SHH信号通路在海马神经可塑性及相关神经系统疾病中的作用

音猬因子（sonic hedgehog,SHH）是一种分泌蛋白质,可在发育过程中控制神经祖细胞、神经元和神经胶质细胞的形成。研究发现,海马是学习和记忆中至关重要的大脑区域,SHH在海马神经元回路的形成和可塑性中发挥重要作用,可介导海马神经的发生和突触的可塑性调节。海马神经元树突中SHH受体的激活是跨神经元信号通路的组成部分,该信号通路可加速轴突的生长并增强谷氨酸从突触前末端的释放。SHH信号通路转导受损可导致中枢神经系统损伤和相关疾病（如自闭症、抑郁症和神经退行性疾病等）发生。因此,控制SHH信号通路转导,如使用SHH通路抑制剂或激动剂可能有助于相关疾病的治疗。综述了SHH信号通路的海马神经可塑性及其在中枢神经系统发育和相关疾病中的影响,以期为阐明SHH信号转导受损导致的海马神经受损和中枢神经系统相关疾病的机制奠定一定的理论依据。     

The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide

Neurodegenerative diseases, a subset of age-driven diseases, have been known to exhibit increased oxidative stress. The resultant increase in reactive oxygen species (ROS) has long been viewed as a detrimental byproduct of many cellular processes. Despite this, therapeutic approaches using antioxidants were deemed unsuccessful in circumventing neurodegenerative diseases. In recent times, it is widely accepted that these toxic by-products could act as secondary messengers, such as hydrogen peroxide (H₂O₂), to drive important signaling pathways. Notably, mitochondria are considered one of the major producers of ROS, especially in the production of mitochondrial H₂O₂. As a secondary messenger, cellular H₂O₂ can initiate redox signaling through oxidative post-translational modifications (oxPTMs) on the thiol group of the amino acid cysteine. With the current consensus that cellular ROS could drive important biological signaling pathways through redox signaling, researchers have started to investigate the role of cellular ROS in the pathogenesis of neurodegenerative diseases. Moreover, mitochondrial dysfunction has been linked to various neurodegenerative diseases, and recent studies have started to focus on the implications of mitochondrial ROS from dysfunctional mitochondria on the dysregulation of redox signaling. Henceforth, in this review, we will focus our attention on the redox signaling of mitochondrial ROS, particularly on mitochondrial H₂O₂, and its potential implications with neurodegenerative diseases.Subject terms: Post-translational modifications, Neurodegenerative diseases     

Impact of oxidative stress on cellular biomechanics and rho signaling in C2C12 myoblasts

Although cells often can tolerate oxidative environments, abnormal oxidative stress has been identified in inflammation, cardiovascular and neurodegenerative diseases, and aging. The impact of oxidative stress on the cellular biomechanics is poorly understood, however. In this study, we used C2C12 myoblasts to investigate the effect of oxidative stress, mimicked by hydrogen peroxide (H₂O₂), on the cell elasticity (i.e., Young?s modulus), viability, and production of intracellular reactive oxygen species (ROS). To better understand the mechanisms underlying the impact of H₂O₂, we examined various effectors of the Rho signaling pathway, which has been shown to play a key role in the control of cell mechanics. H₂O₂ decreased the cell stiffness in a dose-dependent manner, caused cell death, and reduced the RhoA expression that was accompanied by down-regulation of α-actin, cytoskeleton-membrane linker proteins (ezrin–radixin–moesion proteins), and focal adhesion. Modulating the Rho signaling by using a Rho activator partially restored the cell stiffness, enhanced the cell viability, and decreased the intracellular ROS level, suggesting a potential intervention strategy to maintain the cellular biomechanical homeostasis and rescue cell damage in the threat of oxidative stresses.     

Curcumin attenuates endothelial cell oxidative stress injury through Notch signaling inhibition

Previous studies have demonstrated that Notch signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, our aim was to explore the role of the Notch signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of curcumin during (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). DAPT, a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to study Notch activity. Further, HUVECs were exposed to H₂O₂ in the absence or presence of curcumin. DAPT and Notch1 siRNA significantly inhibited OSI and the expression of Notch1 and Hes1. Curcumin conferred a protective effect on the HUVECs against H₂O₂, which was evidenced by improved cell viability, adhesive ability and migratory ability and a decreased apoptotic index, decreased production of reactive oxygen species (ROS) and a reduction in several biochemical parameters. Immunofluorescence and Western blotting analyses demonstrated that H₂O₂ treatment upregulated the expression of Notch1, Hes1, Caspase3, Bax and cytochrome c downregulated the expression of Bcl2, and treatment with curcumin reversed these effects. We demonstrated for the first time that the inhibition of Notch signaling pathway imparts a protective effect against endothelial OSI. The protective effects of curcumin against OSI are at least in part dependent on Notch1 inhibition.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H₂O₂) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca²⁺-free medium or treated with BAPTA showed reduced glutamate-induced H₂O₂ generation, indicating that H₂O₂ generation is Ca²⁺-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H₂O₂ generation and that activation of NMDA and AMPA receptors is involved in this H₂O₂ generation. The Nox4 siRNA reduced NMDA-induced H₂O₂ production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H₂O₂ production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.     

Flavonoid Baicalein Modulates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Mitogen-Activated Protein Kinases Activation and Cell Death in SK-N-MC Cells

It is believed that ROS-induced oxidative stress triggers numerous signaling pathways which are involved in neurodegenerative diseases, including Alzheimer’s disease. To find the effective drugs for neurodegenerative diseases, the deep delve into molecular mechanisms underlie these diseases is necessary. In the current study, we investigated the effects of flavonoid baicalein on H₂O₂-induced oxidative stress and cell death in SK-N-MC cells. Our results revealed that the treatment of SK-N-MC cells with H₂O₂ led to a decrease in cell viability through phosphorylation and activation of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) pathways followed by increase in Bax/Bcl2 ratio and initiation of caspase-dependent apoptotic pathways. In addition, our results showed that the exposure of SK-N-MC cells to H₂O₂ ended up in reduction of glutathione (GSH) levels of SK-N-MC cells via JNK/ERK-mediated down-regulation of γ-glutamyl-cysteine synthetase (γ-GCS) expression. Our results demonstrated that flavonoid baicalein protected against H₂O₂-induced cell death by inhibition of JNK/ERK pathways activation and other key molecules in apoptotic pathways, including blockage of Bax and caspase-9 activation, induction of Bcl-2 expression and prevention of cell death. Baicalein supported intracellular defense mechanisms through maintaining GSH levels in SK-N-MC cells by the removal of inhibition effects of JNK/ERK pathways from γ-GCS expression. In addition, baicalein attenuated lipid and protein peroxidation and intracellular reactive oxygen species in SK-N-MC cells. In accordance with these observations, baicalein can be a promising candidate in antioxidant therapy and designing of natural-based drug for ROS-induced neurodegenerative disorders.     

New Role of JAK2/STAT3 Signaling in Endothelial Cell Oxidative Stress Injury and Protective Effect of Melatonin

Previous studies have shown that the JAK2/STAT3 signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, we explored the role of the JAK2/STAT3 signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of melatonin against (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity, and the results showed that AG490 and JAK2 siRNA inhibited OSI and the levels of p-JAK2 and p-STAT3. HUVECs were then subjected to H₂O₂ in the absence or presence of melatonin, the main secretory product of the pineal gland. Melatonin conferred a protective effect against H₂O₂, which was evidenced by improvements in cell viability, adhesive ability and migratory ability, decreases in the apoptotic index and reactive oxygen species (ROS) production and several biochemical parameters in HUVECs. Immunofluorescence and Western blotting showed that H₂O₂ treatment increased the levels of p-JAK2, p-STAT3, Cytochrome c, Bax and Caspase3 and decreased the levels of Bcl2, whereas melatonin treatment partially reversed these effects. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in a protective effect against endothelial OSI. The protective effects of melatonin against OSI, at least partially, depend upon JAK2/STAT3 inhibition.     

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H₂O₂). Shh alleviated the apoptosis rate of H₂O₂-induced neurons. Shh also increased neuritogenesis injuried by H₂O₂ in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H₂O₂. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H₂O₂ -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.     

MicroRNA-137-3p Protects PC12 Cells Against Oxidative Stress by Downregulation of Calpain-2 and nNOS

The imbalance between excess reactive oxygen species (ROS) generation and insufficient antioxidant defenses contribute to a range of neurodegenerative diseases. High ROS levels damage cellular macromolecules such as DNA, proteins and lipids, leading to neuron vulnerability and eventual death. However, the underlying molecular mechanism of the ROS regulation is not fully elucidated. Recently, an increasing number of studies suggest that microRNAs (miRNAs) emerge as the targets in regulating oxidative stress. We recently reported the neuroprotective effect of miR-137-3p for brachial plexus avulsion-induced motoneuron death. The present study is sought to investigate whether miR-137-3p also could protect PC12 cells against hydrogen peroxide (H₂O₂) induced neurotoxicity. By using cell viability assay, ROS assay, gene and protein expression assay, we found that PC-12 cells exposed to H₂O₂ exhibited decreased cell viability, increased expression levels of calpain-2 and neuronal nitric oxide synthase (nNOS), whereas a decreased miR-137-3p expression. Importantly, restoring the miR-137-3p levels in H₂O₂ exposure robustly inhibited the elevated nNOS, calpain-2 and ROS expression levels, which subsequently improved the cell viability. Furthermore, the suppressive effect of miR-137-3p on the elevated ROS level under oxidative stress was considerably blunted when we mutated the binding site of calpain-2 targted by miR-137-3p, suggesting the critical role of calpain-2 involving the neuroprotective effect of miR-137-3p. Collectively, these findings highlight the neuroprotective role of miR-137-3p through down-regulating calpain and NOS activity, suggesting its potential role for combating oxidative stress insults in the neurodegenerative diseases.

     

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

Protein disulfide isomerase (PDI), a principal endoplasmic reticulum resident oxidoreductase chaperone, is known to play a role in malignancies. This study aims to explore the molecular mechanism by which PDI regulates endoplasmic reticulum stress and the apoptosis signaling pathway in colorectal cancer (CRC). We determined the expression of PDI in CRC tissues and adjacent normal tissues. Gain- and loss- of function assays were conducted to evaluate the effects of PDI on oxidative stress, endoplasmic reticulum stress, and apoptosis in CRC cells, as reflected by hydrogen peroxide (H₂O₂) level and the expression of related proteins. PDI protein expression was upregulated in CRC tissues. Small molecule inhibitor of PDI or PDI knockdown reduced CRC cell viability and induced apoptosis. Overexpression of wild-type PDI augmented the viability of CRC cells and inhibited endoplasmic reticulum stress response and apoptosis. Small molecule inhibitor of PDI or PDI knockdown increased intracellular H₂O₂ level and activated apoptosis signaling pathway, which could be reversed by wild-type PDI restoration. Moreover, the catalytic active site of C-terminal of PDI was found to be indispensable for the regulatory effects of PDI on H₂O₂ levels, apoptosis and cell viability in CRC cells. Collectively, PDI inhibits endoplasmic reticulum stress and apoptosis of CRC cells through its oxidoreductase activity, thereby promoting the malignancy of CRC.     

miR-146a down-regulation alleviates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H₂O₂-induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H₂O₂ to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H₂O₂-induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H₂O₂-induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H₂O₂-induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H₂O₂-induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H₂O₂-induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.     

Peroxisome proliferator-activated receptor-α activation protects against endoplasmic reticulum stress-induced HepG2 cell apoptosis

Live ischemia–reperfusion injury is associated with endoplasmic reticulum (ER) stress-induced apoptosis. Activation of peroxisome proliferator-activated receptor-α (PPARα) may inhibit hepatocyte apoptosis induced by oxidative stress and protect against liver injury. This study aimed to investigate the effects of PPARα activation, through a specific agonist, on ER stress-induced apoptosis in human liver hepatocellular carcinoma (HepG2) cells. HepG2 cells were challenged with H₂O₂ and treated with WY14643, a selective PPARα agonist, in the presence or absence of the PPARα antagonist of MK886. Cell viable assay (MTT) and immunostaining were used to evaluate cell viability. The level of apoptotic cell death was quantified through Annexin V/PI staining. Alanine aminotransferase, asparatate aminotransferase, and malondialdehyde levels were measured to determine the presence of cellular injury and oxidative stress. RT-PCR and Western blot analysis were used to detect mRNA and protein expression of PPARα, BiP, and CHOP. Immunofluorescence was utilized to determine the intracellular localization of CHOP. H₂O₂ and MK886 both reduced the viability of HepG2 cells, increased oxidative stress and apoptosis, up-regulated the BiP and CHOP expression, and induced CHOP translocation from the cytoplasm to the nucleus. Compared with cells treated with H₂O₂ alone, pre-administration of WY14643 increased cell viability, attenuated apoptosis, improved cell function, down-regulated BiP and CHOP expression and inhibited CHOP translocation. The effects of WY14643 were completely abolished using the MK886 antagonist. PPARα activation protects against H₂O₂-induced HepG2 cell apoptosis. The underlying mechanisms may be associated with its activation to suppress excessive ER stress.     

Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro

Carthamus tinctorius L. (safflower) is one of the most commonly used Chinese herbal medicines to prevent and treat cardiac disease in clinical practice. However, the mechanisms responsible for such protective effects remain largely unknown. In this study, we investigated the anti-myocardial ischemia effects of a purified extract of C. tinctorius (ECT) both in vivo and in vitro. An animal model of myocardial ischemia injury was induced by left anterior descending coronary artery occlusion in adult rats. Pretreatment with ECT (100, 200, 400, 600 mg/kg body wt.) could protect the heart from ischemia injury by limiting infarct size and improving cardiac function. In the in vitro experiment, neonatal rat ventricular myocytes were incubated to test the direct cytoprotective effect of ECT against H₂O₂ exposure. Pretreatment with 100–400 μg/ml ECT prior to H₂O₂ exposure significantly increased cell viability as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECT also markedly attenuated H₂O₂-induced cardiomyocyte apoptosis, as detected by Annexin V and PI double labeling with flow cytometry. The intracellular level of reactive oxygen species (ROS) was shown by 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and ECT pretreatment significantly inhibited H₂O₂-induced ROS increase. We made a preliminary examination of the signaling cascade involved in ECT mediated anti-apoptotic effects. Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) blocked the cytoprotective effect conferred by ECT. Taken together, our findings provide the first evidence that the cardioprotective effects of ECT in myocardial ischemia operate partially through reducing oxidative stress induced damage and apoptosis. The protection is achieved by scavenging of ROS and mediating the PI3K signaling pathway.     

Hydrogen Peroxide Induces Lysosomal Protease Alterations in PC12 Cells

Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H₂O₂. Furthermore, activation of CATD occurred following exposure to H₂O₂ and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H₂O₂ exposure, while inhibition of caspase-3 significantly increased cell viability compared to H₂O₂ administration alone. Collectively, our data suggest that H₂O₂ induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations.     

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.