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• W2109127510 abstract "The Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily that has been shown to play an important role in bile acid and cholesterol homeostasis. Here we identify four murine FXR transcripts, derived from a single gene, that encode four isoforms, FXRα1, FXRα2, FXRβ1, and FXRβ2. FXRα and FXRβ differ at their amino terminus, and FXRα1 and FXRβ1 have a four-amino acid residue insertion in the hinge region immediately adjacent to the DNA binding domain. Real time PCR and 5′-rapid amplification of cDNA ends followed by Southern blotting reveal that these four transcripts are expressed differentially in liver, intestine, kidney, adrenals, stomach, fat, and heart. Electrophoretic mobility shift assays demonstrate that FXRα2 and FXRβ2 bind to FXR response elements with a higher affinity as compared with FXRα1 and FXRβ1, suggesting that the four-amino acid insert may affect FXR function. Consistent with this idea, the results of transient transfection experiments demonstrate that the four FXR isoforms differentially transactivated a number of promoter-reporter genes; activation of an ileal bile acid-binding protein promoter-reporter gene varied 20-fold depending on the FXR isoform; the rank order of activation was FXRβ2 > FXRα2 ≫ FXRα1 = FXRβ1. In contrast, SHP reporter or BSEP reporter genes were activated to similar degrees by each of the FXR isoforms. Finally, NIH3T3 cells were stably infected with individual murine FXR isoforms, and the cells were treated with FXR ligands. The endogenous ileal bile acid-binding protein gene was activated by the four FXR isoforms with the same rank order as seen in transfections. This effect was gene-specific, since induction of bile salt export pump mRNA was independent of the FXR isoform. These observations suggest that there are four distinct murine FXR isoforms that differentially regulate gene expression in numerous tissues in vivo. The Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily that has been shown to play an important role in bile acid and cholesterol homeostasis. Here we identify four murine FXR transcripts, derived from a single gene, that encode four isoforms, FXRα1, FXRα2, FXRβ1, and FXRβ2. FXRα and FXRβ differ at their amino terminus, and FXRα1 and FXRβ1 have a four-amino acid residue insertion in the hinge region immediately adjacent to the DNA binding domain. Real time PCR and 5′-rapid amplification of cDNA ends followed by Southern blotting reveal that these four transcripts are expressed differentially in liver, intestine, kidney, adrenals, stomach, fat, and heart. Electrophoretic mobility shift assays demonstrate that FXRα2 and FXRβ2 bind to FXR response elements with a higher affinity as compared with FXRα1 and FXRβ1, suggesting that the four-amino acid insert may affect FXR function. Consistent with this idea, the results of transient transfection experiments demonstrate that the four FXR isoforms differentially transactivated a number of promoter-reporter genes; activation of an ileal bile acid-binding protein promoter-reporter gene varied 20-fold depending on the FXR isoform; the rank order of activation was FXRβ2 > FXRα2 ≫ FXRα1 = FXRβ1. In contrast, SHP reporter or BSEP reporter genes were activated to similar degrees by each of the FXR isoforms. Finally, NIH3T3 cells were stably infected with individual murine FXR isoforms, and the cells were treated with FXR ligands. The endogenous ileal bile acid-binding protein gene was activated by the four FXR isoforms with the same rank order as seen in transfections. This effect was gene-specific, since induction of bile salt export pump mRNA was independent of the FXR isoform. These observations suggest that there are four distinct murine FXR isoforms that differentially regulate gene expression in numerous tissues in vivo. retinoid X receptor, FXR, farnesoid X receptor FXR response element ileal bile acid-binding protein mouse BABP bile salt export pump small heterodimer partner gene-specific primer rapid amplification of cDNA ends electrophoretic mobility shift assay base pairs mouse human adapter primer Nuclear hormone receptors are transcription factors that are involved in numerous processes, including reproduction, development, and general metabolism (1Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1696) Google Scholar). Most of these receptors are comprised of a ligand-independent transcriptional activation function (AF-1) at the amino terminus, a DNA binding domain, a hinge region and a ligand binding domain, a dimerization interface, and a ligand-dependent activation function (AF-2) at the carboxyl terminus (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar, 3Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar). In many cases entry of a specific ligand into the pocket formed by the ligand binding domain results in a conformational change of the receptor, recruitment of co-activators, and transcriptional activation (4Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2063) Google Scholar, 5Kamei Y., Xu, L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. 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A. 1997; 94: 8479-8484Crossref PubMed Scopus (504) Google Scholar, 12Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1108) Google Scholar, 13McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar). Nuclear hormone receptors have been classified into sub-groups depending on whether they bind DNA as homodimers, heterodimers, or monomers (14Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). A few family members have been identified that do not bind DNA directly but instead function by interacting with other transcription factors and altering their activity (15Seol W. Choi H.S. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (446) Google Scholar, 16Ito M., Yu, R. Jameson J.L. Mol. Cell. Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (401) Google Scholar). Nonetheless, the major sub-group contains members that bind to DNA as heterodimers with the common partner, retinoid X receptor (RXR)1 (14Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). The farnesoid X receptor (FXR, NR1H4) falls into this category. FXR was isolated by screening a rat cDNA library using PCR and degenerate primers corresponding to the highly conserved DNA binding domain of nuclear receptors (17Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (978) Google Scholar). Independently, two mouse homologues of rat FXR, termed RIP14-1 and RIP14-2, were isolated using the yeast two-hybrid assay and the human RXR ligand binding domain as bait (18Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). Northern blot assays and in situ hybridization indicate that FXR expression was limited to the liver, small intestine, kidney, and adrenal gland (17Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (978) Google Scholar, 19Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In the initial studies, supraphysiological levels of farnesol were shown to activate the rat (17Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (978) Google Scholar) but not the murine FXR (20Zavacki 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: 7909-7914Crossref PubMed Scopus (88) Google Scholar). In 1999, several groups independently identified bile acids as endogenous ligands that activated FXR at physiological concentrations (21Makishima 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, 22Parks 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, 23Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar). The finding that bile acids not only bound to FXR but that this interaction resulted in recruitment of co-activators (21Makishima 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, 22Parks 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) provides compelling evidence that bile acids are physiologically important hormones that function to activate the FXR/RXR heterodimer. The recent characterization of FXR null mice (24Sinal C., J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar), the synthesis and utilization of a high affinity ligand for FXR (25Goodwin B. Jones S.A. Price R., R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M., E. Maloney P., R. Willson T., M. Kliewer S., A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar), and the identification of a number of FXR target genes provide important insights into the role of FXR in controlling lipid metabolism. FXR target genes include ileal bile acid-binding protein (I-BABP) (21Makishima 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, 26Grober J. Zaghini I. Fujii H. Jones S.A. Kliewer S.A. Willson T.M. Ono T. Besnard P. J. Biol. Chem. 1999; 274: 29749-29754Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), phospholipid transfer protein (27Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 28Urizar N.L. Dowhan D.H. Moore D.D. J. Biol. Chem. 2000; 275: 39313-39317Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), apolipoprotein C-II (29Kast 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: 1720-1728Crossref PubMed Scopus (224) Google Scholar), multidrug resistance-associated protein 2 (ABCC2) (30Kast 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: 2908-2915Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar), the bile salt export pump (BSEP) (31Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. J. Biol. Chem. 2001; 276: 28857-28865Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar), and the small heterodimer partner receptor (SHP) (25Goodwin B. Jones S.A. Price R., R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M., E. Maloney P., R. Willson T., M. Kliewer S., A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 32Lu 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) (for review, see Ref. 33Edwards P.A. Kast H.R. Anisfeld A.M. J. Lipid Res. 2002; 43: 2-12Abstract Full Text Full Text PDF PubMed Google Scholar). These genes are involved in various aspects of bile acid, lipoprotein, and lipid metabolism (33Edwards P.A. Kast H.R. Anisfeld A.M. J. Lipid Res. 2002; 43: 2-12Abstract Full Text Full Text PDF PubMed Google Scholar). The demonstration that FXR null mice are unable to respond appropriately to diets enriched in fat or bile acids (24Sinal C., J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar) further emphasized the critical role of FXR in controlling lipid homeostasis. Two forms of murine FXR (RIP14–1 and RIP14–2) that differ at their amino terminus were originally isolated (18Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). RIP14-2, in contrast to RIP14-1, contained an additional 12 bp that results in the insertion of four amino acids in the hinge region, adjacent to the DNA binding domain. Analysis of the cDNA encoding rat FXR indicates that it does not contain the 12-bp insert but otherwise corresponds to murine RIP14-1. It is not known whether these different isoforms have different functions. Taken together, these results suggested that there might be at least four FXR isoforms that differ either at their amino terminus and/or at the site of the four-amino acid insertion in the hinge region. Because the hinge region is thought to have a role in the DNA binding properties of nuclear receptors (34Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 35Kumar V. Green S. Staub A. Chambon P. EMBO J. 1986; 5: 2231-2236Crossref PubMed Scopus (407) Google Scholar, 36Kumar V. Green S. Stack G. Berry M. Jin J.R. Chambon P. Cell. 1987; 51: 941-951Abstract Full Text PDF PubMed Scopus (1069) Google Scholar), we hypothesized that these different isoforms might differentially bind to DNA and/or differentially activate target genes. The current report provides evidence to support these proposals. C57BL/6J female mice were fed a standard rodent chow diet in a temperature-controlled room (23 °C) on a 12-h light/dark cycle. Eight to 12-week-old wild type mice were sacrificed, and tissues were snap-frozen in liquid nitrogen and stored at −80 °C until use. Four different mouse FXR cDNAs were isolated from the liver tissue using gene-specific primers (GSP1 and GSP2) and adapter primers (ADP1 and ADP2) in the Sure-RACE panels according to the manufacture's protocol (OriGene Technologies, Inc., Rockville, MD). The first round of PCR utilized ADP1 and GSP1 (Fig. 1 A). The generated cDNA was further amplified in a second round of PCR utilizing ADP2 and GSP2. The full-length coding regions of four murine FXR isoforms were amplified by PCR using gene-specific primers and cloned into BamHI/XhoI sites of CMX-PL1 vector to produce expression constructs CMX-FXR-α1, CMX-FXRα2, CMX-FXRβ1, and CMX-FXRβ2. To make retroviral expression constructs, the full-length coding regions of four different isoforms were separately excised from the CMX expression constructs using BamHI/XhoI restriction enzymes and subcloned into BglII/XhoI sites of the MSCV-IRES-neo vector to make constructs MSCV-FXRα1, -FXRα2, -FXRβ1, and -FXRβ2. The mouse BSEP promoter (−1050 to +25) was amplified using gene-specific primers and cloned intoSacI/XhoI-digested-pGL3-LUC vector (Promega) to create pGL3-BSEP-Luc. All the plasmids have been confirmed by sequencing. Plasmids pIBABP1031-Luc and pIBABP142mut-Luc were kind gifts from Dr. David Mangelsdorf (University of Texas Southwestern Medical Center) (21Makishima 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). pGL3-hSHP-Luc was kindly provided by Dr. Bryan Goodwin (GlaxoSmithKline) (25Goodwin B. Jones S.A. Price R., R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M., E. Maloney P., R. Willson T., M. Kliewer S., A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar). The retroviral vector MSCV-IRES-neo plasmid was a gift from Dr. Owen Witte (University of California, Los Angeles). The sources of other plasmids and synthetic ligands have been described elsewhere (30Kast 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: 2908-2915Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Sure-RACE mouse panels (OriGene Technologies) contain double-stranded cDNAs synthesized from 24 tissues. A 5′ adapter, containing sequences corresponding to ADP1 and ADP2, was ligated at the 5′ ends. The cDNAs were amplified using gene-specific primers (GSP1 and GSP2) and adapter-specific primers (ADP1 and ADP2). The PCR products were then isolated on a 1.2% agarose gel and transferred to a nylon membrane, and the membranes were probed with a mouse FXR cDNA probe. The bands corresponding to FXRα or FXRβ were recovered from the gel and cloned into pCR2.1-TOPO vector (Invitrogen). After transformation, the white colonies were patched onto duplicate ampicillin-positive LB plates. The colonies from the plate were first screened to identify whether they represent FXRα or FXRβ using P3 and P4 (see Fig. 1) followed by using P1 (for screening the isoform with 12-bp insert) and then P2 (for screening the isoform without 12-bp insert) (Fig. 1). The sequences for P1 and P2 are 5′-TGGCTGAATGTATGTATACAGGTTTGTTAA-3′ and 5′-ATGTTGGCTGAATGTTTGTTAACTGA-3′, respectively. All positive colonies were further confirmed either by sequencing or PCR. For Northern blot analysis, total RNA was isolated using Trizol reagent (Invitrogen), and 10 μg of RNA was denatured, electrophoresed, transferred to a nylon membrane, and probed with the indicated cDNA probe. Real time PCR was performed essentially as described (37Laffitte B.A. Joseph S.B. Walczak R. Pei L. Wilpitz D.C. Collins J.L. Tontonoz P. Mol. Cell. Biol. 2001; 21: 7558-7568Crossref PubMed Scopus (284) Google Scholar). Briefly, 1 μg of DNase I-treated total RNA was reverse-transcribed with random hexamers using the Taqman reverse transcription kit (Applied Biosystems) according to the manufacturer's protocol. Each amplification mixture (50 μl) contained 50 ng of cDNA, 900 nm forward primer, 900 nm reverse primer, 250 nm fluorogenic probe, and 25 μl of Universal PCR Master Mix (Applied Biosystems). PCR thermocycling parameters were 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Real time PCR was carried out using Applied Biosystems 7700 sequence detector. Samples were analyzed simultaneously for cyclophilin expression. Quantitative expression values were extrapolated from separate standard curves. Each sample was assayed in duplicate and normalized to cyclophilin. The sequences for primers and probes are as follows: FXRα, 5′-TGGGCTCCGAATCCTCTTAGA-3′ (forward primer, F), 5′-TGGTCCTCAAATAAGATCCTTGG-3′ (reverse primer, R), 5′-CCTTGGACATCTCTGGCCCAAAGCA-3′ (Probe, P); FXRβ, 5′-GGGCTTAGAAAATCCAATTCAGATTA-3′ (F), 5′-CGTCCGGCACAAATCCTG-3′ (R), 5′-TCTTCACCACAGCCACCGGCTG-3′ (P); I-BABP, 5′-CAAGGCTACCGTGAAGATGGA-3′ (F), 5′-ACCTCCGAAGTCTGGTGATAGTTG-3′(R), 5′-GGAACTCTGCCACCACCTTGCCA-3′ (P); BSEP, 5′-ACAGAAGCAAAGGGTAGCCATC-3′ (F), GGTAGCCATGTCCAGAAGCAG-3′ (R), CCGCGCCCTCATACGGAAACC-3′ (P); cyclophilin, 5′-GGCCGATGACGAGCCC-3′ (F), 5′-TGTCTTTGGAACTTTGTCTGCAA-3′ (R), 5′-TGGGCCGCGTCTCCTTCGA-3′ (P). All probes were dually labeled at the 5′ end with 6-carboxyfluorescein and at the 3′ end with 6- carboxytetramethylrhodamine. EMSAs were performed essentially as described (27Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Mouse FXR isoforms or human RXRα was synthesized in vitro using the TNT T7-coupled reticulocyte system (Promega). To compare transcription/translation efficiency of the expression constructs expressing different mouse FXR isoforms, equal volumes of 35S-labeled lysates were loaded and separated on an 8% SDS-polyacrylamide gel. The gel was dried and autoradiographed. The bands were quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Binding reactions were carried out in a buffer containing 10 mm HEPES, pH 7.8, 100 mm KCl, 0.2% Nonidet P-40, 6% glycerol, 0.3 mg/ml bovine serum albumin, 1 mm dithiothreitol, 2 μg of poly(dI-dC), 1–3 μl each of in vitro translated receptors and32P end-labeled oligonucleotide. DNA-protein complexes were resolved on a 5% polyacrylamide gel in 0.5× TBE (45 mmTris borate, 1 mm EDTA) at 4 °C. Gels were dried and autoradiographed. The sequences for mouse I-BABP (mI-BABP) probe and hSHP probe were 5′-GTTTTCCTTAAGGTGAATAACCTTGGGGCTC-3′ and 5′-GTACAGCCTGGGTTAATGACCCTGTTTATGC-3′, respectively. CV-1 and HepG2 cells were maintained in modified Eagle's medium, 10% fetal bovine serum. Transient transfections were performed in triplicate in 48-well plates as described (27Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Cells were treated with ligands or vehicle (Me2SO) in super-stripped fetal bovine serum (HyClone, Logan, UT) as indicated in the legends. Luciferase activity was measured and normalized to β-galactosidase activity. To produce stable cell lines, 293T cells were transfected with MSCV-FXRα1, -FXRα2, -FXRβ1, -FXRβ2, or MSCV-neo along with ΨE helper virus. The supernatants of the culture media were then used to infect NIH3T3 cells followed by selection with 800 μg/ml G418 sulfate (Geneticin®, Invitrogen) for 3–4 weeks. The selected stable cells were subsequently treated with the indicated ligands, as described in the legend to Fig. 6. Based on the previous reports on rat (17Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C.W. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (978) Google Scholar) and murine (18Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar) FXR, we hypothesized that there might be four murine FXR isoforms. For clarity, the four isoforms that have been characterized in the present report have been termed FXRα1, FXRα2, FXRβ1, and FXRβ2 (Fig. 1 A). FXRβ2 and FXRα1 correspond to RIP14-1 and RIP14-2, previously identified by Seolet al. (18Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). To identify all possible FXR isoforms we employed 5′-RACE and cDNAs generated from 24 murine tissues that had been ligated to a 5′ adaptor (OriGene Technologies). Gene-specific primers (GSP1 or GSP2 in Fig. 1 A) together with adapter-specific primers (ADP1 or ADP2) were used in a series of PCR reactions to amplify FXR-specific cDNAs (see “Experimental Procedures”). Southern blot analysis identified the PCR products that corresponded to FXR cDNAs, and these were subsequently cloned into pCR2.1-TOPO vector. Radiolabeled oligonucleotides P3 or P4 (Fig. 1 A) were then used to differentiate colonies corresponding to FXRα from FXRβ (data not shown). Filters containing either FXRα- or FXRβ-positive colonies were probed with radiolabeled oligonucleotides P1 or P2 (Fig. 1 A) to distinguish whether these colonies did or did not contain the 12-bp insert (Fig. 2 A). This approach coupled with DNA sequencing of selected inserts identified four murine FXR isoforms (Fig. 1 A). Analysis of the 5′ RACE data and various databases suggests that (i) murine FXR consists of 11 exons and 10 introns, (ii) FXRα transcription is initiated from exon 1, (iii) FXRβ transcription is initiated from exon 3, (iv) FXRα and FXRβ share exons 4∼11, and (v) the 12-bp insert is located at the 3′ terminus of exon 5 (Fig 1; Table I). Thus, alternative splicing between exon 5 (that contains the variable 12 bp) and exon 6 produces FXR isoforms that include (α1, β1) or exclude (α2, β2) the four-amino acid (12 bp) insert (Fig. 1 B). Table I provides details of the genomic organization and intron-exon junctions of the murine FXR gene; the gene encompasses 76,997 bp and contains 11 exons that range in size from 100 to 572 bp and introns that vary from 328 to 16,388 bp. Almost all of the exon/intron boundaries display the canonical GT/AG sequence (Table I). The data were obtained by comparison of the sequence of the FXR cDNA with the publicly available genomic sequence of murine chromosome 10 (www.genome.ucsc.edu). Fig. 1 C illustrates the domain structures of the FXR isoforms and shows that the FXRβ isoforms contain an additional 37 amino acids at the amino terminus that are absent from FXRα. The four-amino acid (MYTG) insert is located in the hinge region, adjacent to the DNA binding domain (Fig. 1 C).Table IGenomic organization of the murine FXR geneExonExon sizeIntron sizeSplice acceptor site (intron-exon)Donor acceptor site (exon-intron)bpbp118416,388 GTCACCCAGGGCAATCCAAG-taagtaccaa21349,602tgccttccag-GATCTTATTTAGCTTATTTG-gtaagttgtc31218,086tccttctcag-TTGCCGTGAACCAGCTAAAG-gtaggtcact437214,615tttacttcag-GTATGCTAACGGCTGCAAAG-gtgagagctt51422,749tctcccacag-GTTTCTTCCGTTGGCTGAAT-gtatgtatac61381,613cccccccaag-GTTTGTTAACGTTTTGCAGG-gtaacagtgc7100328gccagtgtag-GAGAAAACGGAAATAAAATC-gtatgtgctc81014,450taaaatttag-TTAAAAGAAGAAGCTTCCAG-gtattttttt914815,454atcatcatag-GGTTTCAGACCGAAAGAGTG-gtaagtgaca101151,585tcctacatag-GTATCTCTGACTCTCTCCAG-gtaataatgc11572ccccacatag-ACAGACAATATGAATGTAAT Open table in a new tab To determine the relative expression of FXRα1:FXRα2 and FXRβ1:FXRβ2 in different tissues, we transferred ≥33 bacterial colonies that contained DNA corresponding to either FXRα or FXRβ to filters. These filters were probed sequentially with probes P1 and P2 (Fig. 1 A and Fig. 2 A). As illustrated in Fig. 2 A, this approach distinguished between FXRβ colonies that contained (Fig. 2 A, left panel) or did not contain (Fig. 2 A, right panel) the 12-bp insert. Thus, the ratio of FXRβ1:FXRβ2 in the liver was 1:1.9 (Fig. 2 A; Table II). Table IIsummarizes the results obtained from similar assays utilizing cDNAs generated from six tissues. The data indicate that the ratio of FXRα1:FXRα2 and FXRβ1:FXRβ2 in different tissues varies significantly. For example, the ratio of FXRα1:FXRα2 (+12 bp/−12 bp) is 1:51 in the heart and 1:0.75 in the adrenal gland (Table II). Analysis of the PCR products also indicated that some tissues, including the heart, kidney, stomach, and adrenal gland expressed predominantly one FXR isoform, either FXRα or FXRβ (Table II). Because the DNA was analyzed after a PCR amplification step, the ratios of the four FXR isoforms are considered to be semi-quantitative.Table IIRelative expression of FXRα1:FXRα2 and FXRβ1:FXRβ2 in mouse tissuesTissueFXRαFXRβNumber of coloniesRatio of α1/α2Number of coloniesRatio of β1/β2Heart521:51NDNDKidneyNDND351:1.7Liver331:1.2351:1.9StomachNDND351:4.8Small intestine351:1.2341:2.1Adrenal gland351:0.75NDNDColonies (≥33 colonies) were screened for analysis of the relative expression of FXRα1 and FXRα2 or FXRβ1 and FXRβ2 using the method as described in Fig. 2 A. The ratio of FXRα1 to FXRα2 or FXRβ1 to FXRβ2 in different tissues is shown. ND, not detected. Open table in a new tab Colonies (≥33 colonies) were screened for analysis of the relative expression of FXRα1 and FXRα2 or FXRβ1 and FXRβ2 using the method as described in Fig. 2 A. The ratio of FXRα1 to FXRα2 or FXRβ1 to FXRβ2 in different tissues is shown. ND, not detected. To determine the relative expression of FXRα versusFXRβ, total RNA from 13 tissues in C57BL/6J mice (n = 5) fed a normal chow diet was isolated, and real time quantitative PCR was performed. As shown in Fig. 2 B, FXRα and FXRβ were most abundantly expressed in the liver. The liver was the only organ that expressed similar levels of both isoforms. FXRβ was abundantly expressed in ileum, moderately in kidney, and at low levels in stomach, duodenum, and jejunum (Fig. 2 B). FXRα was moderately expressed in ileum and adrenal gland. In addition, heart, lung, and fat contained low but measurable leve" @default.
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