2829 OST Alpha-OST Beta: a Key Membrane Transporter of Bile Acids

[Frontiers in Bioscience 14, 2829-2844, January 1, 2009]

OST alpha-OST beta: a key membrane transporter of bile acids and conjugated steroids

Nazzareno Ballatori1, Na Li1, Fang Fang1, James L. Boyer2, Whitney V. Christian1, Christine L. Hammond1

1Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642, 2Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, CT 06520

TABLE OF CONTENTS

1. Abstract 2. Introduction 3. Bile acid synthesis and disposition 4. Hepatic bile acid transporters 5. Intestinal bile acid transporters 6. Identification of Ost alpha-Ost beta 7. Ost alpha and Ost beta are expressed in most tissues, but are most abundant in tissues involved in bile acid and steroid homeostasis 8. Ost alpha-Ost beta mediates the transport of bile acids, conjugated steroids, and structurally-related molecules: transport occurs by a facilitated diffusion mechanism 9. Heterodimerization of Ost alpha and Ost beta increases the stability of the individual proteins, facilitates their post- translational modifications, and is required for delivery of the Ost alpha-Ost beta complex to the plasma membrane 10. Expression of both Ost genes is positively regulated by bile acids through the bile acid-activated farnesoid X receptor, Fxr 11. Ost alpha-Ost beta is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver 12. Implications for human diseases 13. Summary 14. Acknowledgements 15. References

1. ABSTRACT 2. INTRODUCTION

The organic solute and steroid transporter, Ost The steroid-derived class of compounds, alpha-Ost beta, is an unusual heteromeric carrier that including the bile acids, steroid hormones, and other appears to play a central role in the transport of bile acids, cholesterol metabolites, play critical roles in human conjugated steroids, and structurally-related molecules physiology; however, relatively little is known about the across the basolateral membrane of many epithelial cells. transport proteins that mediate cellular import and export of The transporter’s substrate specificity, transport these molecules. Although it is often assumed that transport mechanism, tissue distribution, subcellular localization, of steroid hormones occurs by simple diffusion, this mode transcriptional regulation, as well as the phenotype of the of transport would largely preclude the ability to regulate recently characterized Ost alpha-deficient mice all strongly intracellular concentrations of these important bioactive and support this model. In particular, the Ost alpha-deficient signaling molecules, and is therefore unlikely to play a mice display a marked defect in intestinal bile acid and significant role in their disposition. The present report conjugated steroid absorption; a decrease in bile acid pool summarizes the evidence for an important role of the organic size and serum bile acid levels; altered intestinal, hepatic solute and steroid transporter, Ost alpha-Ost beta, in the and renal disposition of known substrates of the transporter; disposition of sterols, and focuses on the contribution of Ost and altered serum triglyceride, cholesterol, and glucose alpha-Ost beta to the enterohepatic circulation of bile acids. levels. Collectively, the data indicate that Ost alpha-Ost beta is essential for bile acid and sterol disposition, and 3. BILE ACID SYNTHESIS AND DISPOSITION suggest that the carrier may be involved in human conditions related to imbalances in bile acid or lipid Bile acids are the major products of cholesterol homeostasis. catabolism, and they regulate a multitude of biological

2829 Function and regulation of Ost alpha-Ost beta processes, including hepatic bile secretion and the intestinal In humans, the most important bile acids are absorption of fat and fat-soluble vitamins (1-3). Bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid, also modulate triglyceride, cholesterol, energy, and glucose which differ on the sites of hydroxylation (14). Bile acids homeostasis through their activation of specific receptors may be conjugated with either glycine or taurine, a process and signaling pathways (4). that increases water solubility, preventing passive reabsorption once secreted into the small intestine. As a The liver is the only organ that synthesizes bile result, the concentration of bile acids in the small intestine acids. Approximately 5-10% of the bile acids made in the can stay high enough to form micelles and solubilize lipids liver originate from peripherally obtained precursors and (3). around 90-95% directly from cholesterol within hepatocytes (5). Specific hepatic enzymes oxidize In the intestine, the detergent properties of bile cholesterol, and these initial oxidized products are further acids aid in the solubilization of dietary lipids (especially metabolized to bile acids, which are then secreted into bile fats and lipid soluble vitamins), lipophilic drugs, and and delivered into the intestine (6). Bile acids are defined electrolytes. Bile acids also regulate pancreatic secretions as primary bile acids when they are sythesized in liver, or and the release of gastrointestinal peptides (15,16), activate as secondary bile acids when they are made by bacteria in carboxyl ester lipase (17), and promote the propulsive the intestine. The classical or neutral pathway and the motility of the muscle layer in colon (18,19). In the liver, acidic pathway are responsible for the production of at least bile acids induce bile flow and biliary lipid secretion, and 95% of the bile acids, and these two pathways contribute promote mitosis during hepatic regeneration (3,20). In about equally to bile acid synthesis in humans (5). In the the biliary tract, bile acids solubilize cholesterol, trap neutral pathway, the rate limiting step in bile acid synthesis cholephilic xenobiotics in micelles, stimulate is the addition of a hydroxyl group on position 7 of the bicarbonate secretion via cystic fibrosis transmembrane steroid nucleus by the enzyme cholesterol 7 alpha- conductance regulator (CFTR) and anion exchanger hydroxylase (CYP7A1). Likewise in the acidic pathway, AE2, and promote proliferation when bile duct secretion sterol 27-hydroxylase, CYP27A1, is the rate limiting is obstructed (21,22). In brown adipose tissue, bile enzyme that introduces a hydroxy group to the carbon at acids stimulate thermogenesis by thyroid hormone (23). position 27 of cholesterol. In an alternative pathway, Bile acids also play a role in regulating gene expression cholesterol 24-hydroxylase, CYP46A1, initiates synthesis via nuclear receptors and triggering various signal by converting cholesterol to 24S-hydroxycholesterol in the pathways impinging on numerous cellular processes, brain, which then travels to the liver for its ultimate including apoptosis (24,25). conversion to bile acids. However, CYP46A1 only contributes about 1% to the overall synthesis of bile acids About 90% of the bile acids secreted into the (5,7,8). intestine are reabsorbed into portal blood and are then efficiently taken back up by hepatocytes. The ileum is the Of note, the transcription of CYP7A1 is subject to major site of reabsorption. Bile acids continue to cycle both feedforward and feedback regulation (9). The liver X between the intestine and the liver, creating an receptor alpha (LXR alpha), whose ligands include enterohepatic cycle. Because the majority of the circulating hydroxylated cholesterol metabolites (oxysterols), bile acids are conjugated and/or charged, they are unlikely dimerizes with the retinoid X receptor (RXR), and binds to to cross the plasma membrane by simple diffusion. Thus, its response element on the CYP7A1 promoter, thereby the transcellular movement of the bile aids is mediated by mediating transcriptional activation. Transcription of specialized transporters. CYP7A1 is downregulated by the bile acid-activated farnesoid X receptor (FXR) via a complex molecular 4. HEPATIC BILE ACID TRANSPORTERS mechanism that involves the coordinated regulation of several liver-enriched nuclear receptors (10,11). Bile acid- Hepatocytes import bile acids and other organic activated FXR dimerizes with RXR and transcriptionally solutes from the sinusoidal blood and can transport them activates its target nuclear receptor, the short heterodimer into bile canaliculi (Figure 1). Hepatic bile acid uptake is partner (SHP). SHP represses CYP7A1 indirectly through mediated predominantly (>80%) by the Na+-taurocholate its association with yet another nuclear receptor, the liver co-transporting polypeptide (NTCP/SLC10A1) (2,25-28). receptor homolog-1 (LRH-1) (12). Another major regulator NTCP mediates both conjugated and unconjugated bile of CYP7A1 transcription is fibroblast growth factor 19 acid uptake in a sodium dependent manner with a (FGF19 in humans, and the mouse homologue Fgf15). stoichiometry of 2 Na+ ions for one taurocholate molecule FGF19/Fgf15 originates from ileum and is delivered to (29). NTCP is predicted to have 7 transmembrane (TM) liver via the portal circulation with a pronounced diurnal domains with an intracellular C-terminus and an pattern. When bile acid levels in ileocytes are elevated, this extracellular N-terminus. NTCP gene expression is leads to the FXR-mediated induction of FGF19/Fgf15. suppressed by high levels of bile acids as an adaptive This growth factor is secreted into portal blood, delivered response to reduce bile acid entry into the hepatocytes (30). to the liver where it binds to the hepatic FGFR4/Fgfr4 In hepatocytes, bile acids activate FXR, which induces the receptor, and represses CYP7A1 transcription through a expression of SHP, a blocker of the stimulating effect of mechanism that involves the orphan nuclear receptor SHP retinoic acid receptor and retinoid X receptor RAR/RXR (13). heterodimer on the NTCP promoter (31).

2830 Function and regulation of Ost alpha-Ost beta

Figure 1. The major bile acid transporters in the enterohepatic circulation. Bile acid transport across the basolateral membrane of the hepatocytes is mediated mainly by the Na+-dependent taurocholate cotransporting polypeptide (NTCP) and organic anion transporting polypeptides (OATPs). Bile acids efflux across the basolateral membrane of hepatocytes may occur via the organic solute and steroid transporter (OST alpha-OST beta) and/or the multidrug resistance-associated proteins 3 and 4 (MRP3 and MRP4). The secretion of bile acids across the canalicular membrane into bile occurs via two members of the ATP-binding cassette transporters: the bile acid export pump (BSEP) and MRP2. Bile acids are delivered to the intestinal lumen through bile duct where they aid in emulsifying dietary lipids. Bile acids are actively re-absorbed in the distal ileum (and in cholangiocytes) via Na+-dependent apical sodium dependent bile acid transporter (ASBT), and are effluxed through OST alpha-OST beta.

The sodium-independent organic anion associated protein 2 (MRP2) (2,37) (Figure 1). BSEP is transporting polypeptides, OATPs (e.g., predicted to contain 12 TM domains with two typical OATP1A2/SLC1A2, OATP1B1/SLC1B1, intracellular nucleotide-binding domains for binding and OATP1B3/SLC1B3) also contribute to hepatic bile acid hydrolysis of ATP, whereas MRP2 has 17 TM domains uptake, particularly for unconjugated bile acids (Figure 1) (38,39). BSEP preferentially mediates the transport of (32,33). OATPs are glycoproteins with a common conjugated monovalent bile acids whereas MRP2 exports structure consisting of predicted 12 TM domains where divalent bile acids. Both BSEP and MRP2 expression both the N- and C-termini face intracellularly. Some undergo a positive feed-forward regulation by bile acids via OATP proteins mediate bile acid uptake by exchanging the FXR receptor (40.41). - them with the HCO3 or reduced glutathione (GSH) (34,35). Other major hepatic organic solute transporters include As the bile acids travel along the biliary tree, a organic anion transporter 1 (OAT1) and the organic cation small amount may be reabsorbed by the cholangiocytes, transporters (OCTs/SLC22A) (33,36), although these which express the apical sodium bile acid cotransporter membrane proteins appear to play relatively minor roles in (Asbt/Slc10a2) on their lumenal membranes (42), and Ost bile acid transport. alpha-Ost beta on their basolateral membranes (43). The bile acids that are reabsorbed by cholangiocytes recycle via Hepatic export of bile acids into canalicular bile the peribiliary plexus back to hepatocytes for re-secretion is mediated largely by members of the ATP-binding into bile, a process that is referred to as the cholehepatic cassette (ABC) superfamily, principally the bile salt export shunt pathway. It is not clear to what extent the shunt pump (BSEP/ABCB11) and the multidrug resistance- pathway contributes to the overall hepatobiliary transport

2831 Function and regulation of Ost alpha-Ost beta of bile acids, or to the adaptation to chronic cholestasis due two genes must be co-expressed in order to elicit transport to extrahepatic obstruction (42). activity (52).

Under normal conditions, the efflux of bile acids In 2003, using an enlarged gene and protein across the sinusoidal membrane of hepatocyte is negligible; sequence database, human and mouse orthologues of skate however, in cholestatic conditions, OST alpha-OST beta Ost alpha and Ost beta were identified (54). The amino and the multidrug resistance-associated proteins MRP3 and acid sequence alignments of OST alpha and OST beta from MRP4 are upregulated, and may mediate the export of bile different species are shown in figures 2 and 3. The acids into the blood plasma, although their functions are not predicted human OST alpha protein and the predicted well defined (Figure 1) (2,27,44-46). mouse Ost alpha protein each exhibit 41% amino acid identity with skate Ost alpha, and they also exhibit 83% 5. INTESTINAL BILE ACID TRANSPORTERS amino acid identity with each other. Human OST alpha and mouse Ost alpha are predicted to have 340 amino Once bile reaches the small intestine, most acids and a molecular mass of ~ 37kDa. This protein is (>85%) of the bile acids are reabsorbed and returned to the predicted to have 7 TM domains in all species (Figure liver via portal blood. Bile acid uptake from the intestinal 2). Likewise, the predicted human OST beta and mouse lumen is mediated largely by ASBT/SLC10A2 (Figure 1). Ost beta proteins exhibit 25% and 29% amino acid ASBT represents a highly efficient bile acid uptake identity with skate Ost beta, and they also exhibit 63% transporter that prefers conjugated bile acids (28). Similar amino acid identity with each other (Figure 3). Human to NTCP, it mediates bile acid uptake coupled with Na+ at a OST beta and mouse Ost beta have 128 amino acids and ratio of 2:1 (47), and bile acids induce a negative feedback the predicted molecular mass is ~19 kDa. OST beta regulation of Asbt gene expression (48). Bile acids induce proteins are predicted to have a single TM domain FXR-dependent SHP activation, and SHP in turn represses (Figure 3). LRH-1 dependent activation of ASBT expression (49). In addition to ASBT, Oatp3/Slc21A7 may also contribute to 7. OST ALPHA AND OST BETA ARE EXPRESSED bile acid uptake in the small intestine (50). When ileal bile IN MOST TISSUES, BUT ARE MOST ABUNDANT acid uptake is inhibited, as is the case in Asbt-/- mice (51), IN TISSUES INVOLVED IN BILE ACID AND bile acid levels within the ileocytes decrease, and this STEROID HOMEOSTASIS represses the expression of FGF19/Fgf15. The lower FGF19/Fgf15 levels relieve the inhibition of Cyp7a1 Using quantitative real-time PCR analysis, transcription, and thus bile acid synthesis rate is increased Seward and colleagues (54) demonstrated that OST alpha under these conditions. and OST beta mRNA is widely expressed in human tissues, with the highest expression in small intestine, liver, colon, In contrast to the uptake step, the mechanism of kidney, testes, ovary, and the adrenal gland. The export from enterocytes into the splanchnic circulation was distribution was also confirmed by Northern blotting. undefined until the recent discovery of OST alpha-OST Additional studies revealed significant species differences beta, as described in more detail below. Mrp3 is another in OST protein expression. For example, OST alpha and putative basolateral bile acid exporter in the enterocytes, OST beta mRNA are relatively abundant in human liver, but this protein appears to play a small role in the efflux of whereas, both mouse and rat liver have very low levels of bile acids into mesenteric venous blood (2). these transcripts. Human OST alpha protein is detected in hepatocytes and cholangiocytes, whereas mouse 6. IDENTIFICATION OF OST ALPHA-OST BETA hepatocytes have no detectable Ost alpha protein and mouse cholangiocytes have only moderate expression of In 2001, our laboratory identified a novel organic this protein (43). Relatively high levels of Ost alpha and solute and steroid transporter (Ost) from the little skate Ost beta message and protein are found in mouse small Leucoraja erinacea using expression cloning in Xenopus intestine, colon, and kidney, and the expression is laevis oocytes (52). Unlike mammalian hepatocytes, which especially high in ileum (55). Tissues with high levels of uptake bile acids largely via Ntcp, skate liver does not OST alpha generally also have high levels of OST beta, appear to express an Ntcp transporter. In addition, the little indicating co-expression of these genes (43,54). Both skate synthesizes and excretes sulfated bile alcohols instead transcripts are relatively abundant in tissues that of bile acids, and therefore these animals were thought to also express ASBT/Asbt. have an additional transport mechanism for steroid derived molecules. The tissue distribution of Ost alpha and Ost beta mRNA and protein are also supported by expressed In order to identify a possible sterol transporter, sequenced tag (EST) counts (Table 1). Human OST alpha total mRNA of skate liver was screened for bile acid and OST beta are most abundant in liver, intestine, kidney, (taurocholate) transport activity in Xenopus oocytes, and a testis, mammary gland, uterus, prostate, and thyroid, which cDNA library that included two positive mRNA size are all steroid rich organs (Table 1). This expression fractions (0.6-1.5 kb and 1.2-2.3 kb) was constructed pattern suggests that OST alpha-OST beta may transport (52,53). Using this approach, two distinct genes were steroid-derived compounds in these tissues. In mouse, Ost identified and denoted as organic solute and steroid alpha and Ost beta tissue distribution parallels that of Asbt transporter alpha and beta (Ost alpha and Ost beta). These (Table 1).

2832 Function and regulation of Ost alpha-Ost beta

Figure 2. Amino acid alignments of the deduced OST alpha sequences from multiple species. Reference sequences for the human, mouse, dog, horse, chicken, zebrafish, and skate OST alpha proteins were obtained from the NCBI database and were aligned using the MUSCLE program (multiple sequence comparison by log-expectation; http://www.ebi.ac.uk/Tools/muscle/index.html). The predicted TM domain is boxed. “*” denotes the amino acids that are conserved among the seven species; “:” denotes that conserved substitutions are observed; “.” denotes that semi-conserved substitutions are observed.

In support of a role of the transporter in In addition to bile acids, Ost alpha-Ost beta also basolateral efflux, both Ost proteins are localized to the transports steroids such as estrone 3-sulfate, basolateral membrane of key epithelial cells, including dehydroepiandrosterone 3-sulfate (DHEAS), and digoxin, enterocytes, renal tubular cells, and cholangiocytes (43,55). as well as the eicosanoid PGE2 (43,54). Additional studies are needed to further define the substrate selectivity of Ost 8. OST ALPHA-OST BETA MEDIATES THE alpha-Ost beta. TRANSPORT OF BILE ACIDS, CONJUGATED STEROIDS, AND STRUCTURALLY-RELATED 9. HETERODIMERIZATION OF OST ALPHA AND MOLECULES: TRANSPORT OCCURS BY A OST BETA INCREASES THE STABILITY OF THE FACILITATED DIFFUSION MECHANISM INDIVIDUAL PROTEINS, FACILITATES THEIR POST-TRANSLATIONAL MODIFICATIONS, AND The mechanism of Ost alpha-Ost beta IS REQUIRED FOR DELIVERY OF THE OST mediated transport was identified by Ballatori et al. in ALPHA-OST BETA COMPLEX TO THE PLASMA 2005 (43). Ost alpha-Ost beta mediated uptake of MEMBRANE estrone 3-sulfate was measured in Xenopus laevis oocytes. Transport was shown to be bidirectional and As noted above, in vitro transfection studies unaffected by depletion of intracellular ATP, changes in showed that co-expression of Ost alpha and Ost beta is transmembrane electrolyte concentration gradients, or changes required for delivery of the individual proteins to the in the pH gradient. Ost alpha-Ost beta mediated transport of plasma membrane (52,54). Dawson et al. (55) estrone 3-sulfate was trans-stimulated by known substrates. In demonstrated that co-expression is also required to convert addition, direct efflux of substrates was observed in oocytes the Ost alpha subunit to a mature N-glycosylated, Endo H- expressing Ost alpha and Ost beta. These results resistant form, suggesting that co-expression facilitates the indicate that Ost alpha-Ost beta mediates transport movement of Ost alpha through the Golgi apparatus. Li et through facilitated diffusion, and thus can mediate either al. (56) extended these findings by showing that an Ost efflux or uptake depending on the particular substrate’s alpha-Ost beta heteromeric complex is apparently formed electrochemical gradient (43). in the ER, is modified as it transits through the Golgi

2833 Function and regulation of Ost alpha-Ost beta

Figure 3. Amino acid alignments of the deduced OST beta sequences from multiple species. Reference sequences for the human, mouse, dog, horse, chicken, zebrafish, and skate OST beta proteins were obtained from the NCBI database and were aligned using the MUSCLE program (multiple sequence comparison by log-expectation; http://www.ebi.ac.uk/Tools/muscle/index.html). The predicted TM domain is boxed. “*” denotes the amino acids that are conserved among the seven species; “:” denotes that conserved substitutions are observed; “.” denotes that semi-conserved substitutions are observed.

Table 1. Tissue expression of human OST alpha and OST beta and mouse Ost alpha and Ost beta as indicated by analysis of expressed sequence tag (EST) counts Tissue Transcripts per million Human Mouse OSTalpha OSTbeta ASBT Ostalpha Ostbeta Asbt Liver 28 4 0 9 0 0 Kidney 14 37 61 185 24 24 Intestine 25 25 8 139 442 23 Mammary Gland 6 0 0 0 0 0 Uterus 4 0 0 0 0 0 Testis 54 3 0 0 0 0 Prostate 10 0 0 0 0 0 Thyroid 20 0 0 0 0 0 Thymus 0 0 0 0 24 0 Data were obtained from the UniGene EST Profile Viewer Web site of the National Center for Biotechnology Information in April, 2008 (http://www.ncbi.nlm.nih.gov/UniGene/) apparatus, and is then targeted to the plasma membrane. In partners interact, the two fragments of the fluorescence contrast, when Ost alpha and Ost beta are expressed protein may associate with each other to regenerate a individually, the proteins appear to be targeted for functional fluorophore. BiFC has several features that degradation (56). make it useful to detect protein-protein interactions, including direct visualization of interaction and its Evidence for a direct interaction between Ost subcellular localization in living cells (57,60). alpha and Ost beta was obtained from co- immunoprecipitation and bimolecular fluorescence Co-immunoprecipitation studies using mouse complementation (BiFC) analyses, two powerful ileal proteins and transfected HEK293 cells revealed that approaches for examining protein-protein interactions (56). the two proteins that generate the organic solute transporter In BiFC analysis, two fragments of a fluorescence protein, are able to immunoprecipitate each other, indicating which are non-fluorescent when separated, are fused to the formation of a heteromeric complex (56). Mouse ileal Ost putative interacting protein partners (57-59). When the alpha protein appeared on Western blots largely as bands of

2834 Function and regulation of Ost alpha-Ost beta

Ost beta mRNA levels were maintained, yet Ost beta protein was not detectable, indicating that Ost beta protein is not stable in the absence of Ost alpha. Overall, these findings identify the membrane topology of Ost alpha and Ost beta, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ost alpha and Ost beta increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane (56).

10. EXPRESSION OF BOTH OST GENES IS POSITIVELY REGULATED BY BILE ACIDS THROUGH THE BILE ACID-ACTIVATED FARNESOID X RECEPTOR, FXR

The regulation of OSTs at the cellular and molecular level has been investigated in several studies, and emerging evidence indicates that both subunits are regulated by bile acids through specific transcription factors (Figure 4). Evidence for a critical role of Fxr in the transcriptional regulation of human OST alpha and OST beta and mouse Ost alpha and Ost beta has been provided

by studies using transgenic animals, in vitro transcription of Figure 4. Ost alpha and Ost beta are dynamically the genes, electrophoretic mobility shift assays, and regulated via the Fxr-Shp-Lrh-1 pathway. Bile acids are promoter analysis (46,62-65). In particular, Fxr-/- mice actively transported into enterocytes by Asbt, where they have lower basal levels of Ost alpha and Ost beta bind to and activate Fxr. Fxr forms a heterodimer with Rxr expression in ileum, indicating that Ost alpha and Ost beta alpha, which translocates to the nucleus, binds to FxrEs gene expression is dependent on Fxr 65). Both the thereby priming expression of target genes Ost alpha, Ost biological Fxr agonist, CDCA and the synthetic Fxr beta, and Shp in a positive feedback manner (62,98). Upon agonist, GW4064 induce OST alpha-OST beta mRNA and accumulation of Shp, a non-DNA binding orphan nuclear protein expression in many cell lines, including two human receptor viewed as a transcription inactivator, Shp forms a liver cell lines, HepG2 and Huh7, a human adrenal heterodimer with Lrh-1, an obligatory factor for carcinoma cell line, H295R, and a murine colon transcriptional activation, thereby antagonizing Lrh-1 and adenocarcinoma cell line, CT26 (46,62-64). Reducing ultimately leading to down-regulation of Ost alpha, Ost FXR expression by transfecting Huh7 cells with a pool of beta, and Shp (62,99). Thus, negative regulation is a four FXR-specific siRNAs led to the abolition of CDCA subsidiary mechanism to positive feedback via bile acids. and GW4064 induced OST alpha and OST beta mRNA

expression (63). GW4064 induces Ost alpha and Ost beta 40 and 80 kDa, the latter band is consistent with mRNAs in wild-type mouse adrenal gland organ culture, an Ost alpha homodimer, and both of these bands were but not in the Fxr-/- mice organ culture (65). These findings sensitive to digestion by the glycosidase PNGase F. Ost establish that Fxr is an essential regulator for Ost alpha and beta appeared as bands of 17 and 19 kDa, and these bands Ost beta expression. were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ost alpha and only the 19 kDa form of Ost beta Two groups have reported that farnesoid X were detected among the immunoprecipitated proteins, receptor elements (FXRE) are found in human OST alpha indicating that the interaction between Ost alpha and Ost and OST beta promoters by using different computer based beta is associated with specific post-translational algorithms (63,64). Two putative IR-1/FXREs were processing (56). Co-immunoprecipitation of human OST identified within the OST alpha promoter and one within alpha and OST beta also supported the formation of a the OST beta promoter (Figure 5). In the mouse, one heterodimeric complex (61). potential FXRE sequence within the Ost alpha promoter

and one within the Ost beta promoter has also been Additional evidence for homodimerization of Ost reported (62) (Figure 5). These studies reveal that the alpha and for a direct interaction between Ost alpha and Ost induction of OST alpha and OST beta mRNA expression beta was provided by BiFC analysis of HEK293 cells by FXR is ligand dependent and requires the transfected with Ost alpha and Ost beta tagged with YFP heterodimerization of FXR with RXR alpha. FXR-RXR fragments (56). BiFC analysis and surface immunolabeling alpha heterodimers bind to the putative IR-1/FXREs and of transfected HEK293 cells also indicated that the induce OST gene expression when bile acids and other carboxyl termini of both Ost alpha and Ost beta are facing potential FXR ligands are present (Figure 4). the intracellular space. The interaction between Ost alpha and Ost beta was required not only for delivery of the Although Fxr is the essential regulator, Ost proteins to the plasma membrane, but it increased their alpha-Ost beta expression is fine-tuned by other stability, as noted in transfected HEK293 cells and in transcription factors, including small heterodimer partner tissues from Ost alpha-deficient mice. In Ost alpha-/- mice,

2835 Function and regulation of Ost alpha-Ost beta

(62). These findings reveal an elaborate dual regulatory system mediated by nuclear receptors for more precise regulation of mouse Ost alpha and Ost beta gene expression. They also suggest a potential critical role of Ost alpha-Ost beta in bile acid homeostasis.

Interestingly, mouse Ost alpha and Ost beta may also be coordinately regulated by Fxr/Rxr and Lxr/Rxr (66). Lxr alpha was found to bind to the same IR-1/FxrEs that Fxr/Rxr alpha binds to activate transcription of the murine organic solute transporter. The physiological implication of the regulation by Lxr alpha is unknown, although the oxysterol nuclear receptor is known to dimerize with Rxr alpha and function in cholesterol and bile acid homeostasis (67). Only a few cases of FXREs functioning as LXREs are known; one being the FXRE of the ileal bile acid binding protein (I-BABP), which is an interesting coincidence since I-BABP and the organic solute transporter are regulated by the same pathways (68). Okuwaki and colleagues (66) also elucidated a role for hepatic nuclear factor-4 alpha (HNF-4 alpha), a regulator of lipid and bile acid homeostasis, in transcriptional activation Figure 5. Functional FxrEs and Lrh-1 binding sites in the of the alpha subunit of the murine and human transporter, proximal promoter of murine Ost alpha and Ost beta, and but not the beta subunit. functional FXREs in the proximal promoter of human OST alpha and OST beta. After heterodimerization with RXR Examination into the mechanism of pregnane X alpha, FXR binds to the FXRE cis-acting element with an receptor (Pxr) activation on bile acid toxicity attenuation idealized sequence of an inverted hexameric nucleotide revealed a possible novel negative feedback loop for Fxr repeat consisting of minor variants of two AGGTCA half- regulated genes (69). Pxr is known to regulate the sites separated by one nucleotide (IR-1) (100-102). LRH-1 expression of several enzymes and transporters involved in binds to a consensus sequence of YCAAGGYCR where Y bile acid homeostasis of which Cyp7a1 and Mrp3 are of is any pyridine and R is any purine (103). A: position of note. Teng and Piquette-Miller (69) observed an increase functional FxrEs and Lrh-1 binding sites in the promoters in expression of Ost alpha and Ost beta as well as Mrp3 in of murine Ost alpha and Ost beta relative to putative Pxr-null livers as compared to wild type counterparts, and transcription start sites and sequence comparison of the the Pxr-null mice were less sensitive to cholic acid-induced functional cis-acting elements to optimal binding sequences hepatotoxicity. In cholic acid-fed mice, administration of (62). B: location of human OST alpha and OST beta 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), a functional FXREs with sequence comparison to an Pxr activator, caused a down-regulation of Ost alpha and idealized binding sequence (64). Conservation of the Ost beta and up-regulation of Mrp3 in wild type mice, but murine Lrh-1 binding sites in the promoters of their human not in Pxr-null mice. In the same mice, Fxr levels were counterparts has been indicated although the exact position roughly 50% lower upon PCN supplementation (69). and sequence of the cis-acting elements have not been Cholic acid and its main conjugate, taurocholic acid, are published (62). Therefore, OST alpha and OST beta are substrates of rodent Mrp3 (70,71), and sulfated sterol assumed to be negatively regulated in a similar fashion as conjugates such as dehydroepiandrosterone sulfate their murine orthologues. (DHEAS) and estrone 3-sulfate are known substrates of Ost alpha and Ost beta (43). Interestingly, sulfation is not a (Shp) and liver receptor homolog-1 (Lrh-1) (62). major metabolic pathway in mice (72), therefore the up- One functional Lrh-1 element was identified in the mouse regulation of Mrp3 rather than Ost alpha and Ost beta and Ost alpha promoter region and two were identified in the the subsequent efflux of cholic acid and its conjugates in mouse Ost beta promoter region (Figure 5). Functional response to induced hepatotoxicty following PCN treatment analysis of Lrh-1 in mouse Ost alpha and Ost beta could be indicative of preferential regulation due to promoter regions suggests that mouse Ost alpha and Ost substrate specificity. In mice and probably in humans, beta expression is negatively regulated by bile acids via the Mrp4 may be more important as it prefers sulfated Fxr-Shp-Lrh-1 pathway even though Lrh-1 exerts conjugates, while Mrp3 prefers glucuronide conjugates constitutively positive regulation on mouse Ost alpha and (45). A hypothesized schematic for this novel negative Ost beta expression (Figure 4). The same mechanism is feedback mechanism is illustrated in Figure 6. used to regulate Cyp7a1, the rate-limiting enzyme for bile acid synthesis. The Fxr-Shp-Lrh-1 pathway is thought to be Boyer and colleague (46) found that both OST subordinate under basal conditions in the ileum in Shp-/- alpha and OST beta mRNA and protein expression are mice, where the basal expression level of mouse Ost alpha upregulated in human livers from primary biliary cirrhosis and Ost beta is unchanged compared to the wild type (PBC) patients. PBC is a chronic cholestatic disease mouse, but Ost alpha and Ost beta are lower in Fxr-/- mice characterized by inflammatory destruction of the small bile

2836 Function and regulation of Ost alpha-Ost beta

including its substrate specificity, transport mechanism, tissue distribution, subcellular localization, and transcriptional regulation. To more directly test the functional role of Ost alpha-Ost beta, Ost alpha-/- mice have recently been generated and characterized from two different laboratories, and they have provided important insights into the function of this transporter (80,81). As demonstrated by Li et al. (56), Ost alpha-/- mice lack both Ost alpha and Ost beta proteins because the individual

subunits of this heteromeric transport complex are not Figure 6. Proposed mechanism of a novel negative stable in the absence of their obligate heterodimerization partner. The knockout mice are viable and fertile but feedback pathway for Ost alpha and Ost beta by Pxr. -/- Ligand activation of Pxr, which dimerizes with Rxr alpha, exhibit early growth retardation. Pre-weanling Ost alpha seemingly reduces Fxr levels ultimately down-regulating mice of both sexes are 25% smaller than heterozygous Ost alpha and Ost beta, but simultaneously inducing littermates (80) although this difference disappears after the expression of Mrp3. Mrp3 protein can then mediate bile mice reach adulthood. In addition, both pre-weanling and acid efflux. This suggests a negative feedback loop that is adult Ost alpha-deficient mice display intestinal absent in Pxr-deficient mice thereby explaining the higher hypertrophy. As expected, Ost alpha knockout mice expression levels of Ost alpha and Ost beta under control display impaired trans-ileal taurocholate transport, conditions as compared to wild type animals (69). significantly decreased bile acid pool size, and altered hepatic, renal and intestinal disposition of bile acids ducts within the liver (73,74) resulting in the accumulation (80,91). These data provide evidence that Ost alpha-Ost of hepatoxic bile acids. Because OST alpha-OST beta is an beta is critical for bile acid and steroid homeostasis. important player in bile acid enterohepatic circulation, it might undergo adaptive regulation to protect the liver from The Ost alpha null mice also provide important tissue injury. In the mouse, the first indication that Ost insight into mechanisms of bile acid homeostasis, and alpha-Ost beta is regulated by bile acids was suggested in identify Ost alpha-Ost beta as a potential target for Asbt-/- mice (55). In the cecum and proximal colon of the interrupting the enterohepatic circulation of bile acids. Asbt-/- mouse, Ost alpha and Ost beta are up-regulated; Although there is a significant decrease of bile acid however, they are decreased in the ileum (55). The same absorption in Ost alpha null mice, the fecal excretion of expression pattern of Ost alpha-Ost beta is also observed in bile acids is unchanged. This is due in part to a decrease in male C57BL/6J mice fed with 0.2% dietary cholic acid for hepatic bile acid synthesis and the resulting marked 14 days (62). The decreased ileal expression of Ost alpha- decrease in the bile acid pool size, as illustrated Ost beta in both models can be explained by reduced schematically in figure 7. Interestingly, cholesterol uptake of bile acids into the ileal enterocytes. absorption is less in Ost alpha knockout mice (81). Fecal neutral sterol excretion in the knockout mice is more than A recent study demonstrates that arachidonic the wild type and also the serum cholesterol and acid, the precursor to eicosanoids, strongly induces Ost triglycerides are lower in knockout mice. Thus, OST alpha-Ost beta expression in a new cell line derived from alpha-OST beta might be a potential therapeutic target for an early embryo of Leucoraja erinacea (75). Although the altering lipid levels. function of this transporter in embryo-derived cells is unknown, it may play a role in the disposition of In addition, in contrast to the increase in hepatic eicosanoids or steroid-derived molecules. This conclusion bile acid synthesis seen when intestinal bile acid uptake is interrupted with either bile acid sequestrants or with Asbt is supported by the observation that prostaglandin E2 is a inhibitors, hepatic Cyp7a1 expression is decreased in the substrate for this transporter (52). -/- Ost alpha mice indicating a decrease in bile acid synthesis Although relatively little information is available (Figure 7). As described in more detail below, these on ontogenic changes in Ost alpha and Ost beta expression, contrasting effects are most likely explained by the the available evidence indicates that Ost alpha and Ost beta enhanced intestinal expression of Fgf15, an important expression is quite low in neonatal rats and rabbits and negative regulator of hepatic bile acid synthesis (13), when increases to adult levels shortly after weaning (76-78). Ost alpha-Ost beta function is abrogated. These changes parallel those for many of the genes involved in bile acid homeostasis including Fxr (79). As noted above, bile acid concentrations are normally controlled by a feedback regulatory mechanism, 11. OST ALPHA-OST BETA IS REQUIRED FOR whereby bile acid activation of Fxr represses hepatic BILE ACID AND CONJUGATED STEROID transcription of Cyp7a1 levels, and thus leads to a decrease DISPOSITION IN THE INTESTINE, KIDNEY, AND in bile acid synthesis (Figure 7). Bile acid activation of Fxr LIVER also leads to decreased expression of the bile acid uptake transporters Asbt and Ntcp, and to increased expression of As described above, several lines of evidence the bile acid exporters Bsep and Ost alpha-Ost beta. support the hypothesis that Ost alpha-Ost beta is the key Collectively, these transport proteins, along with the basolateral exporter of bile acids and related molecules, enzyme Cyp7a1, mediate a decrease in intracellular bile

2837 Function and regulation of Ost alpha-Ost beta

Figure 7. Comparison of the enterohepatic circulation of bile acid in Ost alpha+/+ and Ost alpha-/- mice. Bile acids (BA) are taken up from the lumen of the small intestine into ileal enterocytes via Asbt. Once inside the cell, BA bind to Fxr and mediate transcription of genes such as Fgf15. Fgf15 is delivered via portal blood to the hepatocytes where it can bind to Fgfr4. In Ost alpha+/+ mice, Ost alpha-Ost beta effluxes BA from the enterocyte into blood on the basolateral side, and the BA return to the liver via portal circulation. Also depicted are the apically-localized Mrp2 and basolaterally-localized Mrp3 proteins that may also contribute to BA export. BA enter the hepatocyte from the blood via Ntcp, at which point they may bind Fxr and initiate transcription of Shp, which along with Fgf15-Fgfr4 synergistically mediate transcriptional repression of Cyp7a1. Also depicted are Ost alpha-Ost beta and Mrp3 on the sinusoidal membrane mediating low-level transport of their respective substrates back into blood. BA are in turn actively transported across the canalicular membrane by Bsep, or to an extent by Mrp2, directly into bile where they eventually re-enter the intestines thereby completing enterohepatic circulation. In Ost alpha-/- mice, the uptake of BA into the ileal enterocyte results in their accumulation because of the eliminated basolateral efflux mediated by Ost alpha-Ost beta (80,81). The elevated BA levels activate Fxr, leading to a marked up-regulation of Shp, which results in the subsequent down-regulation of Asbt, and Fgf15. Fgf15 in turn down-regulates Cyp7a1 in the liver after binding Fgfr4. The compensatory BA transporters Mrp3 and Mrp2 are up-regulated, which may help eliminate BA from the enterocytes. In the hepatocyte, an up- regulation of Ntcp, Mrp3, Bsep, and Mrp2 is observed in an attempt to escalate cycling of BA due to their increased loss evident from the decreased bile acid pool size coupled with the unchanged fecal excretion rate as compared to wild type. The decreased bile acid pool size is not only due to intestinal malabsorption of BA, but also to the substantial down-regulation of Cyp7a1, a rate-limiting enzyme in bile acid synthesis (80).

2838 [Frontiers in Bioscience 14, 2829-2844, January 1, 2009] acid concentrations back to basal levels. Ileocytes, accumulate in both the ileal lumen and enterocytes of neonatal however, also express Fgf15, another key regulator of bile animals with NEC and the increased bile acid levels are acid synthesis (13). When intracellular bile acid levels in positively correlated with disease severity (76,95). Halpern et ileocytes are elevated (as they are expected to be in the Ost al. (76) found that Asbt was up-regulated at the site of injury in alpha-/- mice), this leads to the Fxr-mediated induction of rats with experimentally-induced NEC and decreased after Fgf15 (13). Fgf15 is then delivered to the liver, where it EGF treatment; however, the ileal bile acid binding protein represses Cyp7a1 expression through a mechanism that was up-regulated only in the NEC and EGF group, and Ost involves Fgf receptor 4 (Fgfr4). Intestinal expression of alpha and Ost beta expression was low in all groups, and only Fgf15 was higher and hepatic expression of Cyp7a1 was slightly increased in the NEC group. The inability to lower in Ost alpha-/- mice, as predicted by this model upregulate Ost alpha and Ost beta expression may have (Figure 7). contributed to the accumulation of toxic levels of bile acids in the ileocytes. These results suggest that bile acids play a role These observations in Ost alpha-/- mice contrast in the development of ileal damage in experimental NEC and with those seen in Asbt-/- mice (51). The absence of Asbt that alterations in bile acid transport in the neonatal ileum may leads to a diminished ability of the enterocytes to take up contribute to disease development. bile acids across their apical membranes, and thus, to relatively low intracellular bile acid levels. These low bile Another disease related to the concentration of acid levels down-regulate Fgf15 expression, and thus bile acids is colon cancer. Epidemiological studies have shown relieve Fgf15-mediated repression of Cyp7a1 transcription. that the concentration of fecal bile acids is positively correlated In addition, the low hepatocellular bile acid levels in Asbt-/- with the colorectal cancer incidence (96). In animal models, mice also relieve the Fxr- and Shp-mediated repression of diversion of bile ducts, small bowel resection, or direct hepatic Cyp7a1 activity, with the net result being a 5-fold installation of bile acids in the large bowel can be tumor increase in Cyp7a1 expression and in bile acid synthesis promoting (97). Because OST alpha-OST beta regulates rate in Asbt-/- mice (51). enterocyte and intestinal lumen bile aids concentrations, altered levels or function of OST alpha-OST beta may be a risk factor 12. IMPLICATIONS FOR HUMAN DISEASES for these diseases, although there is no evidence for this hypothesis as yet. In addition, bile acids are required for Currently, there is no disease that is directly intestinal lipid absorption. Thus, OST alpha-OST beta may associated with OST alpha-OST beta. However, given the also be involved in a variety of human conditions related to central role of OST alpha-OST beta in bile acid imbalances in sterol or lipid homeostasis, although this has not homeostasis, this transporter may be involved in the yet been established. progression of diseases related to bile acid malabsorption, cholestasis, or cholelithiasis. In support of this hypothesis, 13. SUMMARY changes in OST alpha and OST beta gene expression have already been reported in some of these conditions (82-85). Recent studies indicate that Ost alpha-Ost beta is Bile acid malabsorption is observed in a number of human a major basolateral transporter of bile acids and conjugated conditions, including intractable diarrhea, irritable bowel steroids in the intestine, kidney, and liver. Crosstalk syndrome, immunodeficiency virus (HIV) enteropathy, and between multiple signaling pathways and nuclear receptors cystic fibrosis (86-89). On the other hand, too much bile reveals a more intricate means of regulation for OST alpha- acid reabsorption is the cause for constipation (90). It will OST beta than initially thought. The involvement of clearly be of interest to examine the possible contribution several transcription factors, including FXR, RXR alpha, of OST alpha-OST beta to the etiology and/or progression SHP, LRH-1, LXR alpha, HNF-4 alpha, and PXR, paints a of these human conditions. complicated regulatory mechanism that needs further study. Ost alpha-deficient mice display impaired trans-ileal Although bile acids play important roles in many taurocholate transport, significantly decreased bile acid physiological processes, they can also be toxic when pool size, and altered hepatic, renal and intestinal present at high levels. Retention of bile acids in cholestatic disposition of bile acids (80,81). These data provide liver diseases results in progressive liver injury that may evidence that Ost alpha-Ost beta is critical for bile acid and ultimately result in organ failure and the need for liver steroid homeostasis, and suggest that the resulting changes transplantation. The secondary hydrophobic bile acids are in bile acid levels have an effect on serum cholesterol, more toxic than primary bile acids but all can result in triglyceride, and glucose levels. In addition, the results apoptosis and or necrosis of liver tissue (91). Bile acids can indicate that targeted inhibition of Ost alpha-Ost beta may also induce oxidative stress, with secondary consequences have advantages over other maneuvers that have been used of inflammation and fibrosis. The rationale for therapy with to interrupt the enterohepatic circulation of bile acids. ursodeoxycholic acid in cholestatic diseases is to reduce the hydrophobicity of the circulating bile acid pool. Another 14. ACKNOWLEDGEMENTS disease that may be caused by elevated bile acid concentrations is necrotizing enterocolitis (NEC), a disease This work was supported in part by National that affects thousands of newborns each year in the United Institute of Health Grants DK067214 and DK48823, and States alone, and with a mortality rate ranging from 10% to National Institute of Environmental Health Sciences 50% (92-94). NEC remains a major cause of morbidity and Training Grant ES07026 and Center Grants ES03828 and mortality in prematurely born infants. Bile acids ES01247.

2839 Function and regulation of Ost alpha-Ost beta

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Key Words: Conjugated Steroids, Bile Acid Transport, FXR, Ost Alpha-Ost Beta, Ost alpha-/- mice, Review

Send correspondence to: Ned Ballatori, Department of Environmental Medicine, Box EHSC, University of Rochester School of Medicine, 575 Elmwood Avenue, Rochester, NY 14642, Tel: 585-275-0262; Fax: 585-256- 2591, E-mail: [email protected] http://www.bioscience.org/current/vol14.htm

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