Farnesoid X Receptor Responds to Bile Acids and Represses Cholesterol 7α-Hydroxylase Gene (CYP7A1) Transcription

Diane Stroup,John Y.L. Chiang,Cary Weinberger,Rhonda Kimmel

doi:10.1074/jbc.275.15.10918

Abstract

Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

Highlights

From the ‡Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272 and ¶NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Chinese hamster ovary (CHO) cells were transfected with rat farnesoid X receptor (FXR) and mouse RXR␣ using a reporter plasmid that contains five copies of an ecdysone response element fused to the upstream of the mouse mammary tumor virus promoter/CAT reporter gene
Glyco- and tauro- conjugates of DCA and chenodeoxycholic acid (CDCA) were inactive, as were cholic acid (CA; 3␣, 7␣, 12␣) and its taurine and glycine conjugates (TCA and GCA), taurolithocholic acid (TLCA, 3␣), taurohyodeoxycholic acid (THDCA, 3␣, 6␣), and ursodeoxycholic acid (UDCA; 3␣, 7␤) and its conjugate, tauroursodeoxycholic acid (TUDCA). These data are in agreement with those observed in CV-1 cells that hydrophobic bile acids (CDCA and DCA) are more active FXR ligands than hydrophilic bile acids [12,13,14]

Summary

Introduction

From the ‡Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272 and ¶NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709. Deletion analysis revealed that the bile acid response element located between nucleotides ؊148 and ؊128 was the FXR response element, but RXR␣/FXR did not bind to this sequence These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Two bile acid response elements, BARE-I and BARE-II, have been identified previously [2, 3] These DNA sequences contain AGGTCA direct repeats similar to the elements recognized by nuclear receptors, which regulate transcription of target genes in response to ligands such as steroids and thyroid hormones, retinoids, and fatty acids. The studies presented here attempted to dissect the mechanism of transcriptional repression of the CYP7A1 by the bile acid-activated FXR

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