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Originally published In Press as doi:10.1074/jbc.M601984200 on March 20, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13092-13102, May 12, 2006
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17beta-Estradiol Protects against Oxidative Stress-induced Cell Death through the Glutathione/Glutaredoxin-dependent Redox Regulation of Akt in Myocardiac H9c2 Cells*

Yoshishige Urata{ddagger}12, Yoshito Ihara{ddagger}1, Hiroaki Murata{ddagger}, Shinji Goto{ddagger}, Takehiko Koji§, Junji Yodoi, Satoshi Inoue||, and Takahito Kondo{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, the §Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, the Department of Biological Responses, Institute for Viral Research, Graduate School of Medicine, Kyoto University, 53 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8397, Japan, and the ||Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Received for publication, March 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226–50233). Estrogens, such as 17beta-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor {alpha} (ER{alpha}). However, the role of the ERbeta-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERbeta from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as {gamma}-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both {gamma}-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERbeta is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERbeta-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERbeta. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is a principle cause of the development of aging and diseases such as inflammation, infection, cancer, and cardiovascular disorders (1, 2). Exogenous or endogenous sources of oxidative stress and weakened antioxidative defenses can damage macromolecules such as DNA, lipids, and proteins.

Estrogens play an important role in development, growth, and the differentiation of both female and male secondary sex characteristics (3). Protective effects of estrogen, such as 17beta-estradiol (E2),3 on oxidative stress have been indicated (4). E2 regulates longevity signals to enhance resistance to oxidative stress in mice. Inhibitory effects of E2 on atherosclerosis are mediated by COX-2-derived prostacyclin (5). E2 induces production of antioxidative enzymes, such as superoxide dismutase (6), {gamma}-glutamylcysteine synthetase ({gamma}-GCS), and glutathione S-transferase (7). The effects of E2 are mediated mostly through ER{alpha}, which functions as a ligand-induced transcription factor and belongs to the nuclear receptor superfamily (8). ER{alpha} binds to a variety of ligands and displays tissue-specific effects through estrogen-response element (ERE). When estrogen-responsive genes do not contain EREs, ER{alpha} can up-regulate gene expression through AP-1 and Sp1 sites (9). Another ER, ERbeta, is expressed in cells targeted by E2, including cardiomyocytes (10). However, the role of ERbeta in protection against oxidative stress has not been well studied.

Protein thiols act as redox-sensitive switches and are believed to be a key element in maintaining the cellular redox balance. The redox state of protein thiols is regulated by oxidative stress and redox signaling and important to cellular functions. To maintain the cellular thiol-disulfide redox status, living cells possess two major systems, the thioredoxin (TRX)/TRX reductase system and the glutathione ({gamma}-glutamylcysteinyl glycine, GSH)/glutaredoxin (GRX) system (11). GSH is synthesized in two sequential enzymatic reactions that are each catalyzed by a rate-limiting enzyme, {gamma}-GCS, and GSH synthetase (12). GRX, also known as thioltransferase, was first discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking TRX (13). Oxidized GRX is recycled to the reduced form by GSH with the formation of glutathione disulfide and regeneration of GSH by coupling with NADPH and glutathione disulfide reductase (14). GRX functions via a disulfide exchange reaction by utilizing the active site, Cys-Pro-Tyr-Cys, which specifically and efficiently catalyzes the reduction of protein-SSG mixed disulfide (15). GRX also partially shares its function as a redox sensor with TRX (16, 17). Recently, we have found that GRX protects against oxidative stress-induced cell death from apoptosis by regulating the redox state of Akt (18).

Akt/protein kinase B is a pleckstrin homology domain-containing serine/threonine kinase and a critical component of an intracellular signaling pathway that exerts effects on survival and apoptosis (19). Akt has been found to be responsive to extracellular signaling factors, oxidative and osmotic stress, irradiation, and ischemic stress (20). Akt can phosphorylate Bad, caspase-9, and forkhead-related transcription factors, leading to an inhibition of apoptosis (21). The unphosphorylated form of Akt is virtually inactive, and phosphorylation at Thr308 and Ser473 stimulates its activity. Inactivation of Akt also occurs via dephosphorylation of the two phosphorylation sites by protein phosphatase 2A (PP2A) (22, 23). The activation of Akt contributes to the survival of H2O2-treated cells (24).

It has been reported that the function of ER-mediated transcriptional activity is regulated by redox (25). However, the precise mechanisms of redox regulation in the E2-mediated signal pathways have not been clarified. Here we describe a mechanism for the antiapoptotic effect of E2 through the regulation of the redox state of Akt under oxidative stress. Treatment of cardiac H9c2 cells with E2 for 18 h protected against H2O2-induced apoptosis. E2 induced the expression of GRX and{gamma}-GCS, at least in part, through ERbeta-mediated regulation. Elevated GSH and GRX levels potentiated the redox of Akt on the exposure of cells to H2O2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Anti-PP2A scaffolding A subunit (PR65) antibody was obtained from Santa Cruz Biotechnology. Antibodies against human ER{alpha} (clone ER88) and ERbeta (polyclonal) were from Kyowa Medex (Tokyo, Japan). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG F was purchased from MBL (Nagoya, Japan). HRP-goat anti-mouse IgG F was from Chemicon International (Temocula, CA). Normal goat IgG, normal rabbit IgG, and normal mouse IgG were from Sigma. Anti-Akt and anti-phospho-(Ser473)-Akt antibodies were from Cell Signaling Technology. Anti-PP2A catalytic C subunit antibody was from BD Transduction Laboratories. 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma. 4-Acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) was purchased from Molecular Probes, Inc. (Eugene, OR). H2O2 and CdCl2 were from Wako Pure Chemicals (Osaka, Japan). ICI182,780 and propylpyrazoletriol (PPT) were from Tocris (Ballwin, MO).

Cell Culture—H9c2 cells, a clonal line derived from embryonic rat heart, and human breast cancer SK-BR-3 (SKB3) cells, and MDA-MB-231 (MDA) cells, were obtained from the American Type Culture Collection (CRL-1446). Human breast cancer MCF7 cells were from The Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Japan). H9c2 cells were routinely maintained in Dulbecco's modified Eagle's medium, or MDA and SKB3 cells were maintained in RPMI1640 medium. The cells were supplied with 10% fetal calf serum in a humidified atmosphere of 95% air and 5% CO2 at 37 °C (23).

Cell Viability—Cell viability was determined by a MTT assay as described (26). Briefly, cells (1500–5000) were placed in 100 µl of medium per well in 96-well plates. Four hours after treatment with various concentrations of H2O2, the cells were incubated for 4 h at 37 °C with 3-(4,5-dimethylthiazol-e-yl)-2,5-diphenyltetrazolium bromide (652 µg/ml) and lysed with 100 µl of 20% SDS, 50% N,N-dimethylformamide (pH 4.7) in each well. After an overnight incubation at 37 °C, the absorbance at 570 nm was measured. Wells without cells served as blanks.

Nuclear Condensation—For the histochemical analysis, cells were maintained in a four-well Lab Tec Chamber (Nalge Nunc International, Naperville, IL). After treatment with H2O2, cells were treated with 10 µM Hoechst 33342 for 30 min to estimate the extent of nuclear condensation. They were then washed again with PBS. Fluorescence intensity was examined using an Axioskop2 fluorescence microscope (Carl Zeiss, Jena, Germany), and the findings were analyzed using a charge-coupled device camera (Axio-Cam) and AxioVision software.

Morphological Staining—The immunohistochemical analysis to examine the expression of ER{alpha} and ERbeta was performed as described previously (27). Briefly, cells were fixed with 4% paraformaldehyde in PBS and then preincubated with blocking solution for 1 h at room temperature. For ERbeta 10% normal goat serum and 1% bovine serum albumin in PBS and for ER{alpha} 500 µg/ml of normal goat IgG and 1% bovine serum albumin in PBS were used, respectively. Next, the samples were incubated with the primary antibodies overnight and washed three times with 0.075% Brij 35 in PBS. Then samples were reacted with HRP-goat anti-mouse IgG or HRP-goat anti-rabbit IgG for 1 h at room temperature and washed three times with 0.075% Brij 35 in PBS. HRP sites were visualized with H2O2 and DAB solution or H2O2 and DAB in the presence of nickel and cobalt ions. As a negative control, normal rabbit IgG and normal mouse IgG were used instead of the primary antibodies. The results of immunohistochemistry for ERs were graded as positive or negative, compared with the staining with IgG or serum of a normal rabbit or mouse.

Immunoblot Analysis—Cultured cells were harvested and lysed for 20 min at 4 °C in lysis buffer as described previously (17). The supernatants obtained by centrifugation of the lysates at 8000 x g for 15 min were used in subsequent experiments. Protein concentrations were determined using a BCA assay kit (Pierce). Protein samples were electrophoresed on 10, 12.5, or 15% SDS-polyacrylamide gels under reducing conditions, except for thiol-modified protein samples. The proteins in the gels were transferred onto nitrocellulose membranes. The membranes were blocked in Tris-buffered saline (10 mM Tris-HCl (pH 7.5) and 0.15 M NaCl; TBS) containing 0.05% Tween 20 (v/v) (TBST) and 5% (w/v) nonfat dry milk and then reacted with primary antibodies in TBST containing 3% (w/v) bovine serum albumin overnight with constant agitation at 4 °C. After several washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-IgG antibodies. Proteins in the membranes were then visualized using the enhanced chemiluminescence detection kit (Amersham Biosciences) according to the manufacturer's instructions.

Akt Activity Assay—Akt activity was assayed using an Akt assay kit (Cell Signaling Technology) according to the manufacturer's directions with GSK3{alpha}/beta fusion protein (GSK3{alpha}/beta) as a substrate. Phosphorylation of GSK3{alpha}/beta was assessed by immunoblot analysis using a specific antibody. Briefly, Akt was immunoprecipitated from cell lysates using the anti-Akt antibody, and then the immunoprecipitates were incubated at 30 °C for 30 min in an assay mixture containing GSK3{alpha}/beta. Phosphorylated proteins were separated by 12.5% SDS-PAGE and then transferred to nitrocellulose membranes to detect phosphorylated GSK3{alpha}/beta using an anti-phosphorylated GSK3{alpha}/beta antibody.

Protein Phosphatase Assay—PP2A activity was assayed spectrophotometrically using a Ser/Thr phosphatase assay kit 1 (Upstate%20Biotechnology">Upstate Biotechnology, Inc.) according to the manufacturer's instructions. The phosphopeptide RKpTIRR and p-nitrophenyl phosphate were used as phosphatase substrates.

Determination of Redox States—The redox states of proteins were assessed by modifying free thiol with AMS (28). Briefly, after incubation with or without H2O2, cell lysates or proteins were treated with trichloroacetic acid at a final concentration of 7.5% to denature and precipitate the proteins as well as to avoid any subsequent redox reactions. The protein precipitates were collected by centrifugation at 12,000 x g for 10 min at 4 °C. The pellets were rinsed in acetone and centrifuged twice and then dissolved in a buffer containing 50 mM Tris-HCl (pH 7.4), 1% SDS, and 15 mM AMS. Proteins were then separated by 10% SDS-PAGE without using any reducing agents and blotted to nitrocellulose membranes. Proteins in the membranes were visualized by immunoblotting as described above.

Northern Blot Analysis—A 764-bp DNA fragment (bp 865–1628) of full-length {gamma}-GCS heavy subunit cDNA was obtained by digestion with PstI (29). The probes were radiolabeled with 32P using a random primer labeling kit (Takara Biomedicals, Shiga, Japan). The isolation of cytoplasmic RNA and Northern blotting were essentially performed as described by Sambrook et al. (30). Isolated RNAs (30 µg) were electrophoresed on a 1% agarose gel containing 0.6 M formaldehyde, transferred to a nylon membrane, and then hybridized with 32P-labeled probes. Autoradiographed membranes were analyzed using a BAS5000 bioimage analyzer (Fuji Photo Film). A specific system for the amplification of mRNA was also used: an mRNA-selective PCR kit (Takara-Biomedicals; distributed by BioWhittaker, Europe). It had a total volume of 25 µl, comprising 12.5 µl of 2x buffer II, 5 mM MgCl2, 0.5 µM PE1 and PE2, 1 mM each dNTP, 0.4 units of RNAsin (40 units/µl), 0.5 units of Rtase XL (5 units/µl), and 0.5 units of optimized Taq. As material, 1 µg of total RNA extracted from the cells was used. The 330-bp oligonucleotides for GRX (rat GRX sequence, accession number AF167981 [GenBank] ) were obtained using as a sense primer 5'-GCA TGG CTC AGG AGT TTG TGA ACT GCA AGA TTC AG-3' and, as an antisense primer, 5'-CCT TTC ATA ACT GCA GAG CTC CAA TCT GCT TCA GC-3'. The 410-bp oligonucleotides for beta-actin (rat beta-actin sequence, accession number BC063166 [GenBank] ) were obtained using 5'-GAG CTA TGA GCT GCC TGA CG-3' and 5'-AGC ATT TGC GGT GCA CGA TG-3'. The 410-bp oligonucleotides for beta-actin (human beta-actin sequence, accession number NM_001101 [GenBank] .2) were obtained using 5'-GAG CTA CGA GCT GCC TGA CG-3' and 5'-AGC ATT TGC GGT GCA CGA TG-3'. The 325-beta oligonucleotides for ER{alpha} (rat ER{alpha} sequence, accession number AY280663 [GenBank] .1) and 280-bp oligonucleotides for ERbeta (rat ERbeta sequence, accession number U57439 [GenBank] ) were obtained using 5'-GCT CTT GAC AAA CCC A-3' and 5'-GCG GCG TTG AAC TCG TAG-3' and 5'-GGC TGA GGA AAG CAC CTG TC-3' and 5'-GCG GCG TTG AAC TCG TAG-3', respectively. Similarly, the sense and antisense primers for the oligonucleotides for human ER{alpha} (accession number NM_000125 [GenBank] .2) were 5'-GAC TAT GCT TCA GGC TAC C-5' and 5'-GGT TCC TGT CCA AGA GCA AG-3', whereas those for human ERbeta (accession number NM_001437 [GenBank] .1) were 5'-GTG GTC CAT CGC CAG TTA TC-3' and 5'-GCA CTT CTC TGT CTC CGC AC-3', respectively. The RT-amplification was carried out as follows: 30 min at 50 °C for the reverse transcription, denaturation for 5 min at 95 °C, and then a succession of 23 (35 for ERs) cycles as follows: 30 s at 95 °C, 40 s at 65 °C (55 °C for ERs), and 90 s at 72 °C. Amplification took place after 5 min at 72 °C.

Determination of Cellular Glutathione Levels—Total GSH and GSSG levels were measured using a total glutathione quantification kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer's directions. Briefly, 5,5'-dithiobis-(2-nitrobenzoic acid) and GSH react to generate 2-nitro-5-thiobenzoic acid. The concentration of GSH in the sample solution was determined by measuring absorbance at 412 nm. For quantification of GSSG, cell lysates were treated with 2-vinylpyridine and triethanolamine to block the sulfhydryl residue of GSH. GSSG in the sample solution was reduced to GSH using a reducing mixture containing GSSG reductase and NADPH, and the levels of GSSG were determined photometrically as for GSH.

3-Phosphoinositide-dependent Protein Kinase-1 Activity—3-Phosphoinositide-dependent protein kinase-1 (PDK1) activity was estimated using an assay kit according to the manufacturer's instructions (Upstate%20Biotechnology">Upstate Biotechnology). Briefly, recombinant human active PDK1 (Upstate%20Biotechnology">Upstate Biotechnology) protein was incubated first with inhibitors for 30 min at 30 °C and then with inactive glucocorticoid-inducible kinase 1 (SGK1) for 30 min at 30 °C. Next, the PDK1-dependent SGK1 kinase activity was estimated by incubating the reaction mixture with glycogen synthase kinase 3 (GSK3) peptide as a substrate for 10 min at 30 °C in the presence of [{gamma}-32P]ATP.

Generation of Luciferase Reporter Constructs—A 2.0-kb fragment of the human GRX gene promoter (–2023 to –22) (31) was amplified by PCR using Pfu turbo DNA polymerase (Stratagene). The primers used were as follows: a forward primer (5'-GGA CTG AGT GAG AGG CAG ACA ATA GTC TCC-3') and a reverse primer (5'-CGG GAA GAA TCC TCA GTT GCA GGT ATT GCT TGG-3'). The PCR product was subcloned into pUC18 to obtain pUC18-pro-GRX. PUC18-pro-GRX was digested with HindIII, and the resulting fragment containing the promoter region from –2023 to –22 was inserted into the HindIII site of the reporter vector pGL3-Basic (Stratogene) to give pGL3-pro-GRX. To generate a deleted version of the luciferase reporter construct (pGL3-pro-GRX-del), pGL3-pro-GRX was digested with KpnI and PvuII (Takara Biomedicals). Site-directed mutagenesis for luciferase vectors was performed with pGL3-pro-GRX (–2023 to –22) as a template by using a QuikChange site-directed mutagenesis kit (Stratagene). The oligonucleotides used were as follows: electrophoretic response element (EpRE)-like 1 forward (5'-GCT CCC CCT CCG GGA CTC AGA ATC TGG-3') and EpRE-like 1 reverse (5'-CCA GAT TCT GAG TCC CGG AGG GGG AGC-3'). The nucleotide sequence was confirmed by sequencing with an ALFexpress II system (Amersham Biosciences).

Luciferase Activity Assay—Each vector was introduced into H9c2 cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 12 h of transfection, cells were harvested for 24 h and then treated with E2 (100 nM) or left untreated for 18 h. Then luciferase activity was assayed with cellular extracts by using a dual luciferase reporter assay system (Promega).

Electrophoretic Mobility Shift Assay—The electrophoretic mobility shift assay for the GC box and EpRE-like 1 element was performed as described (32). Briefly, oligonucleotides were annealed to double-stranded oligonucleotides and then labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Oligonucleotides specific for the GC box and EpRE-like 1 element were prepared according to the nucleotide sequence of the human GRX promoter region. Oligonucleotides used were as follows: EpRE-like 1 element, 5'-CCC TCC GTG ACT CAG AAT CTG GCT TTC-3'; mutated EpRE-like 1 element, 5'-CCC TCC GGG ACT GTA AGC ACT TTA TGC TTC-3'. Binding reactions were performed in 15 µl of reaction mixture (25 mM Tris, pH 7.0, 6.25 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM KCl, and 10% glycerol) containing 10 µg of nuclear extract and 25 ng of labeled oligonucleotide. For the supershift assay, specific antibodies were added to the reaction mixture during the binding reaction for 30 min.

Statistical Analysis—Data were presented as means ± S.D. Differences were examined by using Student's t test. A value of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ERs—The expression of ERs in H9c2 cells was estimated immunohistochemically and genetically. Fig. 1 shows the results of the immunohistochemical analysis. Unlike MCF7 cells, which are known to express both ER{alpha} (Fig. 1A) and ERbeta (Fig. 1B), H9c2 cells expressed ERbeta (Fig. 1F) but not ER{alpha} (Fig. 1E). Fig. 1I shows the results of the RT-PCR analysis. ERbeta mRNA but not ER{alpha} mRNA was detected in H9c2 cells. On the other hand, both ER mRNAs were detected in MCF7 cells.


Figure 1
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  		FIGURE 1.
Immunohistochemical analysis for ERs. The expression of ERs was examined by immunohistochemical analysis. A–D, MCF7 cells were treated with antibody against ER{alpha} (A) and ERbeta (B). E–H, H9c2 cells were treated with antibody against ER{alpha} (E) and ERbeta (F). As a negative control, normal mouse IgG (C and G) or normal rabbit IgG (D and H) was used. The gene expression of ERs was examined by RT-PCR analysis (I) using sense-primers for rat ER{alpha} and -beta mRNAs in H9c2 cells and those for human mRNA in MCF7 cells.

 
Cytoprotective Effect of E2 on Oxidative Stress—We tested the cytoprotective effect of E2 on oxidative stress-induced apoptosis in H9c2 cells. Hydrogen peroxide induces apoptosis or early mitochondrial dysfunction in cardiac H9c2 cells (32, 33). Since 10% fetal calf serum, required for maintaining cultured cells, reduces oxidative stress modification of cells, in order to observe the effect of E2 on H2O2-induced oxidative stress, the concentration of fetal calf serum in the medium was changed from 10 to 0.5% in the experiments that followed. As shown in Fig. 2A, the cell viability decreased by H2O2, as assessed photometrically with the MTT assay. The cell viability upon treatment with 100 µM H2O2 for 4 h was ~27% of the control. Prior treatment of the cells with 100 and 200 nM E2 for 18 h prevented the H2O2 (100 µM)-induced cell damage by 1.4-fold and 1.8-fold of the control level, respectively (Fig. 2B). The increase in cell viability caused by E2 observed in 18 h continued until 24 h and then declined until 36 h (Fig. 2C). Morphologically, H2O2-induced DNA condensation was observed (Fig. 3, A versus E). E2 protected against DNA condensation (Fig. 3, B versus F). ICI182,780, an ER antagonist, abolished the protective effect of E2 (Fig. 3, C versus G). PPT (0.5 mM), a specific inhibitor of ER{alpha}, had no apparent influence on the protective effect of E2 (Fig. 3, D versus H). These results suggest that the protective effect against oxidative stress observed on treatment of the cells with E2 for 18 h involves transcriptional regulation mediated by ERbeta through a genomic pathway in this cell line. Unless otherwise indicated, subsequent experiments on the effect of E2 were done by incubating the cells with 100 nM E2 for 18 h.


Figure 2
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  		FIGURE 2.
Cytoprotective effect of E2 on H2O2-induced apoptosis. A, viability of H9c2 cells exposed to H2O2. Cells were treated with H2O2 (0–100 mM) for 4 h, and viability was estimated by the MTT assay. B, effect of E2 on H2O2-induced apoptosis. Cells were treated with various concentrations (0–200 nM) of E2 for 18 h and then with 100 mM H2O2 for 4 h. C, time course of the cytoprotective effect of E2. Cells were treated with 100 nM E2 for 0–36 h and then with 100 mM H2O2 for 4 h. The bars express viability (percentage) compared with the cells without H2O2. The data are mean ± S.D. of three independent analyses. *, p < 0.05 compared with untreated cells; **, p < 0.05 compared with cells with H2O2 without E2.

 


Figure 3
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  		FIGURE 3.
H2O2-induced nuclear condensation. Nuclear condensation was estimated from the PI staining. Cells were treated with 100 nM E2 for 18 h with or without ICI182,780 (1 µM), a specific inhibitor of ERs, or 0.5 µM PPT (ER{alpha} agonist) and then with 100 µM H2O2 for 4 h. A–D, control cells; B, E2; C, E2 and ICI182,780; D, E2 and PPT. E–H, cells treated with H2O2; F, E2; G, E2 and ICI182,780; H, E2 and PPT.

 
E2 Stimulated the Activity of Akt in Response to H2O2—The Akt cascade is known to mediate the survival function. The Akt signal is involved in both the genomic (34) and the nongenomic pathway of E2 (35). We tested the involvement of Akt in the cytoprotective effect of E2 in ERbeta-positive H9c2 cells. Phosphorylation of Akt (Ser473) was promoted by H2O2 in 10 min by 1.7-fold, and the control level was reached in 60 min (Fig. 4, A and B). Prior treatment with E2 for 18 h resulted in a further increase in the H2O2-induced phosphorylation of Akt in 10 min by 4.1-fold, and the phosphorylation continued until 30 min (Fig. 4, A and B, lanes 6 versus lanes 2 and lanes 7 versus lanes 3, respectively). ICI182,780 abolished the effect of E2 (Fig. 4, C and D, lanes 6 versus lanes 4). The H2O2-induced enhancement of Akt activity estimated using GSK3{alpha}/beta as a substrate was increased by E2 (Fig. 4, E and F, lanes 5 versus lanes 2 and lanes 6 versus lanes 3, respectively), concomitant with the increase in the phosphorylation of Akt. The activity of PDK1, upstream of Akt, was stimulated by H2O2; however, E2 had no apparent effect on the activity of PDK1 (Fig. 4G). The phosphorylation of Akt is regulated by PP2A (18). The activity of PP2A assayed spectrophotometrically using RKpTIRR and p-nitrophenylphosphate as substrates was not affected by H2O2 and E2 (Fig. 4H). The data suggest that the change in the activity of PP2A is not involved in the up-regulation of the phosphorylation of Akt by E2.


Figure 4
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  		FIGURE 4.
Involvement of phosphorylation of Akt in the cytoprotective effect of E2 against H2O2-induced apoptosis. A, time course of Akt phosphorylation in H9c2 cells under oxidative stress. C, cells were treated with 100 nM E2 in the presence or absence of 1 µM ICI182,780 for 18 h and then with 100 µM H2O2 for the period indicated. Phosphorylation of Akt was detected by immunoblot analysis using specific antibodies as described under "Materials and Methods." B and D, band intensity was estimated densitometrically, and the phosphorylation rates are expressed as the intensity of phosphorylated Akt relative to total Akt (Akt-p/Akt). E, activity of GSK3{alpha}/beta. The kinase activity of Akt was measured based on the phosphorylation of GSK3{alpha}/beta as described under "Materials and Methods." F, band intensity was estimated densitometrically, and the phosphorylation rates are expressed as arbitrary units. G, the activity of PDK1. The experimental conditions are the same as in E. H, the activity of PP2A. The activity of PP2A was measured as described under "Materials and Methods." The data are the mean ± S.D. of three independent analyses (B, D, and F). *, p < 0.05 compared with cells without E2 at each time point; **, p < 0.05 compared with cells with H2O2 and E2 without ICI182,780; ***, p < 0.05 compared with cells with H2O2 without E2 at each time point.

 
It has been reported that inactive Akt develops a redox-sensitive intramolecular disulfide bond close to its activation loop (18), and recently we found that the redox state of Akt is modulated by H2O2 (19). Fig. 5A shows the redox state of Akt assessed by modifying free thiol with AMS. In control cells, Akt existed mostly in an oxidized form (lane 1). Treatment of cells with H2O2 resulted in a further increase in an oxidized form of Akt (lanes 2 and 3). In the cells treated with E2 for 18 h, Akt existed more in a reduced form (lane 4). The reduced form of Akt, once decreased by H2O2 for 30 min, was restored again in 60 min (lanes 5 and 6). The data suggested that E2 maintains Akt in a reduced form under oxidative stress. The redox state of Akt is regulated by the GSH/GRX system, and this system protects cells against H2O2-induced apoptosis by preventing the association of Akt with PP2A (19). Then we estimated the effect of E2 on the phosphorylation of Akt in the presence of buthionine sulfoximine (BSO), a specific inhibitor of {gamma}-GCS, or cadmium, an inhibitor of GRX. {gamma}-GCS is a rate-limiting enzyme of GSH synthesis. The effect of E2 on the phosphorylation was abolished both by BSO (Fig. 5B) and by cadmium (Fig. 5C). These results suggest that E2 increases the levels of GSH/GRX to protect cells against oxidative stress.

E2 Induces the Expression of {gamma}-GCS and GRX—We tested if E2 increases the levels of GSH and GRX. E2 increased the levels of GSH (Fig. 6A). The level of GSH was 24.8 ± 4.0 nmol/106 cells in control cells and 38.5 ± 5.2 nmol/106 cells in cells treated with 100 nM E2 for 18 h. The level of GSSG in the control cells was ~2 nmol/106 cells and was not changed by E2 treatment (data not shown). The expression of {gamma}-GCS was up-regulated by 100 nM E2 by 1.6-fold in 18 h (Fig. 6, B and C). Similarly, 100 nM E2 increased the expression of GRX by 1.8-fold in 18 h (Fig. 6, D and E). ICI182,780 abolished the E2-dependent up-regulation of GSH synthesis as well as GRX synthesis (Fig. 6, A–E). It is suggested that the redox state of Akt is regulated by an E2-dependent enhancement of the GRX/GSH system.


Figure 5
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  		FIGURE 5.
E2 retains the redox state of Akt. A, the redox state of Akt was assessed based on mobility shifts of these proteins in an immunoblot analysis as described under "Materials and Methods." The positions of reduced (Red) and oxidized (Ox) proteins are indicated. The data are from a typical analysis. B, effect of modification of the redox on the phosphorylation of Akt was estimated, using 200 µM BSO, a specific inhibitor of {gamma}-GCS (lanes 5 and 6), and 2.5 µM cadmium, an inhibitor of GRX (lanes 7 and 8). C, the activity of Akt phosphorylation is shown as relative intensity in the absence (open bar) and presence of H2O2 (closed bar). The data are the mean ± S.D. of three independent analyses. *, p < 0.05 compared with cells with H2O2 and E2 without inhibitors.

 
The Gene Promoter Activity of GRX Is Regulated by E2 via an EpRE-like Element—As reported by Montano et al. (7), the expression of the {gamma}-GCS heavy (catalytic) subunit is up-regulated by E2 via an EpRE (5'-G/A)TGACNNNGC(G/A)-3'), not by an ERE. To investigate the mechanism of the transcriptional regulation of GRX by E2, we used a 2.0-kb genomic fragment containing the promoter region of GRX inserted into a luciferase vector, pGL3 Basic. The promoter region contains no apparent ERE or EpRE. There were two EpRE-like sites (EpRE-like 1 (–1380 to –1370; GTGACTCAGAA) and EpRE-like 2 (–347 to –337; GTGAGTAAGCA)) and Sp1 (–1217 to –1208, GCCCCGCCTC). The luciferase activity of the cells previously treated with E2 for 18 h was almost lost when the EpRE-like 1 site was deleted or mutated (Fig. 7). Deletion of EpRE-like 2 or Sp1 had no apparent effect on the E2-induced up-regulation of the luciferase activity.


Figure 6
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  		FIGURE 6.
GSH synthesis and GRX. Effects of E2 on levels of GSH, the {gamma}-GCS heavy subunit, and GRX were estimated in the presence or absence of ICI182,780, as described under "Materials and Methods." A, cells were treated with 100 nM E2 for 0–24 h, and the levels of GSH in the cell lysates were estimated. Cells were incubated with 100 nM E2 for 6 h for the analysis of the expression of the {gamma}-GCS heavy subunit by Northern blotting (B) and that of GRX by RT-PCR (D). The expression of {gamma}-GCS was expressed as relative intensity (percentage of control) (C), and that of GRX was expressed as the intensity of GRX/beta-actin (E). Each datum is a mean ± S.D. of three independent analyses. *, p < 0.05 compared with untreated cells.

 
E2 Up-regulated the ERbeta-EpRE-like 1 Complex Formation—To investigate the importance of the EpRE-like elements in the E2-induced expression of GRX, an electrophoretic mobility shift assay was performed with nuclear extracts from the cells treated with E2 for 18 h using 32P-labeled oligonucleotides designed for EpRE-like 1. As shown in Fig. 8, a protein-DNA complex of EpRE-like 1 (lane 2) increased by E2 (lane 3) and appeared in the presence of an excess of unlabeled probe (lane 4), or 32P-labeled probe with the disabled mutant for EpRE-like 1 (lane 5). The addition of the anti-ERbeta antibody caused the ERbeta-DNA-binding complex to disappear (lane 12), indicating the involvement of ERbeta as a transcription factor that bound to the EpRE-like 1 site. The EpRE-like 1 of GRX did not bind with Nrf2, Sp1, c-Jun, or c-Fos (lanes 9–11), different from the EpRE site of the {gamma}-GCS heavy subunit (7). On the other hand, neither EpRE-like 2 site nor the Sp1 site was stimulated by E2 (data not shown).


Figure 7
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  		FIGURE 7.
The EpRE-like element is important for the E2-dependent induction of the GRX promoter in H9c2 cells. Left, schematic representation of luciferase vector constructs for the human GRX promoter. Each luciferase vector construct was generated as described under "Materials and Methods." Right, luciferase activity of the vector constructs for the human GRX gene promoter in H9c2 cells. The cells were transiently transfected with the GRX promoter-luciferase gene fusion plasmids. After the transfection, luciferase activity was assayed with cellular extracts as described under "Materials and Methods." Each value represents the mean of at least three independent experiments, and the S.D. was always within 10% of the mean. *, p < 0.05 compared with control.

 


Figure 8
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  		FIGURE 8.
The EpRE-like 1 element is responsive to E2 in electrophoretic mobility shift assays. H9c2 cells were incubated with 100 nM E2 for 18 h, and the nuclear extracts were prepared as described under "Materials and Methods." 32P-Labeled oligonucleotides specific to EpRE-like elements 1 and 2 of the GRX gene promoter were prepared and incubated with each nuclear extract and then subjected to a 5% nondenatured PAGE. In lanes 1, 5, and 6, the nuclear extract (NE) was free. In lanes 6, 8, and 14, 32P-labeled mutant oligonucleotides were used. In lanes 9–12, +Ab, specific antibodies were added to the reaction mixture during the binding reaction for the supershift assay. Arrowhead, protein-DNA complex.

 
Important Role of ERbeta in Other Cells—To further confirm the role of ERbeta in protection against oxidative stress through redox regulation of Akt, we employed human breast cancer cells, SK-BR-3 and MDA-MB-231 cells. As shown in Fig. 9A, an RT-PCR analysis revealed that these cells mainly expressed ERbeta mRNA. A stimulatory effect of E2 on the activity of Akt was observed in these cells (Fig. 9, B and C). However, ICI182,780 abolished the protective effect of E2 (Fig. 9, B and C). E2 induced the expression of GRX (Fig. 9, D and E). The results suggested that the cytoprotective effect of E2 is mediated through redox regulation of Akt activity in ERbeta-expressing cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ERbeta-mediated Cytoprotection against Oxidative Stress—Estrogenic hormones are required for the growth and differentiation of female reproductive tissues, contribute to male fertility, and play a role in maintaining cardiovascular, skeletal, and neural cell functions (9). Estrogen has been widely used to regulate fertility, relieve postmenopausal symptoms, and decrease the incidence and recurrence of mammary tumors. The ERs were the first members of the nuclear receptor family to be identified. ER{alpha} has been well characterized and plays a major role in E2-mediated genomic actions in both reproductive and nonreproductive tissues. ER{alpha}-mediated cytoprotection against oxidative stress-induced cell damage has been reported in neurological cells (33, 34) and breast cancer cells (35). On the other hand, the role of ERbeta is not well understood. A report using microarray analyses showed that most of the genes regulated by ERbeta are distinct from those regulated by ER{alpha} in response to E2 and selective estrogen receptor modulators (36). ERbeta regulates plasminogen activator inhibitor-1 in endothelial cells, and a clinical evaluation of ERbeta was suggested as a prognostic or predictive factor of drug resistance in breast cancer (37). These results suggest a significant role for ERbeta in the regulation of cellular function, although the function of ERbeta and its precise mechanism are still unclear (3). Thus, this is the first report aimed at the significant role of ERbeta-mediated signals of E2 in redox regulation in response to oxidative stress.


Figure 9
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  		FIGURE 9.
Protective effect of E2 in other cell lines. The effect of E2 was studied using ERbeta-expressing human breast cancer SK-BR-3 cells and MDA-MB-231 cells. A, expression of ERs. The gene expression of ERs was estimated by RT-PCR analysis as described in the legend to Fig. 1. B, phosphorylation of Akt. The effect of E2 on the phosphorylation of Akt under oxidative stress was estimated by immunoblot analysis using specific antibodies as described under "Materials and Methods." C, band intensity was estimated densitometrically, and the phosphorylation rates are expressed as the relative intensity of phosphorylated Akt to total Akt (Akt-p/Akt). The data are the mean ± S.D. of three independent analyses (B and D). D, gene expression of GRX. The effect of E2 on levels of the GRX was estimated as described under "Materials and Methods." E, band intensity was estimated densitometrically and expressed as the intensity of GRX/beta-actin. Each datum is a mean ± S.D. of three independent analyses. *, p < 0.05 compared with cells with H2O2 without E2; **, p < 0.05 compared with cells with E2 without ICI182,780.

 
Involvement of Akt in the Cytoprotection of E2 Mediated by ERbeta—The importance of Akt has been suggested in the cytoprotective effect of E2 against oxidative stress. This effect of E2 was rapid and nongenomic in neurological cells (38), vascular endothelial cells (39), and ovarian cancer cells (40). On the other hand, Stoica et al. (41) reported that ER{alpha}-mediated signals up-regulated the expression of Akt in ER{alpha}-positive MCF-7 breast cancer cells. They also demonstrated that Akt-mediated signals up-regulated the expression of ER{alpha} in these cells, suggesting that Akt plays a central role in the growth and survival of breast cancer cells; however, the mechanism by which Akt is activated by E2 was not fully characterized.

In the present study, we were interested in the possible involvement of Akt signals in the ERbeta-mediated anti-apoptotic effect against oxidative stress. We employed H9c2 cells that apparently express only ERbeta (Fig. 1). We found that 1) H2O2-induced apoptosis was prevented when the cells were incubated with E2 for over 18 h; 2) the anti-oxidative effect of E2 was mediated by a genomic pathway through ERbeta; and 3) E2 retained the level of phosphorylated Akt in response to H2O2 via the GSH/GRX system.

Role of the GSH/GRX System in ERbeta-mediated Akt Signals—We reported previously a role for the GRX/GSH system in the regulation of Akt phosphorylation (19). Akt is a Ser/Thr protein kinase with anti-apoptotic and oncogenic activities. Akt is activated through a growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase pathway (21). The unphosphorylated form of Akt is virtually inactive, and phosphorylation at Thr308 and Ser473 stimulates its activity. Inactivation of Akt also occurs via dephosphorylation of the two phosphorylation sites by PP2A (23, 24). The activation of Akt contributes to the survival of H2O2-treated cells (25). H2O2 induces oxidation of Akt at Cys297 and Cys311, and the oxidized form of Akt can be dephosphorylated by PP2A (19). PP2A is a major Ser/Thr phosphatase implicated in the regulation of many cellular processes, including the regulation of different signal transduction pathways, cell cycle progression, DNA replication, gene transcription, and protein translation (42). Yasukawa et al. have reported that Akt is also inactivated by S-nitrosylation at Cys224 in NO donor-treated cells (43). Furthermore, we recently reported that the phosphorylation of Akt is down-regulated by cytoplasmic calcium (32). Calcium induced the expression of the PP2A catalytic subunit mediated by cAMP via the cAMP-response element. In the present study, the activity and the expression of the anti-PP2A catalytic C subunit did not change upon treatment with E2 in H9c2 cells (Fig. 4H), suggesting that the modulation of calcium levels may not be involved. Inactivation by ROS of protein phosphatases, such as protein-tyrosine phosphatase 1B (44), mitogen-activated protein kinase (MAPK) phosphatases (45), and PP2A (46), has been reported. In the present study, the activity of PP2A was not changed by H2O2 (Fig. 4H), suggesting that inactivation of PP2A by ROS is not involved. The redox state of Akt is regulated by GSH/GRX (19). Oxidation of Akt at Cys297 and Cys311 facilitates the association of PP2A, leading to the dephosphorylation of Akt. However, the activity of Akt is not affected by the oxidation. In the present study, oxidation of Akt was observed in the medium with 0.5% fetal calf serum in the absence of H2O2, and after the treatment with H2O2, the oxidation of Akt continued for 60 min. In such conditions, E2 maintained Akt in the reduced form (Fig. 5). This suggested that E2 potentiates the functions of the GSH/GRX system. The GSH/GRX system regulates many signals, such as ASK-1, NF1, PTP1B, protein kinase C, and protein kinase A (49). The present study indicates for the first time that ERbeta-mediated signaling via E2 up-regulates the activity of the GSH/GRX system to stimulate Akt and protects cells against oxidative stress.

Up-Regulation of {gamma}-GCS and GRX by E2—The ER{alpha}-mediated expression of antioxidants in response to oxidative stress has been reported. Genomic effects on the expression of antioxidant enzymes reported were Mn-SOD (4, 6), Cu,Zn-SOD (6), COX-1 (47), and COX-2 (5). Induction of GRX expression by E2 was reported in bovine aortic endothelial cells (48) and in female mice (34). These reports suggested a potential contribution of TRX and GRX to the protection of cells against oxidative stress. As to ERbeta, the expression of {gamma}-GCS induced by E2 was reported to be mediated by ERbeta (7) in breast epithelial cell lines.

In the present study, we found that the expression of both GRX and {gamma}-GCS is up-regulated by E2 in ERbeta-expressing cells. Data on the induction of the {gamma}-GCS heavy subunit (Fig. 6, B and C) together with an increase in the level of GSH obtained here (Fig. 6A) is consistent with such a contribution. Furthermore, E2 up-regulated the expression of GRX (Fig. 6, D and E). Elevated levels of both GSH and GRX were necessary to retain the reduced form of Akt. BSO abolished the effect of E2 on the phosphorylation of Akt (Fig. 5B), and cadmium also abolished the effect of E2 (Fig. 5C). The up-regulation of {gamma}-GCS as well as GRX expression by E2 was abolished by ICI182,780 (Fig. 6, A–E), suggesting involvement of the ERbeta-mediated genomic effect of E2. The possible role of ERbeta in the cytoprotection against oxidative stress was supported by the results obtained using other ERbeta-expressing cells (Fig. 9). Although involvement of ER{alpha} in the cytoprotective effect of E2 cannot be ruled out in these cells, it is suggested that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERbeta. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy. A difference in the distribution of ER{alpha} and ERbeta was reported (3, 27, 50, 51). ER{alpha} and ERbeta differ in the distribution in tissue cells and how they regulate cell proliferation and apoptosis, which may provide some insight into the tissue-specific functions and interplay between the two receptors.

The role of the GSH/GRX redox system in the antiapoptotic effect of E2 was studied further. In the present study, we found that the induction of GRX expression by E2 is mediated by an EpRE-like 1 element (Figs. 7 and 8). The human GRX promoter employed here possessed no apparent ERE or EpRE but had two EpRE-like sites. Interestingly, one of these sites, EpRE-like 1, bound to ERbeta and promoted the transcriptional activity of GRX. Transcription of the GRX gene was increased by E2 but decreased by anti-ERbeta antibody. However, EpRE-like 1 did not bind to Nrf-2 or AP-1. This element may be a novel kind of ERE. In summary, E2 has a cytoprotective effect against oxidative stress in H9c2 cells expressing ERbeta. The genomic effect of E2 on the GSH/GRX redox system potentiates Akt activity, a mechanism that may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for scientific research from the Ministry of Health, Labor, and Welfare of Japan (H15-Choju-015), by the Technology through the 21st Century Center of Excellence program, and by a research grant for health sciences from the Japanese Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF167981 [GenBank] , BC063166 [GenBank] , NM_001101 [GenBank] .2, AY280663 [GenBank] .1, U57439 [GenBank] , NM_000125 [GenBank] .2, and NM_001437 [GenBank] .1. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel.: 81-95-849-7099; Fax: 81-95-849-7100; E-mail: urata{at}net.nagasaki-u.ac.jp.

3 The abbreviations used are: E2, 17beta-estradiol; ER, estrogen receptor; ERE, estrogen-response element; GRX, glutaredoxin; {gamma}-GCS, {gamma}-glutamylcysteine synthetase; PP2A, protein phosphatase 2A; MTT, 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyltetrazolium bromide; HRP, horseradish peroxidase; PPT, propylpyrazoletriol; TBS, Tris-buffered saline; PDK1, 3-phosphoinositide-dependent protein kinase-1; TRX, thioredoxin; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; PBS, phosphate-buffered saline; RT, reverse transcription; BSO, buthionine sulfoximine; EpRE, electrophoretic response element. Back


   ACKNOWLEDGMENTS
 
We are grateful to Takaaki Kohno for excellent technical assistance.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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