Direct Association with Thioredoxin Allows Redox Regulation of Glucocorticoid Receptor Function
1999; Elsevier BV; Volume: 274; Issue: 5 Linguagem: Inglês
10.1074/jbc.274.5.3182
ISSN1083-351X
AutoresYuichi Makino, Noritada Yoshikawa, Kensaku Okamoto, Kiichi Hirota, Junji Yodoi, Isao Makino, Hirotoshi Tanaka,
Tópico(s)Heat shock proteins research
ResumoThe glucocorticoid receptor (GR) is considered to belong to a class of transcription factors, the functions of which are exposed to redox regulation. We have recently demonstrated that thioredoxin (TRX), a cellular reducing catalyst, plays an important role in restoration of GR function in vivo under oxidative conditions. Although both the ligand binding domain and other domains of the GR have been suggested to be modulated by TRX, the molecular mechanism of the interaction is largely unknown. In the present study, we hypothesized that the DNA binding domain (DBD) of the GR, which is highly conserved among the nuclear receptors, is also responsible for communication with TRX in vivo. Mammalian two-hybrid assay and glutathione S-transferase pull-down assay revealed the direct association between TRX and the GR DBD. Moreover, analysis of subcellular localization of TRX and the chimeric protein harboring herpes simplex viral protein 16 transactivation domain and the GR DBD indicated that the interaction might take place in the nucleus under oxidative conditions. Together these observations indicate that TRX, via a direct association with the conserved DBD motif, may represent a key mediator operating in interplay between cellular redox signaling and nuclear receptor-mediated signal transduction. The glucocorticoid receptor (GR) is considered to belong to a class of transcription factors, the functions of which are exposed to redox regulation. We have recently demonstrated that thioredoxin (TRX), a cellular reducing catalyst, plays an important role in restoration of GR function in vivo under oxidative conditions. Although both the ligand binding domain and other domains of the GR have been suggested to be modulated by TRX, the molecular mechanism of the interaction is largely unknown. In the present study, we hypothesized that the DNA binding domain (DBD) of the GR, which is highly conserved among the nuclear receptors, is also responsible for communication with TRX in vivo. Mammalian two-hybrid assay and glutathione S-transferase pull-down assay revealed the direct association between TRX and the GR DBD. Moreover, analysis of subcellular localization of TRX and the chimeric protein harboring herpes simplex viral protein 16 transactivation domain and the GR DBD indicated that the interaction might take place in the nucleus under oxidative conditions. Together these observations indicate that TRX, via a direct association with the conserved DBD motif, may represent a key mediator operating in interplay between cellular redox signaling and nuclear receptor-mediated signal transduction. Gene expression is regulated via interactions between factors, including DNA-binding proteins, coactivators/corepressors, histones, and DNA, and it allows fine tuning of essential cellular processes;e.g. proliferation, growth, differentiation, energy metabolism, and stress responses (1Kadonaga J.T. Cell. 1998; 92: 307-313Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Among others, redox regulation has now been considered to be one of the important determinants for activity of transcription factors and subsequent gene expression; DNA binding activity of a growing number of transcription factors, including AP-1 (2Abate C. Patel L. Rauscher III, F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1375) Google Scholar, 3Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1269) Google Scholar), NFκB (4Matthews J.R. Wakasugi N. Virelizier J.-L. Yodoi J. Hay R.T. 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A. 1992; 89: 7531-7535Crossref PubMed Scopus (81) Google Scholar), TTF-1 (12Kambe F. Nomura Y. Okamoto T. Seo H. Mol. Endocrinol. 1996; 10: 801-812PubMed Google Scholar), and Ets (13Wasylyk C. Wasylyk B. Nucleic Acids Res. 1993; 21: 523-529Crossref PubMed Scopus (51) Google Scholar), has been shown to be regulated by thiol-redox controlling systems. Aside from chemical oxidants/reductants, however, it largely remains to be elucidated which endogenous factor might be involved in redox regulation of transcription factors. A cellular reducing catalyst thioredoxin (TRX) 1The abbreviations used are: GR, glucocorticoid receptor; TRX, thioredoxin; DBD, DNA binding domain; GST, glutathioneS-transferase; VP16, viral protein 16; Ref-1, redox factor-1; NLS, nuclear localization signal; LBD, ligand binding domain; GFP, green fluorescent protein; GRE, glucocorticoid response element; PBS, phosphate-buffered saline; NFκB, nuclear factor κB. is a small protein with a molecular mass of 13 kDa, and acts as a potent disulfide reductase for a variety of target proteins (14Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 15Holmgren A. Structure. 1995; 3: 239-243Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Recently, TRX has been shown to interact, directly or indirectly, with several transcription factors. For example, TRX facilitates the DNA binding and transcriptional activities of NFκB by reducing Cys62 in the DNA binding loop of p50 subunit (4Matthews J.R. Wakasugi N. Virelizier J.-L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (726) Google Scholar, 16Okamoto T. Ogiwara H. Hayashi T. Mitsui A. Kawabe T. Yodoi J. Int. Immunol. 1992; 4: 811-819Crossref PubMed Scopus (133) Google Scholar). TRX has also been suggested to participate in redox regulation of AP-1 via interaction with another reducing catalyst, redox factor-1 (Ref-1) (17Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (723) Google Scholar), which reduces conserved cysteine residues within the DNA binding domains of Fos and Jun (18Xanthoudakis S. Miao G. Wang F. Pan Y.-C.E. Curran T. EMBO J. 1992; 11: 3323-3335Crossref PubMed Scopus (823) Google Scholar, 19Xanthoudakis S. Curran T. EMBO J. 1992; 11: 653-665Crossref PubMed Scopus (600) Google Scholar, 20Xanthoudakis S. Miao G.G. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 23-27Crossref PubMed Scopus (319) Google Scholar). TRX has thus been suggested as a candidate endogenous molecule operating in the redox-regulation of gene expression via modulation of many transcription factors. The glucocorticoid receptor (GR) is a ligand-inducible transcription factor that belongs to the superfamily of the nuclear receptors, comprising a central DNA binding domain (DBD), nuclear localization signals (NLSs), a ligand binding domain (LBD), and several transactivation functions (21Beato M. Herrlich P. Schütz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1638) Google Scholar, 22Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar, 23Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar). After binding hormone and dissociation of heat shock proteins, the GR translocates into the nucleus, thereby communicating with basal transcriptional machinery, coactivators, other transcription factors, and DNA and modulating target gene expression to produce pleiotropic glucocorticoid hormone actions (24Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2058) Google Scholar, 25Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1996; 83: 835-839Abstract Full Text PDF Scopus (6088) Google Scholar, 26Katzenellenbogen J.A. O'Malley B.W. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 119-131Crossref PubMed Scopus (526) Google Scholar). Numerous biochemical studies have demonstrated that GR function in vitro is subject to redox modulation, via reversible modification of functionally and structurally critical cysteine residues within the GR; oxidative treatment of the GR reduces both ligand binding activity (27Chakraborti P.K. Garabedian M.J. Yamamoto K.R. Simons Jr., S.S. J. Biol. Chem. 1992; 267: 11366-11373Abstract Full Text PDF PubMed Google Scholar, 28Simons S.S.J. Pratt W.B. Methods Enzymol. 1995; 251: 406-422Crossref PubMed Scopus (44) Google Scholar) and binding to DNA cellulose (29Hutchison K.A. Matic G. Meshinchi S. Bresnick E.H. Pratt W.B. J. Biol. Chem. 1991; 266: 10505-10509Abstract Full Text PDF PubMed Google Scholar,30Bodwell J.E. Holbrook N.J. Munck A. Biochemistry. 1984; 23: 1392-1398Crossref PubMed Scopus (59) Google Scholar). We have previously demonstrated that metal ions that have high affinity for thiols interfere GR functions in living cells, plausibly via similar modification of cysteine thiols (31Makino Y. Tanaka H. Dahlman-Wright K. Makino I. Mol. Pharmacol. 1996; 49: 612-620PubMed Google Scholar). Moreover, a recent study has shown that cellular redox state is an important determinant of GR function in vivo and that TRX is implicated in redox regulation of GR function; GR-mediated gene expression is suppressed by oxidative treatment of cells, which overexpression of TRX counteracts (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). Suggested mechanisms are 1) that TRX may preserve ligand binding activity of the GR, in accordance with previous biochemical observations showing that ligand binding activity of cytosolic GR is maintained by the presence of TRX systems (TRX and TRX reductase) (33Grippo J.F. Tienrungroj W. Dahmer K.M. Housley P.R. Pratt W.B. J. Biol. Chem. 1983; 258: 13658-13664Abstract Full Text PDF PubMed Google Scholar,34Grippo J.F. Holmgren A. Pratt W.B. J. Biol. Chem. 1985; 260: 93-97Abstract Full Text PDF PubMed Google Scholar), and 2) that the nuclear translocation, DNA binding, and transactivation of the GR may also be influenced by TRX. The precise mechanisms of molecular interplay between the GR and TRX, however, are not yet well understood. To explore the molecular mechanism of redox regulation of GR function with particular reference to its interaction with TRX, we here report that the conserved DBD of the GR, independent of the LBD, is a target for redox regulation by TRX. Mechanistically, direct association between the DBD and TRX in the nucleus was shown by mammalian two-hybrid and in vitro protein-protein interaction assays. Thus, we suggest that TRX may play a critical role allowing cellular redox potential to modulate steroid hormone receptor-mediated gene expression. COS7, CV-1, and HeLa cells were obtained from RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), pH 7.0, supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.) and antibiotics. The human GR overexpressing (300,000–500,000 molecules/cell) Chinese hamster ovary-pMTGR cells (35Alksnis M. Barkhem T. Strömstedt P.-E. Ahola H. Kutoh E. Gustafsson J.-Å. Poellinger L. Nilson S. J. Biol. Chem. 1991; 266: 10078-10085Abstract Full Text PDF PubMed Google Scholar), were kindly provided by Dr. S. Nilsson (Karo Bio, Huddinge, Sweden) and maintained in Ham's F-12 medium (Life Technologies, Inc.) supplemented with antibiotics and 10% heat-inactivated fetal calf serum in the presence of cadmium and zinc ions, each at a concentration of 40 μm. In all experiments, serum was stripped with dextran-coated charcoal, and cells were cultured in a humidified atmosphere at 37 °C with 5% CO2. Diamide and dexamethasone were purchased from Sigma. Other chemicals were from Wako Pure Chemical (Osaka, Japan). Recombinant TRX was produced according to the method described previously and kindly provided by Ajinomoto Co. Inc., Basic Research Laboratory (Kawasaki, Japan) (36Tagaya Y. Wakasugi H. Masutani H. Nakamura S. Iwata S. Mitsui A. Fujii S. Wakasugi N. Tursz T. Yodoi J. Mol. Immunol. 1990; 27: 1279-1289Crossref PubMed Scopus (62) Google Scholar). Monoclonal antibody against the carboxyl-terminal sequence of TRX was prepared as described previously (37Tagaya Y. Okada M. Sugie K. Kasahara T. Kondo N. Hamuro J. Brown N. Arai K.-I. Yokota T. Wakasugi H. Yodoi J. EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (516) Google Scholar). Anti-GST polyclonal antibody was obtained from Amersham Pharmacia Biotech. All enzymes were purchased from TaKaRa Syuzo (Kyoto, Japan). The expression vectors for the wild-type and mutant GR, RShGRα and I550, respectively, have been described elsewhere (38Rangarajan P.N. Umesono K. Evans R.M. Mol. Endocrinol. 1992; 6: 1451-1457Crossref PubMed Scopus (143) Google Scholar) and were kindly supplied by Dr. R. M. Evans (Salk Institute, La Jolla, CA). The expression plasmids for TRX and antisense TRX, pcDSRαADF and pASADF, respectively, have also previously been described (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). To construct expression plasmids for fusion protein VP16 transactivation domain and the DBD of the human GR with or without constitutive NLS, NL1, the DNA fragments encoding 129 amino acids (serine 403 to leucine 532) or 87 amino acids (serine 403 to alanine 490) of the human GR were amplified by polymerase chain reaction with appropriate flanking sequences for enzymatic cleavage and inserted into the BamHI site of the parent pCMX-VP16 (39Perlman T. Rangarajan P.N. Umesono K. Evans R.M. Genes Dev. 1993; 7: 1411-1422Crossref PubMed Scopus (333) Google Scholar), resulting in pCMX-VP16-GR DBD and pCMX-VP16-GR DBDΔNL1, respectively. Construction of the expression plasmid for the fusion protein of the DNA binding domain of GAL4 (40Ma J. Ptashne M. Cell. 1987; 51: 113-119Abstract Full Text PDF PubMed Scopus (498) Google Scholar) and TRX has been previously described (17Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (723) Google Scholar). The expression plasmids for the green fluorescent protein (GFP)-fused chimeric protein, pCMX-GFP-VP16-GR DBD and pCMX-GFP-VP16-GR DBDΔNL1, were made by inserting a polymerase chain reaction-cloned DNA fragments encoding VP16-GR DBD and VP16-GR DBDΔNL1, respectively, into the pCMX-GFP vector (41Ogawa H. Inoue S. Tsujii F.I. Yasuda K. Umesono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11899-11903Crossref PubMed Scopus (235) Google Scholar). The glucocorticoid-responsive reporter construct pGRE-Luc (31Makino Y. Tanaka H. Dahlman-Wright K. Makino I. Mol. Pharmacol. 1996; 49: 612-620PubMed Google Scholar) and the GAL4-responsive luciferase reporter tk-GALpx3-Luc (17Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (723) Google Scholar) were previously described. The β-galactosidase expression plasmid pCH110 (Amersham Pharmacia Biotech) was used as an internal control for transfection efficiency when appropriate. Partially purified GR was prepared from Chinese hamster ovary-pMTGR whole cell extract essentially as described by Cairns et al. (42Cairns W. Cairns C. Pongratz I. Poellinger L. Okret S. J. Biol. Chem. 1991; 266: 11221-11226Abstract Full Text PDF PubMed Google Scholar). Briefly, whole cell extracts were prepared in the presence of molybdate and chromatographed through a phosphocellulose column. The flow-through material was then applied to a DEAE-Sepharose column, and the absorbed material was eluted with 200 mm NaCl. Salt and molybdate were removed from the pooled, eluted material by chromatography on Sephadex G-25. After transformation (25 °C for 60 min), the receptor fraction was further purified by fast protein liquid anion-exchange Mono Q chromatography (Amersham Pharmacia Biotech). Fractions containing receptor were identified by ligand binding and specific DNA binding assays. These fractions contained 10–20% pure receptor and were used for protein-DNA interaction experiments. Electrophoretic mobility shift assay was carried out as described previously (43Tanaka H. Makino Y. Dahlman-Wright K. Gustafsson J.-Å. Okamoto K. Makino I. Mol. Pharmacol. 1995; 48: 938-945PubMed Google Scholar). Briefly, partially purified GR (usually 10 ng of protein per reaction) was incubated with 0.2 ng of 32P-labeled GRE oligonucleotide (5′-CGAGTAGCTAGAACAGACTGTTCTGAGG-3′) probe in a 20-μl reaction mixture containing 5 mm HEPES, pH 7.9, 60 mm KCl, 2.5 mm EDTA, 2.5 mmMgCl2, 10 mm spermidine, 0.25 mmdithiothreitol, 10% glycerol, and 100 ng of poly(dI-dC) (Amersham Pharmacia Biotech) for 15 min on ice. The reaction mixture was loaded onto a 4% nondenaturing polyacrylamide gel containing 0.25× TBE (1× TBE is 89 mm Tris borate, 89 mm boric acid, and 2 mm EDTA). The gels were run at 350 V for 2 h and dried. Results were visualized by autoradiography. Cells grown on 8-chambered sterile glass slides (Nippon Becton & Dickinson, Tokyo, Japan) were fixed for immunostaining using a freshly prepared solution of 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS) overnight at 4 °C. Immunocytochemistry was carried out as described previously with minor modification (44Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). Briefly, after fixation, cells were washed five times with PBS at room temperature and incubated with anti-human TRX monoclonal mouse antibody at 1 μg/ml in PBS containing 0.1% Triton X-100 for 9 h at 4 °C. The cells were then again washed five times with PBS and incubated with biotinylated rabbit anti-mouse IgG antibody at a dilution of 1:200 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. The cells were then washed a further five times with PBS and incubated with fluorescein isothiocyanate-conjugated streptavidin at a dilution of 1:100 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. The cells were then washed a final five times with PBS and mounted with GEL/MOUNTTM (Biomeda Co. Ltd., Foster City, CA) for examination on a laser scanning microscope (Zeiss LSM 510, Karl Zeiss Jena GmbH, Jena, Germany). Transient transfection was performed as described previously (31Makino Y. Tanaka H. Dahlman-Wright K. Makino I. Mol. Pharmacol. 1996; 49: 612-620PubMed Google Scholar). Briefly, cells were plated on plastic culture dishes (IWAKI Glass, Funabashi, Japan) to 30–50% confluence and washed with PBS three times, and medium was replaced with Opti-MEM (Life Technologies, Inc.). Plasmid mixture was mixed with TransIT-LT1 transfection reagent (Pan Vera Corp., Madison, WI) and added to the culture. After 6 h of incubation, the medium was replaced with fresh Dulbecco's modified Eagle's medium supplemented with 2% dextran-coated charcoal-stripped fetal calf serum, and the cells further cultured in the presence or absence of various ligands for 24 h. After normalization of transfection efficiency by β-galactosidase expression, luciferase enzyme activity was determined in a luminometer (Berthold GmbH & Co. KG, Bad Wildbad, Germany) essentially as described before (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). For the construction of the expression plasmid for GST-GR DBD fusion protein, the DNA fragment encoding the DNA binding domain (serine 403 to leucine 532) of the human GR was amplified by polymerase chain reaction and ligated in frame into the BamHI site of the pGEX4T-3 plasmid (Amersham Pharmacia Biotech). GST fusion protein was expressed inEscherichia coli BL21 (DE3) (Stratagene, La Jolla, CA) by induction with 0.1 mmisopropyl-β-d-thiogalactopyranoside. The cell pellets were suspended in PBS containing 1 mm ZnCl2, 1% Triton X-100, and 5 mm dithiothreitol and subsequently sonicated. Lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and supernatants were incubated with 200 μg of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) at room temperature for 30 min. Beads were washed three times with PBS, and bound proteins were eluted with 1 ml of elution buffer (10 mm glutathione, 50 mm Tris, pH 8.0, 1 mm dithiothreitol). Eluted proteins were dialyzed against PBS containing 1 mm dithiothreitol before storage at −80 °C. The protein was characterized by Western blot analysis. Bacterially expressed GST-GR DBD fusion protein (10 μg) or GST (5 μg) was incubated with 45 μg of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) in 250 μl of PBS at room temperature for 30 min. After being washed three times, the beads were incubated with 10 μg of recombinant TRX in PBS containing 3% bovine serum albumin and 0.1% Triton X-100, with or without 5 mm diamide at room temperature for 30 min. The beads were then washed five times, and bound proteins were eluted by boiling in 30 μl of 2× SDS loading buffer (20% v/v glycerol, 4.6% w/v SDS, 0.125 m Tris-HCl, pH 6.8, 4% 2-mercaptoethanol), and separated by polyacrylamide gel electrophoresis. The proteins were then electrically transferred onto polyvinylidine difluoride membrane (Bio-Rad) and probed with appropriate antibodies. Antigen-antibody complexes were detected with the ECL Western blot detection kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. As described above, we hypothesized that TRX may preserve GR-dependent gene expression under oxidative conditions (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). To further explore this hypothesis, we manipulated cellular TRX levels by means of transfection of either sense or antisense TRX expression plasmids and tested glucocorticoid-dependent reporter gene expression under oxidative conditions. HeLa cells were transfected with the GR expression plasmid, the glucocorticoid-responsive reporter plasmid, and either TRX or antisense TRX expression plasmid, and cultured in the presence of 100 nm dexamethasone and 1 mmH2O2 as indicated (Fig.1). As reported previously (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar), treatment with H2O2 resulted in an approximately 3-fold decrease in hormone induction response (lanes 1–3). When TRX was overexpressed, the repression effect of H2O2 was dose-dependently abolished (lanes 4–6, hatched columns). In contrast, reduction of cellular TRX levels, accomplished by expression of antisense TRX (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar, 45Yokomizo A. Ono M. Nanri H. Makino Y. Ohga T. Wada M. Okamoto T. Yodoi J. Kuwano M. Kohno K. Cancer Res. 1995; 55: 4293-4296PubMed Google Scholar), resulted in increased sensitivity to H2O2 and a further decrease in hormone induction (lanes 4–6, filled columns). This indicates that cellular TRX levels are an important determinant of glucocorticoid-mediated gene expression in oxidative conditions. We next studied the subcellular localization of TRX in HeLa cells using indirect immunofluorescent analysis. HeLa cells were cultured in the presence or absence of H2O2 for 2 h and then fixed for immunodetection of TRX. In the absence of H2O2, TRX is mainly found in the cytoplasm, with some cells showing partial nuclear fluorescence (Fig. 2, left panel). However, in the presence of H2O2, the majority of the cells showed nuclear-predominant TRX fluorescence (right panel), indicating that TRX translocates into the nucleus under oxidative conditions. If TRX translocates into the nucleus under oxidative conditions, it is likely that TRX interacts with the GR in the nucleus to restore the transactivational function of the GR. The GR mutant I550 (which lacks the ligand binding domain, is constitutively present in the nucleus even in the absence of hormone (46Jewell C.M. Webster J.C. Burnstein K.L. Sar M. Bodwell J.E. Cidlowski J.A. J. Steroid Biochem. Mol. Biol. 1995; 55: 135-146Crossref PubMed Scopus (55) Google Scholar), and acts as a ligand-independent transcriptional activator (47Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (678) Google Scholar)) was shown to be sensitive to oxidative stress (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). Indeed, suppression of I550-mediated gene expression either by treatment with H2O2 (Fig.3 A) or antisense TRX expression (Fig. 3 B) is similar to that seen for wild-type GR (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar), further evidence for the nuclear location of the GR-TRX interaction. Electrophoretic mobility shift assay using either partially purified full-length GR (Fig. 4) or the recombinant GR DBD (32Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar) revealed that sequence-specific DNA binding activity of the GR is abolished by addition of the oxidative reagent diamide, and progressively restored by addition of recombinant TRX. Based on these results and the fact that the GR DBD contains several cysteine residues as a part of the zinc finger structures (22Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar), we speculated that the DBD of the GR could be one of the targets of TRX in the nucleus. To further examine this possibility, we constructed expression plasmids for fusion proteins of the GR DBD with or without the constitutive NLS (NL1) plus the activation domain of the herpes simplex virus VP16 protein, VP16-GR DBD and VP16-GR DBDΔNL1, respectively (Fig. 5 A). Note that transactivation domain of VP16 does not contain cysteine residues (48Triezenberg S.J. Kingsbury R.C. McKnight S.L. Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (595) Google Scholar). When these chimeric proteins were expressed as a fusion protein with GFP in COS7 cells, VP16-GR DBD was shown to be constitutively localized in the nucleus, whereas VP16-GR DBDΔNL1 was exclusively cytoplasmic (Fig. 5 B). Fig. 5 C shows that VP16-GR DBD but not VP16-GR DBDΔNL1 acts as a GRE-specific constitutive transcriptional activator. Neither VP16-GR DBDΔNL1, VP16, nor a fusion protein of the VP16 activation domain plus GAL4 DBD (VP16-GAL4) transactivates the GRE-driven reporter plasmid (Fig.5 C).Figure 5A, schematic presentation of fusion proteins VP16-GR DBD and VP16-GR DBDΔNL1. B, subcellular localization of GFP-VP16-GR DBD and GFP-VP16-GR DBDΔNL1. COS7 cells were transfected with 5 μg of GFP-VP16-GR DBD or GFP-VP16-GR DBDΔNL1 expression plasmids. After 24 h of transfection, subcellular localization of expressed chimeric proteins was examined by fluorescence microscopy. C, VP16-GR DBD activates gene expression through sequence-specific interaction with GRE in a ligand-independent manner. COS7 cells were grown in 60-mm-diameter culture dishes and transfected with 2 μg of reporter plasmids pGRE-Luc or tk-GALpx3-Luc and 100 ng of the expression plasmids for VP16-GR DBD, VP16-GR DBDΔNL1, VP16-GAL4, and VP16. The cells were harvested at 24 h after transfection, and cellular luciferase activity was determined as described under "Experimental Procedures." Expressed luciferase activities when the cells were transfected with each reporter plasmid alone served as controls (columns 1 and 6). Results are plotted as mean ± S.D. of three experiments. D,H2O2-mediated repression and TRX-mediated restoration of the GR DBD-GRE interaction in living cells. COS7 cells were transfected with 2 μg of pGRE-Luc reporter, 100 ng of VP16-GR DBD expression plasmid, and various amounts of TRX expression plasmid pcDSRαADF, as indicated. The cells were incubated with increasing concentrations of H2O2 for 24 h, and then cellular luciferase activity was measured as described under "Experimental Procedures." Results are expressed as fold induction compared with the cells without either cotransfection of any expression plasmids or treatment with H2O2. Data represent the mean ± S.D. of three independent experiments.View Large Image Figure ViewerDownload (PPT) VP16-GR DBD can thus be used in monitoring of the influence of TRX on the GR DBD and its interaction with GRE in the nucleus. To this end, VP16-GR DBD and TRX were coexpressed in COS7 cells, and the cells were cultured in the absence or pr
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