The cellular resistance against oxidative stress (H2O2) is independent of neutral trehalase (Ntc1p) activity in Candida albicans

The protective role of trehalose against oxidative stress caused by hydrogen peroxide in Candida albicans has been investigated in the homozygous mutant ntc1Delta/ntc1Delta, disrupted in the NTC1 gene, which encodes the neutral (cytosolic) trehalase (Ntc1p). After a severe oxidative exposure (50 mM H(2)O(2)), both parental (CAI-4) and ntc1Delta/ntc1Delta exponential-phase cells stored large amounts of intracellular trehalose. In turn, the degree of cell survival was roughly equivalent in both strains, although slightly higher in ntc1Delta/ntc1Delta cultures. The mechanism of 'adaptive tolerance' was functional in the two strains. Thus, a gently oxidative pretreatment (5 mM H(2)O(2)) increased the recovery of cellular viability when it was followed by a severe challenge (50 mM H(2)O(2)); this phenomenon was accompanied by a significant elevation of the endogenous trehalose content. Oxidative stress also induced specific activation of the antioxidant enzymes catalase and glutathione reductase upon gentle oxidative treatment (5 mM H(2)O(2)), whereas superoxide dismutase activity was only activated upon prolonged exposure. Taken together, these results strongly suggest that in C. albicans neutral trehalase activity does not play an essential role in the protective response against oxidative stress. They also suggest that a diminished Ntc1p activity might favour the growth of C. albicans cells subjected to a strong oxidative exposure.     

Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage

Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks.     

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.     

The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.     

Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.     

In vivo protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress mediated by 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH) or Fe(2+)/H(2)O(2): insight into mechanisms of neuroprotection and relevance to oxidative stress-related neurodegenerative disorders

Ferulic acid ethyl ester (FAEE) is an ester derivative of ferulic acid, the latter known for its anti-inflammatory and antioxidant properties. Previous studies from our laboratory have shown that ferulic acid protects synaptosomal membrane system and neuronal cell culture systems against hydroxyl and peroxyl radical oxidation. FAEE is lipophilic and is able to penetrate lipid bilayer. Previous studies reported that FAEE reduces Alzheimer's amyloid beta peptide Abeta(1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture by direct radical scavenging and by inducing certain antioxidant proteins. In the present study we tested the hypothesis that FAEE would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes were isolated from the gerbils that were previously injected intraperitoneally (i.p.) with FAEE or DMSO and were treated with oxidants, Fe(2+)/H(2)O(2) or 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH). Synaptosomes isolated from the gerbil previously injected i.p. with FAEE and treated with Fe(2+)/H(2)O(2) and AAPH showed significant reduction in reactive oxygen species (ROS), levels of protein carbonyl, protein bound 4-hydroxynonenal (HNE, a lipid peroxidation product), and 3-nitrotyrosine (3-NT, another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to Fe(2+)/H(2)O(2) or AAPH induced oxidative stress in synapotosomes isolated from the brain of gerbils that were previously injected with DMSO. The synaptosomes isolated from gerbil pre-injected with FAEE and subsequently treated with AAPH or Fe(2+)/H(2)O(2) showed induction of heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) but reduced inducible nitric oxide synthase (iNOS) levels. These results are discussed with reference to potential use of this lipophilic antioxidant phenolic compound in the treatment of oxidative stress-related neurodegenerative disorders.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H(2)O(2), interferon (INF)-gamma and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H(2)O(2), INF-gamma or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H(2)O(2), IL-6 and IL-10. Also, H(2)O(2) induced serine (Ser)-727 phosphorylation of Stat1 but not Stat3. The degree of Tyr-701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr-701 but not Ser-727, Stat1 phosphorylation. Also, AG490 inhibited Tyr-705 Stat3 phosphorylation induced by H(2)O(2) and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-gamma, IL-6 and H(2)O(2). These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H(2)O(2), which further shows that H(2)O(2) and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H(2)O(2)-induced cell death.     

Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564-6569). We confirmed that mutants lacking a V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2Delta mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.     

A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals

The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins. Here we report a new role for trehalose in protecting cells against oxygen radicals. Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron). When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly. Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells. Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2). Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage. After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells. Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger. Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.     

蓝莓提取物对H2O2诱导的体外培养的大鼠海马神经元氧化损伤的保护作用

目的：探讨蓝莓提取物对过氧化氢致大鼠海马神经元氧化应激损伤的减缓作用。方法：将培养7d的海马神经细胞分为8组：①过氧化氢组（H2O2）：培养液中加入50μmol/L的H2O2,作用24h。②不同剂量蓝莓提取物预处理组（BE＋H2O2）：在加入H2O2前24h,分别加入0.01、0.1、1.0、10.0、20.0和40.0μg/mlBE。③空白对照组（Control）：处理步骤同上组,但每次处理物质均为等量的培养液。通过测定细胞存活率和上清液中乳酸脱氢酶（LDH）的活性确定减轻海马神经元损伤的蓝莓提取物的适宜浓度。并检测细胞内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性以及细胞凋亡率的变化。结果：①蓝莓提取物组（0.1,1.0和10.0μg/mlBE）LDH活性显著低于H2O2组,细胞存活率由H2O2组的57.44%分别上升至78.42%、87.71%、72.40%;1μg/ml蓝莓提取物组对H2O2诱导的海马神经细胞氧化应激损伤的保护作用最好。②1μg/ml蓝莓提取物组海马神经细胞培养上清液中MDA含量及细胞凋亡率显著低于H2O2组,SOD活性显著高于H2O2组。结论：适宜剂量的蓝莓提取物对氧化应激损伤的海马神经元有一定的保护作用,其机制可能与抑制海马神经元凋亡、增强神经细胞的抗氧化功能有关。     

Hydrogen peroxide (H2O2) and methyl-beta-cyclodextrin (MPCD) down regulate caveolin expression in human lens epithelial cells (HLECs)

Oxidative stimulation induced by hydrogen peroxide (H2O2) on human epithelial cells (HLECs) was performed to observe the effects on cell viability, caveolin expression, and cholesterol depletion in HLECs caused by methyl-beta-cyclodextrin (MbetaCD) was also studied. SRA01/04 HLECs were exposed to H2O2 or MbetaCD of various concentrations and durations. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the effect of H2O2 on the viability of SRA01/04 HLECs. The distributions of caveolins after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Immunoblotting was performed to analyze alterations of caveolins expression. We observed that the viability of SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer was significantly reduced (*p < 0.05, F = 11.63). Laser scanning microscopy showed immunofluorescent caveolins in SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer, caveolins were largely confined to intracellular domains. Western blots showed both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5 or 1.0 mM H2O2 for 30 min were significantly reduced, compared with the untreated (*p < 0.05, F = 6.149, or *p < 0.05, F = 14.489 respectively). In addition, the membrane and total caveolin protein level after treated with 0.1 mM (*p < 0.05, F = 6.843, or *p < 0.05, F = 7.944 respectively) H2O2 for different durations also down regulated. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. This study concludes that the responses of HLECs to oxidative stress may include down regulation of caveolin and phosphorylation of caveolin-1 on Tyr14, and that MbetaCD also down regulates caveolin while depleting cholesterol in HLECs.     

Base excision repair enzyme endonuclease III suppresses mutagenesis caused by 8-hydroxy-dGTP

To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP were introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei) and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. The mutations induced by exogenous 2-hydroxy-dATP were similar in all of the strains tested. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 2-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.     

Changes in gene expression induced by H(2)O(2) in cardiac myocytes

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.     

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK)

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.