Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.
REFERENCES AND NOTES
Russell D. W., Setchell K. D. R., Biochemistry 31, 4737 (1992).
Gong Y. Z., Everett E. T., Schwartz D. A., Norris J. S., Wilson F. A., Proc. Natl. Acad. Sci. U.S.A. 91, 4741 (1994);
Fujita M., et al., Eur. J. Biochem. 233, 406 (1995);
Kanda T., et al., FEBS Lett. 384, 131 (1996).
Kanda T., et al., Biochem. J. 330, 261 (1998).
Mangelsdorf D. J., et al., Cell 83, 835 (1995).
Lehmann J. M., et al., J. Biol. Chem. 270, 12953 (1995).
Forman B. M., et al., Cell 81, 687 (1995).
Seol W., Choi H.-S., Moore D. D., Mol. Endocrinol. 9, 72 (1995).
Zavacki A. M., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 7909 (1997).
A. M. Zavacki, unpublished data.
Sippel C. J., Dawson P., Shen T., Perlmutter D. H., J. Biol. Chem. 272, 18290 (1997);
Wong M. H., Oelkers P., Dawson P. A., ibid. 270, 27228 (1995).
Craddock A. L, et al., Am. J. Physiol. 274, G157 (1998).
Crestani M., Karam W. G., Chiang J. Y. L., Biochem. Biophys. Res. Commun. 198, 546 (1994);
Twisk J., et al., Eur. J. Biochem. 228, 596 (1995).
Glass C. K., Rose D. W., Rosenfeld M. G., Curr. Opin. Cell Biol. 9, 222 (1997);
Moras D., Gronemeyer H., ibid. 10, 384 (1998).
Le Douarin B., et al., EMBO J. 15, 6701 (1996);
Heery D. M., Kalkhoven E., Hoare S., Parker M. G., Nature 387, 733 (1997);
Krey G., et al., Mol. Endocrinol. 11, 779 (1997).
Soini E., Hemmila I., Dahlen P., Ann. Biol. Clin. 48, 567 (1990);
Gudgin Dickson E. F., Pollak A., Diamandis E. P., J. Photochem. Photobiol. 27, 3 (1995).
Zhou G., et al., Mol. Endocrinol. 12, 1594 (1998);
Paige L., et al., Proc. Natl. Acad. Sci. U.S.A. 96, 3999 (1999).
Mallory A., Kern F., Smith J., Savage D., Gastroenterology 64, 26 (1973);
Setchell K. D. R., et al., ibid. 112, 226 (1997).
D. J. Parks, unpublished data.
Makishima M., et al., Science 284, 1362 (1999).
S. A. Kliewer, J. M. Lehmann, T. M. Willson, ibid., p. 757.
Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Expression plasmids for the human nuclear receptor–GAL4 chimeras were generated by amplification of the cDNA encoding the putative LBDs and insertion into a modified pSG5 expression vector (Stratagene) containing the GAL4 DNA binding domain (amino acids 1 to 147) and the simian virus 40 (SV40) Tag nuclear localization signal (5).
Riddihough G., Pelham H. R. B., EMBO J. 6, 3729 (1987).
DNA encoding the human FXR LBD (amino acids 222 to 472; GenBank ) was inserted into the pRSET-A expression vector (Invitrogen) and expressed in bacteria. The transformed cells were grown for 12 hours at 25°C, cooled to 9°C, maintained at this temperature until they reached a cell density, as measured by optical density (OD) at 600 nm, of OD600 = 14 , induced with 0.25 mM isopropyl β-d-thiogalactoside to a final cell density of OD600 = 16, and harvested by centrifugation. Filtered bacterial lysate was applied to an affinity column of Ni2+-charged chelation Sepharose (Amersham Pharmacia Biotechnology, 25 mM tris-HCl, pH 7.2, 150 mM NaCl, and 50 mM imidazole). Protein was eluted with 365 mM imidazole after washing with buffer containing 95 mM imidazole, and further purified by size exclusion chromatography with Superdex S-75 resin (Amersham Pharmacia Biotechnology). The FXR LBD was biotinylated with NHS-LC-biotin reagent (Pierce).
The FRET ligand-sensing assay was performed by incubating 10 nM of the biotinylated FXR LBD that was labeled with streptavidin-conjugated allophycocyanin (Molecular Probes) and 10 nM of the SRC1 peptide [amino acids 676 to 700, 5′-biotin-CPSSHSSLTERHKILHRLLQEGSPS-CONH2] (21) (SynPEP) that was labeled with streptavidin-conjugated europium chelate (Wallac), in 50 mM tris pH 8, 50 mM KCl, 0.1 mg/ml bovine serum albumin, 1 mM EDTA, and 10 mM dithiothreitol, in the presence of test compound for 2 hours at 22°C. Data were collected with a Wallac Victor fluorescence reader in a time-resolved mode. The relative fluorescence was measured at 665 nM, and the indicated values were calculated by subtracting the fluorescence obtained in the absence of test compound from the value obtained in the presence of test compound. Values are expressed as the means ± SD derived from three independent experiments.
We thank P. A. Dawson for providing the IBAT expression plasmid pCMV-HISBT, B. W. O'Malley, S. Y. Tsai, M.-Y. Tsai, and M. C. Lewis for critically reading the manuscript. Supported in part by NIH grant RO1 DK53366 to D.D.M. and National Institute of Diabetes and Digestive and Kidney Diseases grant F32 DK09793 to A.M.Z.
Volume 284 | Issue 5418
21 May 1999
21 May 1999
Received: 2 March 1999
Accepted: 28 April 1999
Published in print: 21 May 1999
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