Search

NOX enzymes are major ROS generators in pathological states and represent a promising pharmacological target. In this issue of Chemistry & Biology, Smith and colleagues developed an original and elegant cell-free assay to identify specific Nox2 inhibitors in a high throughput manner., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During evolution, genetic networks are rewired through strengthening or weakening their interactions to develop new regulatory schemes. In the galactose network, the GAL1/GAL3 paralogues and the GAL2 gene enhance their own expression mediated by the Gal4p transcriptional activator. The wiring strength in these feedback loops is set by the number of Gal4p binding sites. Here we show using synthetic circuits that multiplying the binding sites increases the expression of a gene under the direct control of an activator, but this enhancement is not fed back in the circuit. The feedback loops are rather activated by genes that have frequent stochastic bursts and fast RNA decay rates. In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes. Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

In mammals, the NADPH oxidase family of enzymes comprises seven members: NOXs 1-5, DUOX1, and DUOX2. All of these enzymes function to move an electron across cellular membranes, transferring it to oxygen to generate the superoxide anion. This generation of reactive oxygen species has important physiological and pathophysiological roles. NOX5 is perhaps the least well understood of these NOX isoforms, in part because the gene is not present in mice or rats. In recent years, however, there has been a rapid increase in our understanding of the NOX5 gene, the structural and biochemical aspects of the NOX5 enzyme, the role NOX5 plays in health and disease, and the development of novel NOX inhibitors. This review takes a look back at some historical aspects of the discovery of NOX5 and summarizes our current understanding of the enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Myocardial ischemia is a major cause of morbidity and mortality in the world. Although restoration of blood flow after prolonged ischemia is essential for cardiomyocytes salvation and to limit myocardial damage and cardiac dysfunction, reperfusion itself exacerbates myocardial injury. Considerable evidence attributes reactive oxygen species (ROS), produced either by the myocardium itself or by infiltrating inflammatory cells, as an early event in this process. Once produced, ROS can lead to cellular damage through a number of pathways including direct damage to membranes and proteins or indirect damage through the activation of pro-apoptotic pathways. While using antioxidants to scavenge free radicals or targeting the sources of ROS, such as xanthine oxidase, may be potential attractive approaches to reduce myocardial reperfusion injury, clinical trials using antioxidant therapies have been largely disappointing. Neither oxidant scavengers like N-acetylcysteine and vitamins E and C, nor xanthine oxidase inhibitor allopurinol have provided indisputable evidence of a clinical benefit despite numerous favourable studies in animal models. Evidence to support a role of ROS in myocardial injury reperfusion is strong, but the clinical approach used has so far been inadequate. Absence of optimal pharmacology, variation in end-points used and low specificity of the compounds used have often been pointed out. In addition, the efficacy of antioxidants is often evaluated based on indirect biomarkers, which are prone to variation. Thus, clinical trials could be improved by the standardisation of the methods to measure oxidative stress and their impact on prognosis outcome.

Background: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood., Experimental Approach: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins., Key Results: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC₅₀ values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC₅₀ of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively., Conclusions and Implications: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Subanesthetic doses of NMDA receptor antagonist ketamine induce schizophrenia-like symptoms in humans and behavioral changes in rodents. Subchronic administration of ketamine leads to loss of parvalbumin-positive interneurons through reactive oxygen species (ROS), generated by the NADPH oxidase NOX2. However, ketamine induces very rapid alterations, in both mice and humans. Thus, we have investigated the role of NOX2 in acute responses to subanesthetic doses of ketamine. In wild-type mice, ketamine caused rapid (30 min) behavioral alterations, release of neurotransmitters, and brain oxidative stress, whereas NOX2-deficient mice did not display such alterations. Decreased expression of the subunit 2A of the NMDA receptor after repetitive ketamine exposure was also precluded by NOX2 deficiency. However, neurotransmitter release and behavioral changes in response to amphetamine were not altered in NOX2-deficient mice. Our results suggest that NOX2 is a major source of ROS production in the prefrontal cortex controlling glutamate release and associated behavioral alterations after acute ketamine exposure. Prolonged NOX2-dependent glutamate release may lead to neuroadaptative downregulation of NMDA receptor subunits.

The superoxide-generating NADPH oxidase NOX1 is thought to be involved in signaling by the angiotensin II-receptor AT1R. However, underlying signaling steps are poorly understood. In this study, we investigated the effect of AngII on aortic smooth muscle from wild-type and NOX1-deficient mice. NOX1-deficient cells showed decreased basal ROS generation and did not produce ROS in response to AngII. Unexpectedly, AngII-dependent Ca(2+) signaling was markedly decreased in NOX1-deficient cells. Immunostaining demonstrated that AT1R was localized on the plasma membrane in wild-type, but intracellularly in NOX1-deficient cells. Immunohistochemistry and immunoblotting showed a decreased expression of AT1R in the aorta of NOX1-deficient mice. To investigate the basis of the abnormal AT1R targeting, we studied caveolin expression and phosphorylation. The amounts of total caveolin and of caveolae were not different in NOX1-deficient mice, but a marked decrease occurred in the phosphorylated form of caveolin. Exogenous H(2)O(2) or transfection of a NOX1 plasmid restored AngII responses in NOX1-deficient cells. Based on these findings, we propose that NOX1-derived reactive oxygen species regulate cell-surface expression of AT1R through mechanisms including caveolin phosphorylation. The lack cell-surface AT1R expression in smooth muscle could be involved in the decreased blood pressure in NOX1-deficient mice.

NOX NADPH oxidases are electron-transporting membrane enzymes whose primary function is the generation of reactive oxygen species (ROS). ROS produced by NOX enzymes show a variety of biologic functions, such as microbial killing, blood pressure regulation, and otoconia formation. Strong evidence suggests that NOX enzymes are major contributors to oxidative damage in pathologic conditions. Blocking the undesirable actions of NOX enzymes, therefore, is a therapeutic strategy for treating oxidative stress-related pathologies, such as ischemia/reperfusion tissue injury, and neurodegenerative and metabolic diseases. Most currently available NOX inhibitors have low selectivity, potency, and bioavailability, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. This review has two main purposes. First, we describe a systematic approach that we believe should be followed in the search for truly selective NOX inhibitors. Second, we present a critical review of small-molecule NOX inhibitors described over the last two decades, including recently published patents from the pharmaceutical industry. Structures, activities, and in vitro/in vivo specificity of these NOX inhibitors are discussed. We conclude that NOX inhibition is a pertinent and promising novel pharmacologic concept, but that major efforts will be necessary to develop specific NOX inhibitors suited for clinical application.

Background: Social stress leads to oxidative stress in the central nervous system, contributing to the development of mental disorders. Loss of parvalbumin in interneurons is an important feature of these diseases. We studied the role of the superoxide-producing nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) in rats exposed to social isolation., Methods: Male rats were kept for 7 weeks in group or in social isolation (n = 6-10 per group). Behavioral tests, immunohistochemistry, and analysis of NOX2 expression were performed at the end of social isolation. Apocynin was given in the drinking water (5 mg/kg/day)., Results: NOX2 was below detection level in the brains of control animals, whereas it was highly expressed in isolated rats, particularly in nucleus accumbens and prefrontal cortex. Indirect markers of oxidative stress (oxidized nucleic acid 8-hydroxy-2'-deoxyguanosine, redox-sensitive transcription factor c-fos, and hypoxia-inducible factor-1alpha) were increased after social isolation in brain areas with high NOX2 expression. An increase in immunoreactive microglia suggested that oxidative stress could be in part due to NOX2 activation in microglia. In response to social isolation, rats showed increased locomotor activity, decreased discrimination, signs of oxidative stress in neurons, and loss of parvalbumin-immunoreactivity. Treatment of isolated rats with the antioxidant/NOX inhibitor apocynin prevented the behavioral and histopathological alterations induced by social isolation., Conclusions: Our data suggest that NOX2-derived oxidative stress is involved in loss of parvalbumin immunoreactivity and development of behavioral alterations after social isolation. These results provide a molecular mechanism for the coupling between social stress and brain oxidative stress, as well as potential new therapeutic avenues.

NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation., ((c) 2009 Wiley-Liss, Inc.)

Chronic granulomatous disease (CGD) is an immunodeficiency caused by the lack of the superoxide-producing phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, CGD patients not only suffer from recurrent infections, but also present with inflammatory, non-infectious conditions. Among the latter, granulomas figure prominently, which gave the name to the disease, and colitis, which is frequent and leads to a substantial morbidity. In this paper, we systematically review the inflammatory lesions in different organs of CGD patients and compare them to observations in CGD mouse models. In addition to the more classical inflammatory lesions, CGD patients and their relatives have increased frequency of autoimmune diseases, and CGD mice are arthritis-prone. Possible mechanisms involved in CGD hyperinflammation include decreased degradation of phagocytosed material, redox-dependent termination of proinflammatory mediators and/or signaling, as well as redox-dependent cross-talk between phagocytes and lymphocytes (e.g. defective tryptophan catabolism). As a conclusion from this review, we propose the existence of ROS high and ROS low inflammatory responses, which are triggered as a function of the level of reactive oxygen species and have specific characteristics in terms of physiology and pathophysiology.

The expression of human neuronal protein 22 (hNP22) is up-regulated in the superior frontal cortex of chronic alcoholics. hNP22 shares significant homology with a number of proteins implicated in bundling of actin filaments. In addition, it contains domains similar to those found in microtubule-associated proteins. We investigated the ability of hNP22 to induce cytoskeletal changes by overexpression in Chinese hamster ovary cells. Overexpression of hNP22 resulted in process formation in these cells that increased upon treatment with cytochalasin D, an actin depolymerising agent. Transfection of mutant hNP22 containing either a deletion of the putative actin-binding domain or deletion of a consensus protein kinase C (PKC) phosphorylation site (Ser-180) failed to induce process formation. In contrast, a mutation to mimic persistent PKC phosphorylation resulted in a cellular morphology similar to that seen in wild-type hNP22 transfections. This observation suggests that hNP22 requires phosphorylation at Ser-180 by PKC to induce cytoskeletal rearrangements. hNP22 was also observed to colocalise with actin and tubulin in processes of transfected cells. An hNP22-specific antibody specifically immunoprecipitated a complex including tubulin from human brain indicating that hNP22 binds directly to microtubules. Taken together, this data suggests that NP22 is part of a signaling complex that associates with cytoskeletal elements to regulate neuronal morphology.

To understand the function of the myelin oligodendrocyte glycoprotein (MOG), a myelin specific protein of the central nervous system, transgenic mice were produced. The transgene is a fusion gene containing 1.9 kb of murine myelin basic protein promoter, 430 bp of rat MOG cDNA in the reverse orientation and 4.5 kb of human proteolipid protein gene. In spite of high expression of antisense MOG mRNA in the oligodendrocytes, MOG synthesis was not inhibited in transgenic mice. This lack of inhibition of MOG underlines the difficulties encountered with antisense transgenic strategies.