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.

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. Downregulation 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. INTRODUCTIONReactive oxygen species (ROS) are generated either in a nonregulated manner as side products of several enzymatic systems (e.g., cyclooxygenases, nitric oxide [NO] synthases, mitochondrial cytochromes) or in a regulated way as main products of superoxide producing enzymes, the NADPH oxidases. In the mouse, the family of NADPH oxidases includes NOX1, NOX2 (gp91 phox ), NOX3, and NOX4.Excessive cellular generation of ROS, such as superoxide anions (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ), is potentially destructive and is used by phagocytes to kill invading microorganisms. Under normal conditions, scavenging mechanisms (e.g., superoxide dismutase, catalase, glutathioneglutathione peroxidase system) remove excessive amounts of ROS. Under stress conditions, however, the production of ROS may exceed the reducing capacity of the cell and damage cellular functions. Small amounts of ROS, on the other hand, can function as intracellular second messengers and activate signaling cascades involved in growth and differentiation of many cell types (for review see Rhee, 1999;Laloi et al., 2004). For example, the MAP kinase-signaling pathway is sensitive to ROS (Torres and Forman, 2003). Moreover, distinct signaling pathways have differential sensitivity to oxidative stress, leading to dose-dependent effect on, for example, cardiomyocytes on which ROS can induce hypertrophy or apoptosis (Kwon et al., 2003). Transcription factors such as NF-B, p53, and AP-1 are redox-sensitive and can be directly modified by ROS, providing a link with the control of gene expression (Morel and Barouki, 1999).Cardiac differentiation can be studied by differentiating mouse embryonic stem cells (ESC) into embryoid bodies (EB), where the appearance of spontaneously beating cardiomyocytes is observed after 7-8 d of culture. This system thus provides a unique experimental model to study the role of ROS and ROS-generating enzymes in the regulation of cardiomyocyte growth and differentiation in vitro. Previous reports have shown a link between ROS and...

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 (O2.-) 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.

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