Salvianolic acid B inhibits hydrogen peroxide-induced endothelial cell apoptosis through regulating PI3K/Akt signaling

Background

Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H₂O₂) is implicated in the pathogenesis of cerebrovascular disorders.

Methodology and Principal Findings

By examining the effect of Sal B on H₂O₂-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H₂O₂-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H₂O₂ induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) blocked ERK activation caused by H₂O₂ and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H₂O₂-induced apoptosis, suggesting that Sal B prevents H₂O₂-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.

Significance

Our findings provide the first evidence that H₂O₂ induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H₂O₂-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H₂O₂ generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.

     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H₂O₂ and cDNA microarray technique was used to produce gene expression profiles. We found that H₂O₂ treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF- B, ERK, JNK, p38, PKC and INF- . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.Y. Zhang and C.C. Fong contributed equally to this work.     

Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling

Reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelial (RPE) cells, which is known as one major cause of age-related macular degeneration. Salvianolic acid A (Sal A) is the main effective aqueous extract of Salvia miltiorrhiza. The aim of this study was to test the potential role of Sal A against oxidative stress in cultured RPE cells and to investigate the underlying mechanistic signaling pathways. We observed that Sal A significantly inhibited hydrogen peroxide (H₂O₂)-induced primary and transformed RPE cell death and apoptosis. H₂O₂-stimulated mitogen-activated protein kinase activation, ROS production, and subsequent proapoptotic AMP-activated protein kinase activation were largely inhibited by Sal A. Further, Sal A stimulation resulted in a fast and dramatic activation of Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, followed by phosphorylation, accumulation, and nuclear translocation of the NF-E2-related factor 2 (Nrf2), along with increased expression of the antioxidant-response element-dependent gene heme oxygenase-1 (HO-1). Both Nrf2 and HO-1 were required for Sal A-mediated cytoprotective effect, as Nrf2/HO-1 inhibition abolished Sal A-induced beneficial effects against H₂O₂. Meanwhile, the PI3K/Akt/mTORC1 chemical inhibitors not only suppressed Sal A-induced Nrf2/HO-1 activation, but also eliminated its cytoprotective effect in RPE cells. These observations suggest that Sal A activates the Nrf2/HO-1 axis in RPE cells and protects against oxidative stress via activation of Akt/mTORC1 signaling.     

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells

Abstract

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and hydroxyl radical (HO^?), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO^? than with H₂O₂. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H₂O₂ or HO^? stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.     

Cell Hypertrophy and MEK/ERK Phosphorylation are Regulated by Glyceraldehyde-Derived AGEs in Cardiomyocyte H9c2 Cells

Diabetic cardiomyopathy has been shown to promote hypertrophy, leading to heart failure. Recent studies have reported a correlation between diabetic cardiomyopathy and oxidative stress, suggesting that the accumulation of advanced glycation end products (AGEs) induces the production of reactive oxygen species (ROS). In a clinical setting, AGEs have been shown to increase the risk of cardiovascular disease; however, the relationship between AGEs and cardiac hypertrophy remains unclear. This study sought to identify the role of AGEs in cardiac hypertrophy by treating H9c2 cells with glyceraldehyde-derived AGEs (200 μg/ml) or H₂O₂ (50 μM) for 96 h. Our results demonstrate that AGEs significantly increased protein levels and cell size. These effects were effectively blocked with PD98059 (10 μM; MEK/ERK inhibitor) pretreatment, suggesting that AGEs caused cell hypertrophy via the MEK/ERK pathway. We then treated cells with AGEs and H₂O₂ for 0–120 min and employed the Odyssey infrared imaging system to detect MEK/ERK phosphorylation. Our results show that AGEs up-regulated MEK/ERK phosphorylation. However, this effect was blocked by NAC (5 mM; ROS inhibitor), indicating that AGEs regulate MEK/ERK phosphorylation via ROS. Our findings suggest that glyceraldehyde-derived AGEs are closely related to cardiac hypertrophy and further identify a molecular mechanism underlying the promotion of diabetic cardiomyopathy by AGEs.     

Indirubin-3-Oxime Prevents H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H₂O₂)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H₂O₂ exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H₂O₂. In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H₂O₂-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H₂O₂-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H₂O₂-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling

Excessive generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases, including atherosclerosis, hypertension, and vascular complications of diabetes. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully characterized. Hydrogen peroxide (H₂O₂), a ROS, has been shown to activate several signaling protein kinases, such as extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (PKB) in different cell types, notably in vascular smooth muscle cells. Because these pathways regulate cellular mitogenesis, migration, proliferation, survival, and death responses, their aberrant activtion has been suggested to be a potential mechanism of ROS-induced pathologies. The upstream elements responsible for H₂O₂-induced ERK1/2 and PKB activation remain poorly characterized, but a potential role of receptor and nonreceptor protein tyrosine kinases (PTKs) as triggers that initiate such events has been postulated. Therefore, the aim of this review is to highlight the involvement of receptor and nonreceptor PTKs in modulating H₂O₂-induced ERK1/2 and PKB signaling.     

Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK–ERK pathway

Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H₂O₂)-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H₂O₂; instead, it inhibited GJIC synergistically with H₂O₂. AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK–ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary antioxidants on carcinogenesis may be paradoxical.     

Saikosaponin-D Reduces H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced PC12 Cell Apoptosis by Removing ROS and Blocking MAPK-Dependent Oxidative Damage

Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H₂O₂-induced apoptosis. The non-toxic level of SSD significantly mitigated the H₂O₂-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H₂O₂-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H₂O₂-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H₂O₂-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H₂O₂-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.     

Arsenic trioxide induces apoptosis through JNK and ERK in human mesothelioma cells

Malignant mesothelioma is an aggressive tumor of serosal surfaces, which is refractory to current treatment options. Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia, and also to inhibit proliferation of several solid tumors including hepatoma, esophageal, and gastric cancer in vitro. Here we found that As₂O₃ inhibited cell viability of a mesothelioma cell line, NCI‐H2052. As₂O₃ induced apoptosis of NCI‐H2052 cells, which was accompanied by activation of c‐Jun NH₂‐terminal kinase (JNK)1/2, extracellular signal‐regulated kinase (ERK)1/2, and caspase‐3. zVAD‐fmk, a broad‐spectrum caspase inhibitor, inhibited As₂O₃‐induced apoptosis and activation of caspase‐3, but not that of JNK1/2 and ERK1/2. Small interfering RNAs (siRNAs) targeting JNK1/2 suppressed As₂O₃‐induced caspase‐3 activation and apoptosis, indicating that JNK1/2 regulate As₂O₃‐induced apoptosis though caspase cascade. Furthermore, JNK1 siRNA abrogated As₂O₃‐induced JNK2 phosphorylation and JNK2 siRNA abrogated As₂O₃‐induced JNK1 phosphorylation, suggesting that JNK1 and JNK2 interact with each other. Moreover, JNK1 siRNA, but not JNK2 siRNA, abrogated As₂O₃‐induced ERK1/2 phosphorylation. JNK2 siRNA together with PD98059, a specific MAPK/ERK kinase inhibitor, suppressed As₂O₃‐induced apoptosis more significantly than JNK2 siRNA alone. These results indicated that As₂O₃ induces apoptosis of NCI‐H2052 cells mainly through JNK1/2 activation, and that ERK1/2 is involved in As₂O₃‐induced apoptosis when JNK1/2 are inactivated. J. Cell. Physiol. 226: 762–768, 2011. © 2010 Wiley‐Liss, Inc.     

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.