Botond Banfi

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47 phox and p67 phox subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47 phox and p67 phox are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47 phox homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67 phox homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47 phox was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC(50)>100 muM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O(2)(-) within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform.

Reduced Airway Surface pH Impairs Bacterial Killing in the Porcine Cystic Fibrosis Lung

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO3− transport. Without CFTR, airway epithelial HCO3− secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.

Decreased blood pressure in NOX1-deficient mice

To understand the role of the superoxide‐generating NADPH oxidase NOX1 in the vascular system, we have generated NOX1‐deficient mice. NOX1‐deficient mice had a moderately decreased basal blood pressure. In response to angiotensin II they showed an almost complete loss of the sustained blood pressure response, while the initial increase was conserved. NOX1‐deficient mice showed a marked reduction in aortic media hypertrophy. Angiotensin II‐induced smooth muscle cell proliferation was conserved, but there was a marked decrease in extracellular matrix accumulation. Our results establish a role for NOX1 in blood pressure regulation and vascular angiotensin II response.

Nox2 and Rac1 Regulate H2O2-Dependent Recruitment of TRAF6 to Endosomal Interleukin-1 Receptor Complexes

ABSTRACT Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) have been implicated in the regulation of signal transduction. However, the cellular mechanisms that link Nox activation with plasma membrane receptor signaling remain poorly defined. We have found that Nox2-derived ROS influence the formation of an active interleukin-1 (IL-1) receptor complex in the endosomal compartment by directing the H2O2-dependent binding of TRAF6 to the IL-1R1/MyD88 complex. Clearance of both superoxide and H2O2 from within the endosomal compartment significantly abrogated IL-1β-dependent IKK and NF-κB activation. MyD88-dependent endocytosis of IL-1R1 following IL-1β binding was required for the redox-dependent formation of an active endosomal receptor complex competent for IKK and NF-κB activation. Small interfering RNAs to either MyD88 or Rac1 inhibited IL-1β induction of endosomal superoxide and NF-κB activation. However, MyD88 and Rac1 appear to be recruited independently to IL-1R1 following ligand stimulation. In this context, MyD88 binding was required for inducing endocytosis of IL-1R1 following ligand binding, while Rac1 facilitated the recruitment of Nox2 into the endosomal compartment and subsequent redox-dependent recruitment of TRAF6 to the MyD88/IL-1R1 complex. The identification of Nox-active endosomes helps explain how subcellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.

Electron currents generated by the human phagocyte NADPH oxidase

Electron transport across biological membranes is a well-known feature of bacteria, mitochondria and chloroplasts, where it provides motive forces for vectorial transport processes. In contrast, electron transport is generally not found in the plasma membrane of eukaryotic cells, possibly because it would interfere with electric processes at the plasma membrane. An exception is provided by the phagocyte NADPH oxidase, which generates superoxide () through electron transfer from cytosolic NADPH to extracellular oxygen. The enzyme is essential for host defence, and patients with chronic granulomatous disease, who lack the functional enzyme, suffer from severe infections,. It has been suggested that electron transfer by the NADPH oxidase might be electrogenic. Here we demonstrate, using the whole-cell patch-clamp technique, the generation of electron currents by the NADPH oxidase in human eosinophil granulocytes. The currents were absent in granulocytes of sufferers of chronic granulomatous disease and under conditions of low oxygen. Generation of electron currents across the plasma membrane of eukaryotic cells has not been observed previously and might be — independently of the generation of superoxide — a physiologically relevant function of the phagocyte NADPH oxidase.

Mechanisms of Vascular Smooth Muscle NADPH Oxidase 1 (Nox1) Contribution to Injury-Induced Neointimal Formation

Objective—Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models. Methods and Results—Wire injury–induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1y/− mice, but there was little difference in TgSMCnox1 mice compared with wild-type (WT) mice. Proliferation and migration were reduced in cultured Nox1y/− VSMCs and increased in TgSMCnox1 cells. TgSMCnox1 cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1y/− cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1y/− cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1y/− VSMCs. Conclusions—These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease

Reactive oxygen species (ROS) are at the centre of many physiological and pathological processes. NOX1, a ROS‐producing NADPH oxidase, is highly expressed in the colon but its function in colonic physiology or pathology is still poorly understood. It has been suggested to play a role in host defence, but also in cell growth and possibly malignant transformation. In this study we characterized NOX1 expression in human colon samples derived from healthy control subjects and patients with colon cancer or inflammatory bowel disease (IBD). NOX1 mRNA expression was assessed by dot‐blot hybridization, real‐time PCR and in situ hybridization, using samples derived from surgical specimens from patients undergoing colon resection. In normal tissues, NOX1 expression was low in the ileum, intermediate in the right colon, and high in the left colon (p = 0.0056 right vs. left colon). NOX1 mRNA levels were not influenced by factors linked to colon tumourigenesis, such as age or sex. Moreover, there was no statistical difference in NOX1 expression between samples derived from adenomas, well differentiated or poorly differentiated colon adenocarcinomas. At a cellular level, NOX1 was highly expressed in colon epithelial cells, both within the crypts and on the luminal surface. In addition, a population of lymphocytes, particularly in the appendix, showed NOX1 expression. Lymphocytes in lesions of Crohns disease and ulcerative colitis were also strongly positive for NOX1. In conclusion, NOX1 is an enzyme that is constitutively expressed in colon epithelium and is not associated with tumourigenesis. Its distribution in crypts and on the luminal surface, as well as its left‐to‐right gradient in the colon, suggests a role in host defence function. In addition to the known epithelial localization, we define lymphocytes as a novel site of NOX1 expression, where it may potentially be involved in the pathogenesis of inflammatory bowel diseases. Copyright

A key role for NOX4 in epithelial cell death during development of lung fibrosis.

UNLABELLED The pathogenesis of pulmonary fibrosis is linked to oxidative stress, possibly generated by the reactive oxygen species (ROS) generating NADPH oxidase NOX4. Epithelial cell death is a crucial early step in the development of the disease, followed only later by the fibrotic stage. We demonstrate that in lungs of patients with idiopathic lung fibrosis, there is strong expression of NOX4 in hyperplastic alveolar type II cells. AIM To study a possible causative role of NOX4 in the death of alveolar cells, we have generated NOX4-deficient mice. RESULTS Three weeks after administration of bleomycin, wild-type (WT) mice developed massive fibrosis, whereas NOX4-deficient mice displayed almost normal lung histology, and only little Smad2 phosphorylation and accumulation of myofibroblasts. However, the protective effects of NOX4 deficiency preceded the fibrotic stage. Indeed, at day 7 after bleomycin, lungs of WT mice showed massive increase in epithelial cell apoptosis and inflammation. In NOX4-deficient mice, no increase in apoptosis was observed, whereas inflammation was comparable to WT. In vitro, NOX4-deficient primary alveolar epithelial cells exposed to transforming growth factor-β(1) did not generate ROS and were protected from apoptosis. Acute treatment with the NOX inhibitors also blunted transforming growth factor-β(1)-induced apoptosis. CONCLUSION ROS generation by NOX4 is a key player in epithelial cell death leading to pulmonary fibrosis.

Novel Mechanism of Activation of NADPH Oxidase 5 CALCIUM SENSITIZATION VIA PHOSPHORYLATION

In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC50 of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr494 and Ser498. Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr494 and Ser498 to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.