consumption rates (OCR) were measured in mitochondria energized with pyruvate + malate as substrates for 3 different respiratory states using an Oroboros Oxygraph‐2k respirometer. This includes a) leak state (energized mitochondria in the absence of ADP), b) ADP‐stimulated state, and c) uncoupled state (energized mitochondria in the presence of an uncoupler FCCP).ResultsA biphasic pattern of ADP‐stimulated OCR with progressive uncoupling was observed in both the kidney (OM & cortex) and heart mitochondria. In kidney, mitochondrial efficiency for ATP synthesis was increased in the early phase of hypertension (3 & 7 days) but was severely compromised in the established phase of hypertension (14 & 21 days). Cardiac mitochondria also showed a similar pattern, however the reduced mitochondrial efficiency for ATP synthesis occurred at a later time, specifically at day 21. This decreased mitochondrial efficiency was hypothesized to be associated with higher levels of uncoupling, ROS production, and oxidative stress in both organs due to mitochondrial ROS‐cellular ROS crosstalk. To test this, experiments were performed in SS rats with double knock out (DKO) of the Nox family of NADPH oxidases: Nox4 and p67phox cytosolic subunit of Nox2 (SSp67phox−/−/Nox4−/−) and determined the role of mitochondrial ROS in stimulating cellular ROS production, particularly by membrane Noxs, and vice versa. Specifically, the DKO SS rats were fed a HS diet and isolated mitochondrial OCR was studied at days 7 and 14. DKO SS rats fed a HS diet showed no significant differences in mitochondrial OCR as compared to the control group (LS diet) in the kidney (OM & cortex) and heart.ConclusionOur data suggest that a HS diet in SS rats results initially in increased efficiency of renal cortical and medullary mitochondrial energy production during the 1st week of a HS diet followed by an enhanced ROS production with mitochondrial uncoupling and reduced efficiency of ATP production by the 3rd week. Similar changes occurred in the heart but 1–2 weeks following the changes observed in the kidney.Support or Funding InformationP01‐GM066730, P01‐HL116264

Dual Character of Reactive Oxygen, Nitrogen, and Halogen Species: Endogenous Sources, Interconversions and Neutralization.

Oxidative stress resulting from accumulation of reactive oxygen, nitrogen, and halogen species (ROS, RNS, and RHS, respectively) causes the damage of cells and biomolecules. However, over the long evolutionary time, living organisms have developed the mechanisms for adaptation to oxidative stress conditions including the activity of the antioxidant system (AOS), which maintains low intracellular levels of RONS (ROS and RNS) and RHS. Moreover, living organisms have adapted to use low concentrations of these electrophiles for the regulation of cell functions through the reversible post-translational chemical modifications of redox-sensitive amino acid residues in intracellular effectors of signal transduction pathways (protein kinases and protein phosphatases), transcription factors, etc. An important fine-tuning mechanism that ensures involvement of RONS and RHS in the regulation of physiological processes is interconversion between different reactive species. This review focuses on the complex networks of interacting RONS and RHS types and their endogenous sources, such as NOX family of NADPH oxidases, complexes I and III of the mitochondrial electron transport chain, NO synthases, cytochrome P450-containing monooxygenase system, xanthine oxidoreductase, and myeloperoxidases. We highlight that kinetic parameters of reactions involving RONS and RHS determine the effects of these reactive species on cell functions. We also describe the functioning of enzymatic and non-enzymatic AOS components and the mechanisms of RONS and RHS scavenging under physiological conditions. We believe that analysis of interactions between RONS and relationships between different endogenous sources of these compounds will contribute to better understanding of their role in the maintenance of cell redox homeostasis as well as initiation and progression of diseases.

NADPH oxidase 4 and its role in the cardiovascular system.

The heart relies on complex mechanisms that provide adequate myocardial oxygen supply in order to maintain its contractile function. At the cellular level, oxygen undergoes one electron reduction to superoxide through the action of different types of oxidases (e.g. xanthine oxidases, uncoupled nitric oxide synthases, NADPH oxidases or NOX). Locally generated oxygen-derived reactive species (ROS) are involved in various signaling pathways including cardiac adaptation to different types of physiological and pathophysiological stresses (e.g. hypoxia or overload). The specific effects of ROS and their regulation by oxidases are dependent on the amount of ROS generated and their specific subcellular localization. The NOX family of NADPH oxidases is a main source of ROS in the heart. Seven distinct Nox isoforms (NOX1–NOX5 and DUOX1 and 2) have been identified, of which NOX1, 2, 4 and 5 have been characterized in the cardiovascular system. For the purposes of this review, we will focus on the effects of NADPH oxidase 4 (NOX4) in the heart.

NADPH Oxidase 4 Expression Is Increased through TRPV4 Channel in D. farinae‐Induced Airway Remodeling and TGF‐β1‐Mediated Fibroblast Differentiation into Myofibroblasts

NADPH oxidase 4 (NOX4) is a member of the NOX family of NADPH oxidases, encoded by NOX4 gene and has been implicated in TGF‐β1‐induced human cardiac fibroblast to myofibroblast differentiation. Further, NOX4 has been shown to potentiate lung fibroblast differentiation in vitro and its transcript level is enhanced in fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) patients compared to normal ones. Our lab has previously identified that Transient Receptor Potential Vanilloid 4 (TRPV4) TRPV4, a mechano‐sensitive ion channel is involved in D. farinae‐induced airway remodeling during asthma in vivo and myofibroblast differentiation in vitro. However, the role of NOX4 in asthma, its interaction with TRPV4 and the mechanism of action still remains elusive. In the present study, we show that NOX4 is up‐regulated during lung remodeling in asthma and fibroblast differentiation in vitro in a TRPV4‐dependent pathway.Using NOX4 antagonist and NOX4 siRNA, we demonstrate that NOX4 is involved in TGFβ‐1 induced α smooth muscle actin (α SMA) and fibronectin (FN) expression in human lung fibroblasts (HLF). Further, TGFβ‐1 treatment up‐regulated NOX4 gene expression as determined by real time PCR.. Further, to investigate the effect of TRPV4 on NOX4, we inhibited TRPV4 channel using a pharmacological antagonist RN 1734, and this led to a reduction in TGFβ‐1‐mediated NOX4 expression and differentiation. Interestingly, NOX4 transcripts were up‐regulated in lung tissues in response to D. farinae in an asthma model. More importantly, TRPV4 KO mice which were protected from D. farinae‐induced asthma also exhibited lower levels of NOX4 gene expression, suggesting that TRPV4 promotes airway remodeling through NOX4. Our results unravel a unique role for NOX4 in TRPV4 mediated fibroblast differentiation and lung remodeling and can be used as a new therapeutic target in asthma treatment.

Dexmedetomidine Protects against Transient Global Cerebral Ischemia/Reperfusion Induced Oxidative Stress and Inflammation in Diabetic Rats

BackgroundTransient global cerebral ischemia/reperfusion (I/R) is a major perioperative complication, and diabetes increases the response of oxidative stress and inflammation induced by I/R. The objective of this study was to determine the protective effect of dexmedetomidine against transient global cerebral ischemia/reperfusion induced oxidative stress and inflammation in diabetic rats.MethodsSixty-four rats were assigned into four experimental groups: normoglycemia, normoglycemia + dexmedetomidine, hyperglycemia, and hyperglycemia + dexmedetomidine and all subsequent neurological examinations were evaluated by a blinded observer. Damage to the brain was histologically assessed using the TUNEL staining method while western blotting was used to investigate changes in the expression levels of apoptosis-related proteins as well as the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1). Water content in the brain was also analyzed. In addition, hippocampal concentrations of malondialdehyde (MDA) and Nox2 (a member of the Nox family of NADPH oxidases), and the activity of superoxide dismutase and catalase were analyzed. Finally, changes in serum concentrations of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were detected.ResultsResults showed that diabetes increased brain water content, the number of apoptotic neurons, early neurological deficit scores, oxidative stress (MDA and Nox2) and inflammation (pro-inflammatory cytokines including TNF-α and IL-6) levels following transient global I/R injury, but that these symptoms were attenuated following administration of dexmedetomidine.ConclusionsThese findings suggest that dexmedetomidine can significantly alleviate damage resulting from I/R, and this mechanism may be related to a reduction in both oxidative stress and inflammation which is normally associated with I/R.

NADPH oxidases: an overview from structure to innate immunity-associated pathologies.

Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), play important roles in immunity, cell growth, and cell signaling. In excess, however, ROS are lethal to cells, and the overproduction of these molecules leads to a myriad of devastating diseases. The key producers of ROS in many cells are the NOX family of NADPH oxidases, of which there are seven members, with various tissue distributions and activation mechanisms. NADPH oxidase is a multisubunit enzyme comprising membrane and cytosolic components, which actively communicate during the host responses to a wide variety of stimuli, including viral and bacterial infections. This enzymatic complex has been implicated in many functions ranging from host defense to cellular signaling and the regulation of gene expression. NOX deficiency might lead to immunosuppression, while the intracellular accumulation of ROS results in the inhibition of viral propagation and apoptosis. However, excess ROS production causes cellular stress, leading to various lethal diseases, including autoimmune diseases and cancer. During the later stages of injury, NOX promotes tissue repair through the induction of angiogenesis and cell proliferation. Therefore, a complete understanding of the function of NOX is important to direct the role of this enzyme towards host defense and tissue repair or increase resistance to stress in a timely and disease-specific manner.

Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor

Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47phox, NOXO1/p67phox, and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1–Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isolated Nox4 produces H2O2 and 10% forms superoxide. The kinetic mechanism of H2O2 formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H2O2; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H2O2 formed. Critically, Nox4 has an unusually high Km for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2–3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H2O2 as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO2.

NADPH Oxidases in Redox Regulation of Cell Adhesion and Migration

Adhesion and migration induced by cytokines or growth factors are well-organized processes in cellular motility. Reactive oxygen species (ROS) are specifically produced by the Nox family of NADPH oxidases. The signal transduction of migration and adhesion depends on ROS produced by Nox enzymes and factors that initiate migration and adhesion and stimulate cellular ROS formation. The identification of molecular targets of ROS formation in the signal transduction of adhesion and migration is still in its beginnings, but a site and isoform-specific contribution of Nox enzymes to this process becomes apparent. Nox-derived ROS, therefore, act as second messengers that are specifically modifying signaling proteins involved in adhesion and migration. Individual protein targets of Nox-mediated redox signaling in different cell types and tissues will be identified. Isoform-specific Nox inhibitors will be developed to modulate the ROS-dependent component of migration and adhesion. These compounds might be suited to elicit differential effects between pathophysiologic and physiologic adhesion and migration.

NADPH Oxidases: A Perspective on Reactive Oxygen Species Production in Tumor Biology

Reactive oxygen species (ROS) promote genomic instability, altered signal transduction, and an environment that can sustain tumor formation and growth. The NOX family of NADPH oxidases, membrane-bound epithelial superoxide and hydrogen peroxide producers, plays a critical role in the maintenance of immune function, cell growth, and apoptosis. The impact of NOX enzymes in carcinogenesis is currently being defined and may directly link chronic inflammation and NOX ROS-mediated tumor formation. Increased interest in the function of NOX enzymes in tumor biology has spurred a surge of investigative effort to understand the variability of NOX expression levels in tumors and the effect of NOX activity on tumor cell proliferation. These initial efforts have demonstrated a wide variance in NOX distribution and expression levels across numerous cancers as well as in common tumor cell lines, suggesting that much remains to be discovered about the unique role of NOX-related ROS production within each system. Progression from in vitro cell line studies toward in vivo tumor tissue screening and xenograft models has begun to provide evidence supporting the importance of NOX expression in carcinogenesis. A lack of universally available, isoform-specific antibodies and animal tumor models of inducible knockout or over-expression of NOX isoforms has hindered progress toward the completion of in vivo studies. In vivo validation experiments and the use of large, existing gene expression data sets should help define the best model systems for studying the NOX homologues in the context of cancer.

Abstract 621: The G Protein-Coupled Estrogen Receptor GPER is Required for Induction of Nox Activity in Mice

The Nox family of NADPH oxidases is a major source of reactive oxygen species (ROS) such as superoxide (O 2 - ) and hydrogen peroxide (H 2 O 2 ), which importantly influence cardiovascular function. Vasoactive agonists such as angiotensin II can stimulate Nox-dependent ROS production, whereas endogenous estrogens act as antioxidants under certain conditions. We hypothesized that the intracellular transmembrane G protein-coupled estrogen receptor GPER is involved in the regulation of Nox activity. Using GPER -/- and WT mice, we determined the effect of angiotensin II on O 2 - production in isolated vascular smooth muscle cells (VSMC) by lucigenin-enhanced chemiluminescence, and on contractile responses in renal arteries prepared for recording of isometric tension. The contribution of NADPH oxidases was assessed using its specific inhibitor gp91ds-tat (3 μmol/L). Angiotensin II (100 nmol/L) acutely increased O 2 - production in WT VSMC, an effect blocked by gp91ds-tat (6.6±2.1 AU vs. -0.8±2.2 AU after 20 min, n =4-5, p <0.05). Surprisingly, angiotensin II was unable to induce O 2 - formation in GPER -/- VSMC (-0.2±1.0 AU vs. 6.6±2.1 AU, n =5, p <0.05 vs. WT). Following administration of NADPH (100 μmol/L), maximal NADPH oxidase activity in GPER -/- VSMC was also reduced by 73% (442±185 AU vs. 1617±468 AU, n =5, p <0.05 vs. WT), indicating that the induction of Nox activity is largely impaired by GPER deficiency. In line with these findings, contraction to angiotensin II was 1.9-fold less potent in GPER -/- renal arteries (35±4% vs. 66±11% of contraction to 60 mmol/L KCl, n =5, p <0.05 vs. WT); this difference was abolished by gp91ds-tat, which decreased the contractile response in WT by 47% ( p <0.05), but had no effect in GPER -/- arteries. In conclusion, these data indicate that GPER is required for induction of vascular Nox activity in mice, and suggest that estrogen-dependent regulation of ROS production may not be limited to antioxidative effects.

Regulation of NOXO1 activity through reversible interactions with p22 and NOXA1.

Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22phox for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22phox. Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22phox and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

Upregulation of Nox4 by Hypertrophic Stimuli Promotes Apoptosis and Mitochondrial Dysfunction in Cardiac Myocytes

NADPH oxidases are a major source of superoxide (O(2)(-)) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood. The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart. Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging. Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O(2)(-) production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone- and diphenyleneiodonium-sensitive O(2)(-) production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4. Upregulation of Nox4 by hypertrophic stimuli and aging induces oxidative stress, apoptosis and LV dysfunction, in part because of mitochondrial insufficiency caused by increased O(2)(-) production and consequent cysteine oxidation in mitochondrial proteins.

Molecular mechanisms of hypertension: role of Nox family NADPH oxidases

Molecular mechanisms contributing to the pathoetiology of hypertension are complex, involving many interacting systems such as signaling through G protein-coupled receptors, the renin-angiotensin system, vascular inflammation and remodeling, vascular senescence and aging and developmental programming, as highlighted in the current issue of the journal. Common to these systems is NADPH oxidase-derived reactive oxygen species (ROS). This editorial highlights current concepts relating to the production of ROS in hypertension and focuses on the Nox family NADPH oxidases, major sources of free radicals in the cardiovascular and renal systems. ROS play a major role as intracellular signaling molecules to regulate normal biological cellular responses. In pathological conditions, loss of redox homeostasis contributes to vascular oxidative damage. Recent evidence indicates that specific enzymes, the Nox family of NADPH oxidases, have the sole function of generating ROS in a highly regulated fashion in physiological conditions, and that in disease states, hyperactivation of Noxes contributes to oxidative stress and consequent cardiovascular and renal injury. The Nox family comprises seven members, Nox1-Nox7. Nox1, Nox2 (gp91phox-containing NADPH oxidase), Nox4 and Nox5 have been identified in the cardiovascular-renal systems and have been implicated in the pathophysiology of cardiovascular and renal disease. Noxes, which are differentially regulated in hypertension, are major sources of cardiovascular and renal oxidative stress. This has evoked considerable interest because of the possibilities that therapies targeted against specific Nox isoforms to decrease ROS generation or to increase nitric oxide availability or both may be useful in minimizing vascular injury and renal dysfunction, and thereby prevent or regress target organ damage associated with hypertension.

Effects of Quercetin on Gene and Protein Expression of NOX and NOS after Myocardial Ischemia and Reperfusion in Rabbit

Previous studies have suggested that reactive oxygen species (ROS), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) are involved in the pathophysiology of myocardial ischemia-reperfusion injury (MIRI). The NOX family of NADPH oxidases share the capacity to generate superoxide and ROS. Several studies have demonstrated that quercetin possesses a protective effect against MIRI. Our aim is to investigate the effects of quercetin on NOX2, eNOS, and iNOS after MIRI in rabbits. New Zealand rabbits were subjected to 30 min of myocardial ischemia followed by 12 h of reperfusion. They were then randomly assigned to four experimental groups: control, I/R (ischemia/reperfusion), quercetin (Que), I/R + Que. Gene and protein expression of NOX2, eNOS, and iNOS were compared. Both in real-time PCR and in the Western blotting studies, myocardial ischemia-reperfusion-induced NOX2 and iNOS expression were enhanced (P < 0.01) but eNOS mRNA and protein expression in I/R hearts were not significantly different from those in control (P < 0.01). Administration of quercetin reduced NOX2, eNOS, and iNOS mRNA and protein expression both in control and in I/R heart (P < 0.01). Gene and protein expression of NOX2 and iNOS were increased after MIRI. Quercetin not only inhibited myocardial ischemia-reperfusion-induced NOX2 and iNOS mRNA and protein expression but also inhibited eNOS mRNA and protein expression.

NOX family NADPH oxidases in liver and in pancreatic islets: a role in the metabolic syndrome and diabetes?

The incidence of obesity and non-esterified ('free') fatty acid-associated metabolic disorders such as the metabolic syndrome and diabetes is increasing dramatically in most countries. Although the pathogenesis of these metabolic disorders is complex, there is emerging evidence that ROS (reactive oxygen species) are critically involved in the aberrant signalling and tissue damage observed in this context. Indeed, it is now widely accepted that ROS not only play an important role in physiology, but also contribute to cell and tissue dysfunction. Inappropriate ROS generation may contribute to tissue dysfunction in two ways: (i) dysregulation of redox-sensitive signalling pathways, and (ii) oxidative damage to biological structures (DNA, proteins, lipids, etc.). An important source of ROS is the NOX family of NADPH oxidases. Several NOX isoforms are expressed in the liver and pancreatic beta-cells. There is now evidence that inappropriate activation of NOX enzymes may damage the liver and pancreatic beta-cells. In the context of the metabolic syndrome, the emerging epidemic of non-alcoholic steatohepatitis is thought to be NOX/ROS-dependent and of particular medical relevance. NOX/ROS-dependent beta-cell damage is thought to be involved in glucolipotoxicity and thereby leads to progression from the metabolic syndrome to Type 2 diabetes. Thus understanding the role of NOX enzymes in liver and beta-cell damage should lead to an increased understanding of pathomechanisms in the metabolic syndrome and diabetes and may identify useful targets for novel therapeutic strategies.

Positive regulation of the NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F

The generation of reactive oxygen species (ROS) by the NOX family of NADPH oxidases is known to be involved in the regulation of many physiological cellular functions. Unlike other members of this family, NOX4 generates ROS constitutively without the need for a stimulus. The activity of NOX4 is known to be regulated, at least in part, at the level of mRNA expression. However, nothing is known of the molecular mechanisms which underlie its transcriptional regulation. We have therefore determined the transcriptional initiation site of NOX4 in vascular smooth muscle cells (VSMC) and identified NOX4 genomic sequences necessary to effect high levels of expression of a linked luciferase reporter gene in both rat and mouse VSMCs. A potential binding site for members of the E2F family of transcription factors was identified, and electrophoretic mobility-shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays confirmed that this site binds E2F1 both in vitro and in vivo. siRNA against E2F1 decreased NOX4 promoter activity, while site-specific mutation of the core-binding site both downregulated the NOX4 promoter and abolished transregulation by E2F1. These data therefore demonstrate that E2F factor(s) are positive regulators of NOX4 transcription in VSMCs.

Regulation of NOX1 expression by GATA, HNF-1α, and Cdx transcription factors

NOX1, a member of the NOX family of NADPH oxidases, is expressed primarily in colon epithelium, where it may function in host defense and growth regulation. We investigated factors responsible for its transcriptional regulation in vitro and its expression in vivo. Analysis of promoter constructs in the CaCo2 cell line identified a complex element between - 422 and - 291 critical for promoter activity. This element contained four sites that bound GATA-4, -5, and -6 in vitro with varied affinities. One site also bound the caudal-related homeodomain proteins Cdx1 and Cdx2, whereas another also bound hepatocyte nuclear factor-1α (HNF-1α). GATA-6, HNF-1α, and Cdx2 also bound to this region in the intact chromatin of CaCo2 cells. These factors demonstrated cooperativity when transactivating the NOX1 promoter. NOX1 mRNA was detected in human colon epithelial cells along the crypt-villus axis. A gradient of NOX1 mRNA expression was seen in the colons of normal as well as germ-free mice, with significantly higher levels in distal compared with proximal segments. The expression gradients of NOX1 mRNA in the colon paralleled those of GATA-6, HNF-1α, and Cdx1. These data indicate that developmental, tissue-restricted transcription factors play a key role in NOX1 regulation in vivo.

The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the phagocyte NADPH oxidase itself (NOX2/gp91(phox)), the homologs are now referred to as the NOX family of NADPH oxidases. These enzymes share the capacity to transport electrons across the plasma membrane and to generate superoxide and other downstream reactive oxygen species (ROS). Activation mechanisms and tissue distribution of the different members of the family are markedly different. The physiological functions of NOX family enzymes include host defense, posttranlational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. NOX enzymes also contribute to a wide range of pathological processes. NOX deficiency may lead to immunosuppresion, lack of otoconogenesis, or hypothyroidism. Increased NOX activity also contributes to a large number or pathologies, in particular cardiovascular diseases and neurodegeneration. This review summarizes the current state of knowledge of the functions of NOX enzymes in physiology and pathology.

NADPH oxidases: New kids on the block

Reactive oxygen species (ROS) play a pivotal role in many physiological processes including host defense, hormone biosynthesis, fertilization and cellular signaling. Altered production of ROS has been implicated in the development of immunodeficiency, hypothyroidism and cardiovascular pathologies. In the last few years, several enzymes were identified at the molecular level, which are now thought to be responsible for ROS production observed in diverse tissues. These enzymes show a high degree of homology to the phagocytic NADPH oxidase and are now designated the Nox family of NADPH oxidases. This review updates our knowledge on six new members of the Nox family: Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2.

The NADPH Oxidase NOX4 Drives Cardiac Differentiation: Role in Regulating Cardiac Transcription Factors and MAP Kinase Activation

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.