Glucocorticoid Ligands Specify Different Interactions with NF-κB by Allosteric Effects on the Glucocorticoid Receptor DNA Binding Domain
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m407309200
ISSN1083-351X
AutoresHelen Garside, Adam Stevens, Stuart Farrow, Claire Normand, Benoit Houle, Andrew Berry, Barbara Maschera, David Ray,
Tópico(s)NF-κB Signaling Pathways
ResumoGlucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-κB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-κB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-κB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-κB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-κB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-κB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-κB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-κB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication. Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-κB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-κB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-κB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-κB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-κB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-κB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-κB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-κB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication. Glucocorticoids (GCs) 1The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; NRE, NF-κB response element; RHD, Rel homology domain; GST, glutathione S-transferase; Dex, dexamethasone; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TNF, transforming growth factor; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; BRET, bioluminescence resonance energy transfer; YFP, yellow fluorescence protein; EYFP, enhanced YFP; RID, receptor-interacting domain; DBD, DNA binding domain; LBD, ligand-binding domain.1The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; NRE, NF-κB response element; RHD, Rel homology domain; GST, glutathione S-transferase; Dex, dexamethasone; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TNF, transforming growth factor; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; BRET, bioluminescence resonance energy transfer; YFP, yellow fluorescence protein; EYFP, enhanced YFP; RID, receptor-interacting domain; DBD, DNA binding domain; LBD, ligand-binding domain. activate the cytosolic glucocorticoid receptor (GR), which translocates to the nucleus to regulate gene expression. The anti-inflammatory activities of GCs are caused, in part, by transrepression of the proinflammatory transcription factor NF-κB. The GR has distinct functional domains, an N-terminal transactivation domain (AF-1), a central DNA binding domain (DBD), and a C-terminal domain that includes ligand binding (LBD) and transactivation activities (AF-2) (1Hollenberg S.M. Weinberger C. Ong E.S. Cerelli G. Oro A. Lebo R. Thompson E.B. Rosenfeld M.G. Evans R.M. Nature. 1985; 318: 635-641Crossref PubMed Scopus (1430) Google Scholar, 2Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (672) Google Scholar, 3Iniguez-Lluhi J.A. Lou D.Y. Yamamoto K.R. J. Biol. Chem. 1997; 272: 4149-4156Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, although the domains can function separately, there is evidence for functional interdependence within the intact GR, which may be necessary for authentic activity (4Hittelman A.B. Burakov D. Iniguez-Lluhi J.A. Freedman L.P. Garabedian M.J. EMBO J. 1999; 18: 5380-5388Crossref PubMed Scopus (239) Google Scholar). For example, the conformation of the DBD, induced by either DNA sequence or protein binding, can determine whether transcription is induced or repressed (5Lefstin J.A. Yamamoto K.R. Nature. 1998; 392: 885-888Crossref PubMed Scopus (436) Google Scholar). The chemical structure of glucocorticoid ligands alter the function of the receptor as they induce conformational changes to the LBD that specify whether co-activators or co-repressors are recruited (6Bledsoe R.K. Montana V.G. Stanley T.B. Delves C.J. Apolito C.J. McKee D.D. Consler T.G. Parks D.J. Stewart E.L. Willson T.M. Lambert M.H. Moore J.T. Pearce K.H. Xu H.E. 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Endocrinology. 1993; 133: 728-740Crossref PubMed Scopus (61) Google Scholar); however, when RU486 is bound, the position of helix 12, as shown by the recent crystal structure, is altered and partially blocks the co-activator pocket (7Kauppi B. Jakob C. Farnegardh M. Yang J. Ahola H. Alarcon M. Calles K. Engstrom O. Harlan J. Muchmore S. Ramqvist A.K. Thorell S. Ohman L. Greer J. Gustafsson J.A. Carlstedt-Duke J. Carlquist M. J. Biol. Chem. 2003; 278: 22748-22754Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). This leads to the recruitment of co-repressors, such as NCoR, and not co-activators, such as SRC1, which are recruited to the agonist bound GR (12Stevens A. Garside H. Berry A. Waters C. White A. Ray D. Mol. Endocrinol. 2003; 17: 845-859Crossref PubMed Scopus (47) Google Scholar, 13Schulz M. Eggert M. Baniahmad A. Dostert A. Heinzel T. Renkawitz R. J. Biol. Chem. 2002; 277: 26238-26243Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, the effects of these ligand-induced conformational changes on the function of the DNA binding domain have not been fully explored. The mechanism of NF-κB repression by GR has yet to be fully defined, but it is known that GR and the p65 subunit of NF-κB physically interact (14Caldenhoven E. Liden J. Wissink S. Van de S.A. Raaijmakers J. Koenderman L. Okret S. Gustafsson J.A. Van der Saag P.T. Mol. Endocrinol. 1995; 9: 401-412Crossref PubMed Google Scholar, 15Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (913) Google Scholar, 16Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 17McKay L.I. Cidlowski J.A. Mol. Endocrinol. 1998; 12: 45-56Crossref PubMed Scopus (304) Google Scholar) with the GR DBD binding to the Rel homology domain (RHD) of p65 (18Nissen R.M. Yamamoto K.R. Genes Dev. 2000; 14: 2314-2329Crossref PubMed Scopus (454) Google Scholar). This interaction occurs on the NF-κB response element resulting in "tethering" of the GR to DNA. GR repression of p65 requires the LBD, in addition to the DBD (19McKay L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar). A repressor protein or complex, recruited to the tethered GR, has been proposed, with evidence implicating GRIP-1, a member of the p160 family (18Nissen R.M. Yamamoto K.R. Genes Dev. 2000; 14: 2314-2329Crossref PubMed Scopus (454) Google Scholar, 20Rogatsky I. Zarember K.A. Yamamoto K.R. EMBO J. 2001; 20: 6071-6083Crossref PubMed Scopus (153) Google Scholar, 21Rogatsky I. Luecke H.F. Leitman D.C. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16701-16706Crossref PubMed Scopus (176) Google Scholar). However, it is not clear if functional differences between agonist and antagonist are due to defective interaction with NF-κB or subsequent defective recruitment of the co-repressor protein. The function of the GR DBD has been thought to be independent of the LBD, but RU486 causes subtle alteration of DNA binding kinetics for the GR compared with dexamethasone (22Pandit S. Geissler W. Harris G. Sitlani A. J. Biol. Chem. 2002; 277: 1538-1543Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In this paper, we explore the influence of ligand on functional interactions between the ligand binding domain and DNA binding domain of the GR. Improved understanding of how GR interaction with p65 occurs and how this can be modulated by ligand has major implications for effective drug design and screening. Here we describe a previously unsuspected role for the GR LBD in regulating recruitment of NF-κB to the GR DNA binding domain. Plasmids—The NRE-luc plasmid consists of three copies of the ICAM1 promoter NF-κB response elements (NREs) upstream of the luciferase gene; it responds to p65 homodimers and has been previously described (27Sheppard K.A. Phelps K.M. Williams A.J. Thanos D. Glass C.K. Rosenfeld M.G. Gerritsen M.E. Collins T. J. Biol. Chem. 1998; 273: 29291-29294Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The p65 RHD was amplified using the HF2 PCR kit (Clontech) and inserted into the pGEX-5X-3 fusion protein expression plasmid (Amersham Biosciences) to give pGST-RHD. The specific primers had BamHI and NotI restriction enzyme sites added for ligation into the vector (forward primer for GST-RHD, 5′-GGATCCTGGACGAACTGTTCCCCCTC-3′; reverse primer, 5′-GCGGCCGCCTTGAAGGTCTCATATGTCCTTTTACG-3′). Full-length p65 was subcloned from p65-GFP into the pEYFP-N1 vector (Clontech) using HindIII and BamHI restriction sites. The p65-GFP vector was kindly donated by Dr. Mike White and has been previously described (23Nelson G. Paraoan L. Spiller D.G. Wilde G.J. Browne M.A. Djali P.K. Unitt J.F. Sullivan E. Floettmann E. White M.R. J. Cell Sci. 2002; 115: 1137-1148Crossref PubMed Google Scholar). SRC1-RID and NCoR-RID were amplified using the HF2 PCR kit (Clontech) and inserted into pEYFP-N1 or pEYFP-C1 vectors, respectively. The specific primers had EcoRI and KpnI restriction enzyme sites added for ligation into the vector (forward primer for SRC1-RID-EYFP, 5′-GGAATTCTACCTAGCAGATTAAATATACAACCA-3′; reverse primer, 5′ GGGGGTACCAGCGTGGGCAGTAACTGATC-3′; forward primer for EYFP-NCoR-RID, 5′-GGGGAATTCCACTTATATTCCTGGTACAC-3′; reverse primer, 5′-GGTGGTACCGTCATCACTATCCGACAG-3′). The Rluc:GR, GR:Rluc, GFP2-p65, and p65-GFP2 plasmids were made by BioSignal Packard Inc. (Cambridge, UK). For the construction of GR:Rluc, the GR coding sequence was amplified from pECFP-GR (forward primer, 5′-CGGATATCGCCACCATGGACTCCAAAG-3′; reverse primer, 5′-CCGCCTAGGGAAAACTACTTTGTCTTCAAAAAAC-3′) and ligated into pCR Blunt II TOPO (Invitrogen) to generate pCR/GR (minus STOP); then a restriction fragment bearing GR-coding sequences (without the STOP codon) was excised from pCR/GR using EcoRV and BamHI and ligated into p(h)Rluc-N2(FG)Kan (Biosignal Packard). For the construction of Rluc:GR plasmid, the GR coding sequence was amplified from pECFP-GR (forward primer, 5′-CGCTCGAGGACTCCAAAGAATCATTAACTC-3′; reverse primer, 5′-CTTCTGTTTCATCAAAAGTGAGGTACCGC-3′) and ligated into pCR Blunt II TOPO (Invitrogen) to generate pCR/GR (minus START); then a restriction fragment bearing GR coding sequences (without the START codon) was excised from pCR/GR using XhoI and KpnI and ligated into p(h)Rluc-C2(FG)Kan (Biosignal Packard). In order to obtain the p65 coding sequence devoid of either the START or STOP codon, two PCRs were performed using the appropriate pairs of primers: 1) forward primer (5′-CCGCTCGAGGACGAACTGTTCCCCCTCATC-3′) and reverse primer (5′-CTGAGTCAGATCAGCTCCTAAAAGCTTGGG-3′) for p65 minus START; 2) forward primer (5′-CCCAAGCTTATGGACGAACTGTTCCCCCTCAT-3′) and reverse primer (5′-CCTGCTGAGTCAGATCAGCTCCGGATCCGCG-3′) for p65 minus STOP. For the construct encoding the p65:GFP2 fusion protein, the enzymes used were HindIII and BamHI, and the recipient vector was pGFP21-C1(FG)Zeo (Biosignal Packard). Similarly, the construct encoding the GFP2:p65 fusion protein was obtained by using XhoI and HindIII, and the recipient vector was pGFP21-N2(FG)Zeo (Biosignal Packard). The pcDNA3-GR plasmid containing full-length wild type GR has been previously described (12Stevens A. Garside H. Berry A. Waters C. White A. Ray D. Mol. Endocrinol. 2003; 17: 845-859Crossref PubMed Scopus (47) Google Scholar). The human GR DNA binding domain/ligand binding domain (DBD/LBD) between codons 289 and 777 was amplified from pcDNA3-GR using the HF2 PCR kit and inserted into the pACT plasmid (Promega Corp. Southampton, UK) to give VP16-DBD/LBD. The specific primers had BamHI and NotI restriction enzyme sites added for ligation into the vector (forward primer for VP16-DBD/LBD, 5′-GGATCCCTGGGGTAATTAAGCAAGAGAAACTGG-3′; reverse primer, 5′-GCGGCCGCCTTTTGATGAAACAGAAGTTTTTTG-3′). The CMV-Renilla plasmid (pRL-CMV) was obtained from Promega and has been previously described (12Stevens A. Garside H. Berry A. Waters C. White A. Ray D. Mol. Endocrinol. 2003; 17: 845-859Crossref PubMed Scopus (47) Google Scholar). All plasmid constructs were sequenced to confirm the presence of the predicted changes and to exclude introduction of errors. Interaction of p65-RHD and the Glucocorticoid Receptor in Vitro— Glutathione S-transferase (GST) and GST-RHD fusion proteins were expressed in Escherichia coli strain DH5α (Invitrogen) and were purified as described (29Widen C. Gustafsson J.A. Wikstrom A.C. Biochem. J. 2003; 373: 211-220Crossref PubMed Scopus (39) Google Scholar). Briefly, a 50-ml culture of the expression vector was stimulated with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside (Sigma) and grown for 3 h. The bacteria were then pelleted and resuspended in NETN buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.01% Nonidet P-40) supplemented with protease inhibitors ("Complete"; Roche Applied Science). The bacteria were treated with lysozyme (100 μg/ml) for 15 min at 4 °C and then disrupted using a 50-watt, 20-kHz sonicator (Jencons, Leighton Buzzard, UK) on full power for 5 × 30 s. The samples were then centrifuged to remove the debris, and glutathione-Sepharose beads (Amersham Biosciences) were added to the supernatant. The fusion proteins were allowed to bind to the beads for 1 h at 4 °C. The beads were then washed three times in NETN plus protease inhibitors and stored at 4 °C. 35S-Labeled GR was synthesized using the TNT kit (Promega) following the manufacturer instructions with the full-length, human GR cDNA in pcDNA3 or VP16-DBD/LBD as template, with no ligand, 10 μm Dex, or 10 μm RU486 and [35S]methionine (Amersham Biosciences). Labeled GR was incubated with the GST proteins bound to glutathione-Sepharose beads for 1 h at 4 °C. The beads were then washed three times in NETN, and boiling in 2× SDS-PAGE sample buffer eluted the bound proteins. Proteins were analyzed by SDS-PAGE, and the gel was stained in Sypro Red to visualize the GST fusion proteins. The 35S-labeled GR was then visualized by exposure of the gel to a phosphor imaging plate (BasIII; Fuji). The Sypro Red-stained acrylamide gels were analyzed under UV light (AlphaImager 2000; Flowgen, Lichfield, UK). The amount of radiolabeled protein present on the gel was quantitated using a phosphor imager (BAS1800; FujiFilm) and Aida 2.0 analysis software (Raytest, GMBH). Transrepression Assay—COS7 cells were seeded at 5 × 104 cells into 24-well plates and were transfected with 2.4 μg of NRE-luc and 1.2 μg of GR construct (pcDNA3-GR or VP16-DBD/LBD) and 7.5 ng of GAL4-p65. Cell lysates were subjected to both firefly and Renilla luciferase assays; firefly luciferase was then corrected for Renilla luciferase expression as previously described (12Stevens A. Garside H. Berry A. Waters C. White A. Ray D. Mol. Endocrinol. 2003; 17: 845-859Crossref PubMed Scopus (47) Google Scholar). Bioluminescence Resonance Energy Transfer (BRET)2 Assay—The day before transfection, HEK 293T cells were seeded at 3 × 106 cells/10-cm tissue culture dish containing DMEM, 2 mm glutamine, and 10% fetal bovine serum. On the day of transfection, the medium was replaced by growth medium supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone), and cells were transfected using LipofectAMINE 2000™ (Invitrogen) according to the manufacturer's instructions. Cells were transfected with GR fused to Renilla luciferase at the C or N terminus and p65 fused to GFP2 at the C or N terminus using RenLuc-GFP2 at different ratios (4:1, 4:4, 1:4, and 0.5:4). As a negative control for interaction, GR-RenLuc was also cotransfected with GFP2-KaiB plasmid. 48 h post-transfection, cells were harvested using Versene (PBS and 0.5 mm EDTA); centrifuged for 5 min at 1200 rpm, resuspended in BRET buffer, Dulbecco's PBS (Invitrogen) supplemented with 2 μg/ml aprotinin (Sigma, Poole, UK); and counted. Cells were recentrifuged and resuspended in BRET buffer to a cell density of 1.5 × 106 cells/ml. 5 × 104 cells were added to each well of a 96-well shallow Optiplate (PerkinElmer BioSignal Inc.) followed by stimuli diluted in BRET buffer so that the final concentrations were 1 μm dexamethasone and 10 ng/ml TNF-α. The treatment was performed at room temperature for 1 h. Triplicate determinations for each condition were prepared. After incubation, the luminescence reaction was started by adding 10 μl of the substrate DeepBlueC™ coelenterazine (PerkinElmer BioSignal), to give a final concentration of 5 μm; plates were read after 20 min on a Fusion Universal Microplate Analyzer (PerkinElmer BioSignal) using the following filter settings: 410 nm for luciferase emission and 515 nm for enhanced yellow fluorescent protein (EYFP) emission. After subtraction of the average counts obtained with untransfected cells from the readings of individual wells, the BRET2 signal was determined as the ratio between GFP2 emission (515 nm) and Renilla luciferase emission (410 nm). BRET1 Assay—The day before transfection, HEK 293T cells were seeded at 2 × 106 cells per 10-cm tissue culture dish containing DMEM, 2 mm glutamine, and 10% fetal bovine serum. Cells were transfected using Fugene 6 transfection reagent following the manufacturer's instructions (Roche Applied Science). The medium on the cells was replaced with 10 ml of Opti-MEM I reduced serum medium (Invitrogen). The transfection solution containing a 1:5 DNA/Fugene ratio, using 150 μl of Opti-MEM I reduced serum medium, 30 μl of Fugene, and 6 μg of total DNA, was then added on the cells after 20 min at room temperature. Cells were cotransfected with C-terminal tagged GR-Renilla and the other protein encoding cDNA fused to EYFP (p65, SRC1, or NCoR) at a ratio of 1:4, 1:4, and 1:8, respectively. After 4 h, medium was replaced by DMEM, 2 mm glutamine, and 10% charcoal/dextran-treated fetal bovine serum (Hyclone). 48 h post-transfection, cells were harvested, resuspended in BRET buffer, and seeded in 96-well plates as for the BRET2 assay. Cells were then treated with GR ligands diluted in BRET buffer so that the final concentration range was between 0.01 and 1000 nm. The incubation was performed at 37 °C in the presence of 5% CO2 for 1 h. Triplicate determinations for each condition were prepared. After incubation, the luminescence reaction was started by adding 10 μl of the substrate coelenterazine, to give a final concentration of 5 μm, and plates were read after 15 min on a Fusion Universal Microplate Analyzer (Packard BioScience, Pangbourne, UK) using the following filter settings: 485 nm for luciferase emission and 535 nm for EYFP emission. The BRET ratio was then corrected for the BRET signal obtained from cells transfected with the Renilla-fused protein alone (Ren) determined as follows: BRET ratio = (emission 535 nm-emission 485 nm × cf)/emission 485 nm, where cf = emission 535 nm Renilla alone/emission 485 nm Renilla alone. Curves were then constructed, and EC50 values were calculated using a four-parameter curve fit. IL-8 ELISA—HeLa cells were cultured in DMEM (Invitrogen) supplemented with 10% charcoal/dextran-treated fetal calf serum (Hyclone) for 48 h. Cell-conditioned medium was then collected from HeLa cells treated with 0.5 ng/ml TNF-α, and a dose response of either Dex or RU486 (1–1000 μm) for 16 h. IL-8 concentration was measured using an IL-8 ELISA kit (R & D Systems), following the manufacturer's instructions. Immunofluorescence of Endogenous Protein—HeLa cells were seeded into each well of 8-chamber slides at 30,000 cells/chamber using DMEM supplemented with Glutamax-1 (Invitrogen Life Technologies) and 3% charcoal/dextran-stripped serum. 24 h later, the cells were treated with TNF-α (10 ng/ml) and either 100 nm Dex or 100 nm RU486 for 1 h. Cells were then washed with PBS and fixed for 15 min at room temperature with 3.7% paraformaldehyde. After fixing, cells were washed three times with TD buffer (10 mm Tris-HCl, pH 8, 150 mm NaCl) and treated with blocking buffer (TD plus 1% bovine serum albumin, 0.2% Triton X-100) for 1 h at room temperature. A 1:200 dilution of primary antibody, P20 sc-1002 for the GR, sc-109-G for p65 (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in washing buffer (TD plus 1% bovine serum albumin, 0.05% Triton X-100), was added for 2 h at room temperature. Cells were then washed three times with wash buffer, and a 1:200 dilution of the appropriate secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 568 rabbit anti-goat IgG) (Molecular Probes, Inc., Eugene, OR) in washing buffer was added for 1 h at room temperature. The cells were then washed three times and mounted on slides using Citifluor (glycerol/PBS) (Citifluor Ltd.). Coverslips were sealed and stored at 4 °C. Images were taken using a Leica TCS-4D confocal microscope (Leica Microsystems, Heidelberg, Germany) using a ×63 water immersion objective. To visualize Alexa 488, cells were excited at 488 nm using an argon laser, and emission was collected using a band pass filter of 525 ± 25 nm and a dichroic beam splitter of 500. Alexa 568 cells were viewed using an excitation filter of 568 nm, and emission was collected using a 590-nm long pass filter. Immunofluorescence of Overexpressed YFP Constructs—HeLa cells were pelleted, resuspended in medium supplemented with 10% charcoal/dextran-stripped serum (Hyclone), and seeded onto 22-mm glass coverslips at a density of 3 × 105 cells/slide. Cells were transfected with 1 μg of either p65-EYFP, SRC1-RID-EYFP, or EYFP-NCoR-RID using Fugene 6 transfection reagent (Roche Applied Science). 24 h post-transfection, cells were visualized on a Leica TCS-4D confocal microscope, using a × 63 water immersion objective. Cells were excited with an argon laser at 488 nm, and emission was collected using a band pass filter of 525 ± 25 nm. Chromatin Immunoprecipitation—HeLa cells were cultured for 3 days in DMEM supplemented with 10% charcoal/dextran-stripped fetal bovine serum (Hyclone) to 95% confluence in T75 flasks (∼ 5 × 106 cells). Cells were treated with vehicle, TNF-α (10 ng/ml), or TNF-α and GR ligand (1 μm) for 2 h and then washed twice with PBS. 1% formaldehyde was then added, and cells were incubated at room temperature for 10 min. Glycine (final concentration 0.125 m) was then added for 5 min to quench the formaldehyde. The protocol described by Shang et al. (24Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar) was then followed. Briefly, cells were then washed twice with ice-cold PBS, collected into 100 mm Tris-HCl (pH 9.4), 10 mm dithiothreitol, incubated for 15 min at 30 °C, and centrifuged for 5 min at 2000 × g. Cells were then sequentially washed with 1 ml of ice-cold PBS, buffer I (0.25% Triton X-100, 10 mm EDTA, 0.5 mm EGTA, 10 mm HEPES, pH 6.5), and buffer II (200 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 10 mm HEPES, pH 6.5). Cells were resuspended in 0.5 ml of lysis buffer containing 1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, 1× protease inhibitor mixture (Roche Applied Science), and sonicated three times for 30 s each at the half-power setting (Jencons) followed by centrifugation for 10 min. Supernatants were collected and made up to 1 ml in lysis buffer, and 100 μl was set aside to represent input. The input sample was heated at 65 °C overnight to reverse the cross-links, and then DNA was extracted using the DNeasy tissue kit (Qiagen). The remaining supernatant was immunocleared with 2 μg of sheared salmon sperm DNA, 20 μl of preimmune serum, and 25 μl of protein G-Sepharose (Santa Cruz Biotechnology) for 2 h at 4 °C. Immunoprecipitation was performed overnight at 4 °C, using either 2.5 μg of GR M20 or 2 μg of p65 sc-109 antibody (Santa Cruz Biotechnology). After immunoprecipitation, 25 μl of protein A-Sepharose and 2 μg of salmon sperm DNA were added, and the incubation was continued for another 2 h. Precipitates were then washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), and buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1). Precipitates were then washed three times with TE buffer and extracted twice with 1% SDS, 0.1 m NaHCO3. Eluates were pooled and heated at 65 °C overnight to reverse the formaldehyde cross-linking. DNA fragments were purified using the DNeasy tissue kit (Qiagen). Following DNA extraction, real time quantitative PCR was performed (Stratagene M3000), using the SYBR Green master mix (Sigma), 0.4 μm primers for promoter regions of the IL-8 and U6SnRNA genes, which have been previously described (18Nissen R.M. Yamamoto K.R. Genes Dev. 2000; 14: 2314-2329Crossref PubMed Scopus (454) Google Scholar) (IL-8 promoter region –121/+61, forward (5′-GGGCCATCAGTTGCAAATC-3′) and reverse (5′-TTCCTTCCGGTGGTTTCTTC-3′); U6SnRNA promoter region –248/+85, forward (5′-GGCCTATTTCCCATGATTCC-3′) and reverse (5′-ATTTGCGTGTCATCCTTGC-3′) and 5 μl of extracted DNA product. Amounts of DNA precipitated in each experiment were calculated by comparison with standard curve values obtained from amplification reactions carried out with serial dilutions of genomic DNA. These values were then corrected for the amount of DNA precipitated from the IgG control and the amount of DNA present in the input sample. To control the specificity of the amplification and to ensure that only one product was generated, products were examined with both a dissociation curve program and by gel electrophoresis. Statistics—Where appropriate, data were analyzed using analysis of variance followed by the Bonferoni post hoc test. Dexamethasone but Not RU486 Repressed IL-8 Production—To investigate the ability of GR ligands to repress p65-mediated transactivation, HeLa cells were treated for 16 h with TNF-α (0.5 ng/ml) in the presence or absence of steroid. IL-8 was measured in cell-conditioned medium by ELISA. TNF-α treatment increased IL-8 concentration 6.5-fold; this was significantly suppressed by Dex. In contrast, RU486 did not significantly repress IL-8 secretion (Fig. 1). Co-localization of GR and NF-κB p65 in Glucocorticoidtreated Cells—Both Dex and RU486 allow GR translocation, and both promote DNA binding (22Pandit S. Geissler W. Harris G. Sitlani A. J. Biol. Chem. 20
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