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

Abstract

The organic solute and steroid transporter, Ost alpha-Ost beta, is an unusual heteromeric carrier that appears to play a central role in the transport of bile acids, conjugated steroids, and structurally-related molecules across the basolateral membrane of many epithelial cells. The transporter's substrate specificity, transport mechanism, tissue distribution, subcellular localization, transcriptional regulation, as well as the phenotype of the recently characterized Ost alpha-deficient mice all strongly support this model. In particular, the Ost alpha-deficient mice display a marked defect in intestinal bile acid and conjugated steroid absorption; a decrease in bile acid pool size and serum bile acid levels; altered intestinal, hepatic and renal disposition of known substrates of the transporter; and altered serum triglyceride, cholesterol, and glucose levels. Collectively, the data indicate that Ost alpha-Ost beta is essential for bile acid and sterol disposition, and suggest that the carrier may be involved in human conditions related to imbalances in bile acid or lipid homeostasis.

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.

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.

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.

Figure 4. Ost alpha and Ost beta are dynamically regulated via the Fxr-Shp-Lrh-1 pathway

Bile acids are actively transported into enterocytes by Asbt, where they bind to and activate Fxr. Fxr forms a heterodimer with Rxr alpha, which translocates to the nucleus, binds to FxrEs thereby priming expression of target genes Ost alpha, Ost beta, and Shp in a positive feedback manner (62,98). Upon accumulation of Shp, a non-DNA binding orphan nuclear receptor viewed as a transcription inactivator, Shp forms a heterodimer with Lrh-1, an obligatory factor for transcriptional activation, thereby antagonizing Lrh-1 and ultimately leading to down-regulation of Ost alpha, Ost beta, and Shp (62,99). Thus, negative regulation is a subsidiary mechanism to positive feedback via bile acids.

Figure 5. Functional FxrEs and Lrh-1 binding sites in the proximal promoter of murine Ost alpha and Ost beta, and functional FXREs in the proximal promoter of human OST alpha and OST beta

After heterodimerization with RXR alpha, FXR binds to the FXRE cis-acting element with an idealized sequence of an inverted hexameric nucleotide repeat consisting of minor variants of two AGGTCA half-sites separated by one nucleotide (IR-1) (–102). LRH-1 binds to a consensus sequence of YCAAGGYCR where Y is any pyridine and R is any purine (103). A: position of functional FxrEs and Lrh-1 binding sites in the promoters of murine Ost alpha and Ost beta relative to putative transcription start sites and sequence comparison of the functional cis-acting elements to optimal binding sequences (62). B: location of human OST alpha and OST beta functional FXREs with sequence comparison to an idealized binding sequence (64). Conservation of the murine Lrh-1 binding sites in the promoters of their human counterparts has been indicated although the exact position and sequence of the cis-acting elements have not been published (62). Therefore, OST alpha and OST beta are assumed to be negatively regulated in a similar fashion as their murine orthologues.

Figure 6. Proposed mechanism of a novel negative feedback pathway for Ost alpha and Ost beta by Pxr

Ligand activation of Pxr, which dimerizes with Rxr alpha, seemingly reduces Fxr levels ultimately down-regulating Ost alpha and Ost beta, but simultaneously inducing expression of Mrp3. Mrp3 protein can then mediate bile acid efflux. This suggests a negative feedback loop that is absent in Pxr-deficient mice thereby explaining the higher expression levels of Ost alpha and Ost beta under control conditions as compared to wild type animals (69).

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).