Functional Modulation of the Glucocorticoid Receptor and Suppression of NF-κB-dependent Transcription by Ursodeoxycholic Acid

Ursodeoxycholic acid (UDCA) is the current mainstay of treatment for various liver diseases including primary biliary cirrhosis. UDCA acts as a bile secretagogue, cytoprotective agent, immunomodulator, and inhibitor of cellular apoptosis. Despite this cumulative evidence of the cytoprotective and immunosuppressive effects of UDCA, both the target molecule and pathway of UDCA action remain unknown. We previously described that, in the absence of glucocorticoid ligand, UDCA activates the glucocorticoid receptor (GR) into DNA binding species but does not elicit its transactivational function in a transient transfection assay. Here we further studied the molecular mechanism of UDCA action and revealed that the ligand binding domain of the GR is responsible for UDCA-dependent nuclear translocation of the GR. Indeed, we demonstrated that UDCA acts on the distinct region of the ligand binding domain when compared with the classical GR agonist dexamethasone, resulting in loss of coactivator recruitment and differential regulation of gene expression by the GR. Our data clearly indicated that UDCA, at least in part via activation of the GR, suppresses NF-κB-dependent transcription through the intervention of GR-p65 interaction. Together with the established clinical safety of UDCA, we may propose that UDCA could be a prototypical compound for development of a novel and selective GR modifier. Ursodeoxycholic acid (UDCA) is the current mainstay of treatment for various liver diseases including primary biliary cirrhosis. UDCA acts as a bile secretagogue, cytoprotective agent, immunomodulator, and inhibitor of cellular apoptosis. Despite this cumulative evidence of the cytoprotective and immunosuppressive effects of UDCA, both the target molecule and pathway of UDCA action remain unknown. We previously described that, in the absence of glucocorticoid ligand, UDCA activates the glucocorticoid receptor (GR) into DNA binding species but does not elicit its transactivational function in a transient transfection assay. Here we further studied the molecular mechanism of UDCA action and revealed that the ligand binding domain of the GR is responsible for UDCA-dependent nuclear translocation of the GR. Indeed, we demonstrated that UDCA acts on the distinct region of the ligand binding domain when compared with the classical GR agonist dexamethasone, resulting in loss of coactivator recruitment and differential regulation of gene expression by the GR. Our data clearly indicated that UDCA, at least in part via activation of the GR, suppresses NF-κB-dependent transcription through the intervention of GR-p65 interaction. Together with the established clinical safety of UDCA, we may propose that UDCA could be a prototypical compound for development of a novel and selective GR modifier. ursodeoxycholic acid androgen receptor DNA binding domain fetal calf serum fluorescein isothiocyanate green fluorescent protein glucocorticoid receptor glucocorticoid response element heat shock protein 90 ligand binding domain mineralocorticoid receptor nuclear receptor interaction domain phosphate-buffered saline phorbol 12-myristate acetate progesterone receptor transcription intermediary factor 2 4-morpholinepropanesulfonic acid Ursodeoxycholic acid (UDCA)1 is the current mainstay of treatment for primary biliary cirrhosis, which is a chronic cholestatic liver disease characterized by the destruction of biliary epithelial cells (i.e. cholangiocytes), presumably by autoimmune mechanism(s) (1Makino I. Tanaka H. J. Gastroenterol. Hepatol. 1998; 13: 659-664Crossref PubMed Scopus (40) Google Scholar, 2Gershwin M.E. Ansari A.A. Mackay I.R. Nakanuma Y. Nishio A. Rowley M.J. Coppel R.L. Immunol. Rev. 2000; 174: 210-225Crossref PubMed Scopus (258) Google Scholar, 3Heathcote E.J. Hepatology. 2000; 31: 1005-1013Crossref PubMed Scopus (393) Google Scholar). This hydrophilic bile acid is reported to induce biochemical, histological, and prognostic improvement in patients with primary biliary cirrhosis in the virtual absence of adverse reactions (3Heathcote E.J. Hepatology. 2000; 31: 1005-1013Crossref PubMed Scopus (393) Google Scholar). UDCA acts as a bile secretagogue and cytoprotective agent (1Makino I. Tanaka H. J. Gastroenterol. Hepatol. 1998; 13: 659-664Crossref PubMed Scopus (40) Google Scholar) and exerts diverse immunomodulatory actionsin vitro: suppression of immunoglobulin, interleukin-2, interleukin-4, and interferon-γ production from lymphocytes; attenuation of major histocompatibility complex expression on hepatocytes and cholangiocytes; increase in natural killer cell activity; and inhibition of eosinophil degranulation (1Makino I. Tanaka H. J. Gastroenterol. Hepatol. 1998; 13: 659-664Crossref PubMed Scopus (40) Google Scholar, 4Lacaille F. Paradis K. Hepatology. 1993; 18: 165-172PubMed Google Scholar, 5Nishigaki Y. Ohnishi H. Moriwaki H. Muto Y. Dig. Dis. Sci. 1996; 41: 1487-1493Crossref PubMed Scopus (38) Google Scholar, 6Yamazaki K. Suzuki K. Nakamura A. Sato S. Lindor K.D. Batts K.P. Tarara J.E. Kephart G.M. Kita H. Gleich G.J. Hepatology. 1999; 30: 71-78Crossref PubMed Scopus (67) Google Scholar, 7Yoshikawa M. Tsujii T. Matsumura K. Yamao J. Matsumura Y. Kubo R. Fukui H. Ishizaka S. Hepatology. 1992; 16: 358-364Crossref PubMed Scopus (228) Google Scholar, 8Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Crossref PubMed Scopus (139) Google Scholar, 9Hirano F. Tanaka H. Makino Y. Okamoto K. Makino I. J. Gastroenterol. 1996; 31: 55-60Crossref PubMed Scopus (20) Google Scholar). Recently, it has been shown that UDCA inhibits cellular apoptosis via stabilization of the mitochondria membrane (10Botla R. Spivey J.R. Aguilar H. Bronk S.F. Gores G.J. J. Pharmacol. Exp. Ther. 1995; 272: 930-938PubMed Google Scholar, 11Rodrigues C.M. Fan G. Ma X. Kren B.T. Steer C.J. J. Clin. Invest. 1998; 101: 2790-2799Crossref PubMed Scopus (468) Google Scholar). Despite this cumulative evidence of the cytoprotective and immunosuppressive effects of UDCA, both the target molecule and pathway of UDCA action remain unknown. The glucocorticoid receptor (GR) is a member of the nuclear receptors and an important transcriptional regulator involved in widely diverse physiological functions such as control of embryonic development, cell differentiation, and metabolic homeostasis (12Gustafsson J.A. Carlstedt-Duke J. Poellinger L. Okret S. Wikstrom A.C. Bronnegard M. Gillner M. Dong Y. Fuxe K. Cintra A. Endocr. Rev. 1987; 8: 185-234Crossref PubMed Scopus (365) Google Scholar, 13Reichardt H.M. Tronche F. Berger S. Kellendonk C. Schutz G. Adv. Pharmacol. 2000; 47: 1-21Crossref PubMed Scopus (56) Google Scholar). Moreover, therapeutic activities of glucocorticoids are believed to inevitably be mediated by the GR (14Boumpas D.T. Chrousos G.P. Wilder R.L. Cupps T.R. Balow J.E. Ann. Intern. Med. 1993; 119: 1198-1208Crossref PubMed Scopus (668) Google Scholar). The nuclear receptors share several structural features (e.g. the ligand binding domain (LBD), DNA binding domain (DBD), and several transactivation domains (15Lee K.C. Lee Kraus W. Trends Endocrinol. Metab. 2001; 12: 191-197Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar)). Concerning the GR, the NH2-terminal domain activation function-1 contains sequences responsible for activation of target genes and presumably interacts with the components of the basal transcription machinery and/or with cofactors and other transcription factors, largely in a cell- or tissue-specific context. The central part of the receptor constitutes the DBD, which participates in receptor dimerization, nuclear translocation, and transactivation. The structural motif of the DBD is two zinc fingers formed by the coordination of four cysteines to one zinc atom. Adjacent to the second zinc finger, the amino acids responsible for the nuclear localization, the nuclear localization signal, exist. The carboxyl-terminal portion of the receptor includes the LBD and the sequences for heat shock protein 90 (hsp90) binding, nuclear translocation, dimerization, and transactivation. The COOH-terminal transcriptional activation domain is hormone-dependent and termed activation function-2. The very COOH-terminal portion of the receptor, activation function-2 core, serves as a molecular switch that recruits coactivator proteins and activates the transcription of target genes when flipped into the active conformation by hormone binding (12Gustafsson J.A. Carlstedt-Duke J. Poellinger L. Okret S. Wikstrom A.C. Bronnegard M. Gillner M. Dong Y. Fuxe K. Cintra A. Endocr. Rev. 1987; 8: 185-234Crossref PubMed Scopus (365) Google Scholar, 16Evans R.M. Recent Prog. Horm. Res. 1989; 45: 1-22PubMed Google Scholar, 17Kumar R. Thompson E.B. Steroids. 1999; 64: 310-319Crossref PubMed Scopus (319) Google Scholar, 18Picard D. Kumar V. Chambon P. Yamamoto K.R. Cell Regul. 1990; 1: 291-299Crossref PubMed Scopus (221) Google Scholar). On the other hand, the GR can also mutually interfere with other signaling pathways such as those mediated by the transcription factor NF-κB (19Gottlicher M. Heck S. Herrlich P. J. Mol. Med. 1998; 76: 480-489Crossref PubMed Scopus (323) Google Scholar), which is an inducible transcription factor that regulates expression of various genes involved in inflammation and immune responses (20Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4341) Google Scholar, 21Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar, 22Baldwin Jr., A.S. J. Clin. Invest. 2001; 107: 3-6Crossref PubMed Google Scholar). NF-κB consists of a dimer from five related proteins, most typically a heterodimer composed of p65/RelA and p50 subunits. The regulation of NF-κB is achieved through interaction with an inhibitory protein known as IκB that binds to NF-κB and sequesters it in the cytoplasm. Once cells are stimulated with inducers such as proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1), two serine residues of the IκB protein are phosphorylated by IκB kinases. Phosphorylation of IκB targets it for ubiquitination and subsequent degradation by the 26 S proteasome and renders the nuclear localization signal of NF-κB unmasked. Then NF-κB translocates from the cytoplasm into the nucleus and regulates the transcription of target genes (23Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (1016) Google Scholar, 24Ghosh S. Immunol. Res. 1999; 19: 183-189Crossref PubMed Scopus (118) Google Scholar). In addition to this “classical” milieu, recent reports have suggested that several alternative pathways lead not only to activation but also to repression of NF-κB (25Uranishi H. Tetsuka T. 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Despite possible therapeutic antagonism of NF-κB by the GR in inflammatory disorders, however, side effects such as hypothalamic-pituitary-adrenal axis insufficiency, diabetes, altered lipid metabolism, osteoporosis, steroid myopathy, and infectious and neuropsychiatric complications limit the therapeutic use of the classical glucocorticoid agonists (14Boumpas D.T. Chrousos G.P. Wilder R.L. Cupps T.R. Balow J.E. Ann. Intern. Med. 1993; 119: 1198-1208Crossref PubMed Scopus (668) Google Scholar). In this line, dissociation of glucocorticoid-dependent transactivation and transrepression may lead to the development of better tolerated drugs (20Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4341) Google Scholar). Already several compounds have been reported to exhibit strong inhibition of NF-κB but weak induction of the GRE-dependent reporter gene; however, clinical application of those compounds is still pending (33Adcock I.M. Nasuhara Y. Stevens D.A. Barnes P.J. Br. J. Pharmacol. 1999; 127: 1003-1011Crossref PubMed Scopus (105) Google Scholar, 34Vayssiere B.M. Dupont S. Choquart A. Petit F. Garcia T. Marchandeau C. Gronemeyer H. Resche-Rigon M. Mol. Endocrinol. 1997; 11: 1245-1255Crossref PubMed Scopus (300) Google Scholar, 35Hofmann T.G. Hehner S.P. Bacher S. Droge W. Schmitz M.L. FEBS Lett. 1998; 441: 441-446Crossref PubMed Scopus (50) Google Scholar, 36Belvisi M.G. Wicks S.L. Battram C.H. Bottoms S.E. Redford J.E. Woodman P. Brown T.J. Webber S.E. Foster M.L. J. Immunol. 2001; 166: 1975-1982Crossref PubMed Scopus (173) Google Scholar). We previously described that UDCA, without direct binding to the GR, activates the GR into DNA binding species but does not elicit its transactivational function in a transient transfection assay (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). Moreover, we predicted that the target domain in the GR of UDCA might be the LBD (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). Here we further studied the molecular mechanism of UDCA action and revealed that the LBD is responsible for UDCA-dependent nuclear translocation of the GR. Indeed, it is suggested that UDCA interacts with the distinct region of the LBD when compared with a classical GR agonist dexamethasone, resulting in differential regulation of gene expression by the GR. Our data clearly indicated that UDCA-activated GR suppresses NF-κB-dependent transcription via interaction with the p65 subunit. Taking into consideration the established clinical safety of UDCA, we propose that UDCA could be a prototypical compound for the development of a novel and selective GR modifier. UDCA was donated by Mitsubishi-Tokyo Pharmaceutical Co., Tokyo, Japan. Dexamethasone was purchased from Sigma, and other chemicals were purchased from Wako Pure Chemical (Osaka, Japan) unless otherwise specified. Antibodies against the GR (PA1–512) and hsp90 (3B6 and 3G3) were purchased from Affinity Bioreagents (Golden, CO), and those against TIF2 (sc-6264), p65 (sc-372), p50 (sc-1190), and IκBα (sc-371) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated IκBα and paxillin were from New England Biolabs (Beverly, MA) and Transduction Laboratories (Lexington, KY), respectively. The expression plasmids for the chimeric protein of GFP and the human GR, mineralocorticoid receptor (MR), progesterone receptor (PR), and androgen receptor (AR) were kindly provided by Drs. H. Ogawa (Kyoto University) (for GR and MR), G. Hager (National Institutes of Health) (for PR), and J. Palvimo (Helsinki University) (for AR). The expression plasmid for TIF2, pSG5-TIF2, was a generous gift from Dr. P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). The expression plasmid for the fusion protein of the Gal4 DBD and the LBD of the GR, pCMX-Gal4-GR LBD, and that for the VP16 activation domain (VP16AD), pCMX-VP16AD, and the Gal4-driven luciferase reporter plasmid, ptk-GALpx3-LUC, were from Dr. Kazuhiko Umesono (Kyoto University, Kyoto, Japan). To construct the chimeric plasmids for NF-κB p65 and the DBD of Gal4 (amino acids 1–147), the fragments containing cDNA encoding either wild-type (amino acids 1–549), the NH2-terminal half (residues 1–285), or the COOH-terminal half (residues 286–549) of mouse p65 were generated by polymerase chain reaction using pCAGGS-p65 (a gift from Dr. H. Handa, Tokyo Institute of Technology, Yokohama, Japan) as a template. Those fragments were then cloned into EcoRI and EcoRV sites of Gal4 DBD expression plasmid pCMX-Gal4 (a gift from K. Umesono) in frame, resulting in pCMX-Gal4-p65/1–549, pCMX-Gal4-p65/1–285, and pCMX-Gal4-p65/286–549, respectively. To construct an expression plasmid for the chimeric protein of VP16AD and nuclear receptor interaction domain (NID) of TIF2, the DNA fragment encoding 173 amino acids (glutamic acid 594 to leucine 766) of the human TIF2 was amplified by polymerase chain reaction using pSG5-TIF2 as a template, and this fragment was inserted into the parent pCMX-VP16AD plasmid, resulting in VP16AD-TIF2/NID. All plasmids constructed as above were verified by sequencing. The glucocorticoid-responsive reporter plasmid pGRE-Luc was described elsewhere (38Makino 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 (163) Google Scholar). COS7, CV-1 and HeLa cells were obtained from the RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in Dulbecco's modified Eagle's medium (Iwaki Glass, Chiba, Japan). CHO-K1 cells were obtained from the RIKEN Cell Bank and maintained in Ham's F-12 medium (Iwaki Glass). All media used in this study were phenol red-free and supplemented with 10% fetal calf serum (FCS) and antibiotics. Serum steroids were stripped from FCS with dextran-coated charcoal, and cells were cultured in a humidified atmosphere at 37 °C with 5% CO2 unless otherwise specified. Cells grown on eight-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 (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar) with small modification. Briefly, cells were washed with PBS at room temperature and incubated with appropriate antibodies at 2 μg/ml in PBS containing 0.1% Triton X-100 for 9 h at 4 °C. The cells were washed and incubated with biotinylated second antibody from donkeys (Amersham Pharmacia Biotech) at a dilution of 1:200 in PBS containing 0.1% Triton X-100 for 1 h at room temperature, and then the cells were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidin at a dilution of 1:100 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Finally, the cells were mounted with GEL/MOUNT™ (Biomeda Co. Ltd., Foster, CA) and then examined by an Olympus Fluoview microscope (Olympus, Tokyo, Japan) equipped with an FITC filter set. Before transfection, cell culture medium was replaced with OPTI-MEM medium lacking phenol red (Life Technologies, Inc.). Plasmid mixture containing pGRE-Luc in the presence or absence of the GR expression plasmid was mixed with TransIT-LT1 reagent (Panvera Corp., Madison, WI) and added to the culture. The total amount of plasmid was kept constant by adding an irrelevant plasmid (pGEM3Z was used unless otherwise specified). After 6 h of incubation, the medium was replaced with fresh Dulbecco's modified Eagle's medium supplemented with 2% dextran-coated charcoal-treated FCS, and the cells were further cultured in the presence or absence of various ligands for 24 h. Luciferase enzyme activity was determined using a luminometer (Berthold GmbH & Co. KG, Bad Wildbad, Germany) essentially as described before (38Makino 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 (163) Google Scholar). For analysis of nuclear translocation of the GFP-GR, we transiently expressed GFP-tagged human GR or its mutants in COS7 cells as previously described (39Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The cells were cultured on the silane-coated coverslips in 6-cm diameter plastic dishes, and the medium was changed to OPTI-MEM medium lacking phenol red before transfection. The plasmid mixture containing 6 μg of the expression plasmids was mixed with 12 μl of TransIT-LT1 reagent and added to the culture. After 6 h of incubation, the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% dextran-coated charcoal-treated FCS, and the cells were cultured at 37 °C. GFP was expressed at detectable levels between 24 and 72 h after transfection. Routinely, cells were used for further experiments 48 h after transfection. After various treatments, cells were examined using an Olympus Fluoview microscope enclosed by an incubator and equipped with a heating stage and an FITC filter set. Quantitative assessment of the subcellular localization of expressed GFP fusion proteins was performed according to the method of Okamotoet al. (39Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In brief, subcellular localization analysis of GFP-tagged proteins was performed by blinded observers who were asked to classify ∼200 GFP-positive cells. The GFP fluorescence-positive cells were classified into four different categories: N< C for cytoplasmic dominant fluorescence;N = C, cells having equal distribution of fluorescence in the cytoplasmic and nuclear compartments;N > C for nuclear dominant fluorescence;N for exclusive nuclear fluorescence. Whole cell extract was prepared by lysing cells in 25 mm N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 8.2), 1 mm EDTA, 50 mm NaCl, 2.5 mm molybdate, and 10% glycerol. Immunoprecipitation experiments, with either the anti-hsp90 IgM antibody 3G3 (Affinity Bioreagents) or control mouse IgM antibody TEPC 183 (Sigma), were carried out as described previously (39Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Briefly, goat anti-mouse IgM (Sigma) was coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) by incubating in the coupling buffer (0.1 mNaHCO3, 0.5 m NaCl, pH 8.3) overnight at 4 °C. 35 μg of either the monoclonal anti-hsp90 IgM antibody or control mouse IgM antibody was then incubated with 80 μl of a 1:1 suspension of the goat anti-mouse IgM antibody coupled with Sepharose in MENG buffer (25 mm Mops (pH 7.5), 1 mm EDTA, 0.02% NaN3, 10% glycerol) on ice for 90 min. This Sepharose-adsorbed material was then pelleted and washed successively once with 1 ml of MENG buffer containing 0.5 m NaCl and twice with MENG buffer containing 20 mm sodium molybdate. After brief centrifugation, the pellet was resuspended in 80 μl of MENG buffer containing 20 mm sodium molybdate, 2 mm dithiothreitol, 0.25 m NaCl, and 2.5% (w/v) bovine serum albumin. In immunoprecipitation experiments, 66 μg of cellular protein was added to the resuspension. The reaction mixtures were incubated on ice for 90 min, after which Sepharose beads were pelleted by centrifugation and washed three times with MENG buffer containing 20 mm sodium molybdate and 2 mmdithiothreitol. Immunoprecipitated proteins were eluted by boiling in SDS sample buffer, analyzed by SDS-polyacrylamide gel electrophoresis, and electrically transferred to an Immobilon-NC Pure nitrocellulose membrane (Millipore Corp., Bedford, MA). Subsequently, immunoblotting was performed with a monoclonal anti-GFP antibody (CLONTECH Laboratories, Palo Alto, CA) diluted at 1:500 followed by horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (Amersham Pharmacia Biotech) diluted at 1:750. In parallel, 20 μg of whole cell extract was independently used for immunodetection of the GFP-GR and hsp90. Western immunoblot analysis for detection of hsp90 was performed in the same membrane, after stripping off the immune complex for the detection of the GFP-GR, using monoclonal mouse anti-hsp90 immunoglobulin G antibody 3B6 (Affinity Bioreagents) diluted at 1:500 followed by horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin diluted at 1:750. Antibody-protein complexes were visualized using the enhanced chemiluminescence method according to the manufacturer's protocol (Amersham Pharmacia Biotech). As described in the Introduction, we previously suggested that, although UDCA does not bind to the GR, the receptor translocates into the nucleus in the presence of UDCA in GR-overexpressing CHO cells (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). In the present study, we first addressed the specificity of such UDCA action and examined the effect of treatment with UDCA on subcellular localization of various steroid receptors, all of which are believed to be predominantly docked in the cytoplasm in the absence of cognate ligands. For this purpose, we transfected the expression plasmids for GFP-tagged MR, PR, and AR as well as GFP-GR and microscopically observed their subcellular localization after 6-h treatment with their cognate ligands (100 nm) or 200 μm UDCA. As shown in Fig. 1, some of those receptors showed weak nuclear fluorescence in the absence of ligand; however, treatment with their ligands promoted complete nuclear condensation of green fluorescence, indicating that the chimeric proteins between GFP and these nuclear receptors are capable of ligand-dependent nuclear localization. Treatment with UDCA did not significantly influence the subcellular localization of either the MR, PR, or AR; however, it preferentially induced nuclear localization of the GR (Fig. 1). To further confirm UDCA's effect on subcellular trafficking of the GR, we transfected COS7 cells with the GR expression plasmid pCMX-GR and cultured cells in the presence of UDCA for the indicated periods of time. Immunocytochemical analysis revealed that expressed GR localized in the cytoplasm in the absence of ligand (data not shown) and rapidly translocated into the nucleus after the addition of dexamethasone (Fig. 2). In the presence of UDCA, the GR revealed nuclear localization in a concentration- and time-dependent manner at a slower rate when compared with that of dexamethasone-induced translocation (Fig. 2). We previously predicted that the LBD is involved in UDCA action on the GR (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). To confirm this, we tested the effect of treatment with UDCA on the subcellular localization of the chimeric protein of Gal4 DBD and the COOH-terminal half of the GR, Gal4-GR LBD, which encompasses the NH2-terminal nuclear localization signal, NL1, and the entire LBD of the GR. After transfection of this Gal4-GR LBD expression plasmid into COS7 cells, we immunocytochemically examined the effect of treatment with UDCA on the subcellular localization of the expressed protein using anti-Gal4 antibody. In the absence of ligand, Gal4-GR LBD exclusively showed cytoplasmic localization (data not shown). After treatment with dexamethasone, Gal4-GR LBD moved into the nucleus in a time-dependent manner (Fig. 3 A). Indeed, treatment with UDCA promoted nuclear translocation of the Gal4-GR LBD even in the absence of dexamethasone (Fig. 3 A). When the transactivational potential of this chimeric protein was assessed in a transient transfection assay, dexamethasone, not UDCA, induced expression of the reporter gene (Fig. 3 B), as in the case of the wild-type GR (37Tanaka H. Makino Y. Miura T. Hirano F. Okamoto K. Komura K. Sato Y. Makino I. J. Immunol. 1996; 156: 1601-1608PubMed Google Scholar). Next, we constructed the expression plasmids for a chimeric protein of GFP and GR, the COOH-terminal end of the LBD of which was deleted to assess the region responsible for UDCA action (Fig. 4 A). It is believed that association of hsp90 is a prerequisite for cytoplasmic docking of the GR. In this line, we examined the interaction between GFP-tagged GR mutants and hsp90 using an immunoprecipitation assay, for a start. After transfection of these expression plasmids for GFP-tagged GR, whole cell extracts were prepared and immunoprecipitated with anti-hsp90 antibody, and then complex formation between the GR and hsp90 was analyzed by Western blot as described under “Experimental Procedures.” As shown in Fig. 4 B, all expressed mutant proteins, as well as the GFP-GR, interacted with hsp90 in the absence of ligand, with sligh