Role of Glutaredoxin in Metabolic Oxidative Stress
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m206826200
ISSN1083-351X
AutoresJae J. Song, Juong G. Rhee, Mohan Suntharalingam, Susan A. Walsh, Douglas R. Spitz, Yong J. Lee,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoEpitope-tagged glutaredoxin (GRX) was utilized to determine the role of GRX in oxidative stress-induced signaling and cytotoxicity in glucose-deprived human cancer cells (MCF-7/ADR and DU-145). GRX-overexpressing cells demonstrated resistance to glucose deprivation-induced cytotoxicity and decreased activation of c-Jun N-terminal kinase (JNK1). Deletion mutants showed the C-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) bound GRX, and glucose deprivation disrupted binding. Treatment withl-buthionine-(S,R)-sulfoximine reduced glutathione content by 99% and prevented glucose deprivation-induced dissociation of GRX from ASK1. A thiol antioxidant,N-acetyl-l-cysteine, or overexpression of an H2O2 scavenger, catalase, inhibited glucose deprivation-induced dissociation of GRX from ASK1. GRX active site cysteine residues (Cys22 and Cys25) were required for dissociation of GRX from ASK1 during glucose deprivation. Kinase assays revealed that SEK1 and JNK1 were regulated in an ASK1-dependent fashion during glucose deprivation. Overexpression of GRX or catalase inhibited activation of ASK1-SEK1-JNK1 signaling during glucose deprivation. These results demonstrate that GRX is a negative regulator of ASK1 and dissociation of GRX from ASK1 activates ASK1-SEK1-JNK1 signaling leading to cytotoxicity during glucose deprivation. These results support the hypothesis that the GRX-ASK1 interaction is redox sensitive and regulated in a glutathione-dependent fashion by H2O2. Epitope-tagged glutaredoxin (GRX) was utilized to determine the role of GRX in oxidative stress-induced signaling and cytotoxicity in glucose-deprived human cancer cells (MCF-7/ADR and DU-145). GRX-overexpressing cells demonstrated resistance to glucose deprivation-induced cytotoxicity and decreased activation of c-Jun N-terminal kinase (JNK1). Deletion mutants showed the C-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) bound GRX, and glucose deprivation disrupted binding. Treatment withl-buthionine-(S,R)-sulfoximine reduced glutathione content by 99% and prevented glucose deprivation-induced dissociation of GRX from ASK1. A thiol antioxidant,N-acetyl-l-cysteine, or overexpression of an H2O2 scavenger, catalase, inhibited glucose deprivation-induced dissociation of GRX from ASK1. GRX active site cysteine residues (Cys22 and Cys25) were required for dissociation of GRX from ASK1 during glucose deprivation. Kinase assays revealed that SEK1 and JNK1 were regulated in an ASK1-dependent fashion during glucose deprivation. Overexpression of GRX or catalase inhibited activation of ASK1-SEK1-JNK1 signaling during glucose deprivation. These results demonstrate that GRX is a negative regulator of ASK1 and dissociation of GRX from ASK1 activates ASK1-SEK1-JNK1 signaling leading to cytotoxicity during glucose deprivation. These results support the hypothesis that the GRX-ASK1 interaction is redox sensitive and regulated in a glutathione-dependent fashion by H2O2. Glucose deprivation has been shown to cause metabolic oxidative stress characterized by increases in steady state levels of intracellular prooxidant production (presumably hydroperoxides) and cytotoxicity in human breast carcinoma MCF-7/ADR cells (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Although the cellular source of prooxidant production is uncertain, recent studies (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 3Lee Y.J. Chen J.C. Amoscato A.A. Bennouna J. Spitz D.R. Sunthralingam M. Rhee J.G. J. Cell Sci. 2001; 114: 677-684PubMed Google Scholar) have suggested that mitochondria may represent a major source of prooxidant production during glucose deprivation. Overexpression of the mitochondrial bcl-2 protein appeared to limit localization of prooxidant production to mitochondria and protected cells from oxidative stress-induced cytotoxicity during glucose deprivation (3Lee Y.J. Chen J.C. Amoscato A.A. Bennouna J. Spitz D.R. Sunthralingam M. Rhee J.G. J. Cell Sci. 2001; 114: 677-684PubMed Google Scholar). These results suggested that prooxidant production seen during glucose deprivation is most likely due to the metabolism of fatty acids and amino acids via the tricarboxylic acid (TCA) cycle leading to the production of NADH and FADH2 as the source of electrons for mitochondrial electron transport chain-mediated prooxidant production during respiration (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar,4Lehninger A.L. Biochemistry. Worth Publishers Inc., New York1976: 456Google Scholar). Glucose deprivation of human cancer cells also resulted in an increase in steady state levels of intracellular glutathione disulfide (GSSG) (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar). Cells appeared to respond to glucose deprivation-induced metabolic oxidative stress by attempting to increase the synthesis of glutathione (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar, 6Lee Y.J. Galoforo S.S. Sim J.E. Ridnour L.A. Choi J. Forman H.J. Corry P.M. Spitz D.R. Free Radic. Biol. Med. 2000; 28: 575-584Crossref PubMed Scopus (26) Google Scholar), but in the absence of substrates necessary for the regeneration of NADPH, glutathione could be maintained in the reduced state (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar). In support of this notion, glutamate rescued the cells from glucose deprivation-induced cytotoxicity and suppressed prooxidant production, presumably via the capacity of glutamate dehydrogenase to generate NADPH during the conversion of glutamate to α-ketoglutarate (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 4Lehninger A.L. Biochemistry. Worth Publishers Inc., New York1976: 456Google Scholar). Therefore, during glucose deprivation, mitochondrial metabolism could result in an increase in steady state levels of intracellular prooxidants due to decreased peroxide scavenging by pyruvate and the glutathione/glutathione peroxidase/glutathione reductase system (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar). Since the discovery that glucose deprivation-induced oxidative stress causes cytotoxicity in MCF-7/ADR cells (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), several studies have focused on determining the role of signal transduction pathways in the biological response (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar, 6Lee Y.J. Galoforo S.S. Sim J.E. Ridnour L.A. Choi J. Forman H.J. Corry P.M. Spitz D.R. Free Radic. Biol. Med. 2000; 28: 575-584Crossref PubMed Scopus (26) Google Scholar). Several researchers had previously demonstrated that reactive oxygen species (ROS) 1The abbreviations used for: ROS, reactive oxygen species; JNK1, c-Jun N-terminal kinase; PBS, phosphate-buffered saline; SAPK, stress-activated protein kinase; ASK1, apoptosis signal-regulating kinase 1; SEK1, stress-activated protein kinase/extracellular-signal regulated kinase kinase; GRX, glutaredoxin; TRX, thioredoxin; HA, hemagglutinin; BSO, l-buthionine-(S,R)-sulfoximine; GST, glutathioneS-transferase; MOI, multiplicity of infection; NAC, N-acetyl-l-cysteine. could act as intracellular second messengers for inducing apoptosis (7Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2104) Google Scholar, 8Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar). During glucose deprivation, supplementation of intracellular reduced thiol pools with N-acetyl-l-cysteine (NAC) was found to suppress metabolic oxidative stress-induced cellular responses including cytotoxicity, cytoskeletal reorganization, and stress-activated protein kinase (SAPK) activation (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 5Blackburn R.V. Spitz D.R. Liu X. Galoford S.S. Sim J.E. Ridnour L.A. Chen J.C. Davis B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (140) Google Scholar, 10Song S.H. Lee K.H. Kang M.S. Lee Y.J. Free Radic. Biol. Med. 2000; 29: 61-70Crossref PubMed Scopus (7) Google Scholar). These results coupled with results using dominant negative c-Jun N-terminal kinase (JNK1) suggested that glucose deprivation-induced SAPK activation was in part responsible for cytotoxicity (6Lee Y.J. Galoforo S.S. Sim J.E. Ridnour L.A. Choi J. Forman H.J. Corry P.M. Spitz D.R. Free Radic. Biol. Med. 2000; 28: 575-584Crossref PubMed Scopus (26) Google Scholar). Recent studies have also shown that pro-apoptotic signaling originating from the mitochondria is mediated by the JNK pathway (11Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1550) Google Scholar) possibly through Bid cleavage, cytochrome c release, and/or mitochondrial membrane depolarization. However, a fundamental question, that remains unanswered, is how glucose deprivation-induced metabolic oxidative stress is sensed resulting in the activation of these signal transduction pathways. Growing evidence has indicated that cellular oxidation/reduction (redox) status regulates various aspects of cellular function including signal transduction (12Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (1001) Google Scholar). Cellular redox status is sensed and maintained in a non-equilibrium steady state by intracellular redox-regulating molecules including thioredoxin (TRX) and glutaredoxin (GRX) (12Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (1001) Google Scholar). GRX, also known as thioltransferase, is a small (12 kDa) dithiol protein that has been shown to be involved in regulating various cellular functions, including redox regulation of certain enzyme activities (13Hirota K. Matsui M. Murata M. Takashima Y. Cheng F.S. Itoh T. Fukuda K. Yodoi J. Biochem. Biophys. Res. Commun. 2000; 274: 177-182Crossref PubMed Scopus (171) Google Scholar). GRX was originally discovered in mutantEscherichia coli that lacked TRX but had a fully active NADPH-dependent ribonucleotide reductase system (14Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2275-2279Crossref PubMed Scopus (364) Google Scholar, 15Wells W.W. Yang Y. Deits T.L. Adv. Enzymol Relat. Areas Mol. Biol. 1993; 66: 149-201PubMed Google Scholar, 16Padilla C. Martinez-Galisteo E. Barcena J.A. Spyrou G. Holmgren A. Eur. J. Biochem. 1995; 227: 27-34Crossref PubMed Scopus (75) Google Scholar, 17Powis G. Briehl M. Oblong J. Pharmacol. Ther. 1995; 68: 149-173Crossref PubMed Scopus (197) Google Scholar). GRX is reduced by glutathione, which in turn is reduced by NADPH and glutathione reductase (15Wells W.W. Yang Y. Deits T.L. Adv. Enzymol Relat. Areas Mol. Biol. 1993; 66: 149-201PubMed Google Scholar). Since previous studies had demonstrated that glucose deprivation-induced oxidative stress increased intracellular GSSG (1Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (289) Google Scholar, 6Lee Y.J. Galoforo S.S. Sim J.E. Ridnour L.A. Choi J. Forman H.J. Corry P.M. Spitz D.R. Free Radic. Biol. Med. 2000; 28: 575-584Crossref PubMed Scopus (26) Google Scholar), it is possible that GRX could sense changes in the redox state of GSH/GSSG and be involved in altering signal transduction pathways resulting in biological responses. Recent studies have also revealed that TRX binds directly to the N-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) and acts as a negative regulator by inhibiting ASK1 kinase activity and cytotoxicity (9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar). It is therefore possible that GRX could act in a similar redox sensing capacity to regulate glucose deprivation-induced cytotoxicity. The current study tested the hypothesis that a glutathione-dependent glucose deprivation-induced disruption of the physical interaction between GRX, and ASK1 mediates the activation of the ASK1-MEK-SAPK signaling pathway resulting in cytotoxicity. The results indicate that glucose deprivation causes the glutathione-dependent, redox-sensitive dissociation of GRX from the C-terminal region of ASK1 leading to the activation of ASK1-SEK1-JNK1 signal transduction pathway and cell death. Furthermore, the overexpression of GRX was found to protect cells from glucose deprivation-induced cell death by inhibiting the activation of the ASK1-SEK1-JNK1 signal transduction pathway. These results demonstrate that GRX can mediate both the sensing of, as well as protection from, metabolic oxidative stress in an ASK1-SEK1-JNK1-dependent fashion during glucose deprivation. Multidrug-resistant human breast carcinoma (MCF-7/ADR) cells or human prostate adenocarcinoma (DU-145) cells were cultured in McCoys' 5A medium with 10% iron-supplemented bovine calf serum (HyClone, Logan, UT) or Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (HyClone), respectively and 26 mm sodium bicarbonate for monolayer cell culture. The cells were maintained in a humidified atmosphere containing 5% CO2 and air at 37 °C. Cells were rinsed three times with phosphate-buffered saline (PBS) and then exposed to glucose-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum (Invitrogen). Cells were treated withl-buthionine-(S,R)-sulfoximine (BSO) obtained from Sigma Chemical Co. by aspirating the medium and replacing it with medium containing the drug. Cells were washed with ice cold PBS, scraped into cold PBS, and centrifuged at 4 °C for 5 min at 400 × g to obtain cell pellets, which were frozen at −80 °C. Pellets were then thawed and homogenized in 50 mm potassium phosphate buffer, pH 7.8 containing 1.34 mm diethylenetriaminepentaacetic acid. Total glutathione content was determined by the method of Anderson (18Anderson M.E. Greenwald R.A. Handbook of methods for oxygen radical research. CRC Press, Boca Raton, FL1985: 317-323Google Scholar). Reduced and oxidized glutathione were distinguished by addition of 2 μl of a 1:1 mixture of 2-vinylpyridine and ethanol per 30 μl of sample followed by incubation for 1.5 h and assay as previously described by Griffith (19Griffith O.W. Anal. Biochem. 1980; 106: 207-212Crossref PubMed Scopus (4060) Google Scholar). All biochemical determinations were normalized to protein content using the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). For survival determination, cells were trypsinized, counted, and plated at appropriate dilutions. After 1–2 weeks of incubation at 37 °C, colonies were stained and counted. For the construction of the hemagglutinin (HA)-tagged GRX expression vector pcDNA3-HA-GRX, a 320-bp DNA fragment encoding the GRX gene was amplified from pBluescript containing the cDNA insert for human glutaredoxin (a gift from Dr. J. J. Mieyal, Case Western Reserve University) by PCR techniques. Briefly, the 5′-primer oligonucleotides (5′-TTAAGAATTCGCTCAAGAGTTTGTGAACTGC, which annealed to the 5′-end of the GRX gene and introduced anEcoRI site) and 3′-primer oligonucleotides (5′-GAGCTCTAGATTACTGCAGAGCTCCAATCTG, complementary to the 3′ terminus of the GRX gene, but inserting an XbaI site) were used as primers. The final reaction concentrations were 5′-primer, 0.2 μm; 3′-primer, 0.2 μm and 1.5 mm MgCl2, 0.2 mm dNTP, 10 ng pBluehGRX, TaqDNA polymerase, 5 units/reaction, 1× reaction buffer. PCR was performed with 33 cycles (94 °C, 1 min; 45 °C, 2 min; 72 °C for 2 min) with initial incubation at 94 °C for 1 min, and final extension at 72 °C for 10 min. The resultant PCR product was digested with EcoRI and XbaI and then cloned into EcoRI/XbaI-cut pcDNA3-HA. pcDNA3-His-GRX was made by inserting EcoRI fragment from pQE-GRX into EcoRI-cut pcDNA3. For the construction of adenoviral vectors, the CRE-Lox recombination system was employed (21Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). pAdlox-His-GRX was made by inserting HindIII/XbaI fragment from pcDNA3-His-GRX intoHindIII/XbaI-cut pAdlox shuttle vector (21Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar) containing N-terminal fused form with hexahistidine (His6) tag. A-His-tagged 324-bp human TRX gene was isolated from pcDNA-His-TRX (a gift from Dr. J. Yodoi, Kyoto University, Japan) digestion with EcoRI and cloned into the EcoRI site of pAdlox shuttle vector. pcDNA3-HA-ASK1, pcDNA3-HA-ASK1ΔN (N-terminal region-truncated ASK1), pcDNA3-HA-ASK1ΔC (C-terminal region-truncated ASK1) were kindly provided by Dr. Ichijo (Tokyo Medical and Dental University, Tokyo, Japan). pAdlox-HA-ASK1 and the other types of ASK1 were made by inserting theSpeI/XbaI fragment from pcDNA3-HA-ASK1 intoXbaI-cut pAdlox. The complete shuttle vector was co-transfected into Cre8 cells with ψ5 viral genomic DNA for homologous recombination as described below. The adenovirus construct containing the transgene for human catalase was kindly provided by Dr. Beverly Davidson, Director, Gene Transfer Vector Core, University of Iowa. All other recombinant adenoviruses were constructed by employing the Cre-lox recombination system (21Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). The selective cell line CRE8 has a β-actin-based expression cassette driving a Cre recombinase gene with an N-terminal nuclear localization signal stably integrated into 293 cells. Transfections were done by using Lipofectin™ Reagent (Invitrogen). 5 × 105 cells were split into a 6-well plate 1 day before transfection. For the production of recombinant adenovirus, 2 μg ofSfiI/ApaI-digested Adlox/ASK1 fragment orSfiI-digested Adlox/GRX (or TRX) fragment and 2 μg of ψ5 viral genomic DNA were cotransfected into CRE8 cells. The recombinant adenoviruses were generated by intermolecular homologous recombination between the shuttle vector and ψ5 viral DNA. A new virus has an intact packaging site and carries a recombinant gene. Plaques were harvested, analyzed, and purified. The insertion of HA-ASK1, His-GRX, or His-TRX to adenovirus was confirmed by Western blotting after infection of corresponding recombinant adenovirus into MCF-7/ADR cells. The QuikChange site-directed mutagenese kit (Stratagene, La Jolla, CA) was used to make point mutations. Listed below are the various primers, which were used for converting two cysteine residues (Cys22, Cys25) to serine in GRX to create one point mutant as well as double mutant: sense primer oligonucleotides (5′-CAAGCCCACCTCCCCGTACTGC-3′) and antisense primer oligonucleotides (5′-GCAGTACGGGGAGGTGGGCTTG-3′) for C22S; sense primer oligonucleotides (5′-CTGCCCGTACTCCAGGAGGG-3′) and antisense primer oligonucleotides (5′-CCCTCCTGGAGTACGGGCAG-3′) for C25S; sense primer oligonucleotides (5′-CCCACCTCCCCGTACTCCAGGAGGGC-3′) and antisense primer oligonucleotides (5′-GCCCTCCTGGAGTACGGGGAGGTGGG-3′) for C22S/C25S. The PCR reaction was prepared by adding 5 μl of 10× reaction buffer, 50 ng of dsDNA template (pAdlox-His-GRX), 125 ng of each sense primer, 125 ng of each antisense primer, 1 μl of dNTP mixture, double-distilled water to a final volume of 50 μl, and 1 μl ofPfu Turbo DNA polymerase (2.5 units/μl). PCR was performed with 12 cycles (95 °C, 30 s; 55 °C, 1 min; 68 °C for 12 min) with initial incubation at 95 °C for 30 s. Following temperature cycling, the reaction was placed on ice for 2 min to cool the reaction. After PCR, 1 μl of DpnI restriction enzyme (10 units/μl) was added directly to each amplification reaction and incubated at 37 °C for 1 h to digest the parental supercoiled dsDNA. The DpnI-treated dsDNA was transformed intoEpicurian coli XL1-Blue supercompetent cells. Colonies were selected and each plasmid (pAdlox-His-GRX) was digested withHindIII/XbaI. Its 400-bp fragment was subcloned into pBluescript SK(−). Each pBluescript SK(−)/His-GRX was sequenced using T7 primer to confirm mutation. Cells were transfected with 20 μg of pcDNA3-HA-GRX by using Lipofectin™ Reagent (Invitrogen). For stable transfection, cells were incubated for 48 h, and then selected with 400 μg/ml geneticin (Invitrogen) for 4 days, followed by continued growth in the presence of 200 μg/ml geneticin to obtain colonies suitable for isolation. To examine the interaction between ASK1 and GRX, adenovirus of HA-tagged ASK1 (Ad.HA-ASK1), HA-tagged ASK1ΔN (Ad.HA-ASK1ΔN), or HA-tagged ASK1ΔC (Ad.HA-ASK1ΔC) at an MOI of 10 and His-tagged GRX (Ad.His-GRX) at an MOI of 30 were co-infected into MCF-7/ADR cells in 10-cm culture plates. For immunoprecipitation, cells were lysed in buffer containing 150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 10 mm EDTA, 1% Triton X-100, 1% deoxycholate, 1 mm phenylmethylsulfonyl fluoride (PMSF), 80 μm aprotinin, 2 mm leupeptin, and the lysates were incubated with 2 μg of anti-penta-His mouse IgG1 (Qiagen, Valencia, CA) for 2 h. After the addition of protein G/A-agarose (Calbiochem, Darmstadt, Germany), the lysates were incubated for an additional 2 h. The beads were washed three times with the lysis buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted with rat anti-HA (clone 3F10, Roche Diagnostics) or mouse anti-penta-His (Qiagen). The proteins were detected with the enhanced chemiluminescence reaction (Amersham Biosciences). To confirm the interaction between endogenous ASK1 and GRX, cell lysates were immunoprecipitated with 2 μg of anti-GRX (American Diagnostica Inc., Greenwich, CT) followed by immunoblotting with anti-ASK1 (Alexis Corp., Lausen, Switzerland) or anti-GRX antibody. To examine the effect of glucose deprivation on the interaction of ASK1 with GRX, Ad.HA-ASK1 and Ad.His-GRX co-infected MCF-7/ADR cells were rinsed three times with PBS and then exposed to glucose-free medium with 10% dialyzed bovine calf serum for various times. The plasmid containing GST-human JNK1 for bacterial fusion protein was constructed in pGEX-4T-1 by inserting the HindIII/XbaI fragment followed by Klenow treatment from pcDNA3-JNK1. The expression of GST-JNK1 protein was confirmed by Western blotting and purified by using glutathione-Sepharose 4B (Amersham Biosciences). GST-SEK1 was purified from 10 plates of 293 cells transfected with the pEBG/SEK1 (kindly provided by J. M. Kyriakis, Massachusetts General Hospital, Charlestown, MA), and the purification step was performed as described (22Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). MCF-7/ADR cells were co-infected with 10 MOI of Ad.HA-ASK1 and various amounts of Ad.His-GRX (0, 30, or 300 MOI) for 24 h. After 24 h, cells were lysed in a buffer solution containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EGTA, 10 mm NaF, 1% Triton X-100, 0.5% deoxycholate, 3 mm dithiothreitol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and protein inhibitor mixture solution (Sigma Chemical Co.). Cell extracts were clarified by centrifugation, and the supernatants immunoprecipitated with mouse anti-HA antibody (12CA5, Roche Diagnostics) and protein A-agarose (Invitrogen). The beads were washed twice with a solution containing 150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 5 mm EGTA, 2 mm dithiothreitol and 1 mm phenylmethylsulfonyl fluoride, and washed once with the kinase buffer solution, and then they were subjected to kinase assays. To measure immune complex activity, 0.2 μg of GST-SEK1 is first incubated with the immune complexes for 10 min at 30 °C in a final volume of 25 μl of a solution containing 20 mm Tris-HCl (pH 7.5), 20 mm MgCl2, and 100 μm ATP, and subsequently with 1 μg of GST-JNK1 for 10 min at 30 °C. Thereafter, the activated complex is subjected to SDS-PAGE, and the phosphorylated JNK1 is analyzed by rabbit anti-ACTIVE JNK antibody (Promega, Madison, WI). To determine the amount of ASK1 protein in the same sample, the upper part of the SDS-PAGE (>116 kDa) was cut out and immunoblotted with the rat anti-HA (Roche Diagnostics, rat IgG1) antibody. Cell lysates were subjected to electrophoresis on 12% polyacrylamide gels containing SDS under reducing conditions, and the proteins in the gels were transferred onto a polyvinylidine difluoride (PVDF) membrane. The membranes were incubated with 7% (w/v) skim milk in PBST (PBS containing 0.1% Tween 20, v/v) and then reacted with primary antibodies. Polyclonal rabbit anti-ACTIVE JNK and anti-catalase antibodies were obtained from Promega and Calbiochem (San Diego, CA), respectively. Monoclonal mouse anti-actin antibody was purchased from ICN (Costa Mesa, CA). After washing three times with PBST, the membranes were incubated with horseradish peroxidase-conjugated anti-IgG. Proteins in the membranes were then visualized using the enhanced chemiluminescence (ECL) reagent (Amersham Biosciences) as recommended by the manufacturer. Since GRX is thought to protect against oxidative stress by maintaining the functional redox state of proteins in the face of oxidant insult, the effect of enforced overexpression of GRX on glucose deprivation-induced cytotoxicity was determined. MCF-7/ADR cells were stably transfected with a control plasmid (Fig. 1 A,Cont) or the pcDNA3-HA-GRX plasmid containing the HA-tagged GRX cDNA (Fig. 1 A, GRX1 andGRX2). Transfectants (Cont, GRX1, and GRX2) were exposed for 4 h to glucose-free medium and then plated for clonogenic survival (Fig. 1 B). Fig. 1 shows cells that overexpressed GRX were resistant to glucose deprivation-induced cell killing as assayed by clonogenic survival. We previously reported that glucose deprivation resulted in the activation of the SAPK signal transduction pathway, which led to increases in oxidative stress and cytotoxicity (6Lee Y.J. Galoforo S.S. Sim J.E. Ridnour L.A. Choi J. Forman H.J. Corry P.M. Spitz D.R. Free Radic. Biol. Med. 2000; 28: 575-584Crossref PubMed Scopus (26) Google Scholar). To determine if overexpression of GRX might protect cells from glucose deprivation-induced cytotoxicity by preventing the activation of SAPK signal transduction, GRX-overexpressing MCF-7/ADR cells were exposed to glucose-free medium, and SAPK activation was determined. Western blots using an antibody specific for the activated form of JNK1 (anti-ACTIVE JNK antibody) showed glucose deprivation-induced JNK activation (Fig.2) within 5 min that was maintained for more than 2 h, in cells containing the control p
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