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Identification of DRIP205 as a Coactivator for the Farnesoid X Receptor*

  • Inés Pineda Torra
    Affiliations
    Department of Microbiology and Urology, New York University School of Medicine, New York, New York 10016
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  • Leonard P. Freedman
    Affiliations
    Department of Molecular Endocrinology, Merck Research Laboratories, West Point, Pennsylvania 19486
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  • Michael J. Garabedian
    Correspondence
    To whom correspondence should be addressed: Dept. of Microbiology and Urology, NYU School of Medicine, 550 First Ave., MSB 235, New York, NY 10016. Tel.: 212-263-7662; Fax: 212-263-8276;
    Affiliations
    Department of Microbiology and Urology, New York University School of Medicine, New York, New York 10016
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  • Author Footnotes
    * This work was supported by National Institutes of Health grants (to M. J. G. and L. P. F.) and by a grant from the American Cancer Society (to M. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      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)
      The 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.
      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 (
      • Vlahcevic Z.R.
      • Pandak W.M.
      • Stravitz R.T.
      ). 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 (
      • Love M.W.
      • Dawson P.A.
      ).
      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 (
      • Chiang J.Y.
      ). FXR is expressed in tissues exposed to bile acids, including the liver and intestine, and in kidney and adrenal cortex (
      • Forman 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.
      ,
      • Lu T.T.
      • Makishima M.
      • Repa J.J.
      • Schoonjans K.
      • Kerr T.A.
      • Auwerx J.
      • Mangelsdorf D.J.
      ). CDCA is the most active FXR ligand (
      • Makishima 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.
      ,
      • Parks 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.
      ,
      • Makishima M.
      • Lu T.T.
      • Xie W.
      • Whitfield G.K.
      • Domoto H.
      • Evans R.M.
      • Haussler M.R.
      • Mangelsdorf D.J.
      ). FXR can also be activated by DCA, CA, and lithocholic acid and their conjugated derivatives, albeit to a lesser extent. FXR is activated by bile acids and regulates transcription by binding as a heterodimer with RXR to response elements (FXREs) within the regulatory regions of target genes (
      • Chiang J.Y.
      ). Most FXREs consist of an inverted repeat of the canonical hexanucleotide core motif AGGTCA spaced by one base pair (IR-1). However, the FXR/RXR heterodimer can also bind to other response elements both in vitro (
      • Laffitte B.A.
      • Kast H.R.
      • Nguyen C.M.
      • Zavacki A.M.
      • Moore D.D.
      • Edwards P.A.
      ) and in vivo (
      • Song C.S.
      • Echchgadda I.
      • Baek B.S.
      • Ahn S.C.
      • Oh T.
      • Roy A.K.
      • Chatterjee B.
      ,
      • Kast H.R.
      • Goodwin B.
      • Tarr P.T.
      • Jones S.A.
      • Anisfeld A.M.
      • Stoltz C.M.
      • Tontonoz P.
      • Kliewer S.
      • Willson T.M.
      • Edwards P.A.
      ,
      • Pineda Torra I.
      • Claudel T.
      • Duval C.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ,
      • Prieur X.
      • Coste H.
      • Rodriguez J.C.
      ). In addition, FXR can bind as a monomer to induce the uridine 5′-diphosphate-glucuronosyltransferase 2B4 gene promoter (UGT2B4) (
      • Barbier O.
      • Torra I.P.
      • Sirvent A.
      • Claudel T.
      • Blanquart C.
      • Duran-Sandoval D.
      • Kuipers F.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ) and inhibit the apolipoprotein AI promoter (
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ).
      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 (
      • Chiang J.Y.
      ). Bile acid-activated FXR regulates bile acid transport and uptake by modulating the expression of a number of ATP-binding cassette transporters (
      • Kast H.R.
      • Goodwin B.
      • Tarr P.T.
      • Jones S.A.
      • Anisfeld A.M.
      • Stoltz C.M.
      • Tontonoz P.
      • Kliewer S.
      • Willson T.M.
      • Edwards P.A.
      ,
      • Sinal C.J.
      • Tohkin M.
      • Miyata M.
      • Ward J.M.
      • Lambert G.
      • Gonzalez F.J.
      ,
      • Ananthanarayanan M.
      • Balasubramanian N.
      • Makishima M.
      • Mangelsdorf D.J.
      • Suchy F.J.
      ,
      • Huang L.
      • Zhao A.
      • Lew J.L.
      • Zhang T.
      • Hrywna Y.
      • Thompson J.R.
      • de Pedro N.
      • Royo I.
      • Blevins R.A.
      • Peláez F.
      • Wright S.D.
      • Cui J.
      ). FXR induces enzymes involved in bile acid (
      • Song C.S.
      • Echchgadda I.
      • Baek B.S.
      • Ahn S.C.
      • Oh T.
      • Roy A.K.
      • Chatterjee B.
      ,
      • Pircher P.C.
      • Kitto J.L.
      • Petrowski M.L.
      • Tangirala R.K.
      • Bischoff E.D.
      • Schulman I.G.
      • Westin S.K.
      ) and glucuronidation (
      • Barbier O.
      • Torra I.P.
      • Sirvent A.
      • Claudel T.
      • Blanquart C.
      • Duran-Sandoval D.
      • Kuipers F.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ). In addition, FXR modulates triglyceride and lipoprotein metabolism by regulating the expression of a number of apolipoproteins (
      • Prieur X.
      • Coste H.
      • Rodriguez J.C.
      ,
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ,
      • Kast H.R.
      • Nguyen C.M.
      • Sinal C.J.
      • Jones S.A.
      • Laffitte B.A.
      • Reue K.
      • Gonzalez F.J.
      • Willson T.M.
      • Edwards P.A.
      ,
      • Claudel T.
      • Inoue Y.
      • Barbier O.
      • Duran-Sandoval D.
      • Kosykh V.
      • Fruchart J.
      • Fruchart J.C.
      • Gonzalez F.J.
      • Staels B.
      ,
      • Mak P.A.
      • Laffitte B.A.
      • Desrumaux C.
      • Joseph S.B.
      • Curtiss L.K.
      • Mangelsdorf D.J.
      • Tontonoz P.
      • Edwards P.A.
      ) and lipoprotein-remodeling enzymes (
      • Urizar N.L.
      • Dowhan D.H.
      • Moore D.D.
      ).
      Transcriptional regulation by nuclear receptors involves the binding and recruitment of coactivators and corepressors to target gene promoters (
      • McKenna N.J.
      • O'Malley B.W.
      ). Recruitment of different coactivators of the p160 family in response to bile acids has been shown to correlate with the expression of bile acid-regulated FXR target genes (
      • Makishima 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.
      ,
      • Parks 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.
      ,
      • Wang H.
      • Chen J.
      • Hollister K.
      • Sowers L.C.
      • Forman B.M.
      ,
      • Bramlett K.S.
      • Yao S.
      • Burris T.P.
      ). This class of coactivators is thought to function, at least in part, via nucleosome remodeling by its intrinsic histone acetyltransferase activities, which allows in turn the recruitment of the general transcriptional machinery and subsequent formation of a functional preinitiation complex (
      • Rachez C.
      • Freedman L.P.
      ). A distinct coactivator, the vitamin D receptor (VDR)-interacting protein (DRIP)/thyroid hormone receptor (TR)-associated protein complex, is composed of at least 15 different subunits, does not possess intrinsic histone acetyltransferase activity, and appears to link the receptor directly to the core promoter and the general transcription factor machinery (
      • Rachez C.
      • Freedman L.P.
      ). This complex is anchored to nuclear receptors mostly in a ligand-dependent manner by a single component referred to as PPAR-binding protein/DRIP205/TRAP220 (with the exception of the glucocorticoid receptor (GR) (
      • Hittelman A.B.
      • Burakov D.
      • Iniguez-Lluhi J.A.
      • Freedman L.P.
      • Garabedian M.J.
      )). Initially reported as a coactivator for TR and VDR (
      • Fondell J.D.
      • Ge H.
      • Roeder R.G.
      ,
      • Rachez C.
      • Suldan Z.
      • Ward J.
      • Chang C.P.
      • Burakov D.
      • Erdjument-Bromage H.
      • Tempst P.
      • Freedman L.P.
      ), DRIP205 is now known to bind to a number of nuclear receptors, including PPARα, PPARγ, RXRα, RAR-related receptor α, and hepatocyte nuclear factor 4α (
      • Ren Y.
      • Behre E.
      • Ren Z.
      • Zhang J.
      • Wang Q.
      • Fondell J.D.
      ,
      • Zhu Y.
      • Qi C.
      • Jain S.
      • Rao M.S.
      • Reddy J.K.
      ,
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ,
      • Yuan C.X.
      • Ito M.
      • Fondell J.D.
      • Fu Z.Y.
      • Roeder R.G.
      ,
      • Atkins G.B.
      • Hu X.
      • Guenther M.G.
      • Rachez C.
      • Freedman L.P.
      • Lazar M.A.
      ,
      • Maeda Y.
      • Rachez C.
      • Hawel III, L.
      • Byus C.V.
      • Freedman L.P.
      • Sladek F.M.
      ,
      • Malik S.
      • Wallberg A.E.
      • Kang Y.K.
      • Roeder R.G.
      ) and the steroid receptors ER, GR, and androgen receptor (
      • Hittelman A.B.
      • Burakov D.
      • Iniguez-Lluhi J.A.
      • Freedman L.P.
      • Garabedian M.J.
      ,
      • Burakov D.
      • Wong C.W.
      • Rachez C.
      • Cheskis B.J.
      • Freedman L.P.
      ,
      • Kang Y.K.
      • Guermah M.
      • Yuan C.X.
      • Roeder R.G.
      ,
      • Wang Q.
      • Sharma D.
      • Ren Y.
      • Fondell J.D.
      ).
      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 (
      • Zhu Y.
      • Qi C.
      • Jia Y.
      • Nye J.S.
      • Rao M.S.
      • Reddy J.K.
      ,
      • Ito M.
      • Yuan C.X.
      • Okano H.J.
      • Darnell R.B.
      • Roeder R.G.
      ,
      • Crawford S.E.
      • Qi C.
      • Misra P.
      • Stellmach V.
      • Rao M.S.
      • Engel J.D.
      • Zhu Y.
      • Reddy J.K.
      ,
      • Landles C.
      • Chalk S.
      • Steel J.H.
      • Rosewell I.
      • Spencer-Dene B.
      • Lalani E.N.
      • Parker M.G.
      ). A more recent study describing the partial rescue of embryos by tetraploid aggregation demonstrates that DRIP205 plays a critical role in both normal placental function and cardiovascular development before embryonic day 11.5 and is also essential in hepatic development thereafter (
      • Landles C.
      • Chalk S.
      • Steel J.H.
      • Rosewell I.
      • Spencer-Dene B.
      • Lalani E.N.
      • Parker M.G.
      ). 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 (
      • Ito M.
      • Yuan C.X.
      • Okano H.J.
      • Darnell R.B.
      • Roeder R.G.
      ). DRIP205-null cells also exhibit defective adipogenesis and PPARγ-dependent activation, which can be rescued by ectopic DRIP205 expression (
      • Ge K.
      • Guermah M.
      • Yuan C.X.
      • Ito M.
      • Wallberg A.E.
      • Spiegelman B.M.
      • Roeder R.G.
      ).
      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.

      EXPERIMENTAL PROCEDURES

      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 (
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ). 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 (
      • Burakov D.
      • Wong C.W.
      • Rachez C.
      • Cheskis B.J.
      • Freedman L.P.
      ,
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      ). For electrophoretic mobility shift assay analysis, proteins bound to glutathione-Sepharose beads were eluted as described (
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ). 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 (
      • Pineda Torra I.
      • Claudel T.
      • Duval C.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ). 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 (
      • Pineda Torra I.
      • Claudel T.
      • Duval C.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ) 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 (
      • Bonazzi A.
      • Mastyugin V.
      • Mieyal P.A.
      • Dunn M.W.
      • Laniado-Schwartman M.
      ).

      RESULTS

      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 (
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      )) 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 (
      • Makishima 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.
      ,
      • Parks 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.
      ,
      • Bramlett K.S.
      • Yao S.
      • Burris T.P.
      ), 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.
      Figure thumbnail gr1
      Fig. 1Interaction between FXR and DRIP205 is induced by bile acids. A–C, in vitro translated, full-length 35S-labeled FXR was assayed for interaction with bacterially expressed GST-SRC1 (aa 613–773), GST-DRIP205 (aa 527–774), or GST. Pull-down assays were carried out in the presence or absence of the indicated bile acids. Bile acids in B and C were used at a concentration of 200 μm. Bound FXR was resolved by SDS-PAGE and visualized by autoradiography. 10% of input 35S-labeled FXR used per incubation reaction is shown. TCDCA, tauro-CDCA; GCDCA, glyco-CDCA. D, gel mobility shift analysis was performed with in vitro translated FXR and RXR and GST-DRIP205 (aa 527–774), GST-SRC1, or GST together with an IR-1 radiolabeled oligonucleotide as a probe. Binding reactions were performed in the presence of 100 μm CDCA.
      The ability of other bile acids to mediate the recruitment of DRIP205 was also examined. To this end, pull-down experiments were carried out in the presence of vehicle (–) or saturating concentrations of CDCA, DCA, and CA and the conjugated derivatives, tauro-CDCA and glyco-CDCA (Fig. 1, B and C). DCA and CA, which have been previously shown to enhance FXR activity, albeit to a lesser extent compared with CDCA (
      • Makishima 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.
      ,
      • Parks 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.
      ,
      • Makishima M.
      • Lu T.T.
      • Xie W.
      • Whitfield G.K.
      • Domoto H.
      • Evans R.M.
      • Haussler M.R.
      • Mangelsdorf D.J.
      ), were weaker inducers of the interaction between DRIP205 and FXR (Fig. 1B). Furthermore, the taurine and glycine conjugates of CDCA, which activate FXR and induce the association of FXR with SRC-1 (
      • Makishima 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.
      ,
      • Parks 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.
      ), also promote the recruitment of DRIP205 to FXR. Therefore DRIP205 interaction with FXR correlates with agonist potency.
      The ability of DRIP205 to interact with FXR was also assessed in the context of a FXR/RXR heterodimer bound to a radiolabeled IR-1-type FXRE (Fig. 1C). Gel mobility shift assays were performed using in vitro translated FXR and RXR and either GST alone or GST-DRIP205 in the presence of CDCA. GST-SRC1 was employed as a positive control for the assay. In the presence of both SRC-1 and DRIP205, but not GST alone, a strong supershift of the FXR/RXR heterodimer on the FXRE was observed, which further demonstrated the association between DRIP205 and FXR in response to CDCA. Because the SRC-1 fragment utilized in these experiments is smaller than the DRIP205 fragment, the complex formed between SRC-1 and FXR/RXR/FXRE migrates faster in the gel compared with the DRIP205 complex.
      DRIP205 Enhances FXR Transcriptional Activity in an AF2-dependent Manner via the NR1 Box—The DRIP205 component of the DRIP/thyroid hormone receptor-associated protein coactivator complex has been shown to interact with the AF2 region of the ligand-binding domain of nuclear receptors via LXXLL motifs, also known as nuclear receptor interaction domains or NR boxes. DRIP205 contains two consensus NR boxes termed NR1 and NR2 (
      • Ren Y.
      • Behre E.
      • Ren Z.
      • Zhang J.
      • Wang Q.
      • Fondell J.D.
      ,
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      ). DRIP205 was previously reported to bind to VDR and TR through the NR2 (
      • Yuan C.X.
      • Ito M.
      • Fondell J.D.
      • Fu Z.Y.
      • Roeder R.G.
      ,
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      ) and to ER and PPARγ through the NR1 (
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ,
      • Burakov D.
      • Wong C.W.
      • Rachez C.
      • Cheskis B.J.
      • Freedman L.P.
      ). To investigate the contribution of each NR box to DRIP205 binding to FXR, GST pull-down assays were performed employing DRIP205 missense mutants in which the last two leucine residues in each LXXLL motif were mutated to alanine (NR1mut and NR2mut) (Fig. 2). Similar to ER, an intact NR1 was necessary for the CDCA-dependent interaction of DRIP205 to FXR, whereas binding through NR2 appeared to be dispensable (Fig. 2). Thus, different nuclear receptors employ distinct modes of interaction with DRIP205.
      Figure thumbnail gr2
      Fig. 2DRIP205 binds FXR through the NR1 box. GST pull-down assays were carried out using in vitro translated 35S-labeled FXR and bacterially expressed GST-DRIP205 (aa 527–974), GST-DRIP205 NR1 or NR2 mutants, or GST in the presence or absence of 200 μm CDCA. Lower panel, a schematic representation of GST-DRIP205 (aa 527–974) and the mutants GST-DRIP205-mutNR1 and GST-DRIP205-mutNR2, which contain point mutations in either NR1 or NR2 boxes, respectively, changing each LXXLL motif to LXXAA. Black and hatched boxes depict wild type and mutated NR boxes, respectively.
      The AF2 function of FXR is essential for the response to bile acids (
      • Zavacki A.M.
      • Lehmann J.M.
      • Seol W.
      • Willson T.M.
      • Kliewer S.A.
      • Moore D.D.
      ). To examine whether DRIP205 binds FXR via interaction with the AF2 domain of the receptor, in vitro binding assays using a full-length FXR construct bearing a point mutation in the AF2 domain, FXRW469A, were performed. This mutated receptor was previously shown to be compromised in its ability to transactivate a bile salt export pump (BSEP) promoter-driven reporter compared with the wild type receptor (
      • Ananthanarayanan M.
      • Balasubramanian N.
      • Makishima M.
      • Mangelsdorf D.J.
      • Suchy F.J.
      ). As shown in Fig. 3A, the AF2-mutated FXR was unable to bind SRC-1 or DRIP205, whereas under identical conditions the wild type receptor was able to bind to DRIP205 in a CDCA-dependent fashion, indicating that the DRIP205-FXR interaction is mediated through contacts with residues within the AF2 of FXR.
      Figure thumbnail gr3
      Fig. 3FXR functionally interacts with DRIP205 in an AF2-dependent manner. A, in vitro translated 35S-labeled wild type FXR or FXRW469A carrying a point mutation in the AF2, were incubated with GST-SRC1 (aa 613–773), GST-DRIP205 (aa 527–774), or GST. Pull-down assays were performed in the presence or absence of 200 μm CDCA. 10% of input of each 35S-labeled FXR protein used per incubation reaction is shown. B, COS-1 cells were transfected with a luciferase reporter plasmid containing a multimerized FXRE (IR-1-TA-Luc) (100 ng) together with pcDNA3-FXR or pCMX-FXRW469A and pcDNA3-RXR expression vectors (30 ng each) and increasing amounts of pcDNA3-DRIP205 (30, 100, and 300 ng) with or without 25 μm CDCA. Shown is the luciferase activity (mean ± S.D.) in relative light units (RLU).
      We next sought to determine whether the interaction observed in vitro between FXR and DRIP205 is functionally relevant in mammalian cells. Therefore we examined the effect of DRIP205 on FXR-mediated transcriptional activity. COS-1 cells were transiently cotransfected with DRIP205 and FXR expression vectors in the absence or presence of CDCA. Transcription was monitored as the activity of a luciferase reporter containing three IR-1-type FXREs. As shown in Fig. 3B, CDCA treatment led to a 30-fold activation of the reporter activity, which was further increased in a dose-dependent fashion by addition of increasing concentrations of DRIP205 (3-, 4-, and 9-fold, respectively). In the absence of CDCA, DRIP205 enhanced FXR activity by about 4-fold at the maximum DRIP205 concentration, likely reflecting a weaker ligand-independent recruitment of DRIP205 in this cellular context. By contrast, no enhancement by DRIP205 was observed when FXR was substituted by the AF2 mutated FXRW469A, which is consistent with the absence of interaction between this FXR mutant receptor and DRIP205 in vitro (Fig. 3A). Similar expression levels of FXRW469A and wild type FXR were verified by Western blot analysis (data not shown). These results indicate that DRIP205 can function as a coactivator for FXR in an AF2-dependent manner.
      DRIP205 Coactivation of FXR Is Dependent on FXR Heterodimerization Status—Because FXR has been reported to mediate bile acid regulation of a subset of FXR target genes in a manner that is independent of its heterodimerization with RXR (
      • Barbier O.
      • Torra I.P.
      • Sirvent A.
      • Claudel T.
      • Blanquart C.
      • Duran-Sandoval D.
      • Kuipers F.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ,
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ), the requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was investigated. Toward this end, a previously characterized heterodimerization-deficient FXR mutant (FXRL433R) was employed (
      • Barbier O.
      • Torra I.P.
      • Sirvent A.
      • Claudel T.
      • Blanquart C.
      • Duran-Sandoval D.
      • Kuipers F.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ,
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ). In marked contrast with wild type FXR, the FXRL433R mutant was unable to bind SRC-1 or DRIP205 in pull-down assays (Fig. 4A). To assess whether this lack of interaction is relevant in a cellular context, transient transfection assays in COS-1 cells using an IR-1-driven heterologous promoter were performed. As expected from published reports (
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ), FXRL433R showed lower transactivation activity than wild type FXR in the presence of CDCA (Fig. 4B). Consistent with the in vitro assays, DRIP205 failed to enhance the activity of this FXR mutant on the IR-1-driven reporter both in the presence and absence of bile acid. Expression levels of FXRL433R and wild type FXR were similar as verified by Western blot analysis (data not shown). Next, the coactivation potential of DRIP205 on FXR transcriptional activity in the absence of cotransfected RXR was analyzed. In cells incubated with vehicle or CDCA, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression even at high concentrations, whereas similar amounts of DRIP205 could effectively induce FXR/RXR activity. Taken together, these results indicate that the heterodimerization status of FXR is critical for coactivation by DRIP205.
      Figure thumbnail gr4
      Fig. 4DRIP205 induction of FXR transcriptional activity is dependent on FXR heterodimerization status. A, in vitro translated 35S-labeled wild type FXR or FXRL433R carrying a point mutation in helix 10 were incubated with GST-SRC1 (aa 613–773), GST-DRIP205 (aa 527–774), or GST. Pull-down assays were performed in the presence or absence of 200 μm CDCA. 10% of input of each 35S-labeled FXR protein used per incubation reaction is shown. B, COS-1 cells were transfected with IR-1-TA-Luc (100 ng), pcDNA3-FXR or pCMX-FXRL433R and pcDNA3-RXR (30 ng each), and pcDNA3-DRIP205 (100 ng) with or without 50 μm CDCA. Shown is the luciferase activity (mean ± S.D.) in relative light units (RLU). C, COS-1 cells were transfected with IR-1-TA-Luc (100 ng), pcDNA3-FXR, and pcDNA3-RXR (30 ng each) and increasing amounts of pcDNA3-DRIP205 (30, 100, and 300 ng) in the presence or absence of 25 μm CDCA. Shown is the luciferase activity (mean ± S.D.) in relative light units (RLU).
      DRIP205 Modulates CDCA-induced FXR Target Gene Expression in HepG2 Cells—To determine the functional significance of the induction of CDCA-activated FXR transcriptional activity by DRIP205, the effect of changes in DRIP205 levels on CDCA-regulated gene expression was assessed by transient overexpression and RNA interference studies. First, HepG2 cells were transfected with vector only (pcDNA) or a DRIP205 expression vector (DRIP205) in the presence of siRNA duplexes (Fig. 5A). A pool of siRNA duplexes specific for DRIP205 (iD205) or a nonspecific siRNA pool (iCTRL) used as a negative control was employed. Transfected cells were then treated with vehicle or CDCA, and thereafter RNA levels of known FXR target genes were monitored by real time-PCR analysis. DRIP205 mRNA expression increased 16-fold in cells transfected with the DRIP205 expression construct with or without CDCA (Fig. 5A, inset). This was accompanied by almost a 3-fold induction in the basal and CDCA response of kininogen (Fig. 5A), a recently described FXR target gene involved in vasodilation and anticoagulation processes that was shown to be highly responsive to bile acid concentrations in HepG2 cells. Cotransfection of DRIP205 together with siRNA duplexes specific for DRIP205 (iD205) led to nearly a 50% inhibition in DRIP205 mRNA levels compared with the observed expression of DRIP205 in cells transfected with nonspecific siRNA duplexes (iCTRL) (Fig. 5A, inset). siRNA-mediated inhibition of DRIP205 resulted in a reduction of kininogen mRNA expression in vehicle and CDCA-treated cells of about 40%. Therefore regulation of ectopic DRIP205 levels modulates the mRNA expression of CDCA-induced FXR target genes.
      Figure thumbnail gr5
      Fig. 5Inhibition of DRIP205 expression by RNA interference modulates CDCA induction of FXR target genes. A, HepG2 cells were transfected with 500 ng of pcDNA3-DRIP205 (DRIP205) or pcDNA3 alone (pcDNA). Cells transfected with DRIP205 were cotransfected with either 300 pmol of a nonspecific pooled siRNA duplex (iCTRL) as a control or a pool of four siRNA duplexes specific for human DRIP205 (iD205). Cells transfected with pcDNA3 alone (pcDNA) were exposed to the buffer in which the siRNAs were dissolved. After transfection, cells were treated with 50 μm CDCA for 36 h and subsequently harvested for RNA extraction. mRNA expression was analyzed by real time PCR. Kininogen (KNG) and DRIP205 mRNA levels (inset) are shown normalized to 28 S mRNA levels and are expressed as a percentage of mRNA levels in cells transfected with pcDNA3 in the presence of CDCA. For DRIP205 only the quantification of CDCA-treated expression levels is depicted. B, HepG2 cells were treated as described for A except that transfection was performed in the absence of cotransfected pcDNA3-DRIP205. After transfection, cells were incubated with 50 μm CDCA and harvested for RNA extraction 24 h later. Each condition was evaluated in triplicate. Real time PCR quantification of DRIP205 (D205), DRIP150 (D150), and kininogen expression in CDCA-treated cells is depicted. mRNA levels are shown normalized to 28 S mRNA levels. For each gene, values are expressed as a percentage of mRNA levels in cells transfected with iCTRL and are means ± S.D. (n = 3). C, whole cell extracts were prepared from HepG2 cells treated as described in B. Expression of DRIP205 was analyzed by Western blotting using a DRIP205 or tubulin antibody, which serves as a loading control.
      Finally, the effect of reduced endogenous DRIP205 levels in the expression of kininogen was investigated. In cells incubated with CDCA, mRNA and protein expression of DRIP205 was specifically reduced by about 40% by iD205 (Fig. 5, B and C), whereas the levels of DRIP150, another subunit of the DRIP coactivator complex, and SRC-1 (data not shown) remained unchanged. Reduction in DRIP205 expression resulted in decreased mRNA levels of kininogen by 40%. Thus these data demonstrate that reduction of endogenous DRIP205 expression modulates the response of FXR target genes to CDCA in HepG2 cells and underscore the importance of DRIP205 coactivation of FXR in CDCA-regulated gene expression.

      DISCUSSION

      FXR is a key regulator of bile acid and cholesterol homeostasis as well as triglyceride metabolism (
      • Chiang J.Y.
      ). As is the case for several other nuclear receptors, FXR transcriptional activity has been shown to be modulated by coactivators, a group of non-DNA-binding proteins that are recruited through conserved LXXLL motifs or NR boxes (
      • McKenna N.J.
      • O'Malley B.W.
      ). Although not exclusively, coactivators have been shown to interact with the AF2 domain of nuclear receptors. Upon such interaction, a ligand-dependent shift in the position of several critical helices in the AF2 domain occurs thereby stabilizing the contacts established between the receptor and coregulator and subsequently enhancing the transcriptional activity of the receptor.
      Besides SRC-1 and transcriptional intermediary factor 2/glucocorticoid receptor-interacting protein-1, both members of the p160 family of cofactors, no other coactivators have been reported to interact physically and/or functionally with FXR. In the present study we report the ligand- and AF2-dependent interaction between DRIP205 and FXR. Similar to the DRIP205 recruitment by PPARγ (
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ) or ER (
      • Burakov D.
      • Wong C.W.
      • Rachez C.
      • Cheskis B.J.
      • Freedman L.P.
      ), binding of DRIP205 to FXR appears to be mediated through contacts between the NR1 motif in the DRIP subunit. In contrast, an intact NR2 is required for the interaction between DRIP205 and other nuclear receptors, including VDR, TR, and PPARα (
      • Ren Y.
      • Behre E.
      • Ren Z.
      • Zhang J.
      • Wang Q.
      • Fondell J.D.
      ,
      • Yuan C.X.
      • Ito M.
      • Fondell J.D.
      • Fu Z.Y.
      • Roeder R.G.
      ,
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      ). Thus it is evident that differential preference for the LXXLL motifs in the recruitment of nuclear receptors exists. However, the predominance of one NR box over the other does not appear to be imposed by the class (steroid, non-steroid, homodimer, or heterodimer with RXR) to which a given receptor belongs. The observed DRIP205 ligand-dependent interaction with FXR in vitro is shown to be functional because DRIP205 enhances FXR transcriptional activity up to 9-fold in transient transfection assays in mammalian cells in response to bile acids. This induction is well within the range observed for other nuclear receptors such as TR, VDR, PPARγ, and HNF4 (
      • Ren Y.
      • Behre E.
      • Ren Z.
      • Zhang J.
      • Wang Q.
      • Fondell J.D.
      ,
      • Yang W.
      • Rachez C.
      • Freedman L.P.
      ,
      • Yuan C.X.
      • Ito M.
      • Fondell J.D.
      • Fu Z.Y.
      • Roeder R.G.
      ,
      • Atkins G.B.
      • Hu X.
      • Guenther M.G.
      • Rachez C.
      • Freedman L.P.
      • Lazar M.A.
      ,
      • Rachez C.
      • Gamble M.
      • Chang C.-P.B.
      • Atkins G.B.
      • Lazar M.A.
      • Freedman L.P.
      ).
      With the exception of the GR (
      • Hittelman A.B.
      • Burakov D.
      • Iniguez-Lluhi J.A.
      • Freedman L.P.
      • Garabedian M.J.
      ), previous studies support the role of DRIP205 as the main anchor of the DRIP complex to liganded nuclear receptors (
      • Yuan C.X.
      • Ito M.
      • Fondell J.D.
      • Fu Z.Y.
      • Roeder R.G.
      ,
      • Kang Y.K.
      • Guermah M.
      • Yuan C.X.
      • Roeder R.G.
      ,
      • Rachez C.
      • Lemon B.D.
      • Suldan Z.
      • Bromleigh V.
      • Gamble M.
      • Näär A.M.
      • Erdjument-Bromage H.
      • Tempst P.
      • Freedman L.P.
      ). However, DRIP205 is not the only component of the complex that has been shown to modulate the transcriptional activity of nuclear receptors. For instance, the DRIP150 subunit binds directly to the AF1 domain of the GR in a ligand-independent manner thereby inducing GR transactivation activity (
      • Hittelman A.B.
      • Burakov D.
      • Iniguez-Lluhi J.A.
      • Freedman L.P.
      • Garabedian M.J.
      ). Additionally, the DRIP100 subunit has been shown to enhance TR and VDR ligand-dependent transcription, although this subunit does not interact directly with these nuclear receptors and likely mediates its effects through binding to DRIP205 (
      • Zhang J.
      • Fondell J.D.
      ). Preliminary studies show that in GST pull-down assays DRIP150 binds directly to FXR in a ligand-independent manner.
      I. Pineda Torra, L. P. Freedman, and M. J. Garabedian, unpublished observations.
      However, the characterization of this interaction as well as the specific contribution of this or other DRIP subunits to the overall FXR ligand-dependent and -independent transcriptional activity awaits further investigation.
      The FXR/RXR heterodimer was initially described as permissive, in which RXR binds its cognate ligand and induces the activity of bile acid-activated FXR on an IR-1 FXRE thereby enhancing FXR target gene expression (
      • Kast H.R.
      • Nguyen C.M.
      • Sinal C.J.
      • Jones S.A.
      • Laffitte B.A.
      • Reue K.
      • Gonzalez F.J.
      • Willson T.M.
      • Edwards P.A.
      ,
      • Grober J.
      • Zaghini I.
      • Fujii H.
      • Jones S.A.
      • Kliewer S.A.
      • Willson T.M.
      • Ono T.
      • Besnard P.
      ). Recently, however, antagonism of bile acid-induced BSEP expression by RXR ligands was described (
      • Kassam A.
      • Miao B.
      • Young P.R.
      • Mukherjee R.
      ). BSEP down-regulation by rexinoids was shown to be because of a decrease in FXR/RXR binding to the BSEP IR-1-like response element together with an impaired recruitment of coactivators. These authors (
      • Kassam A.
      • Miao B.
      • Young P.R.
      • Mukherjee R.
      ) suggest that FXR/RXR may actually be a conditionally permissive heterodimer, in which RXR and its ligands may induce one set of genes while repressing another. Thus the sequence and structure of an FXRE may dictate the regulation by RXR agonists. To date, only two FXR target genes have been reported to be regulated by bile acids in an RXR-independent manner, the UGT2B4 and the apoAI genes involved in bile acid and lipoprotein metabolism, respectively (
      • Barbier O.
      • Torra I.P.
      • Sirvent A.
      • Claudel T.
      • Blanquart C.
      • Duran-Sandoval D.
      • Kuipers F.
      • Kosykh V.
      • Fruchart J.C.
      • Staels B.
      ,
      • Claudel T.
      • Sturm E.
      • Duez H.
      • Pineda Torra I.
      • Sirvent A.
      • Kosykh V.
      • Fruchart J.C.
      • Dallongeville J.
      • Hum D.W.
      • Kuipers F.
      • Staels B.
      ). Our results demonstrate the requirement for the FXR/RXR heterodimer in the activation of bile acid-induced FXR by DRIP205 on an IR-1 FXRE. This is because the activity of either the FXRL433R mutant or FXR alone is not affected by this cofactor in transient transfection assays in the absence of cotransfected RXR (Fig. 4). Thus, on an IR-1 the heterodimer is not only necessary for FXR DNA binding but also, as previously shown (
      • Makishima 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.
      ,
      • Parks 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.
      ), for its full activation by bile acids, which is likely the result of optimal coactivator recruitment by the heterodimer. Interestingly, the requirement for RXR heterodimerization does not seem to be exclusive of DRIP205 binding because SRC-1 recruitment by FXRL433R is also impaired in vitro (Fig. 4), although the functional relevance of this observation remains to be addressed.
      FXR was able to bind both SRC-1 and DRIP205 in solution even in the absence of co-expressed RXR in vitro (Fig. 4A, top panel), whereas its activity was not enhanced by DRIP205 without RXR cotransfection (Fig. 4C). This may be explained by the allosteric effects exerted by the DNA response element on the recruitment of DRIP205 and other coactivators by FXR. Moreover, the sequence and structure of the FXRE may also affect the dependence on the heterodimerization with RXR. Therefore, it will be interesting to determine whether RXR is needed for DRIP205 activation in the context of other FXREs that are distinct from an IR-1. For instance the everted repeat FXRE (ER-8) in the multidrug resistance-associated protein 2 promoter (
      • Kast H.R.
      • Goodwin B.
      • Tarr P.T.
      • Jones S.A.
      • Anisfeld A.M.
      • Stoltz C.M.
      • Tontonoz P.
      • Kliewer S.
      • Willson T.M.
      • Edwards P.A.
      ) or the IR-0 FXRE in the dehydroepiandrosterone sulfotransferase promoter (
      • Song C.S.
      • Echchgadda I.
      • Baek B.S.
      • Ahn S.C.
      • Oh T.
      • Roy A.K.
      • Chatterjee B.
      ) has been reported to be bound exclusively by the FXR/RXR heterodimer and would be predicted to be modulated by DRIP205. On the contrary, in the case of genes containing FXREs that are bound by FXR only, we would expect such genes (e.g. the UGT2B4 and the apoAI) to be less sensitive to DRIP205. Future experiments will be required to define the differential effects of DRIP205 activation on the modulation of distinct FXREs.
      Despite the large number of reports establishing the role of coactivators in nuclear receptor function at the promoter level, the contribution of these proteins to the action of nuclear receptors in vivo remains largely unresolved. Only a few studies have addressed the importance of DRIP205 activity in vivo, and it is of note that specific studies of the in vivo relevance of DRIP205 in the activity of a given nuclear receptor were rendered difficult because disruption of the murine DRIP205 gene causes embryonic death (
      • Zhu Y.
      • Qi C.
      • Jia Y.
      • Nye J.S.
      • Rao M.S.
      • Reddy J.K.
      ,
      • Ito M.
      • Yuan C.X.
      • Okano H.J.
      • Darnell R.B.
      • Roeder R.G.
      ). For some receptors, in vivo studies did not confirm preceding in vitro work showing recruitment of DRIP205. For instance, DRIP205 was initially demonstrated to bind to both TR and RAR (
      • Ren Y.
      • Behre E.
      • Ren Z.
      • Zhang J.
      • Wang Q.
      • Fondell J.D.
      ). However, later studies revealed that TR-but not RARα-driven transcriptional activation is defective in cultured mouse embryonic fibroblasts derived from DRIP205 knock-out embryos suggesting that DRIP205 may be dispensable for RAR signaling at least in these cells (
      • Ito M.
      • Yuan C.X.
      • Okano H.J.
      • Darnell R.B.
      • Roeder R.G.
      ). In the present study we sought to determine whether DRIP205 is required for FXR-mediated transcription in mammalian cells. FXR target gene expression in response to bile acids was shown to be significantly affected in HepG2 cells in which DRIP205 expression was selectively induced by overexpression or reduced by RNA interference (Fig. 5). Thus, our results support a functionally relevant role for DRIP205 as a bile acid-dependent FXR coactivator. Similar approaches have been used to establish the role of p160 coactivators in estradiol-induced ER target gene expression (
      • Cavarretta I.T.
      • Mukopadhyay R.
      • Lonard D.M.
      • Cowsert L.M.
      • Bennett C.F.
      • O'Malley B.W.
      • Smith C.L.
      ,
      • Shang Y.
      • Brown M.
      ). Remarkably, even though DRIP205 levels were only reduced by about 50%, bile acid induction of kininogen was significantly decreased suggesting that even relatively subtle changes in the intracellular concentration of DRIP205 may have profound effects on FXR/RXR gene regulation. This further suggests that alternative coactivator pathways may not be as relevant for bile acid signaling through FXR. Nevertheless, further experiments will be required to more precisely define the contribution of DRIP205 compared with other coactivators and other subunits in the DRIP205 complex. Taken together, our data demonstrate a role for DRIP205 as a FXR coactivator and its functional importance in modulating FXR target gene expression in response to bile acids.

      Acknowledgments

      We are grateful to Thierry Claudel, Bart Staels, and David Mangelsdorf for their kind gifts of plasmids and to members of the Freedman and Garabedian laboratories for help and stimulating discussions. We especially thank Thierry Claudel for critically reading the manuscript.

      References

        • Vlahcevic Z.R.
        • Pandak W.M.
        • Stravitz R.T.
        Gastroenterol. Clin. North Am. 1999; 28: 1-25
        • Love M.W.
        • Dawson P.A.
        Curr. Opin. Lipidol. 1998; 9: 225-229
        • Chiang J.Y.
        Endocr. Rev. 2002; 23: 443-463
        • Forman 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: 687-693
        • Lu T.T.
        • Makishima M.
        • Repa J.J.
        • Schoonjans K.
        • Kerr T.A.
        • Auwerx J.
        • Mangelsdorf D.J.
        Mol. Cell. 2000; 6: 507-515
        • Makishima 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: 1362-1365
        • Parks 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: 1365-1368
        • Makishima M.
        • Lu T.T.
        • Xie W.
        • Whitfield G.K.
        • Domoto H.
        • Evans R.M.
        • Haussler M.R.
        • Mangelsdorf D.J.
        Science. 2002; 296: 1313-1316
        • Laffitte B.A.
        • Kast H.R.
        • Nguyen C.M.
        • Zavacki A.M.
        • Moore D.D.
        • Edwards P.A.
        J. Biol. Chem. 2000; 275: 10638-10647
        • Song C.S.
        • Echchgadda I.
        • Baek B.S.
        • Ahn S.C.
        • Oh T.
        • Roy A.K.
        • Chatterjee B.
        J. Biol. Chem. 2001; 276: 42549-42556
        • Kast H.R.
        • Goodwin B.
        • Tarr P.T.
        • Jones S.A.
        • Anisfeld A.M.
        • Stoltz C.M.
        • Tontonoz P.
        • Kliewer S.
        • Willson T.M.
        • Edwards P.A.
        J. Biol. Chem. 2002; 277: 2908-2915
        • Pineda Torra I.
        • Claudel T.
        • Duval C.
        • Kosykh V.
        • Fruchart J.C.
        • Staels B.
        Mol. Endocrinol. 2003; 17: 259-272
        • Prieur X.
        • Coste H.
        • Rodriguez J.C.
        J. Biol. Chem. 2003; 278: 25468-25480
        • Barbier 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: 1926-1940
        • Claudel 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: 961-971
        • Sinal C.J.
        • Tohkin M.
        • Miyata M.
        • Ward J.M.
        • Lambert G.
        • Gonzalez F.J.
        Cell. 2000; 102: 731-744
        • Ananthanarayanan M.
        • Balasubramanian N.
        • Makishima M.
        • Mangelsdorf D.J.
        • Suchy F.J.
        J. Biol. Chem. 2001; 276: 28857-28865
        • Huang L.
        • Zhao A.
        • Lew J.L.
        • Zhang T.
        • Hrywna Y.
        • Thompson J.R.
        • de Pedro N.
        • Royo I.
        • Blevins R.A.
        • Peláez F.
        • Wright S.D.
        • Cui J.
        J. Biol. Chem. 2003; 278: 51085-51090
        • Pircher P.C.
        • Kitto J.L.
        • Petrowski M.L.
        • Tangirala R.K.
        • Bischoff E.D.
        • Schulman I.G.
        • Westin S.K.
        J. Biol. Chem. 2003; 278: 27703-27711
        • Kast H.R.
        • Nguyen C.M.
        • Sinal C.J.
        • Jones S.A.
        • Laffitte B.A.
        • Reue K.
        • Gonzalez F.J.
        • Willson T.M.
        • Edwards P.A.
        Mol. Endocrinol. 2001; 15: 1720-1728
        • Claudel T.
        • Inoue Y.
        • Barbier O.
        • Duran-Sandoval D.
        • Kosykh V.
        • Fruchart J.
        • Fruchart J.C.
        • Gonzalez F.J.
        • Staels B.
        Gastroenterology. 2003; 125: 544-555
        • Mak P.A.
        • Laffitte B.A.
        • Desrumaux C.
        • Joseph S.B.
        • Curtiss L.K.
        • Mangelsdorf D.J.
        • Tontonoz P.
        • Edwards P.A.
        J. Biol. Chem. 2002; 277: 31900-31908
        • Urizar N.L.
        • Dowhan D.H.
        • Moore D.D.
        J. Biol. Chem. 2000; 275: 39313-39317
        • McKenna N.J.
        • O'Malley B.W.
        Cell. 2002; 108: 465-474
        • Wang H.
        • Chen J.
        • Hollister K.
        • Sowers L.C.
        • Forman B.M.
        Mol. Cell. 1999; 3: 543-553
        • Bramlett K.S.
        • Yao S.
        • Burris T.P.
        Mol. Genet. Metab. 2000; 71: 609-615
        • Rachez C.
        • Freedman L.P.
        Curr. Opin. Cell Biol. 2001; 13: 274-280
        • Hittelman A.B.
        • Burakov D.
        • Iniguez-Lluhi J.A.
        • Freedman L.P.
        • Garabedian M.J.
        EMBO J. 1999; 18: 5380-5388
        • Fondell J.D.
        • Ge H.
        • Roeder R.G.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333
        • Rachez C.
        • Suldan Z.
        • Ward J.
        • Chang C.P.
        • Burakov D.
        • Erdjument-Bromage H.
        • Tempst P.
        • Freedman L.P.
        Genes Dev. 1998; 12: 1787-1800
        • Ren Y.
        • Behre E.
        • Ren Z.
        • Zhang J.
        • Wang Q.
        • Fondell J.D.
        Mol. Cell. Biol. 2000; 20: 5433-5446
        • Zhu Y.
        • Qi C.
        • Jain S.
        • Rao M.S.
        • Reddy J.K.
        J. Biol. Chem. 1997; 272: 25500-25506
        • Yang W.
        • Rachez C.
        • Freedman L.P.
        Mol. Cell. Biol. 2000; 20: 8008-8017
        • Yuan C.X.
        • Ito M.
        • Fondell J.D.
        • Fu Z.Y.
        • Roeder R.G.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7939-7944
        • Atkins G.B.
        • Hu X.
        • Guenther M.G.
        • Rachez C.
        • Freedman L.P.
        • Lazar M.A.
        Mol. Endocrinol. 1999; 13: 1550-1557
        • Maeda Y.
        • Rachez C.
        • Hawel III, L.
        • Byus C.V.
        • Freedman L.P.
        • Sladek F.M.
        Mol. Endocrinol. 2002; 16: 1502-1510
        • Malik S.
        • Wallberg A.E.
        • Kang Y.K.
        • Roeder R.G.
        Mol. Cell. Biol. 2002; 22: 5626-5637
        • Burakov D.
        • Wong C.W.
        • Rachez C.
        • Cheskis B.J.
        • Freedman L.P.
        J. Biol. Chem. 2000; 275: 20928-20934
        • Kang Y.K.
        • Guermah M.
        • Yuan C.X.
        • Roeder R.G.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2642-2647
        • Wang Q.
        • Sharma D.
        • Ren Y.
        • Fondell J.D.
        J. Biol. Chem. 2002; 277: 42852-42858
        • Zhu Y.
        • Qi C.
        • Jia Y.
        • Nye J.S.
        • Rao M.S.
        • Reddy J.K.
        J. Biol. Chem. 2000; 275: 14779-14782
        • Ito M.
        • Yuan C.X.
        • Okano H.J.
        • Darnell R.B.
        • Roeder R.G.
        Mol. Cell. 2000; 5: 683-693
        • Crawford S.E.
        • Qi C.
        • Misra P.
        • Stellmach V.
        • Rao M.S.
        • Engel J.D.
        • Zhu Y.
        • Reddy J.K.
        J. Biol. Chem. 2002; 277: 3585-3592
        • Landles C.
        • Chalk S.
        • Steel J.H.
        • Rosewell I.
        • Spencer-Dene B.
        • Lalani E.N.
        • Parker M.G.
        Mol. Endocrinol. 2003; 17: 2418-2435
        • Ge K.
        • Guermah M.
        • Yuan C.X.
        • Ito M.
        • Wallberg A.E.
        • Spiegelman B.M.
        • Roeder R.G.
        Nature. 2002; 417: 563-567
        • Rachez C.
        • Gamble M.
        • Chang C.-P.B.
        • Atkins G.B.
        • Lazar M.A.
        • Freedman L.P.
        Mol. Cell. Biol. 2000; 20: 2718-2726
        • Bonazzi A.
        • Mastyugin V.
        • Mieyal P.A.
        • Dunn M.W.
        • Laniado-Schwartman M.
        J. Biol. Chem. 2000; 275: 2837-2844
        • Zavacki A.M.
        • Lehmann J.M.
        • Seol W.
        • Willson T.M.
        • Kliewer S.A.
        • Moore D.D.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7909-7914
        • Rachez C.
        • Lemon B.D.
        • Suldan Z.
        • Bromleigh V.
        • Gamble M.
        • Näär A.M.
        • Erdjument-Bromage H.
        • Tempst P.
        • Freedman L.P.
        Nature. 1999; 398: 824-828
        • Zhang J.
        • Fondell J.D.
        Mol. Endocrinol. 1999; 13: 1130-1140
        • Grober J.
        • Zaghini I.
        • Fujii H.
        • Jones S.A.
        • Kliewer S.A.
        • Willson T.M.
        • Ono T.
        • Besnard P.
        J. Biol. Chem. 1999; 274: 29749-29754
        • Kassam A.
        • Miao B.
        • Young P.R.
        • Mukherjee R.
        J. Biol. Chem. 2003; 278: 10028-10032
        • Cavarretta I.T.
        • Mukopadhyay R.
        • Lonard D.M.
        • Cowsert L.M.
        • Bennett C.F.
        • O'Malley B.W.
        • Smith C.L.
        Mol. Endocrinol. 2002; 16: 253-270
        • Shang Y.
        • Brown M.
        Science. 2002; 295: 2465-2468