Identification of DRIP205 as a Coactivator for the Farnesoid X Receptor
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m405126200
ISSN1083-351X
AutoresInès Pineda‐Torra, Leonard P. Freedman, Michael J. Garabedian,
Tópico(s)Trace Elements in Health
ResumoFarnesoid X receptor (FXR) is a bile acid sensor that regulates the expression of a number of genes the products of which control bile acid and cholesterol homeostasis; however, the role of DRIP205 in FXR-mediated gene regulation remains unexplored. In this study we demonstrate that DRIP205 binds FXR in a ligand-dependent manner in vitro and in vivo. Glutathione S-transferase pull-down assays showed that DRIP205 binds FXR in response to bile acid ligands in a dose-dependent fashion and that the potency of this interaction is associated with the ability of the ligand to activate FXR. In addition, the FXR-DRIP205 interaction required the presence of an intact LXXLL nuclear receptor box 1 (N-terminal) motif of DRIP205. In gel shift assays FXR was also able to recruit DRIP205 in the context of a DNA-bound FXR/RXR (retinoid X receptor) heterodimer. In transient transfection assays, DRIP205 efficiently enhanced a bile acid-activated FXRE-driven reporter gene in a dose-dependent manner in cells overexpressing FXR/RXR, demonstrating that DRIP205 enhances FXR-mediated transactivation. By contrast, an FXRW469A mutant in the activation function 2 domain that does not bind to DRIP205 was unable to activate ligand-stimulated FXR transcription, indicating that DRIP205 is recruited to activation function 2 of FXR. Requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was evaluated using an RXR heterodimerization-deficient FXR mutant (FXRL433R). FXRL433R was not able to bind to DRIP205 and failed to enhance an FXRE-driven reporter gene. In addition, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression, indicating that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulated the induction of endogenous FXR target gene mRNA expression by ligand. Together, these results demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the importance of DRIP205 in modulating the bile acid response of FXR target genes. Farnesoid X receptor (FXR) is a bile acid sensor that regulates the expression of a number of genes the products of which control bile acid and cholesterol homeostasis; however, the role of DRIP205 in FXR-mediated gene regulation remains unexplored. In this study we demonstrate that DRIP205 binds FXR in a ligand-dependent manner in vitro and in vivo. Glutathione S-transferase pull-down assays showed that DRIP205 binds FXR in response to bile acid ligands in a dose-dependent fashion and that the potency of this interaction is associated with the ability of the ligand to activate FXR. In addition, the FXR-DRIP205 interaction required the presence of an intact LXXLL nuclear receptor box 1 (N-terminal) motif of DRIP205. In gel shift assays FXR was also able to recruit DRIP205 in the context of a DNA-bound FXR/RXR (retinoid X receptor) heterodimer. In transient transfection assays, DRIP205 efficiently enhanced a bile acid-activated FXRE-driven reporter gene in a dose-dependent manner in cells overexpressing FXR/RXR, demonstrating that DRIP205 enhances FXR-mediated transactivation. By contrast, an FXRW469A mutant in the activation function 2 domain that does not bind to DRIP205 was unable to activate ligand-stimulated FXR transcription, indicating that DRIP205 is recruited to activation function 2 of FXR. Requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was evaluated using an RXR heterodimerization-deficient FXR mutant (FXRL433R). FXRL433R was not able to bind to DRIP205 and failed to enhance an FXRE-driven reporter gene. In addition, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression, indicating that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulated the induction of endogenous FXR target gene mRNA expression by ligand. Together, these results demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the importance of DRIP205 in modulating the bile acid response of FXR target genes. Bile acids serve a number of important physiological functions, including the solubilization of cholesterol, fat-soluble vitamins, and other lipids in the intestine. In liver, conversion of cholesterol to the primary bile acids, cholic acid (CA) 1The abbreviations used are: CA, cholic acid; FXR, farnesoid X receptor; RXR, retinoid X receptor; AF, activation function; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; FXRE, farnesoid X response element; GR, glucocorticoid receptor; IR, inverted repeat; VDR, vitamin D receptor; TR, thyroid hormone receptor; PPAR, peroxisome proliferator-activated receptor; RAR, retinoid acid receptor; ER, estrogen receptor; GST, glutathione S-transferase; aa, amino acid; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; NR, nuclear receptor; SRC-1, steroid receptor coactivator-1; BSEP, bile salt export pump; siRNA, small interfering RNA; AR, androgen receptor; UGT2B4, uridine 5′-diphosphate-glucuronosyltransferase 2B4 gene promoter; cds, charcoal-dextran-stripped. and chenodeoxycholic acid (CDCA), involves the so-called neutral and acidic pathways, respectively (1Vlahcevic Z.R. Pandak W.M. Stravitz R.T. Gastroenterol. Clin. North Am. 1999; 28: 1-25Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). CDCA and CA can be further modified by intestinal bacteria to secondary bile acids, principally deoxycholic acid (DCA) and lithocholic acid. More than 95% of all bile acids are present as glycine or taurine conjugates. Conjugation of bile acids increases their solubility while prohibiting free movement of bile acids across cell membranes, and thus tissues involved in the enterohepatic circulation of bile acids harbor transporters to facilitate bile acid uptake and efflux (2Love M.W. Dawson P.A. Curr. Opin. Lipidol. 1998; 9: 225-229Crossref PubMed Scopus (64) Google Scholar). Because of their intrinsic toxicity, bile acids must be tightly regulated. This is accomplished by transcriptionally regulating genes encoding proteins involved in bile acid biosynthesis, uptake, intracellular transport, export, and metabolism. The farnesoid X receptor (FXR, NR1H4) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that has been described as a master regulator of bile acid and cholesterol metabolism and plasma triglyceride concentrations (3Chiang J.Y. Endocr. Rev. 2002; 23: 443-463Crossref PubMed Scopus (389) Google Scholar). FXR is expressed in tissues exposed to bile acids, including the liver and intestine, and in kidney and adrenal cortex (4Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Nooman D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (978) Google Scholar, 5Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). CDCA is the most active FXR ligand (6Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. 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In addition, FXR can bind as a monomer to induce the uridine 5′-diphosphate-glucuronosyltransferase 2B4 gene promoter (UGT2B4) (14Barbier O. Torra I.P. Sirvent A. Claudel T. Blanquart C. Duran-Sandoval D. Kuipers F. Kosykh V. Fruchart J.C. Staels B. Gastroenterology. 2003; 124: 1926-1940Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and inhibit the apolipoprotein AI promoter (15Claudel T. Sturm E. Duez H. Pineda Torra I. Sirvent A. Kosykh V. Fruchart J.C. Dallongeville J. Hum D.W. Kuipers F. Staels B. J. Clin. Investig. 2002; 109: 961-971Crossref PubMed Scopus (289) Google Scholar). FXR acts as a bile acid sensor inducing the transcription of a cohort of genes involved in the reduction of bile acid concentrations within the hepatocyte. Activation of FXR also results in the repression of crucial genes in the bile acid biosynthetic pathway, namely CYP7A1 and CYP8B1 (3Chiang J.Y. Endocr. Rev. 2002; 23: 443-463Crossref PubMed Scopus (389) Google Scholar). 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Burakov D. Iniguez-Lluhi J.A. Freedman L.P. Garabedian M.J. EMBO J. 1999; 18: 5380-5388Crossref PubMed Scopus (243) Google Scholar, 38Burakov D. Wong C.W. Rachez C. Cheskis B.J. Freedman L.P. J. Biol. Chem. 2000; 275: 20928-20934Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 39Kang Y.K. Guermah M. Yuan C.X. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2642-2647Crossref PubMed Scopus (136) Google Scholar, 40Wang Q. Sharma D. Ren Y. Fondell J.D. J. Biol. Chem. 2002; 277: 42852-42858Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In vivo studies have established the essential role of DRIP205 during embryogenesis, and DRIP205-null mice die on embryonic day 11.5 with defects in heart, placenta, liver, central nervous system, and eye (41Zhu Y. Qi C. Jia Y. Nye J.S. Rao M.S. Reddy J.K. J. Biol. Chem. 2000; 275: 14779-14782Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 42Ito M. Yuan C.X. Okano H.J. Darnell R.B. 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Furthermore, mouse primary embryonic fibroblasts devoid of DRIP205 show an impaired cell cycle regulation and a defective TR transcriptional activity, although transcriptional regulation by other regulators that also bind DRIP205, such as RAR, VP-16, and p53, remains unaltered in those cells (42Ito M. Yuan C.X. Okano H.J. Darnell R.B. Roeder R.G. Mol. Cell. 2000; 5: 683-693Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). DRIP205-null cells also exhibit defective adipogenesis and PPARγ-dependent activation, which can be rescued by ectopic DRIP205 expression (45Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Nature. 2002; 417: 563-567Crossref PubMed Scopus (275) Google Scholar). In an effort to define the molecular determinants of FXR transcriptional activity we investigated whether DRIP205 plays a functional role in FXR-mediated gene expression. In the present study we demonstrate that DRIP205 binds FXR in response to bile acids in a bile acid- and dose-dependent fashion. In transient transfection assays, the ligand-activated activity of an FXRE-driven reporter gene is efficiently enhanced by DRIP205 in a way that requires an intact AF2 domain in FXR. Using an RXR heterodimerization-deficient FXR mutant (FXRL433R) we observe that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulates the induction of FXR target gene mRNA expression by CDCA. In concert, these data demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the role of DRIP205 in modulating the bile acid response of FXR target genes. Expression and Purification of Glutathione S-Transferase (GST) Fusion Proteins—GST fusion proteins were expressed in BL21 cells by induction with 0.25 mm isopropyl-β-d-thiogalactopyranoside at 22 °Cas described (33Yang W. Rachez C. Freedman L.P. Mol. Cell. Biol. 2000; 20: 8008-8017Crossref PubMed Scopus (106) Google Scholar). GST-SRC1 (aa 613–773), GST-DRIP205 (aa 527–774), and GST-DRIP205 (aa 527–974), as well as the DRIP205 mutant derivatives GST-DRIP205-mutNR1 and GST-DRIP205-mutNR2, have been described elsewhere (38Burakov D. Wong C.W. Rachez C. Cheskis B.J. Freedman L.P. J. Biol. Chem. 2000; 275: 20928-20934Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Rachez C. Gamble M. Chang C.-P.B. Atkins G.B. Lazar M.A. Freedman L.P. Mol. Cell. Biol. 2000; 20: 2718-2726Crossref PubMed Scopus (184) Google Scholar). For electrophoretic mobility shift assay analysis, proteins bound to glutathione-Sepharose beads were eluted as described (33Yang W. Rachez C. Freedman L.P. Mol. Cell. Biol. 2000; 20: 8008-8017Crossref PubMed Scopus (106) Google Scholar). Equal amounts of proteins used in pull-down and electrophoretic mobility shift assays were ensured by SDS-PAGE and Coomassie blue staining of the proteins. GST Pull-down Assays—GST fusion proteins, GST-SRC1 (aa 613–773), GST-DRIP205 (aa 527–774), GST-DRIP205 (aa 527–974), GST-DRIP205-mutNR1, and GST-DRIP205-mutNR2 immobilized in glutathione-Sepharose beads were preincubated in binding buffer (20 mm Tris, pH 7.9, 170 mm KCl, 20% glycerol, 0.2 mm EDTA, 0.01% Nonidet P-40, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 1 mm dithiothreitol) in the presence of ligands or vehicle for 30 min to 1 h at 4 °C. In vitro translated [35S]methionine-labeled (TnT transcription/translation system, Promega) full-length human FXR, the mutated FXRL433R (pcDNA3-FXR and pcDNA3-FXRL433R), or the mutated FXRW469A (pCMX-FXRW469A) was incubated with the immobilized fusion proteins for 4 h to overnight at 4 °C. Beads were then washed four times in binding buffer containing 0.1% Nonidet P-40, and samples were resolved by SDS-PAGE and visualized by autoradiography. Each assay was performed at least twice with similar results. Cell Culture and Transient Transfections—COS-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were seeded on 24-well plates at a density of 4 × 104 and cells were transfected in phenol red-free, serum-free medium using the cationic polymer Exgen 500 (MBI Fermentas) following the manufacturer's instructions as described (12Pineda Torra I. Claudel T. Duval C. Kosykh V. Fruchart J.C. Staels B. Mol. Endocrinol. 2003; 17: 259-272Crossref PubMed Scopus (351) Google Scholar). After transfection, medium was supplemented with CDCA and 10% FBS, and luciferase activity was assayed 36 h later. Luciferase units were normalized to the protein content in each well as determined by the Bio-Rad protein assay. HepG2 cells were grown at 37 °C in DMEM supplemented with 10% FBS, 1 mm sodium pyruvate, 2 mm glutamine, and 0.1 mm non-essential amino acids in dishes coated with 0.1% gelatin. Electrophoretic Mobility Shift Assays—In vitro translated FXR and RXRα (TnT transcription/translation system, Promega) were incubated in the presence of CDCA (100 μm) in binding buffer (12Pineda Torra I. Claudel T. Duval C. Kosykh V. Fruchart J.C. Staels B. Mol. Endocrinol. 2003; 17: 259-272Crossref PubMed Scopus (351) Google Scholar) for 30 min at 4 °C. Then equal amounts of purified cofactors as GST fusion proteins and a γ-[32P]ATP radiolabeled double-stranded oligonucleotide encompassing the consensus IR-1 response element were added sequentially. After incubation for 30 min at room temperature reactions were loaded on a 6% polyacrylamide nondenaturing gel and separated in 0.25× Tris-borate-EDTA buffer at 4 °C. Gels were dried prior to autoradiography. RNA Interference—A pool of four small interfering RNA (siRNA) duplexes with UU overhangs and a 5′ phosphate on the antisense strand specific for human DRIP205 (SMARTpool-DRIP205) was designed and synthesized (Dharmacon Research, Lafayette, CO) using the SMART selection and SMARTpooling technologies. As a control a nonspecific pooled duplex control (D-001206-13) was employed. For experiments where endogenous expression of DRIP205 was inhibited HepG2 cells were plated in 6-well plates at 50–60% confluency the day before transfection. Cells were transfected twice with a 20-h interval with 300 pmol of siRNA duplexes (150 nm final concentration) using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. After the second transfection, medium was replaced by fresh phenol red-free DMEM supplemented with 10% charcoal-dextranstripped FBS (cds-FBS) (Hyclone) for 4 h. Thereafter, medium was changed to phenol red-free DMEM with 0.5% cds-FBS supplemented with 50 μm CDCA, and cells were harvested for RNA extraction 24 h later. For experiments where ectopic expression of DRIP205 was inhibited HepG2 cells were cultured as described above and cotransfected with 500 ng of pcDNA3-DRIP205 and 200 pmol of siRNA duplexes using LipofectAMINE Plus (Invitrogen). After 4 h, the medium was changed to fresh phenol red-free DMEM supplemented with 10% cds-FBS for 16 h. Then, medium was changed to phenol red-free DMEM with 0.5% cds-FBS supplemented with 50 μm CDCA, and cells were harvested for RNA extraction 36 h later. Western Blot Analysis—HepG2 cells were transfected with 300 pmol of siRNA duplexes using Oligofectamine, and cells were subsequently incubated with CDCA for 24 h as described above. Whole cell extracts were prepared by lysing cells in SKL lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 10 mm NaF, 25 mm ZnCl2, 1× protease inhibitor mixture (Calbiochem), and 1 mm benzamidine) for 30 min at 4 °C. After preclearing the lysates by centrifugation, protein concentration was measured using the Bio-Rad protein assay, and 25 μg of cell extracts were boiled in SDS sample buffer. Protein extracts were separated by 7.5% SDS-PAGE, transferred to Immobilon membrane, and probed with a mouse monoclonal antibody against DRIP205 generated against bacterially expressed human DRIP205. Real Time PCR mRNA Quantification—Total RNA from HepG2 cells was extracted with TRIzol (Invitrogen) as described by the manufacturer. cDNA specific for each gene was subsequently synthesized using the enhanced avian reverse transcriptase (Sigma) and random primer hexamers (Amersham Biosciences) following the manufacturer's instructions. cDNAs were amplified using the SYBR green quantitative PCR kit (Sigma) on a LightCycler (Roche Applied Science). Reactions were carried out in a 20-μl reaction containing a 500 nm concentration of each primer and the SYBR Green Taq ReadyMix for quantitative reverse transcriptase-PCR (Sigma) as recommended by the manufacturer with the following conditions: 95 °C for 2 min followed by 42 cycles of 5 s at 95 °C, 5 s at 55 °C, and 10 s at 72 °C. DRIP205, DRIP150, and kininogen mRNA levels were normalized to 28 S expression. All reverse transcriptase-PCR products were analyzed in a post-amplification fusion curve to ensure that a single amplicon was obtained. The primers used were as follows: kininogen, 5′-GGCTGTGTGCATCCTATATCAACGC-3′ (sense) and 5′-CGGTATCACCATTCCAAAGGGAC-3′ (antisense); DRIP205, 5′-AAACCATTCAAGCCGAC-3′ (sense) and 5′-CGTTCCCTGTGATTTGC-3″ (antisense) (259 bp); and DRIP150, 5′-TCCATACCTACCATCCTCAC-3′ (sense) and 5′-GGACTAAGAGCTACTCTGC-3′ (antisense) (379 bp). Primers used to amplify 28 S have been described elsewhere (47Bonazzi A. Mastyugin V. Mieyal P.A. Dunn M.W. Laniado-Schwartman M. J. Biol. Chem. 2000; 275: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The Nuclear Receptor Coactivator DRIP205 Binds FXR in Response to Bile Acids—To determine whether DRIP205 interacts with FXR in response to ligand, pull-down assays were performed using in vitro translated full-length FXR and a bacterially expressed fragment of DRIP205 (aa 527–774, containing the two described LXXLL motifs or NR boxes (46Rachez C. Gamble M. Chang C.-P.B. Atkins G.B. Lazar M.A. Freedman L.P. Mol. Cell. Biol. 2000; 20: 2718-2726Crossref PubMed Scopus (184) Google Scholar)) as a GST fusion protein (GST-DRIP205) or GST alone. The ability of SRC-1 (aa 613–773), which has been previously reported to bind FXR in a bile acid-inducible manner (6Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2182) Google Scholar, 7Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1857) Google Scholar, 26Bramlett K.S. Yao S. Burris T.P. Mol. Genet. Metab. 2000; 71: 609-615Crossref PubMed Scopus (43) Google Scholar), was also examined. In the presence of increasing concentrations of CDCA, DRIP205 bound FXR in a dose-dependent manner to a similar extent as SRC-1 (Fig. 1A). In contrast, GST alone did not interact with FXR even at the highest concentration of CDCA tested. Thus, like the p160 coactivators, DRIP205 is recruited to FXR in a ligand-dependent manner.
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