Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.     

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H₂O₂), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H₂O₂ for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-γ. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-γ increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-γ. Both stimuli increased H₂O₂ synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H₂O₂ production, whereas Duox2 siRNA markedly reduced basal H₂O₂ production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H₂O₂ production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H₂O₂ levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H₂O₂ synthesis despite the presence of greater amounts of Duox1.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

Potential role of Noxes in the protection of mucosae: H2O2 as abacterial repellent

Duox proteins are members of the NADPH oxidase (Nox) family and are responsible for hydrogen peroxide (H₂O₂) production by various tissue types including bronchial and intestinal mucosae. The antimicrobial killing role of H₂O₂ in leukocytes and macrophages is generally considered as the paradigm of its function. We investigated here the positive role of H₂O₂ in the prevention of cellular invasion by Salmonella. We show that H₂O₂, under conditions that preserved bacterial growth, has a repellent effect on Salmonella motility on agar plates. In addition, H₂O₂ produced by PCCl3, a rat thyroid cell line, reduces bacterial invasion of the cells by around 40%. To test whether the observed phenotype is attributable to H₂O₂ production, we constructed a CHO stable cell line expressing Duox2 protein at the cell surface (CHO-D2). The transfected cells produce a high amount of H₂O₂. Upon infection with Salmonella, the invasion of CHO-D2 cells was reduced by up to 60%. In both PCCl3 and CHO expressing Duox2 cells, normal invasion was restored upon incubation with catalase. Our data suggest that H₂O₂ at reduced concentrations acts as a repellent for bacteria, keeping them away from cells, a situation that could naturally prevent mucosal cells infection in vivo.     

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

In the thyroid gland Duox2-derived H₂O₂ is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2^thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H₂O₂, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

Hydrogen peroxide primes heart regeneration with a derepression mechanism

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H₂O₂ acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H₂O₂ (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H₂O₂ formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H₂O₂ by catalase overexpression markedly impaired cardiac regeneration while exogenous H₂O₂ rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H₂O₂ is an essential and sufficient signal in this process. Mechanistically, elevated H₂O₂ destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H₂O₂ plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H₂O₂ potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.     

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.     

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca²⁺]_i) mobilization and H₂O₂ generation. These results indicated that a testosterone-GPRC6A complex is required for activation of G_q protein, IP₃ generation, and [Ca²⁺]_i mobilization, leading to Duox1 activation. H₂O₂ generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H₂O₂ generation through the activation of Duox1.     

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H₂O₂) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H₂O₂ may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H₂O₂-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H₂O₂, inhibited S. oralis cytotoxicity, and H₂O₂ alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H₂O₂ production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H₂O₂ induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H₂O₂ is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.     

Ovarian Dual Oxidase (Duox) Activity Is Essential for Insect Eggshell Hardening and Waterproofing

In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H₂O₂ and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H₂O₂ that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H₂O₂ generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H₂O₂ injection into the female hemocoel. Taken together, our results show that Duox-generated H₂O₂ fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.     

Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H₂O₂ produced a rapid influx of Mn²⁺ and an increase of intracellular calcium. The H₂O₂ was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca²⁺ to H₂O₂-treated cells suspended in a free Ca²⁺/EGTA buffer resulted in a great increase in [Ca²⁺]_i reflecting influx of Ca²⁺ across the cell membrane. The increase of intracellular calcium was inhibited by Ni²⁺, La³⁺, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H₂O₂ by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H₂O₂). In this study, the functions of NO and H₂O₂ after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H₂O₂ induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H₂O₂ generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H₂O₂ in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H₂O₂ to activate CuZnSOD and APX, which further decreased H₂O₂ level and reduced the cell death caused by wounding.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.