Glutaredoxin Exerts an Antiapoptotic Effect by Regulating the Redox State of Akt

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt. Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt. The redox status of sulfhydryl groups is important to cellular functions such as the synthesis and folding of proteins and regulation of the structure and activity of enzymes, receptors, and transcription factors. To maintain the cellular thiol-disulfide redox status under reducing conditions, living cells possess two major systems, the thioredoxin (TRX) 1The abbreviations used are: TRXthioredoxinGRXglutaredoxinPP2Aprotein phosphatase 2AMTT3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromideAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingLDHlactate dehydrogenaseGSK3glycogen synthase kinase 3GSTglutathione S-transferasemyrmyristoylDTTdithiothreitolAGCthe cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C.1The abbreviations used are: TRXthioredoxinGRXglutaredoxinPP2Aprotein phosphatase 2AMTT3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromideAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingLDHlactate dehydrogenaseGSK3glycogen synthase kinase 3GSTglutathione S-transferasemyrmyristoylDTTdithiothreitolAGCthe cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C./thioredoxin reductase system and the glutathione (GSH)/glutaredoxin (GRX) system (1.Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar).GRX, also known as thioltransferase, was first discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking TRX (2.Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2275-2279Crossref PubMed Scopus (356) Google Scholar). 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-S-S-glutathione mixed disulfide (3.Gravina S.A. Mieyal J.J. Biochemistry. 1993; 32: 3368-3376Crossref PubMed Scopus (276) Google Scholar). Oxidized GRX is selectively recycled to the reduced form by GSH with the formation of glutathione disulfide (GSSG) and regeneration of GSH by coupling with NADPH and GSSG reductase, termed the GSH-regenerating system (4.Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar, 5.Gan Z.R. Wells W.W. J. Biol. Chem. 1986; 261: 996-1001Abstract Full Text PDF PubMed Google Scholar). These characteristic interactions of GRX with GSH distinguish it from TRX, which favors intramolecular disulfide substrates and is turned over by NADPH and thioredoxin reductase independent of GSH. Functional overlap or cross-talk between the two systems, however, has been indicated (6.Prinz W.A. Åslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: 15661-15667Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 7.Casagrande S. Bonetto V. Fratelli M. Gianazza E. Eberini I. Massignan T. Salmona M. Chang G. Holmgren A. Ghezzi P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9745-9749Crossref PubMed Scopus (296) Google Scholar). GRX also partially shares its function as a redox sensor with TRX (8.Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 9.Song J.J. Lee Y.J. Biochem. J. 2003; 373: 845-853Crossref PubMed Scopus (149) Google Scholar). Although GRX has been shown to play an important role in cytoprotection against oxidative stress (10.Luikenhuis S. Perrone G. Dawes I.W. Grant C.M. Mol. Biol. Cell. 1998; 9: 1081-1091Crossref PubMed Scopus (196) Google Scholar, 11.Rodríguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar) and apoptosis (12.Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 13.Daily D. Vlamis-Gardikas A. Offen D. Mittelman L. Melamed E. Holmgren A. Barzilai A. J. Biol. Chem. 2001; 276: 21618-21626Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the precise mechanism of the antiapoptotic effect of GRX has not been fully elucidated.The serine/threonine kinase Akt is a critical component of an intracellular signaling pathway that exerts effects on survival and apoptosis (14.Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). The unphosphorylated form of Akt is virtually inactive, and phosphorylation at Thr-308 and Ser-473 stimulates its activity. Inactivation of Akt also occurs via dephosphorylation of the two phosphorylation sites by protein phosphatase 2A (PP2A) (15.Andjelković M. Jakubowicz T. Cron P. Ming X.F. Han J.W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16.Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Akt activation contributes to the survival of hydrogen peroxide (H2O2)-treated cells (17.Pham F.H. Sugden P.H. Clerk A. Circ. Res. 2000; 86: 1252-1258Crossref PubMed Scopus (114) Google Scholar). Although H2O2 induces the transient activation of Akt following dephosphorylation and degradation (17.Pham F.H. Sugden P.H. Clerk A. Circ. Res. 2000; 86: 1252-1258Crossref PubMed Scopus (114) Google Scholar, 18.Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 19.Martín D. Salinas M. Fujita N. Tsuruo T. Cuadrado A. J. Biol. Chem. 2002; 277: 42943-42952Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the precise mechanism of H2O2-induced dephosphorylation of Akt is not well understood. Recently the crystal structure of an inactive Akt2 kinase domain has been deduced. Inactive Akt2 develops a redox-sensitive disulfide bond in its activation loop (20.Huang X. Begley M. Morgenstern K.A. Gu Y. Rose P. Zhao H. Zhu X. Structure (Camb.). 2003; 11: 21-30Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), which suggests that Akt is a redox-regulated protein.Here we described a novel mechanism for the antiapoptotic effect of GRX via regulation of the redox state of Akt under oxidative stress. An intramolecular disulfide bond formed between Cys-297 and Cys-311 of Akt in cardiac H9c2 cells treated with H2O2. Overexpression of GRX inhibited oxidation of Akt and protected cells from apoptosis.EXPERIMENTAL PROCEDURESReagents—Anti-mouse GRX antibody was affinity-purified from the serum of a rabbit immunized with a C-terminal 16-mer peptide of mouse GRX (mouse GRX-(91–106)). Anti-PP2A scaffolding A subunit (PR65) antibody was obtained from Santa Cruz Biotechnology. Anti-Akt, anti-phospho(Ser-473)-Akt, anti-phospho(Thr-308)-Akt, anti-Akt5G3, and anti-Akt1G1 antibodies were from Cell Signaling Technology. Anti-PP2A catalytic C subunit (PP2Ac) antibody was from BD Transduction Laboratories. Anti-Myc tag antibody, Akt1 cDNA Allelic Pack, and purified recombinant Akt protein (Akt/inactive and Akt/active) were from Upstate Biotechnology. c-Myc monoclonal antibody-agarose beads were purchased from BD Biosciences Clontech. Anti-FLAG M2 mouse monoclonal antibody, GSH, GSSG, NADPH, 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and l-cysteine S-sulfate (Cys-SO3) were from Sigma. 4-Acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was purchased from Molecular Probes. GSSG reductase was from Roche Applied Science. H2O2 and CdCl2 were from Wako Pure Chemicals (Osaka, Japan).Cell Culture—H9c2 cells, a clonal line derived from embryonic rat heart, were obtained from American Type Culture Collection (CRL-1446). H9c2 cells and gene-transfected cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.Vector Construction—Full-length mouse GRX cDNA subcloned into pBluescript SK(+) was obtained as described previously (21.Nakamura T. Ohno T. Hirota K. Nishiyama A. Nakamura H. Wada H. Yodoi J. Free Radic. Res. 1999; 31: 357-365Crossref PubMed Scopus (25) Google Scholar). The mouse GRX open reading frame was amplified by PCR techniques. As the 5′-primer oligonucleotides, 5′-CGGGGATCCATGGCTCAGGAGTTTGTGAACTGC-3′, which annealed to the 5′-end of GRX cDNA and introduced a BamHI site, and as the 3′-primer oligonucleotides, 5′-CTCGAATTCTTATAACTGCAGAGCTCCAATCTG-3′ complementary to the 3′ terminus of the GRX cDNA and inserting an EcoRI site, were used. The amplified DNA fragment was digested with BamHI and EcoRI and then cloned into the pCMV-tag2B expression vector (Stratagene). GRX cDNA accompanied at the 5′-end with the FLAG sequence (5′FLAG-GRX) was digested with NotI and EcoRV and then cloned into NotI/EcoRV-cut pTRE2-Hyg (BD Biosciences Clontech) and termed pTRE2Hyg-GRX. 5′FLAG-GRX was also digested with EcoRI and XhoI and then cloned into EcoRI-XhoI-cut pGEX-6p-1 (Amersham Biosciences) and termed pGEX-GRX. The nucleotide sequences were confirmed by sequencing with an ALFexpress II system (Amersham Biosciences).Site-directed Mutagenesis—The QuikChange XL site-directed mutagenesis kit (Stratagene) was used to make point mutations of cAkt cDNA. The following are the various primers, which were used for converting two cysteine residues (Cys-297 and Cys-311) to serine in cAkt cDNA to create mutants: sense primer oligonucleotide (5′-GACTTCGGGCTGTCCAAGGAGGGGATC-3′) and antisense primer oligonucleotide (5′-GATCCCCTCCTTGGACAGCCCGAAGTC-3′) for C297S; sense primer oligonucleotide (5′-GGGGCCAGGTACTCCGGCGTTCCGGAGAATGTCTTCATAGTGGC-3′) and antisense primer oligonucleotide (5′-GCCACTATGAAGACATTCTCCGGAACGCCGGAGTACCTGGCCCC-3′) for C311S. A double mutant (C297S/C311S) was constructed using the cAkt-C297S single mutant as a DNA template and primers for C311S. These experiments were performed according to the manufacturer's protocol. The nucleotide sequences were confirmed by sequencing with an ALFexpress II system.Gene Transfection and Selection of Cells—Gene transfection was performed using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's protocol. A Tet-On gene expression system (BD Biosciences Clontech) was used to establish the cell line overexpressing GRX. First, H9c2 cells were transfected with the pTet-on regulation vector. Stable transfectants were screened by culturing with 500 μg/ml G418. The cloned G418-resistant cells were then transfected with pTRE2hyg or pTRE2Hyg-GRX using the same procedure as for pTet-on. Stable transfectants were screened with 100 μg/ml hygromycin B. The cloned G418-resistant and hygromycin B-resistant cells were screened for expression of GRX. After screening, cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing 75 μg/ml G418 and 75 μg/ml hygromycin B.Thioltransferase Activity Assay—Thioltransferase activity was assayed as described previously (5.Gan Z.R. Wells W.W. J. Biol. Chem. 1986; 261: 996-1001Abstract Full Text PDF PubMed Google Scholar). In brief, cell lysate or purified mouse GRX was mixed with a reaction buffer consisting of 137 mm Tris-HCl buffer (pH 8.0), 0.5 mm GSH, 1.2 units of GSSG reductase, 2.5 mm Cys-SO3, 0.35 mm NADPH, and 1.5 mm EDTA (pH 8.0). The reaction proceeded at 30 °C, and thioltransferase activity was measured spectrophotometrically at 340 nm. The net enzymatic reaction rate was obtained by subtraction of the non-enzymatic reaction rate from the total rate.Apoptosis Assay—Apoptosis was detected by flow cytometry with the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method using an ApopTag Plus fluorescein in situ apoptosis detection kit (Intergen) as described previously (16.Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Immunoblot Analysis—Cultured cells were harvested and lysed for 20 min at 4 °C in lysis buffer as described previously (16.Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The supernatants obtained by centrifugation of the lysates at 8000 × 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 with the exception of thiol-modified protein samples. The proteins in the gels were transferred onto a nitrocellulose membrane. The membranes were blocked in Tris-buffered saline (10 mm Tris-HCl (pH 7.5) and 0.15 m NaCl; TBS) containing 0.05% (v/v) Tween 20 (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 (ECL) 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 protocol with GSK3α/β fusion protein (GSK3α/β) as a substrate. Phosphorylation of GSK3α/β was assessed by immunoblot analysis using specific antibody.Protein Phosphatase Assay—PP2A activity was assayed spectrophotometrically using the Ser/Thr phosphatase assay kit 1 (Upstate Biotechnology) according to the manufacturer's protocol. The phosphopeptide RKpTIRR (where pT is phosphothreonine) and p-nitrophenyl phosphate were used as phosphatase substrates.Cell Viability Assay—The viability of cultured cells was evaluated using MTT as described previously (22.Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45566) Google Scholar). The cells (5×103) were placed in 100 μl of medium/well in 96-well plates and cultured overnight. After treatment with or without H2O2 for a period of time, 10 μl of 0.5% MTT solution was added, and the cells were incubated for 4 h. The reaction was stopped by adding 100 μl of lysis buffer (20% SDS, 50% N,N-dimethylformamide (pH 4.7)), and then cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader.Lactate Dehydrogenase (LDH) Release Assay—The activity of LDH released into the medium was measured with an MTX-LDH kit (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. The activity of the cytoplasmic enzyme released was shown as a percentage of LDH activity in the medium over the total enzyme activity. Total enzyme activity was determined by measuring the LDH activity in the lysate of cells treated with 0.2% Tween 20, which caused complete cell death.Determination of Redox States—The redox states of proteins were assessed by modifying free thiol with AMS (23.Kobayashi T. Kishigami S. Sone M. Inokuchi H. Mogi T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11857-11862Crossref PubMed Scopus (206) Google Scholar). 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 × g for 10 min at 4 °C, washed with acetone twice, and 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 a nitrocellulose membrane. Proteins in the membranes were then visualized by immunoblotting as described above.Protein Purification—FLAG-tagged mouse GRX was purified with a GST gene fusion system (Amersham Biosciences) according to the manufacturer's protocol. In brief, competent E. coli strain BL-21(DE3) cells were transformed with pGEX-GRX, and expression was induced by adding 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C. GST-fused GRX (GST-GRX) was affinity purified from cell lysates using glutathione-Sepharose 4B, and digested with PreScission protease. The cleaved GST was removed with glutathione-Sepharose 4B.Peroxide Quantification—Peroxide was quantified using the PeroXOquant quantitative peroxide assay (Pierce) according to the manufacturer's instructions. In brief, H2O2 was incubated in buffer containing components of the GSH/GRX system as indicated in Fig.7E at room temperature for 30 min. After a 1:10 dilution of each sample was made, 10 volumes of working reagent was added to 1 volume of diluted sample and mixed well. After incubation at room temperature for 15–20 min, the purple product composed of Fe3+-xylenol orange complex was detected spectrophotometrically at 570 nm.RESULTSEstablishment and Characterization of H9c2 Cells Overexpressing the GRX Gene—To investigate the functional effect of the overexpression of GRX on the intracellular redox state, we constructed a FLAG-tagged GRX gene expression vector and transfected rat cardiac H9c2 cells with it. The Tet-On gene expression system was utilized to obtain H9c2 cells stably overexpressing GRX. After the two-step screening of G418-resistant and hygromycin B-resistant transfectants, the expression level of GRX was characterized immunologically. We obtained three clones (H9c2-GRX22, H9c2-GRX30, and H9c2-GRX49) that overexpressed GRX without doxycycline, so-called leaky expression (Fig. 1A). Although the anti-mouse GRX antibody was useful in detecting rat brain GRX immunohistochemically (24.Takagi Y. Nakamura T. Nishiyama A. Nozaki K. Tanaka T. Hashimoto N. Yodoi J. Biochem. Biophys. Res. Commun. 1999; 258: 390-394Crossref PubMed Scopus (18) Google Scholar), the expression of GRX in parental and mock-transfected H9c2 cells (H9c2-Vector) was immunologically undetectable. We used no doxycycline to induce further expression of GRX in any experiments. These clones have more thioltransferase activity than parental and H9c2-Vector cells (Fig. 1B).Fig. 1Characterization of H9c2 cells overexpressing the GRX gene.A, expression of GRX in mock-transfected (Vector) and GRX gene-transfected H9c2 cells. The expression levels of proteins were estimated by immunoblot analysis using specific antibodies as described under "Experimental Procedures." Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to confirm that equal amounts of protein were loaded. The data represent three independent experiments. B, thioltransferase activity in mock-transfected (Vector) and GRX gene-transfected H9c2 cells was measured as described under "Experimental Procedures." The value represents the mean ± S.D. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpression of GRX Protects H9c2 Cells from H2O2-induced Apoptosis—A lower concentration (–400 μm) of H2O2 induces apoptosis or early mitochondrial dysfunction followed by a loss of plasma membrane integrity in H9c2 cells (25.Turner N.A. Xia F. Azhar G. Zhang X. Liu L. Wei J.Y. J. Mol. Cell. Cardiol. 1998; 30: 1789-1801Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 26.Neuss M. Monticone R. Lundberg M.S. Chesley A.T. Fleck E. Crow M.T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To examine the functional role of overexpressed GRX in protecting H9c2 cells against oxidative stress, mock-transfected and GRX gene-transfected H9c2 cells were treated with H2O2. As shown in Fig. 2A, the MTT assay revealed that 100 μm H2O2 decreased the viability of H9c2-Vector cells in a time-dependent manner but not that of GRX gene-transfected cells. In the LDH release assay, loss of plasma membrane integrity was observed in H9c2-Vector cells treated with H2O2 but not in H9c2-GRX49 cells (Fig. 2B). A TUNEL assay was carried out to clarify whether apoptosis contributed to the cell damage seen in H9c2-Vector cells treated with H2O2. An increase in fluorescence intensity derived from DNA strand breaks was detected in H9c2-Vector cells treated with H2O2 but not in H9c2-GRX49 cells (Fig. 2C). H9c2-GRX22 and H9c2-GRX30 cells showed results similar to H9c2-GRX49 cells in the LDH release assay and TUNEL assay (data not shown).Fig. 2Overexpression of GRX protects H9c2 cells from H2O2-induced apoptosis. Cells were treated with 100 μm H2O2 for the period indicated. Cell damage was assessed photometrically by the MTT assay (A) and LDH release assay (B) as described under "Experimental Procedures." C, apoptosis was evaluated by the TUNEL method as described under "Experimental Procedures." Cells were treated with (thick lines) or without (thin lines) 100 μm H2O2 for 2 h. The values in A and B represent the mean ± S.D. of three independent experiments. The data in C represent three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Involvement of Akt Signaling Pathway in the Protective Effect of GRX against H2O2-induced Apoptosis—The importance of the Akt signaling pathway in protecting cardiomyocytes from apoptosis has been reported (27.Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (728) Google Scholar). We investigated the phosphorylation of Akt immunologically in the cells treated with 100 μm H2O2. In H9c2-Vector cells, Akt activity increased to a maximum 10–30 min after the addition of H2O2 and then returned to basal levels by 60 min. After 120 min of treatment, Akt underwent degradation. On the other hand, a sustained phosphorylation of Akt for at least 240 min was observed without degradation in H9c2-GRX49 cells treated with H2O2 (Fig. 3, A and B).Fig. 3Akt signaling pathway in the protective effect of GRX against H2O2-induced apoptosis.A, time course of Akt phosphorylation in H9c2-Vector and H9c2-GRX49 cells under oxidative stress. Cells were treated with 100 μm H2O2 for the period indicated. Phosphorylation of Akt was detected by immunoblot analysis using specific antibodies as described under "Experimental Procedures." B, the band intensity was estimated densitometrically, and the phosphorylation rates are expressed as the relative intensity of phosphorylated Akt to total Akt (pAkt/Akt). C, pUSEamp(+) or myr-Akt1-pUSEamp(+) was introduced into H9c2 cells as described under "Experimental Procedures." After 48 h, cells were harvested, and overexpression of myrAkt in H9c2-myrAkt cells was detected by immunoblot analysis using specific antibodies as described under "Experimental Procedures." Anti-Akt antibody detected both myrAkt and endogenous Akt in H9c2-myrAkt. D, after 48 h of transfection of pUSEamp(+) or myr-Akt1-pUSEamp(+) as described above, Akt was immunoprecipitated (IP) from cell lysates with anti-Akt monoclonal antibody-conjugated and c-Myc monoclonal antibody-conjugated agarose beads. Akt activity was measured by phosphorylation of GSK3α/β as described under "Experimental Procedures." E, the band intensity was estimated densitometrically. F, apoptosis was evaluated as in Fig. 2C. After 48 h of transfection of pUSEamp(+) or myr-Akt1-pUSEamp(+), cells were treated with (thick lines) or without (thin lines) 100 μm H2O2 for 2 h. The data in A, C, D, and F represent three independent experiments. The values in B and E represent the mean ± S.D. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To clarify the significance of the sustained activation of the Akt signaling pathway in protecting H9c2 cells from apoptosis under oxidative stress, H9c2 cells transfected with myr-Akt1-pUSEamp(+), which expresses an N-terminal myristoylated constitutively active Akt (myrAkt), were treated with 100 μm H2O2 for 2 h, and apoptosis was evaluated by TUNEL assay. Overexpression of myrAkt enhanced intracellular Akt kinase activity (Fig. 3, C, D, and E). Treatment with 100 μm H2O2 induced apoptosis in pUSEamp(+) vector-transfected cells (H9c2-Vector2) but not in cells overexpressing myrAkt (H9c2-myrAkt) (Fig. 3F).H2O2-induced Modulation of the Redox State of Akt Is Suppressed in GRX-overexpressing H9c2 Cells—Recently it has been reported that the inactive Akt2 develops a redox-sensitive intramolecular disulfide bond close to its activation loop (20.Huang X. Begley M. Morgenstern K.A. Gu Y. Rose P. Zhao H. Zhu X. Structure (Camb.). 2003; 11: 21-30Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To examine the redox states of intracellular proteins including Akt under oxidative stress, we utilized the AMS alkylation method. As shown in Fig. 4A, Akt existed in a fully reduced form in H9c2-Vector cells, and H2O2 increased the amount of oxidized Akt in a time-dependent manner. Akt existed partially in the oxidized form in H9c2-GRX49 cells without oxidative stress, but H2O2 did not induce further oxidation of Akt. Neither the PP2Ac nor the PR65 underwent redox regulation by H2O2. Next we constructed three mutants of the Myc-tagged Akt expression vector to confirm whether the two redox-sensitive cysteines in the vicinity of the activation loop of Akt Cys-297 and Cys-311 form a disulfide bond under oxidative stress (Fig. 4B). H9c2-Vector cells were treated with H2O2 after the transfection of these vectors. Two single mutants of Akt in which a single cysteine residue was replaced with serine (C297S or C311S) showed a mobility shift between the reduced (lane 1) and oxidized (lane 2) forms of wild type Akt under non-stressful conditions (lanes 5 and 7). Treatment with H2O2 induced no mobility shift in these single mutants (lanes 6 and 8). The double mutant of Akt (C297S/C311S) (lane 3) showed almost the same mobility shift as the oxidized form of the wild type Akt (lane 2), and H2O2 did not induce a further change in mobility (lane 4). These results clearly demonstrated that Akt developed a disulfide bond between Cys-297 and Cys-311 in the cells under oxidative stress.Fig. 4H2O2-induced modulation of the redox state of Akt.A, cells were treated with 100 μm H2O2 for the period indicated. Proteins from the cells were denatured and modified with AMS as described under "Experimental Procedures." Redox states of proteins were assessed based on mobility shifts of these proteins in immunoblot analysis. The positions of reduced (Red) and oxidized (Ox) proteins are indicated. B, effect of mutation of indicated cysteine residues on the H2O2-induced mobility shift of Akt. Expression vectors for Myc-tagged wild type (wt) and indicated mutants of