Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.     

Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents

Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.     

The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes

Bile acids are synthesized from cholesterol in the liver and are excreted into bile via the hepatocyte canalicular bile salt export pump. After their passage into the intestine, bile acids are reabsorbed in the ileum by sodium-dependent uptake across the apical membrane of enterocytes. At the basolateral domain of ileal enterocytes, bile acids are extruded into portal blood by the heterodimeric organic solute transporter OSTalpha/OSTbeta. Although the transport function of OSTalpha/OSTbeta has been characterized, little is known about the regulation of its expression. We show here that human OSTalpha/OSTbeta expression is induced by bile acids through ligand-dependent transactivation of both OST genes by the nuclear bile acid receptor/farnesoid X receptor (FXR). FXR agonists induced endogenous mRNA levels of OSTalpha and OSTbeta in cultured cells, an effect that was not discernible upon inhibition of FXR expression by small interfering RNAs. Furthermore, OST mRNAs were induced in human ileal biopsies exposed to the bile acid chenodeoxycholic acid. Reporter constructs containing OSTalpha or OSTbeta promoters were transactivated by FXR in the presence of its ligand. Two functional FXR binding motifs were identified in the OSTalpha gene and one in the OSTbeta gene. Targeted mutation of these elements led to reduced inducibility of both OST promoters by FXR. In conclusion, the genes encoding the human OSTalpha/OSTbeta complex are induced by bile acids and FXR. By coordinated control of OSTalpha/OSTbeta expression, bile acids may adjust the rate of their own efflux from enterocytes in response to changes in intracellular bile acid levels.     

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter,OSTalpha-OSTbeta

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.     

Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.     

FXR regulates organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine

Expression of the farnesoid X receptor (FXR; NR1H4) is limited to the liver, intestine, kidney, and adrenal gland. However, the role of FXR in the latter two organs is unknown. In the current study, we performed microarray analysis using RNA from H295R cells infected with constitutively active FXR. Several putative FXR target genes were identified, including the organic solute transporters alpha and beta (OSTalpha and OSTbeta). Electromobility shift assays and promoter-reporter studies identified functional farnesoid X receptor response elements (FXREs) in the promoters of both human genes. These FXREs are conserved in both mouse genes. Treatment of wild-type mice with 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR agonist, induced OSTalpha and OSTbeta mRNAs in the intestine and kidney. Both mRNAs were also induced when wild-type, but not FXR-deficient (FXR-/-), adrenals were cultured in the presence of GW4064. OSTalpha and OSTbeta mRNA levels were also induced in the adrenals and kidneys of wild-type, but not FXR-/-, mice after the increase of plasma bile acids in response to the hepatotoxin alpha-naphthylisothiocyanate. Finally, overexpression of human OSTalpha and OSTbeta facilitated the uptake of conjugated chenodeoxycholate and the activation of FXR target genes. These results demonstrate that OSTalpha and OSTbeta are novel FXR target genes that are expressed in the adrenal gland, kidney, and intestine.     

Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE-/- mice by SC-435

Blocking intestinal bile acid absorption by inhibiting the apical sodium codependent bile acid transporter (ASBT) is a target for increasing hepatic bile acid synthesis and reducing plasma LDL cholesterol. SC-435 was identified as a potent inhibitor of ASBT (IC50 = 1.5 nM) in cells transfected with the human ASBT gene. Dietary administration of 3 mg/kg to 30 mg/kg SC-435 to apolipoprotein E-/- (apoE-/-) mice increased fecal bile acid excretion by >2.5-fold. In vivo inhibition of ASBT also resulted in significant increases of hepatic mRNA levels for cholesterol 7alpha-hydroxylase and HMG-CoA reductase. Administration of 10 mg/kg SC-435 for 12 weeks to apoE-/- mice lowered serum total cholesterol by 35% and reduced aortic root lesion area by 65%. Treatment of apoE-/- mice also resulted in decreased expression of ileal bile acid binding protein and hepatic nuclear hormone receptor small heterodimer partner, direct target genes of the farnesoid X receptor (FXR), suggesting a possible role of FXR in SC-435 modulation of cholesterol homeostasis. In dogs, SC-435 treatment reduced serum total cholesterol levels by 相似文献   

MALDI-TOF mass spectrometry screening of cholelithiasis risk markers in the gene of HNF1alpha

In recent years MALDI-TOF MS gained importance for high-throughput DNA analysis. In the present study this technique was used for the pathogenetic analysis of gallstone disease. The intestinal apical sodium-dependent bile acid transporter (ASBT) shows a genetic association with gallstone disease. ASBT has 3 binding sites in its 5'UTR for hepatocyte nuclear factor 1alpha (HNF1alpha). We hypothesized that genetic alterations in the HNF1alpha gene could influence ASBT expression. The gene HNF1alpha was sequenced in 46 Stuttgart random samples, composed of 16 controls and 30 gallstone patients. Subsequently, two independent cohorts (Stuttgart: 67 gallstones carriers, 109 controls, Leutkirch: 112 gallstone carriers, 99 controls) were screened by MALDI-TOF MS. The subjects were further divided by gender and weight. 24 known polymorphisms and two novel SNPs in the 3'UTR of HNF1alpha were detected (c.*220G>A and c.*1151G>A). After gender-specific sub-division of the pooled cohorts, 4 SNPs resulted in significant differences between male gallstone carriers and male controls (Stuttgart/Leutkirch: rs2255531 OR=2.78; p=0.006, rs1169288 OR=2.13; p=0.032, rs7310409 OR=2.34; p=0.025 and rs1169294 OR=2.13; p=0.031). Two novel variants in the 3'UTR of HNF1alpha were detected and four SNPs of HNF1alpha show a significant association to cholelithiasis in male gallstone patients. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.     

Diminished gene expression of ileal apical sodium bile acid transporter explains impaired absorption of bile acid in patients with hypertriglyceridemia

Patients with type IV hyperlipoproteinemia, particularly those with familial hypertriglyceridemia (FHT), have impaired absorption of bile acid, a defect that may contribute to the hypertriglyceridemia ( J. Lipid Res. 1995. 36: 96;-107). To determine whether this absorption defect is a result of abnormal expression of the ileal apical sodium bile acid transporter (ASBT) gene, we biopsied the terminal ileum at colonoscopy in 28 subjects, 13 with hypertriglyceridemia and 15 control subjects. Of the 13 hypertriglyceridemic subjects, 10 had lipid profiles compatible with FHT (elevated very low density lipoprotein [VLDL] triglycerides with normal LDL cholesterol). ASBT mRNA levels were measured in these biopsies by RNase protection assay, using glyceraldehyde dehydrogenase mRNA as a reference. ASBT protein was quantitated by Western blotting with an antibody to the carboxy-terminal 20 amino acids of the protein. The mean +/- SEM ASBT mRNA level in the control group was 205.7 +/- 19.9 (arbitrary units) compared with 138. 7 +/- 19.1 for all 13 hypertriglyceridemics (P = 0.03) and 141.7 +/- 20.8 in the 10 FHT patients (P = 0.05). Commensurate with these mRNA levels, the mean ASBT protein level in the control group was 126.2 +/- 22.6 versus 58.8 +/- 13.8 in hypertriglyceridemics (P = 0.02) and 61.8 +/- 15.2 in the FHT patients (P = 0.05).We conclude that impaired absorption of bile acid in type IV hypertriglyceridemia results from diminished expression of the ASBT gene in terminal ileum.     

Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor

Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and 0.068 microM for the homologous human and mouse apical transporters, respectively. Hybrid human-mouse and mouse-human transporters were engineered to identify regions involved in this 150-fold difference in the inhibition constant for 2164U90. A mouse-human chimera with only the most C-terminal hydrophobic domain and the C-terminus of the transporter originating from the human variant was found to have a sensitivity to 2164U90 inhibition similar to that of the human transporter. Conversely, a human-mouse hybrid transporter encompassing the same C-terminal region from the mouse sequence but now inserted into the human sequence demonstrated the greater inhibition seen with the mouse wild type ASBT. Amino acid substitutions, individually or in combinations, of six candidate nonconserved residues between mouse and human transporters in this C-terminal domain showed replacements of Thr294 by Ser and Val295 by Ile to be responsible for the difference in the sensitivity toward 2164U90 seen between the species. The hamster apical SBAT encompassing Ser/Ile in these positions shared the lower sensitivity to 2164U90, as seen with the human ASBT, even though it is identical to the mouse SBAT in the remaining four positions of this region. In addition, the rat ASBT which is identical to the mouse ASBT in this domain also had the high sensitivity to 2164U90 inhibition found for the mouse ASBT. Methanethiosulfonates (MTS) are known to inactivate the sodium/bile acid transporters through alkylation of a cysteine in the most C-terminal hydrophobic domain (1). Inactivation of the human ASBT due to MTS modification of cysteine 270 was shown to be largely abolished when the transporter was preincubated with 2164U90, suggesting that the binding of this benzothiazepine is in the vicinity of position 270. Thus, the domain containing the two most C-terminal putative transmembrane regions of the SBATs, H8-H9, previously shown to constitute part of the binding pocket for bile acids, interacts also with the bile acid transport competitive inhibitor, 2164U90.     

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.     

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.     

Effects of bile acids on expression of the human apical sodium dependent bile acid transporter gene

Using a luciferase reporter assay in both LMH cells and Caco2 cells we found that certain bile acids including unconjugated deoxycholic and others transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) at concentrations ranging from 20 to 300 microM. Confirming this effect, addition of deoxycholic acid to fresh human ileal biopsies caused an approximate 40% increase in endogenous ASBT mRNA production. Promoter deletion analysis indicated the effect of bile acids was mediated by a response element located in the downstream half of the 5'-UTR, a region known to contain a retinoic acid (RXR/RAR) response element and an activated protein-1 (AP-1) response element. Site-directed mutagenesis of the RAR/RXR response element actually enhanced response to deoxycholic acid. Site-directed mutagenesis of the downstream AP-1 response element reduced activation by deoxycholic acid while deletion of this response element completely eliminated this response. The epidermal growth factor (EGF) receptor inhibitor, AG1478, completely eliminated the response to bile acid while the mitogen-activated protein extracellular signal-regulated kinase cascade (MEK) inhibitor, U0126, partially inhibited the response to bile acid. These studies demonstrate that certain bile acids stimulate ASBT gene expression acting on the down-stream AP-1 response element via the EGF receptor and MEK cascade.     

Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT

Apical sodium-dependent bile acid transporter (ASBT) is responsible for the absorption of bile acids from the intestine. A decrease in ASBT function and expression has been implicated in diarrhea associated with intestinal inflammation. Whether infection with pathogenic microorganisms such as the enteropathogenic Escherichia coli (EPEC) affect ASBT activity is not known. EPEC is a food-borne enteric pathogen that translocates bacterial effector molecules via type three secretion system (TTSS) into host cells and is a major cause of infantile diarrhea. We investigated the effects of EPEC infection on ileal ASBT function utilizing human intestinal Caco2 cells and HEK-293 cells stably transfected with ASBT-V5 fusion protein (2BT cells). ASBT activity was significantly inhibited following 60 min infection with EPEC but not with nonpathogenic E. coli. Mutations in bacterial escN, espA, espB, and espD, the genes encoding for the elements of bacterial TTSS, ablated EPEC inhibitory effect on ASBT function. Furthermore, mutation in the bacterial BFP gene encoding for bundle-forming pili abrogated the inhibition of ASBT by EPEC, indicating the essential role for bacterial aggregation and the early attachment. The inhibition by EPEC was associated with a significant decrease in the V(max) of the transporter and a reduction in the level of ASBT on the plasma membrane. The inhibition of ASBT by EPEC was blocked in the presence of protein tyrosine phosphatase inhibitors. Our studies provide novel evidence for the alterations in the activity of ASBT by EPEC infection and suggest a possible effect for EPEC in influencing intestinal bile acid homeostasis.     

Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His(100)-Thr(101)-Ser(102) using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112-128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.     

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.     

Degradation of the apical sodium-dependent bile acid transporter by the ubiquitin-proteasome pathway in cholangiocytes

To attenuate injury during cholestasis, adaptive changes in bile acid transporter expression in the liver provide alternative bile acid excretory pathways. Apical sodium-dependent bile acid transporter (ASBT) (SLC10A2), only expressed in the liver on the cholangiocyte apical membrane, is rapidly regulated in response to inflammation and bile acids. Here, we studied the mechanisms controlling ASBT protein levels in cholangiocytes to determine whether ASBT expression is regulated by ubiquitination and disposal through the proteasome. Protein turnover assays demonstrated that ASBT is an unstable and short-lived protein. Treatment with MG-132, a proteasome inhibitor, causes time-dependent increased ASBT levels and increased intracellular accumulation of ASBT. In cells cotransfected with green fluorescent protein-tagged ASBT and hemagglutinin-tagged ubiquitin, we demonstrated coimmunoprecipitation and colocalization of ASBT and ubiquitin. Interleukin-1beta (IL-1beta) induced down-regulation of ASBT is abrogated by a JNK inhibitor and is accompanied by an increase in ASBT polyubiquitin conjugates and a reduced ASBT half-life. In phosphorylation-deficient S335A and T339A mutants, the ASBT half-life is markedly prolonged, IL-1beta-induced ASBT ubiquitination is significantly reduced, and IL-1beta fails to increase ASBT turnover. These results indicate that ASBT undergoes ubiquitin-proteasome degradation under basal conditions and that ASBT proteasome disposal is increased by IL-1beta due to JNK-regulated serine/threonine phosphorylation of ASBT protein at both Ser-335 and Thr-339. These studies are the first report of regulation of a bile acid transporter expression by the ubiquitin-proteasome pathway.