Ileal Bile Acid-binding Protein, Functionally Associated with the Farnesoid X Receptor or the Ileal Bile Acid Transporter, Regulates Bile Acid Activity in the Small Intestine

Keiko Hirota,Tatsuo Kanda,Akiyoshi Fukamizu,Norihiko Furuya,Takeshi Sugaya,Mayuko Nakahara,Ryuichiro Sato,Kentaro Takagaki,Hiroshi Fujii

doi:10.1074/jbc.m507454200

Abstract

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Highlights

This pathway, cholesterol 7␣-hydroxylase (CYP7A1).3 Bile acids returned to the liver induce farnesoid X receptor (FXR) activity and suppress transcription of the CYP7A1 gene by the inhibitory effect of SHP, one of the FXR target genes, on the activity of LRH-1, which positively regulates the gene expression of this enzyme [1, 2]
The present experiment demonstrates that in differentiated Caco-2 cells the expression level of ileal bile acid-binding protein (I-BABP) mRNA is quite low under normal culture conditions and is tremendously augmented by various bile acids, which are known as FXR ligands, but not by the bile acid urosodeoxycholic acid, a non-ligand (Fig. 1A, UDCA)
On the basis of the findings that I-BABP gene expression is exclusively regulated by the function of FXR and that these two proteins are capable of binding the same ligands, bile acids, we first hypothesized negative feedback regulation of FXR activity by I-BABP via a competition for bile acids

Summary

Introduction

This pathway, cholesterol 7␣-hydroxylase (CYP7A1).3 Bile acids returned to the liver induce FXR activity and suppress transcription of the CYP7A1 gene by the inhibitory effect of SHP (small heterodimer partner), one of the FXR target genes, on the activity of LRH-1 (liver receptor homologue-1), which positively regulates the gene expression of this enzyme [1, 2]. We show that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, and stimulates the FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Results

Conclusion