Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter

Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.     

Cholesterol modulates human intestinal sodium-dependent bile acid transporter

Bile acids are efficiently absorbed from the intestinal lumen via the ileal apical sodium-dependent bile acid transporter (ASBT). ASBT function is essential for maintenance of cholesterol homeostasis in the body. The molecular mechanisms of the direct effect of cholesterol on human ASBT function and expression are not entirely understood. The present studies were undertaken to establish a suitable in vitro experimental model to study human ASBT function and its regulation by cholesterol. Luminal membrane bile acid transport was evaluated by the measurement of sodium-dependent 3H-labeled taurocholic acid (3H-TC) uptake in human intestinal Caco-2 cell monolayers. The relative abundance of human ASBT (hASBT) mRNA was determined by real-time PCR. Transient transfection and luciferase assay techniques were employed to assess hASBT promoter activity. Caco-2 cell line was found to represent a suitable model to study hASBT function and regulation. 25-Hydroxycholesterol (25-HCH; 2.5 microg/ml for 24 h) significantly inhibited Na(+)-dependent 3H-TC uptake in Caco-2 cells. This inhibition was associated with a 50% decrease in the V(max) of the transporter with no significant changes in the apparent K(m). The inhibition in hASBT activity was associated with reduction in both the level of hASBT mRNA and its promoter activity. Our data show the inhibition of hASBT function and expression by 25-HCH in Caco-2 cells. These data provide novel evidence for the direct regulation of human ASBT function by cholesterol and suggest that this phenomenon may play a central role in cholesterol homeostasis.     

beta-Klotho and FGF-15/19 inhibit the apical sodium-dependent bile acid transporter in enterocytes and cholangiocytes

beta-Klotho, a newly described membrane protein, regulates bile acid synthesis. Fibroblast growth factor-15 (FGF-15) and FGF receptor-4 (FGFR4) knockout mice share a similar phenotype with beta-Klotho-deficient mice. FGF-15 secretion by the intestine regulates hepatic bile acid biosynthesis. The effects of beta-Klotho and FGF-15 on the ileal apical sodium bile transporter (ASBT) are unknown. beta-Klotho siRNA treatment of the mouse colon cancer cell line, CT-26, and the human intrahepatic biliary epithelial cells (HIBEC) resulted in upregulation of endogenous ASBT expression that was associated with reduced expression of the farnesoid X receptor (FXR) and the short heterodimer partner (SHP). Silencing beta-Klotho activated the ASBT promoter in CT-26, Mz-ChA-1 (human cholangiocarcinoma), and HIBEC cells. Site-directed mutagenesis of liver receptor homolog-1 (mouse) or retinoic acid receptor/retinoid X receptor (RAR/RXR) (human) cis-elements attenuated the basal activity of the ASBT promoter and abrogated its response to beta-Klotho silencing. siSHP, siFXR, or dominant-negative FXR treatment also eliminated the beta-Klotho response. FGF-15 secretion into cell culture media by CT-26 cells was diminished after siFGF-15 or sibeta-Klotho treatment and enhanced by chenodeoxycholic acid. Exogenous FGF-19 repressed ASBT protein expression in mouse ileum, gallbladder, and in HIBEC and repressed ASBT promoter activity in Caco-2, HIBEC, and Mz-ChA-1 cells. Promoter repression was dependent on the expression of FGFR4. These results indicate that both beta-Klotho and FGF-15/19 repress ASBT in enterocytes and cholangiocytes. These novel signaling pathways need to be considered in analyzing bile acid homeostasis.     

Transactivation of the human apical sodium-dependent bile acid transporter gene by human serum

Using a luciferase reporter assay we found that human serum transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) promoter three to fourfold. Confirming this effect, addition of human serum to both Caco-2 cells and fresh human ileal biopsies caused an approximate 2.0-fold increase in endogenous ASBT mRNA production. Alteration of non-esterified fatty acid (NEFA) content and cortisol content did not affect the transactivation potential of serum. Site-directed mutagenesis of response elements for corticosteroid, peroxisome proliferation-activated alpha (PPARalpha), hepatocyte nuclear factor 1alpha (HNF1alpha), and retinoic acid (RAR/RXR) did not affect transactivation potential of serum. Three putative serum response elements (SRE) were identified on the promoter, but all were determined inactive using site-directed mutagenesis and electrophoretic mobility shift assay. Promoter deletion analysis demonstrated that >80% of the response to serum was located within the last 273 bp of the 5'-UTR, an area containing one of two activate protein 1 (AP-1) response elements. Site-directed mutagenesis of this downstream AP-1 response element reduced the effect of serum on the promoter by about 50% while full deletion of the response element completely eliminated the effect of serum. These studies demonstrate that one or more constituents of human stimulate ASBT gene expression largely via the down-stream AP-1 response element.     

Transmembrane helix 1 contributes to substrate translocation and protein stability of bile acid transporter SLC10A2

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) plays a critical role in the enterohepatic circulation of bile acids, as well as in cholesterol homeostasis. ASBT reclaims bile acids from the distal ileum via active sodium co-transport, in a multistep process, orchestrated by key residues in exofacial loop regions, as well as in membrane-spanning helices. Here, we unravel the functional contribution of highly conserved transmembrane helix 1 (TM1) on the hASBT transport cycle. Consecutive cysteine substitution of individual residues along the TM1 helix (Ile(29)-Gly(50)), as well as exofacial Asn(27) and Asn(28), resulted in functional impairment of ～70% of mutants, despite appreciable cell surface expression for all but G50C. Cell surface expression of G50C and G50A was rescued upon MG132 treatment as well as cyclosporine A, but not by FK506 or bile acids, suggesting that Gly(50) is involved in hASBT folding. TM1 accessibility to membrane-impermeant MTSET remains confined to the exofacial half of the helix along a single, discrete face. Substrate protection from MTSET labeling was temperature-dependent for L34C, T36C, and L38C, consistent with conformational changes playing a role in solvent accessibility for these mutants. Residue Leu(30) was shown to be critical for both bile acid and sodium affinity, while Asn(27), Leu(38), Thr(39), and Met(46) participate in sodium co-transport. Combined, our data demonstrate that TM1 plays a pivotal role in ASBT function and stability, thereby providing further insight in its dynamic transport mechanism.