Role of the Glucocorticoid Receptor for Regulation of Hypoxia-dependent Gene Expression
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m302581200
ISSN1083-351X
AutoresTsunenori Kodama, Noriaki Shimizu, Noritada Yoshikawa, Yuichi Makino, Rika Ouchida, Kensaku Okamoto, Tetsuya Hisada, Hiroshi Nakamura, Chikao Morimoto, Hirotoshi Tanaka,
Tópico(s)Hormonal Regulation and Hypertension
ResumoGlucocorticoids are secreted from the adrenal glands and act as a peripheral effector of the hypothalamic-pituitary-adrenal axis, playing an essential role in stress response and homeostatic regulation. In target cells, however, it remains unknown how glucocorticoids finetune the cellular pathways mediating tissue and systemic adaptation. Recently, considerable evidence indicates that adaptation to hypoxic environments is influenced by glucocorticoids and there is cross-talk between hypoxia-dependent signals and glucocorticoid-mediated regulation of gene expression. We therefore investigated the interaction between these important stress-responsive pathways, focusing on the glucocorticoid receptor (GR) and hypoxia-inducible transcription factor HIF-1. Here we show that, under hypoxic conditions, HIF-1-dependent gene expression is further up-regulated by glucocorticoids via the GR. This up-regulation cannot be substituted by the other steroid receptors and is suggested to result from the interaction between the GR and the transactivation domain of HIF-1α. Moreover, our results also indicate that the ligand binding domain of the GR is essential for this interaction, and the critical requirement for GR agonists suggests the importance of the ligand-mediated conformational change of the GR. Because these proteins are shown to colocalize in the distinct compartments of the nucleus, we suggest that these stress-responsive transcription factors have intimate communication in close proximity to each other, thereby enabling the fine-tuning of cellular responses for adaptation. Glucocorticoids are secreted from the adrenal glands and act as a peripheral effector of the hypothalamic-pituitary-adrenal axis, playing an essential role in stress response and homeostatic regulation. In target cells, however, it remains unknown how glucocorticoids finetune the cellular pathways mediating tissue and systemic adaptation. Recently, considerable evidence indicates that adaptation to hypoxic environments is influenced by glucocorticoids and there is cross-talk between hypoxia-dependent signals and glucocorticoid-mediated regulation of gene expression. We therefore investigated the interaction between these important stress-responsive pathways, focusing on the glucocorticoid receptor (GR) and hypoxia-inducible transcription factor HIF-1. Here we show that, under hypoxic conditions, HIF-1-dependent gene expression is further up-regulated by glucocorticoids via the GR. This up-regulation cannot be substituted by the other steroid receptors and is suggested to result from the interaction between the GR and the transactivation domain of HIF-1α. Moreover, our results also indicate that the ligand binding domain of the GR is essential for this interaction, and the critical requirement for GR agonists suggests the importance of the ligand-mediated conformational change of the GR. Because these proteins are shown to colocalize in the distinct compartments of the nucleus, we suggest that these stress-responsive transcription factors have intimate communication in close proximity to each other, thereby enabling the fine-tuning of cellular responses for adaptation. In man, glucocorticoids are secreted from the adrenal glands and act as a peripheral effector of the hypothalamic-pituitary-adrenal (HPA) 1The abbreviations used are; HPA axis, hypothalamic-pituitary-adrenal axis; ADM, adrenomedullin; AF, activation function; ALD, aldosterone; DBD, DNA binding domain; DEX, dexamethasone; GFP, green fluorescent protein; GLUT, glucose transporter; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HIF, hypoxia-inducible factor; HRE, hypoxia response element; LBD, ligand binding domain; MR, mineralocorticoid receptor; NLS, nuclear localization signal; RT-PCR, reverse transcriptase PCR; VEGF, vascular endothelial growth factor.1The abbreviations used are; HPA axis, hypothalamic-pituitary-adrenal axis; ADM, adrenomedullin; AF, activation function; ALD, aldosterone; DBD, DNA binding domain; DEX, dexamethasone; GFP, green fluorescent protein; GLUT, glucose transporter; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HIF, hypoxia-inducible factor; HRE, hypoxia response element; LBD, ligand binding domain; MR, mineralocorticoid receptor; NLS, nuclear localization signal; RT-PCR, reverse transcriptase PCR; VEGF, vascular endothelial growth factor. axis, playing an essential role not only in energy metabolism but in stress response and homeostatic regulation as well. The central perception of stress, thus, is transmitted to peripheral tissues by glucocorticoids via blood stream, thereby enabling coordinated responses for individual adaptation to environments. However, it remains unknown how glucocorticoids finally integrate cellular pathways in harmonization with tissue and systemic responses (1Sapolsky R.M. Romero L.M. Munck A.U. Endocr. Rev. 2000; 21: 55-89Crossref PubMed Scopus (5386) Google Scholar).Glucocorticoids elicit hormone action via binding to their cognate receptor glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily and localizes in the cytoplasm as a latent species. The GR is composed of several functional domains, including AF-1 transactivation domain, DNA binding domain (DBD), nuclear localization signal (NLS), ligand binding domain (LBD), and AF-2. On binding hormone, the GR translocates into the nucleus and modulates gene expression in a variety of ways. The most classical model is that the GR binds as a homodimer to the glucocorticoid response element (GRE) in the promoter region of a target gene and positively regulates its transcription (2Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1630) Google Scholar). On the other hand, the GR also modulates transcription through interaction with other transcription factors and co-regulators (3Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1163) Google Scholar, 4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6028) Google Scholar, 5Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). For example, the anti-inflammatory action of glucocorticoids is believed to be mediated by the interaction between the GR and proinflammatory transcription factors including AP-1 and NF-κB (6Cato A.C. Wade E. Bioessays. 1996; 18: 371-378Crossref PubMed Scopus (296) Google Scholar). Of note, the gene targeting approach has revealed that GR DNA binding is shown not to be essential for survival in mice (7Reichardt H.M. Kaestner K.H. Tuckermann J. Kretz O. Wessely O. Bock R. Gass P. Schmid W. Herrlich P. Angel P. Schutz G. Cell. 1998; 93: 531-541Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Moreover, we have shown that the GR function is also tightly controlled by cellular redox regulators (8Tanaka H. Makino Y. Okamoto K. Vitam. Horm. 1999; 57: 153-175Crossref PubMed Scopus (13) Google Scholar). It therefore is likely that not only direct DNA binding but also modulation of other cellular machinery by the GR may be important for stress response, because cellular stress evokes distinct intracellular signals and alters the gene expression profile via modulation of a battery of transcription factors. Such diversity of mode of GR action, thus, might be one of the molecular bases for rationale interaction between the HPA axis and cellular adaptive responses.Low oxygen availability, hypoxia, can be encountered not only under pathological but also physiological conditions (9Caldwell C.C. Kojima H. Lukashev D. Armstrong J. Farber M. Apasov S.G. Sitkovsky M.V. J. Immunol. 2001; 167: 6140-6149Crossref PubMed Scopus (308) Google Scholar, 10Matherne G.P. Headrick S.D. Coleman Berne R.M. Pediatr. Res. 1990; 28: 348-353Crossref PubMed Scopus (54) Google Scholar, 11Jelkmann W. J. Interferon Cytokine Res. 1998; 18: 555-559Crossref PubMed Scopus (307) Google Scholar, 12Van Belle H. Goossens. F. Wynants J. Am. J. Pathol. 1987; 252: H886-H893Google Scholar, 13Vaupel P. Kalinowski. F. Okunieff P. Cancer Res. 1989; 49: 6449-6465PubMed Google Scholar). It has been reported that the distribution of oxygen tension shows considerable variation among different tissues, and parts of certain tissues including the liver and brain are exposed to hypoxia even under physiological conditions (13Vaupel P. Kalinowski. F. Okunieff P. Cancer Res. 1989; 49: 6449-6465PubMed Google Scholar). When exposed to hypoxia, a variety of cellular responses is generated, leading to cell and tissue adaptation via induction of the expression of a number of genes including those for glucose transporters (GLUTs), vascular endothelial growth factor (VEGF), and adrenomedullin (ADM). Moreover, hypoxia also enhances gene expression of the hematopoietic hormone erythropoietin in the kidney, enabling humans to adapt systemically at high altitudes via increasing blood levels of hemoglobin. These hypoxic responses are controlled mainly at the level of transcription by hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of α and β subunits (Arnt), both of which belong to a family of basic helix-loop-helix PAS (per/arnt/sim) transcription factors. HIF-1 binds to the hypoxia response element (HRE), which was originally identified in the 3′-enhancer region in the erythropoietin gene and later in the promoter region of the genes for VEGF, GLUTs, and ADM as well (14Semenza G.L. Trends Mol. Med. 2002; 8: S62-S67Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 15Huang L.E. Bunn H.F. J. Biol. Chem. 2003; 278: 19575-19578Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The recent discovery of the dioxygenases for oxygen sensing has shed light on the mechanism of oxygen-dependent regulation of HIF-1 activity. Under normoxic conditions, critical proline residues within the oxygen-dependent degradation domain of the HIF-1α proteins are hydroxylated by a certain class of proline hydroxylases, and HIF-1α proteins are targeted for ubiquitination and degradation by the proteasome (15Huang L.E. Bunn H.F. J. Biol. Chem. 2003; 278: 19575-19578Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2697) Google Scholar, 17Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3828) Google Scholar, 18Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4369) Google Scholar). This post-translational modification is inhibited under hypoxic conditions, resulting in stabilization of HIF-1α protein levels. In addition, hypoxia induces the function of the transactivation domains of HIF-1α proteins and enhances their ability to interact with transcriptional coactivator proteins (19Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1259) Google Scholar). Under normoxic conditions, this interaction has been shown to be blocked by the hydroxylation of a conserved asparagine residue within one of the transactivation domains. This asparagine hydroxylation is catalyzed by asparagine hydroxylase, previously identified as FIH, under normoxic conditions and abrogated under hypoxic conditions (20Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1201) Google Scholar).Recently, growing evidence indicates that adaptation to hypoxia is also influenced by the activity of the HPA axis and glucocorticoids. For example, blood levels of cortisol are shown to be elevated via increased secretion of adrenocorticotropine at high altitudes or under intrauterine hypoxic conditions (21Moncloa F. Donayre J. Sobrevilla L.A. Guerra-Garcia R. J. Clin. Endocrinol. Metab. 1965; 25: 1640-1642Crossref PubMed Scopus (23) Google Scholar, 22Gardner D.S. Fletcher A.J. Fowden A.L. Giussani D.A. Endocrinology. 2001; 142: 589-598Crossref PubMed Scopus (49) Google Scholar, 23Larsen J.J. Hansen J.M. Olsen N.V. Galbo H. Dela F. J. Physiol. 1997; 504: 241-249Crossref PubMed Scopus (168) Google Scholar). Moreover, the prophylactic administration of synthetic glucocorticoids dramatically prevents high mountain sickness (24Johnson T.S. Rock P.B. Fulco C.S. Trad L.A. Spark R.F. Maher J.T. N. Engl. J. Med. 1984; 310: 683-686Crossref PubMed Scopus (112) Google Scholar). In rodents, the administration of glucocorticoids significantly reduces brain tissue damage after cerebral ischemia (25Dardzinski B.J. Smith S.L. Towfighi J. Williams G.D. Vannucci R.C. Smith M.B. Pediatr. Res. 2000; 48: 248-255Crossref PubMed Scopus (50) Google Scholar), and stress-induced erythropoiesis under hypoxic conditions is influenced by glucocorticoids (26Bauer A. Tronche F. Wessely O. Kellendonk C. Reichardt H.M. Steinlein P. Schutz G. Beug H. Genes Dev. 1999; 13: 2996-3002Crossref PubMed Scopus (227) Google Scholar). Thus, there appears to be cross-talk between hypoxia-dependent signals and the HPA axis and glucocorticoid system. However, the underlying molecular mechanisms have not yet been explored, especially at the cellular level. Given this fact, we were prompted to investigate the interaction between these important stress-responsive pathways, focusing on the transcription factors GR and HIF-1. Here we have shown that HIF-1-dependent transactivation is up-regulated by glucocorticoids via the GR and that the LBD of the GR may play a critical role in the functional interaction between these stress-responsive transcription factors.EXPERIMENTAL PROCEDURESReagent and Antibodies—Dexamethasone (DEX) and aldosterone (ALD) were purchased from Sigma. RU486 and cortivazol were kindly gifted from Roussel Uclaf and Merck, respectively. Monoclonal anti-HIF-1α antibody Ab463 was purchased from Abcam (Cambridge, UK). Polyclonal anti-rabbit GR antibody PA1-512 was from Affinity Bioreagents (La Jolla, CA). Monoclonal anti-α actinin antibody was from Sigma. Other chemicals were from Wako Pure Chemical (Osaka, Japan) unless specified otherwise.Cell Culture—COS7 and HeLa cells were obtained from the RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics in a humidified atmosphere at 37 °C with 5% CO2 unless specified otherwise. Hypoxic conditions were achieved by incubation of cells in an acryl anaerobic chamber with 1% O2, 5% CO2, and 94% N2. In all experiments, serum steroids were stripped from fetal calf serum with dextran-coated charcoal.RNA Isolation and RT-PCR Analysis—Total RNA was extracted by the spin column method using the SV Total RNA Isolation System (Promega). One-step RT-PCR was carried out using 50 ng of total RNA as a template and the Access Quick RT-PCR system (Promega) in a total volume of 50 μl of mixture containing 5 units of avian myeloblastosis virus reverse transcriptase, 2 units of Tfl DNA polymerase, 1.5 mm MgSO4, 200 μm dNTPs, and sense and antisense primers at 0.25 μm each. Aliquots of the PCR products were electrophoresed in 2% agarose gels and stained with ethidium bromide. The amount of cDNA, as estimated relatively by the intensity of the amplified β-actin signal, was comparable among the preparations. Experiments in the absence of reverse transcriptase were performed as negative control. PCR primer pairs for amplification of each gene are as follows: VEGF: 5′-TGCCTTGCTGCTCTACCTCC-3′ (sense) and 5′-TCACCGCCTCGGCTTGTCAC-3′ (antisense); GLUT3: 5′-GATGCTGGAGAGGTTAAGGT-3′ (sense) and 5′-ACTTCCACCCAGAGCAAAGT-3′ (antisense); ADM: 5′-AAGAAGTGGAATAAGTGGGCT-3′ (sense) and 5′-TGGCTTAGAAGACACCAGAGT-3′ (antisense); β-actin: 5′-CCTCGCCTTTGCCGATCC-3′ (sense) and 5′-GGATCTTCATGAGGTAGTCAGTC-3′ (antisense).Plasmids—The expression plasmids for the wild-type and mutant human GR, pCMX-hGR and pCMX-GR-(1–765), were described previously (27Yoshikawa N. Makino Y. Okamoto K. Morimoto C. Makino I. Tanaka H. J. Biol. Chem. 2002; 277: 5529-5540Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The expression plasmids for human AF-1-deleted mutant GR Δ9–385 and AF-2-deleted I550 and the wild-type human MR, pRShMR, were kindly gifted by Dr. R. M. Evans (Salk Institute, La Jolla, CA) (28Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (674) Google Scholar). The expression plasmid for dimerization-deficient human GR mutant A458T was from Dr. A. C. B. Cato (Forschungszentrum, Karlsruhe, Germany) (29Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (462) Google Scholar). The expression plasmids for the wild-type human HIF-1α, pCMV4-HIF-1α, and the wild-type human Arnt, pCMV4-Arnt and pCMX-GAL4-Arnt, were from Dr. Lorenz Poellinger (Karolinska Institute, Stockholm, Sweden) (30Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (321) Google Scholar). The expression plasmids for the chimeric protein of green fluorescent protein (GFP) and the wild-type human GR and MR, pCMX-GFP-GR (31Okamoto 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 (159) Google Scholar) and pCMX-GFP-MR (27Yoshikawa N. Makino Y. Okamoto K. Morimoto C. Makino I. Tanaka H. J. Biol. Chem. 2002; 277: 5529-5540Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), respectively, were described previously. The expression plasmids for a chimeric protein of GAL4-DBD and the LBD of the human GR (Glu-489 to Lys-777), pCMX-GAL4-GRLBD, and GAL4-responsive reporter plasmid tk-GALpx3-Luc were kindly gifted by Dr. K. Umesono (University of Kyoto, Kyoto, Japan). To construct the expression plasmid for the chimeric protein of GAL4-DBD and the C-terminal truncated GR LBD, pCMX-GAL4-GRLBD-(489–765), the DNA fragments encoding the amino acids Leu-596 to Ser-765 of human GR was amplified using PCR with the appropriate flanking sequences and inserted into PstI-BamHI-opened pCMX-GAL4-GRLBD. The expression plasmid for the chimeric protein of GAL4-DBD and HIF-1α, pCMX-GAL4-HIF-1α, was described previously (32Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar). To construct the expression plasmids for the chimeric protein of NLS obtained from SV40 large tumor antigen and GR LBD-(499–777) and GR LBD-(499–765), pCMX-NLS-GRLBD-(499–777) and pCMX-NLS-GRLBD-(499–765), the DNA fragments encoding the corresponding amino acids of the NLS, were inserted into parent pCMX, resulting in pCMX-NLS. Then the DNA fragment encoding either amino acids 499–777 or 499–765 of the GR LBD was inserted into pCMX-NLS. HIF-1-responsive reporter plasmid pT81/HRE-Luc contains three tandem copies of the erythropoietin HRE in front of the herpes simplex thymidine kinase promoter and the luciferase gene (30Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (321) Google Scholar, 32Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar). The glucocorticoid-responsive reporter plasmid pGRE-Luc were described previously (33Makino 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). All plasmids constructed as described above were verified by sequencing.Transfection and Reporter Gene Assay—Cells were plated on 6-cm-diameter culture dishes to 30–50% confluence, and the medium was replaced with Opti-MEM (Invitrogen). The plasmid mixture was mixed with TransIT-LT1 transfection reagent (Panvera Corp., Madison, WI) and added to the culture. The total amount of plasmids was kept constant by adding an irrelevant plasmid (pGEM7Z was used unless otherwise specified). After 6 h of incubation, the medium was replaced with fresh Dulbecco's modified Eagle's medium with 2% dextran-coated, charcoal-treated fetal calf serum, and the cells were further cultured in various stimulation for 24 h at 37 °C. Luciferase enzyme activity was determined using a luminometer (Promega), and relative light units were normalized to the protein amount determined with protein assay reagent according to the manufacturer's instructions (Pierce).Western Blot Assay—Whole cell extract of HeLa cells was prepared in lysis buffer containing 25 mm Hepes, 100 mm NaCl, 5 mm EDTA, 100 μm orthovanadate, 1 mm dithiothreitol, and 0.5% Triton X-100, pH 7.9, with a proteinase inhibitor mixture on ice for 15 min followed by centrifugation for 20 min at 14,000 rpm. Twenty micrograms of protein of whole cell extract were separated in 8% SDS-polyacrylamide gels and then blotted to nylon membranes. The membranes were blocked in Tris-buffered saline (50 mm Tris-HCl, pH 7.6, 200 mm NaCl) with 5% nonfat dried skim milk. The membranes were probed with anti-HIF-1α antibody diluted 1:1000 in Tris-buffered saline containing 1% nonfat milk at 4 °C overnight. A 1:1000 dilution of anti-mouse Ig-horseradish peroxidase conjugate (Amersham Biosciences) in Tris-buffered saline containing 1% nonfat milk was applied as a second antibody. After detection of proteins, the same membranes were stripped and reprobed for anti-GR antibody as described previously (34Miura T. Ouchida R. Yoshikawa N. Okamoto K. Makino Y. Nakamura T. Morimoto C. Makino I. Tanaka H. J. Biol. Chem. 2001; 276: 47371-47378Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In all experiments, membranes were reprobed for anti-α actinin antibody to verify the equal amount of loading. For visualization of proteins, the ECL detection system was used according to the manufacturer's instructions (Amersham Biosciences).Subcellular Localization Assay of GFP Fusion Proteins in Living Cells—the chimeric proteins of GFP and either GR or MR were transiently expressed in COS7 cells, and assays were performed as described previously (27Yoshikawa N. Makino Y. Okamoto K. Morimoto C. Makino I. Tanaka H. J. Biol. Chem. 2002; 277: 5529-5540Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Briefly, after 6 h of transient transfection of pCMX-GFP-GR or pCMX-GFP-MR, medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% dextran-coated, charcoal-treated fetal calf serum, and the cells were cultured at 37 °C. GFP was expressed at detectable levels between 24 and 72 h after transfection. After various treatments, cells were examined using a laser scanning confocal microscopy with a fluorescein isothiocyanate filter set (Olympus, Tokyo, Japan). Quantitative analysis of localization of GFP-tagged proteins was performed by blinded observers, who counted ∼200 cells in which GFP fluorescence was detected. The GFP fluorescence-positive cells were classified into four different categories: N < C, cytoplasmic dominant fluorescence; N = C, cells having equal distribution of fluorescence in the cytoplasmic and nuclear compartments; N > C, nuclear-dominant fluorescence; and N, exclusively nuclear fluorescence. Then the percentage of N and N > C cells was calculated (31Okamoto 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 (159) Google Scholar). To assess colocalization of the GR and HIF-1α, COS7 cells were transfected with GR and GFP-HIF-1α expression plasmids and exposed to hypoxia in the presence of 100 nm DEX. The GR was detected by an immunofluorescent technique described previously, using anti-GR antibody (Affinity Bioreagents), and HIF-1α was detected using confocal laser microscopy as described above. Images were taken and analyzed using FLUOVIEW computer software (Olympus).RESULTSGlucocorticoids Enhance Hypoxia-inducible Gene Expression—To examine the effect of glucocorticoids on hypoxia-inducible gene expression, we cultured HeLa cells in the presence or absence of 100 nm DEX under normoxic or hypoxic conditions (oxygen concentrations were 21 and 1%, respectively). After total RNA isolation, mRNA expression of HIF-1-target genes, VEGF, ADM, and GLUT3, and β-actin was analyzed using RT-PCR. As shown in Fig. 1A, mRNA expression of β-actin was not altered under these experimental conditions. Under hypoxic conditions, mRNA expression of VEGF, ADM, and GLUT3 was induced. Treatment with DEX did not significantly increase mRNA expression of these HIF-1-target genes at normoxia. Under hypoxic conditions, however, treatment with DEX enhanced hypoxic inducibility of mRNA expression of these HIF-1 target genes by 1.5–3-fold (Fig. 1). We thus were prompted to investigate whether hypoxia-inducible HIF-1 transcriptional activity is modulated in the presence of glucocorticoids.GR Enhances Transactivational Function of HIF-1α without Alteration in Protein Levels of HIF-1α—We then studied the effect of treatment with DEX on hypoxic induction of HRE-driven reporter gene expression, because native promoters of these target genes contain multiple regulatory elements and usage of this minimal reporter construct should bypass otherwise complicated interaction among those elements. After transfection of the HRE-luciferase reporter plasmid, HeLa cells were cultured in the presence or absence of DEX under normoxic or hypoxic conditions. It has already been shown that HeLa cells contain endogenous GR and HIF-1 (35Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar, 36Eggert M. Michel J. Schneider S. Bornfleth H. Baniahmad A. Fackelmayer F.O. Schmidt S. Renkawitz R. J. Biol. Chem. 1997; 272: 28471-28478Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Western blot analysis revealed that protein expression of the GR was almost constant under these experimental conditions (Fig. 2A). Protein levels of HIF-1α were up-regulated under hypoxia due to stabilization and escape from degradation (see the Introduction) but were not affected by treatment with DEX (Fig. 2A). On the other hand, treatment with DEX enhanced hypoxic induction of HRE-driven reporter gene expression in a concentration-dependent manner (Fig. 2A), possibly reflecting the results shown in Fig. 1. This issue was again confirmed in cotransfection experiments in which not only the HRE-luciferase reporter but also the GR expression plasmid pCMX-GR was transfected into HeLa cells; hypoxic induction of HRE-luciferase expression was increased in concert with an increasing dosage of the GR expression plasmid in the presence of DEX (Fig. 2B). This result raised the possibility that glucocorticoids enhance hypoxic induction of HRE-driven gene expression via the GR. Because protein levels of HIF-1α were not affected by treatment with DEX, we tested the effect of DEX and the GR on the transactivation function of HIF-1α. For that purpose, HIF-1α was expressed as a fusion protein with GAL4 DBD (Fig. 3A), and the effect of hypoxia and DEX on GAL4-reporter plasmid was assayed in COS7 cells. When GAL4-HIF-1α was expressed with a GAL4 reporter plasmid, an ∼2.5-fold induction of the reporter gene was observed under hypoxic condition (Fig. 3B). This induction response was not influenced either by treatment with DEX or by ectopic expression of the GR expression plasmid (Fig. 3B). However, when both GAL4-HIF-1α and GR were expressed, hypoxic treatment in the presence of DEX resulted in a robust increase in the induction response of the reporter plasmid, indicating that the GR enhances the transactivational function of HIF-1α in a ligand-dependent fashion. When GAL4-Arnt (Fig. 3A) was cotransfected, Arnt-dependent transactivation was not influenced by either cotransfection of the GR expression plasmid or treatment with DEX (Fig. 3B). We next transfected the expression plasmid for a constitutively active transcriptional activator, HIF-1α-(1–396)-VP16 (Fig. 3A), and HRE-luciferase and examined the effect of treatment with DEX and coexpression of the GR. HIF-1α-(1–396)-VP16 lacks the oxygen-dependent degradation domain of HIF-1α, thereby escaping degradation even under normoxia and docking in the nucleus (37Vincent K.A. Shyu K.G. Luo Y. Magner M. Tio R.A. Jiang C. Goldberg M.A. Akita G.Y. Gregory R.J. Isner J.M. Circulation. 2000; 102: 2255-2261Crossref PubMed Scopus (289) Google Scholar). As expected, this chimeric protein activated reporter gene expression even under normoxic conditions (Fig. 3C). However, neither treatment with DEX nor coexpression of the GR significantly influenced its transactivation function (Fig. 3C), indicating that VP16 cannot be substituted for the transactivation domains of HIF-1α in terms of functional coupling with the GR. Taken together, we may conclude that ligand-bound GR may not affect protein levels of HIF-1α but modulates the transactivational function of HIF-1α and enhances HIF-1-dependent transcription.Fig. 2GR enhances hypoxia-inducible HRE-driven reporter
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