Bile acids undergo extensive enterohepatic cycling. After being synthesized in the liver, bile acids are excreted into bile and the intestine, subsequently re-absorbed into the portal venous blood and finally transported back to the liver. The enterohepatic circulation of bile acids is maintained by bile acid uptake transporters and excretion pumps, located at the basolateral and apical membranes of intestinal, hepatic and renal cells, which are regulated by changes in the intracellular concentration of bile acids .
Any reduction in the regular availability of oxygen in human tissues leads to hypoxia. Various conditions can cause hypoxia or induce hypoxia modulators, such as ischaemia, ischaemic reperfusion injury, cancer and inflammation. In addition, cholestasis induces hypoxia in the liver [2-4] and low oxygen levels regulate bile salt transporters. However the exact mechanism by which hypoxia modulates the expression of these transport proteins has not been elucidated. Furthermore, no inducing effects on the expression of transport proteins have so far been shown in the literature. An important part of the cellular response to the lack of oxygen is mediated by hypoxia-inducible factor 1 (HIF-1). HIF-1 consists of a hypoxia regulated subunit α (HIF-1α) and a constitutively expressed subunit β (HIF-1β), also known as the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Under normoxic conditions HIF-1α is hydroxylated and consequently ubiquitinated, so that the half-life of HIF-1α is heavily reduced by proteasomal degradation. Under low oxygen conditions, the oxygen dependent hydroxylase activity is reduced and HIF-1α is less prone to degradation [5, 6]. Accumulated HIF-1α dimerises with ARNT, translocates to the nucleus and binds to the hypoxia responsive element (HRE) in the promoter region of a given target gene. With very little variation, the core HRE is composed of the sequence ACGTG .
The heterodimeric organic solute transporter α/β (OSTα/β), encoded by the genes SLC51A and SLC51B, respectively, is a bile acid transporter expressed in the liver, intestine and kidney. In hepatocytes the OST heterodimeric transporters are located in the basolateral membrane, where they are assumed to mediate basolateral efflux of organic anions including bile acids, thereby potentially protecting hepatocytes from intracellular accumulation. In enterocytes OSTs are located in the basolateral membrane, where they are responsible for the efflux of bile acids into the portal venous blood [8, 9].
Cholic acid and chenodeoxycholic acid (CDCA) are the two primary bile acids in humans . The nuclear farnesoid X receptor (FXR), also known as the bile acid receptor (BAR), has been identified as an important mediator of CDCA-dependent regulation of gene expression [11, 12]. CDCA is an agonistic ligand of FXR and leads to the regulation of a whole battery of genes involved in bile acid metabolism and transport in both the liver and intestine. We previously showed that CDCA transactivates the OSTα and OSTβ genes via an FXR dependent pathway. .
In this study, we investigated in vitro to what extent hypoxic conditions affect the expression of OSTα/β in the liver using hepatocyte-derived cell lines as well as a chronic renal failure (CRF) rat model. We characterize the transcriptional pathway that mediates hypoxic effects on OSTα/β gene expression. By exposing cells to elevated bile acid concentrations and hypoxic conditions in parallel, we also simulate the combined effects of hypoxia and cholestasis on the transcriptional regulation of OSTα/β.
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Tissue hypoxia in the liver can be caused by several forms of liver injury [2-4]. In cholestasis the toxic effect of bile acids within hepatocytes is prevented by adaptive responses such as induction of bile acid efflux transporters. This study shows that both hypoxia and elevated bile acid levels have the potential to modulate the expression of bile acid transporters via transcription factor mediated pathways, as demonstrated here for the heterodimeric transport protein OSTα-OSTβ. To determine whether hypoxic conditions affect expression at the transcriptional level, relative mRNA levels were measured in both the hepatoma-derived cell-line Huh7 and PHH treated with hypoxia or CDCA, separately or in combination. The inducing effect of CDCA on OSTα-OSTβ expression was reported previously . Here, we show that hypoxia alone is able to increase OSTα and OSTβ mRNA. In Huh7 cells, the combination of CDCA treatment and hypoxia appeared to induce a greater than additive elevation of OSTα mRNA levels, suggesting a possible synergistic effect. In all experiments in which relative OSTα and OSTβ mRNA levels were measured, the induction of VEGFa mRNA was confirmed as a positive control for the effect of hypoxia on gene expression. CDCA alone had no effect on relative VEGFa mRNA levels (data not shown). The inducing effect of hypoxia on OSTα was also evident at the protein level in both Huh7 cells and PHH after 18 h. This was confirmed in the same samples using a second OSTα antibody provided by the laboratory of N. Ballatori (data not shown). Activation of OSTβ gene transcription by CDCA was also observed, as described previously [9, 16]. A significant inducing effect of hypoxia on OSTβ was found in PHH but not Huh7 cells (Fig. 1c and d).
The binding of HIF-1α to the HRE in the OST promoter regions was analysed in vitro by EMSA as shown in Figure 2. HIF-1α binds to the HRE identified in the OSTα promoter and preferentially to one of the three HRE detected in the OSTβ promoter (Fig. 2a). When the core of the HRE was mutated HIF-1α binding was significantly reduced in both OSTα and OSTβ, as shown by the competition EMSA in Figure 2b. Using siRNA to knock-down HIF-1α and FXR, the inducing effects of CDCA and hypoxia on OSTα regulation could be disrupted (Fig. 3a). In contrast to OSTα, no relevant effect of anti-HIF-1α siRNA on OSTβ expression was observed, suggesting that the overall magnitude of OSTβ regulation by HIF-1α may be less than for OSTα.
Dual luciferase assays using wild-type and mutated OSTα promoter constructs confirmed the importance of the predicted HRE for the regulation of OSTα gene expression by hypoxia (Fig. 4a). Interestingly, the induction of the OSTα promoter by CDCA was reduced when an OSTα promoter construct carrying the mutated HRE site was employed. This observation could be explained by the close proximity of the HRE sequence and the FXR binding site. Mutation of the HRE could affect the functionality of the FXR binding site. In the case of the OSTβ promoter, no relevant effect of CDCA and hypoxia on luciferase reporter activity was seen (Fig. 4b), again suggesting that HIF-1α may not activate OSTβ gene transcription to the degree that OSTα gene transcription is induced. The absence of an effect of CDCA on the promoter activity of OSTα and OSTβ in dual luciferase assays (Fig. 4) does not contradict previous results , as the present set-up relied on shorter incubation times and on the activity of endogenously expressed transcription factors only.
Dimerization of OSTα with OSTβ is thought to be essential for transport function [17-19]. Despite the functional synergism, several reports suggest that OSTα and OSTβ appear to be regulated differently. This was concluded from the divergent effects that transcription factors exert on the two promoters, or from the fact that certain transcriptional effects appear to regulate only one of the subunits. [20-23]. It has been shown that OSTβ is important for both the trafficking of OSTα to the plasma membrane as well as for its function . However, the lack of co-immunoprecipitation of the mature, glycosylated form of OSTα upon immunoprecipitation of OSTβ suggests that the primary interaction may occur early in the biosynthetic pathway and may be transient . This would support the mutual independence of the subunits in substrate transport and gives rise to the theory that different dimerization partners exist for the OST subunits. Another indication of the possibility of alternative binding partners or conformations of OSTα is the existence of different splicing variants that have been deposited in the available databases. The exact role of these splice variants has not been investigated.
OSTα-OSTβ is considered to be an efflux system for bile acids across the basolateral membrane of hepatocytes that allows extrusion of bile acids into sinusoidal blood. This could protect hepatocytes from the intracellular accumulation of potentially toxic bile acids, e.g. during cholestatic liver injury or under conditions of hypoxia. Hypoxia caused by arterial liver ischaemia has previously been shown to decrease expression of the Na+-dependent bile acid uptake transporter Ntcp and the bile salt export pump Bsep, which could lead to cholestasis . Induction of OSTα in this setting could represent a rescue mechanism for reducing the intracellular bile acid load when the canalicular efflux pump shows reduced expression.
Chronic renal failure is associated with hypoxia in the kidney. In this study, we show that the previously observed increase in OSTα expression in the liver tissue of rats suffering from CRF  could be attributable to the increased expression of HIF-1α (Fig. 6). It is assumed that chronic hypoxia occurring during CRF is a multifactorial event caused by damage of renal arterioles, a distortion of peritubular capillaries and imminent interstitial fibrosis. Hypoxia itself is able to induce strong profibrogenic effects leading, among other pathways, via induction of HIF-1α dependent signalling to further destruction of peritubular capillaries, fibrotic remodelling of tubular cells and thus, to the further progression of kidney failure [26-28]. Induction of HIF-1α in the liver suggests that hypoxic stimuli occurring during CRF modulate HIF-1α dependent signalling pathways in other tissues as well.
In conclusion, the expression of OSTα and OSTβ is significantly induced by bile acids and hypoxia. While the CDCA-dependent induction of OSTα-OSTβ expression is known to be mediated by FXR, the hypoxia effect is mediated by the transcription factor HIF-1α through newly detected, functionally relevant HIF-1α responsive elements within the OSTα and OSTβ gene promoters. In the case of the OSTα promoter, FXR and HIF-1α bind in close proximity and putatively interact to produce synergistic effects on OSTα expression.
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We thank Christian Hiller for excellent technical assistance. We are grateful to Margot Crucet for assistance with the handling of the hypoxia chamber. We thank Dr N. Ballatori and coworkers for providing an antibody against human OSTα.
Financial support: This work was supported by Swiss National Science Foundation (SNF) grant numbers 320030_144193/1 (to GAKU) and PDFMP3_127259/1 (ProDoc program), the Swiss National Center for Competence in Research NCCR-Kidney.ch, and the International Fellowship Program (grant no. 246539) on Integrative Kidney Physiology and Pathophysiology (IKPP).
Conflicts of interest: The authors do not have any disclosures to report.