Getting the mOST from OST: Role of organic solute transporter, OSTalpha-OSTbeta, in bile acid and steroid metabolism.
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ABSTRACT:
SUBMITTER: Dawson PA
PROVIDER: S-EPMC2911127 | biostudies-literature | 2010 Sep
REPOSITORIES: biostudies-literature
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Getting the mOST from OST: Role of organic solute transporter, OSTalpha-OSTbeta, in bile acid and steroid metabolism.
Dawson Paul A PA Hubbert Melissa L ML Rao Anuradha A
Biochimica et biophysica acta 20100609 9
The organic solute transporter (OST)(alpha)-OST(beta) is an unusual heteromeric carrier expressed in a variety of tissues including the small intestine, colon, liver, biliary tract, kidney, and adrenal gland. In polarized epithelial cells, OSTalpha-OSTbeta protein is localized on the basolateral membrane and functions in the export or uptake of bile acids and steroids. This article reviews recent results including studies of knockout mouse models that provide new insights to the role of OSTalpha ...[more]
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Project description:Blocking intestinal bile acid (BA) absorption by inhibiting or inactivating the apical sodium-dependent BA transporter (Asbt) classically induces hepatic BA synthesis. In contrast, blocking intestinal BA absorption by inactivating the basolateral BA transporter, organic solute transporter alpha-beta (Ost?-Ost?) is associated with an altered homeostatic response and decreased hepatic BA synthesis. The aim of this study was to determine the mechanisms underlying this phenotype, including the role of the farnesoid X receptor (FXR) and fibroblast growth factor 15 (FGF15).BA and cholesterol metabolism, intestinal phenotype, expression of genes important for BA metabolism, and intestinal FGF15 expression were examined in wild type, Ost?(-/-), Fxr(-/-), and Ost?(-/-)Fxr(-/-) mice.Inactivation of Ost? was associated with decreases in hepatic cholesterol 7?-hydroxylase (Cyp7a1) expression, BA pool size, and intestinal cholesterol absorption. Ost?(-/-) mice exhibited significant small intestinal changes, including altered ileal villus morphology, and increases in intestinal length and mass. Total ileal FGF15 expression was elevated almost 20-fold in Ost?(-/-) mice as a result of increased villus epithelial cell number and ileocyte FGF15 protein expression. Ost?(-/-)Fxr(-/-) mice exhibited decreased ileal FGF15 expression, restoration of intestinal cholesterol absorption, and increases in hepatic Cyp7a1 expression, fecal BA excretion, and BA pool size. FXR deficiency did not reverse the intestinal morphological changes or compensatory decrease for ileal Asbt expression in Ost?(-/-) mice.These results indicate that signaling via FXR is required for the paradoxical repression of hepatic BA synthesis but not the complex intestinal adaptive changes in Ost?(-/-) mice.
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Project description:The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E(2) across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 were identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eicosanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules.
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Project description:Organic solute transporter (OST) α/β is a key bile acid transporter expressed in various organs, including the liver under cholestatic conditions. However, little is known about the involvement of OSTα/β in bile acid-mediated drug-induced liver injury (DILI), a major safety concern in drug development. This study investigated whether OSTα/β preferentially transports more hepatotoxic, conjugated, primary bile acids and to what extent xenobiotics inhibit this transport. Kinetic studies with OSTα/β-overexpressing cells revealed that OSTα/β preferentially transported bile acids in the following order: taurochenodeoxycholate > glycochenodeoxycholate > taurocholate > glycocholate. The apparent half-maximal inhibitory concentrations for OSTα/β-mediated bile acid (5 µM) transport inhibition by fidaxomicin, troglitazone sulfate, and ethinyl estradiol were: 210, 334, and 1050 µM, respectively, for taurochenodeoxycholate; 97.6, 333, and 337 µM, respectively, for glycochenodeoxycholate; 140, 265, and 527 µM, respectively, for taurocholate; 59.8, 102, and 117 µM, respectively, for glycocholate. The potential role of OSTα/β in hepatocellular glycine-conjugated bile acid accumulation and cholestatic DILI was evaluated using sandwich-cultured human hepatocytes (SCHH). Treatment of SCHH with the farnesoid X receptor agonist chenodeoxycholate (100 µM) resulted in substantial OSTα/β induction, among other proteomic alterations, reducing glycochenodeoxycholate and glycocholate accumulation in cells+bile 4.0- and 4.5-fold, respectively. Treatment of SCHH with troglitazone and fidaxomicin together under cholestatic conditions resulted in increased hepatocellular toxicity compared with either compound alone, suggesting that OSTα/β inhibition may accentuate DILI. In conclusion, this study provides insights into the role of OSTα/β in preferential disposition of bile acids associated with hepatotoxicity, the impact of xenobiotics on OSTα/β-mediated bile acid transport, and the role of this transporter in SCHH and cholestatic DILI.
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Project description:Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter ?-? (OST?-OST?). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ost?-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids.Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ost?-/- mice at postnatal days 5, 10, 15, 20, and 30. Ost?-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila.As early as day 5, Ost?-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ost?-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ost?-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ost?-/- mice.Early in postnatal development, loss of Ost? leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ost?-Ost? functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.
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Project description:Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ostalpha and Ostbeta, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ostalpha protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Ostalpha homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ostbeta appeared as bands of 17 and 19 kDa, and these bands were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ostalpha, and only the 19 kDa form of Ostbeta, were detected among the immunoprecipitated proteins, indicating that the interaction between Ostalpha and Ostbeta is associated with specific post-translational processing. Additional evidence for homodimerization of Ostalpha and for a direct interaction between Ostalpha and Ostbeta was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ostalpha and Ostbeta tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK-293 cells also indicated that the C-termini of both Ostalpha and Ostbeta are facing the intracellular space. The interaction between Ostalpha and Ostbeta was required not only for delivery of the proteins to the plasma membrane, but it increased their stability, as noted in transfected HEK-293 cells and in tissues from Ostalpha-deficient (Ostalpha-/-) mice. In Ostalpha-/- mice, Ostbeta mRNA levels were maintained, yet Ostbeta protein was not detectable, indicating that Ostbeta protein is not stable in the absence of Ostalpha. Overall, these findings identify the membrane topology of Ostalpha and Ostbeta, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ostalpha and Ostbeta increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.
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Project description:The heteromeric steroid transporter organic solute transporter ?/? (OST?/?, SLC51A/B) was discovered over a decade ago, but its physiological significance in the liver remains uncertain. A major challenge has been the lack of suitable models expressing OST?/?. Based on observations first reported here that hepatic OST?/? is upregulated in nonalcoholic steatohepatitis, the aim of this research was to develop an in vitro model to evaluate OST?/? function and interaction with drugs and bile acids. OST?/? expression in human liver tissue was analyzed by quantitative RT-PCR, Western blotting, and immunofluorescence. Radiolabeled compounds were used to determine OST?/?-mediated transport in the established in vitro model. The effect of bile acids and drugs, including those associated with cholestatic drug-induced liver injury, on OST?/?-mediated transport was evaluated. Expression of OST?/? was elevated in the liver of patients with nonalcoholic steatohepatitis and primary biliary cholangitis, whereas hepatocyte expression of OST?/? was low in control liver tissue. Studies in the novel cell-based system showed rapid and linear OST?/?-mediated transport for all tested compounds: dehydroepiandrosterone sulfate, digoxin, estrone sulfate, and taurocholate. The interaction study with 26 compounds revealed novel OST?/? inhibitors: a biomarker for cholestasis, glycochenodeoxycholic acid; the major metabolite of troglitazone, troglitazone sulfate; and a macrocyclic antibiotic, fidaxomicin. Additionally, some drugs (e.g., digoxin) consistently stimulated taurocholate uptake in OST?/?-overexpressing cells. Our findings demonstrate that OST?/? is an important transporter in liver disease and imply a role for this transporter in bile acid-bile acid and drug-bile acid interactions, as well as cholestatic drug-induced liver injury. NEW & NOTEWORTHY The organic solute transporter OST?/? is highly expressed in hepatocytes of liver tissue obtained from patients with nonalcoholic steatohepatitis and primary biliary cholangitis. OST?/? substrates exhibit rapid, linear, and concentration-driven transport in an OST?/?-overexpressing cell line. Drugs associated with hepatotoxicity modulate OST?/?-mediated taurocholate transport. These data suggest that hepatic OST?/? plays an essential role in patients with cholestasis and may have important clinical implications for bile acid and drug disposition.
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Project description:The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostalpha-Ostbeta exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostalpha-Ostbeta have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostalpha(-/-) mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostalpha(-/-) vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostalpha(-/-)Mrp3(-/-) mice. The bile acid pool size was significantly reduced (>65%) in Ostalpha(-/-) mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostalpha(-/-) mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostalpha-Ostbeta is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.
| S-EPMC2268840 | biostudies-literature
Project description:UnlabelledOrganic solute transporter alpha-beta (Ostalpha-Ostbeta) is a heteromeric bile acid and sterol transporter that facilitates the enterohepatic and renal-hepatic circulation of bile acids. Hepatic expression of this basolateral membrane protein is increased in cholestasis, presumably to facilitate removal of toxic bile acids from the liver. In this study, we show that the cholestatic phenotype induced by common bile duct ligation (BDL) is reduced in mice genetically deficient in Ostalpha. Although Ostalpha(-/-) mice have a smaller bile acid pool size, which could explain lower serum and hepatic levels of bile acids after BDL, gallbladder bilirubin and urinary bile acid concentrations were significantly greater in Ostalpha(-/-) BDL mice, suggesting additional alternative adaptive responses. Livers of Ostalpha(-/-) mice had higher messenger RNA levels of constitutive androstane receptor (Car) than wild-type BDL mice and increased expression of Phase I enzymes (Cyp7a1, Cyp2b10, Cyp3a11), Phase II enzymes (Sult2a1, Ugt1a1), and Phase III transporters (Mrp2, Mrp3). Following BDL, the bile acid pool size increased in Ostalpha(-/-) mice and protein levels for the hepatic basolateral membrane export transporters, multidrug resistance-associated protein 3 (Mrp3) and Mrp4, and for the apical bilirubin transporter, Mrp2, were all increased. In the kidney of Ostalpha(-/-) mice after BDL, the apical bile acid uptake transporter Asbt is further reduced, whereas the apical export transporters Mrp2 and Mrp4 are increased, resulting in a significant increase in urinary bile acid excretion.ConclusionThese findings indicate that loss of Ostalpha provides protection from liver injury in obstructive cholestasis through adaptive responses in both the kidney and liver that enhance clearance of bile acids into urine and through detoxification pathways most likely mediated by the nuclear receptor Car.
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| S-EPMC2939961 | biostudies-literature