Differential vascular functions of Nox family NADPH oxidases

PURPOSE OF REVIEW: Reactive oxygen species have been implicated in the initiation and progression of atherosclerosis. Reactive oxygen species can oxidize lipoproteins, limit the vascular availability of antiatherosclerotic nitric oxide and promote vascular expression of cytokines and adhesion molecules. Nox proteins of the NADPH oxidase family are prominent sources of vascular reactive oxygen species, and Nox protein-dependent reactive oxygen species production has been linked to atherogenesis. Recently, significant progress has been made in the understanding of differences among the Nox proteins. RECENT FINDINGS: Nox proteins exhibit cell-specific expression patterns and divergent molecular mechanisms controlling activity have been identified for individual Nox proteins. These aspects may relate to cellular activation, differentiation, proliferation, angiogenesis and gene expression, and may also be modulated by the functional states of the vessel such as endothelial dysfunction: in quiescent vessels, Nox proteins contribute to signal transduction and to the physiological responses to growth factors such as vascular endothelial growth factor or thrombin. Excessive Nox-dependent reactive oxygen species formation in vascular disease such as hyperlipidemia or diabetes, however, largely contributes to vascular dysfunction resulting in defective angiogenesis and inflammatory activation. SUMMARY: Reactive oxygen species, specifically generated by individual Nox proteins, act as secondary messengers. Selective inhibition of Nox proteins might be a novel approach to prevent and treat cardiovascular diseases.     

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes

Background  

NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.     

Involvement of Nox2 and Nox4 NADPH oxidases in early brain injury after subarachnoid hemorrhage

Oxidative stress is responsible for a poor prognosis of subarachnoid hemorrhage (SAH) patients. Nox2 has been shown to participate in SAH-induced early brain injury (EBI). Nox4 is another major subtype of Nox family widely expressed in central nervous system (CNS). Here, we investigated the role of Nox4 and whether there was a synergistic effect of Nox2 and Nox4 in SAH-induced EBI. Clinical brain biopsies of four patients with traumatic brain injury (TBI) and perihematomal brain tissue from six subjects with SAH were examined. Gp91ds-tat (a specific inhibitor of Nox2), GKT137831 (a specific inhibitor of Nox4), and apocynin (a non-specific Nox inhibitor) were used to test the role of Nox2 and Nox4. The protein levels of Nox2 and Nox4 were elevated in rat neurons and astrocytes at 12?h after SAH, and in cultured brain microvascular endothelial cells at 24?h after exposure to OxyHb. Similarly, there were higher Nox2 and Nox4 protein levels in perihematomal neurons and astrocytes in SAH patients than that in brain tissue from subjects with TBI. In SAH rat model, gp91ds-tat and GKT137831 could reduce SAH-induced neuronal death and degeneration, whereas apocynin did not induce a more intense neuroprotection. Consistently, in in vitro SAH model, siRNA-mediated silencing of Nox2 and Nox4 suppressed the OxyHb-induced neuronal apoptosis, whereas Nox2 and Nox4 co-knockdown also did not show a remarkable overlay effect. In conclusion, Nox4 should contribute to the pathological processes in SAH-induced EBI, and there was not an overlay effect of Nox2 inhibition and Nox4 inhibition on preventing SAH-induced EBI.     

Involvement of Rac1 in activation of multicomponent Nox1- and Nox3-based NADPH oxidases

Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47phox and p67phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22phox binding sites (SH3 domains) of the Nox organizers (p47phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.     

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.     

Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases

Reactive oxygen species (ROS) are important signal transduction molecules in ligand-induced signaling, regulation of cell growth, differentiation, apoptosis and motility. Recently NADPH oxidases (Nox) homologous to Nox2 (gp91phox) of phagocyte cytochrome b558 have been identified, which are an enzymatic source for ROS generation in epithelial cells. This study was undertaken to delineate the requirements for ROS generation by Nox4. Nox4, in contrast to other Nox proteins, produces large amounts of hydrogen peroxide constitutively. Known cytosolic oxidase proteins or the GTPase Rac are not required for this activity. Nox4 associates with the protein p22phox on internal membranes, where ROS generation occurs. Knockdown and gene transfection studies confirmed that Nox4 requires p22phox for ROS generation. Mutational analysis revealed structural requirements affecting expression of the p22phox protein and Nox activity. Mechanistic insight into ROS regulation is significant for understanding fundamental cell biology and pathophysiological conditions.     

The use of model systems to study biological functions of Nox/Duox enzymes

ROS (reactive oxygen species; including superoxide and H202) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H202 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches.     

NOX family NADPH oxidases: not just in mammals

NOX family NADPH oxidases are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). NOX enzymes in mammalian organisms have received most attention. However, NOX enzymes are widely distributed in different kingdoms of life. While they are not found in prokaryotes and most unicellular eukaryotes, they are present in fungi, plants, and animals. The identity of the ancestral NOX is not known, but most likely it: (i) possessed the basic NOX structure consisting of 6 transmembrane domains (containing two assymmetrical hemes) and a long cytoplasmic C-terminal (containing the FAD and NADPH binding sites); and (ii) emerged before the divergence of life into fungi, plants, and animals. During evolution, acquisition of a Ca(2+)-binding EF hand domain by an ancestral NOX, led to NOX5-like isoforms. DUOX isoforms presumably developed from a NOX5-like isoform through the additional acquisition of a peroxidase homology domain. The expression pattern of NOX enzymes is specific to each kingdom of life. Fungi express only ancestral-type isoforms, and plants only NOX5-like isoforms. NOX expression patterns in animals are complex and ancestral NOXes, NOX5-like isoforms and DUOXes are generally found. But there are exceptions; for example rodents lack NOX5 and Caenorhabditis elegans expresses only DUOXes. Biological functions of NOX enzymes include, among others, host defense, post-translational modification of proteins, and regulation cell growth and differentiation. In summary, the invention of NOX enzymes early in the development of life was a success story: there is no evidence of multicellular life without NOX enzymes.     

Regulation of Nox and Duox enzymatic activity and expression

In recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide, and other metabolites) are produced in biological systems. Rather than being simply a by-product of aerobic metabolism, it is now recognized that specific enzymes--the Nox (NADPH oxidase) and Duox (Dual oxidase) enzymes--seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis, and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes.     

Innate host defense: Nox and Duox on phox's tail

Over the past decade, the capacity of non-phagocytic cells to produce superoxide has been largely documented. As in the case of the well-characterized phagocytic cells context, superoxide formation in non-phagocytic cells depends on the activity of membrane bound NADPH oxidase enzymes. Six mammalian homologues of the classical phagocytic Nox2 enzyme have been described to date, named Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2, which exhibit similar and specific structure and regulation features. Their biological functions are still poorly understood and were initially mostly deduced from their specific tissue expression profiles. However, recent functional data have emerged that suggest the involvement of several of these isoforms in the innate host response to invading microorganisms, including innate immune and proinflammatory responses. Nox2 is well characterized as a key player in the bacterial killing process that takes place in phagocytes. Here, we will discuss the recent advances that revealed alternative roles of Nox1, Nox4, Duox1 and Duox2 isoforms in other aspects of the innate host defense. In particular, we will focus on their implication in the signaling following pathogen recognition by toll like receptors and in the modulation of dendritic cell functions, two key aspects of innate immunity. Moreover, the potential role of Nox/Duox enzymes in the innate response to virus infections will be discussed.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses

The human genome is continuously exposed to such potentially deleterious agents as the highly reactive molecules known as reactive oxygen species (ROS). ROS include superoxide anions (O(2)(-)) and hydrogen peroxide (H(2)O(2)). Over the last decade, the ROS-generating NADPH oxidases (NOXs) have been recognized as one of the main sources of ROS production in numerous human cell types. In addition to regulating normal physiological redox-dependent processes, the NOXs are involved in cellular oxidative stress. In contrast to the other NOXs, the NADPH oxidase NOX4 exists in the immediate environment of the nucleus. There is accumulating evidence for the involvement of NOX4-derived ROS in genomic instability as well as in cancer and other inflammation-related diseases. We recently showed that NOX4 plays a critical role in oncogenic Ras-induced DNA damage. Here we reflect upon the growing awareness of NOX4, review its role in inducing genomic instability, and call attention to its possible role in nuclear redox-sensitive mechanisms underlying DNA-damage signaling and repair.     

The microbicidal and cytoregulatory roles of NADPH oxidases

Despite their prominent microbicidal roles, NADPH oxidases (NOXs) have been recently found to regulate a wide variety of physiological activities. Through generation of reactive oxygen species (ROS), NOXs actively participate in cellular activities, including NET formation, inflammasome activation and wound sensing. The microbicidal and cytoregulatory roles of NOXs are contrasted in this review.     

The role of NADPH oxidases in diabetic cardiomyopathy

Systemic changes during diabetes such as high glucose, dyslipidemia, hormonal changes and low grade inflammation, are believed to induce structural and functional changes in the cardiomyocyte associated with the development of diabetic cardiomyopathy. One of the hallmarks of the diabetic heart is increased oxidative stress. NADPH-oxidases (NOXs) are important ROS-producing enzymes in the cardiomyocyte mediating both adaptive and maladaptive changes in the heart. NOXs have been suggested as a therapeutic target for several diabetic complications, but their role in diabetic cardiomyopathy is far from elucidated. In this review we aim to provide an overview of the current knowledge regarding the understanding of how NOXs influences cardiac adaptive and maladaptive processes in a “diabetic milieu”. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H₂O₂ remain obscure. Here we demonstrate sustained live responses of H₂O₂ to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H₂O₂. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H₂O₂. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H₂O₂, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H₂O₂, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.     

A burst of plant NADPH oxidases

Reactive oxygen species (ROS) are highly reactive molecules able to damage cellular components but they also act as cell signalling elements. ROS are produced by many different enzymatic systems. Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), are the most thoroughly studied enzymatic ROS-generating systems and our understanding of their involvement in various plant processes has increased considerably in recent years. In this review we discuss their roles as ROS producers during cell growth, plant development and plant response to abiotic environmental constraints and biotic interactions, both pathogenic and symbiotic. This broad range of functions suggests that RBOHs may serve as important molecular 'hubs' during ROS-mediated signalling in plants.     

N-linked glycosylation of the superoxide-producing NADPH oxidase Nox1

Nox1 is a membrane-integrated protein that belongs to the Nox family of superoxide-producing NADPH oxidases. Here we show that human Nox1 undergoes glycosylation at Asn-162 and Asn-236 in the second and third extracellular loops, respectively. Simultaneous threonine substitution for these residues completely abrogates the glycosylation, but does not prevent Nox1 from forming a heterodimer with p22^phox, trafficking to the cell surface, or producing superoxide. In the absence of p22^phox, Nox1 is transported to the plasma membrane mainly as a form with high mannose N-glycans, although their conversion into complex N-glycans is induced by expression of p22^phox. These findings indicate that glycosylation and subsequent N-glycan maturation of Nox1 are both dispensable for its cell surface recruitment. Superoxide production by unglycosylated Nox1 is largely dependent on p22^phox, which is abrogated by glutamine substitution for Pro-156 in p22^phox, a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22^phox directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.