Clinical application of transcriptional activators of bile salt transporters.

Hepatobiliary bile salt (BS) transporters are critical determinants of BS homeostasis controlling intracellular concentrations of BSs and their enterohepatic circulation. Genetic or acquired dysfunction of specific transport systems causes intrahepatic and systemic retention of potentially cytotoxic BSs, which, in high concentrations, may disturb integrity of cell membranes and subcellular organelles resulting in cell death, inflammation and fibrosis. Transcriptional regulation of canalicular BS efflux through bile salt export pump (BSEP), basolateral elimination through organic solute transporters alpha and beta (OSTα/OSTβ) as well as inhibition of hepatocellular BS uptake through basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP) represent critical steps in protection from hepatocellular BS overload and can be targeted therapeutically. In this article, we review the potential clinical implications of the major BS transporters BSEP, OSTα/OSTβ and NTCP in the pathogenesis of hereditary and acquired cholestatic syndromes, provide an overview on transcriptional control of these transporters by the key regulatory nuclear receptors and discuss the potential therapeutic role of novel transcriptional activators of BS transporters in cholestasis.

Introduction

Cholestatic liver injury comprises a wide range of genetic or acquired disorders of bile formation and/or flow ultimately resulting in intrahepatic and systemic accumulation of bile salts (BSs) (Lindblad et al., 1977; Setchell et al., 1997). Primary or secondary dysfunctions of specific hepatocellular transport systems as well as mechanical obstruction/destruction of the bile duct system are central mechanisms causing impaired elimination of biliary constituents. Elevated levels of BSs may cause liver damage due to their lipid solubilizing, proinflammatory and proapoptotic properties (Allen et al., 2010; Faubion et al., 1999; Graf et al., 2002; Guicciardi and Gores, 2002; Krahenbuhl et al., 1994; Reinehr et al., 2003). Since intracellular BS content is regulated by complex mechanisms involving BS uptake, synthesis, detoxification and export, transcriptional stimulation or inhibition of specific transport mechanisms may be critical in limiting intrahepatic retention of BSs and hepatocyte damage. The canalicular BS export pump (BSEP), basolateral efflux transporter organic solute transporters alpha and beta (OSTα/OSTβ) as well as the BS uptake system Na+-taurocholate cotransporting polypeptide (NTCP) are the major regulators of intracellular BS load and have emerged as promising drug targets in cholestasis.

Functional role of BSEP in health and disease

Canalicular excretion of BSs constitutes the rate-limiting step in hepatic BS excretion and represents the driving force for their enterohepatic circulation (Stieger and Beuers, 2011). BSEP (ABCB11), previously also known as sister of p-glycoprotein (sPgp) is a member of the canalicular ATP-binding cassette (ABC) transporter superfamily and represents the major canalicular BS export system (Childs et al., 1995; Gerloff et al., 1998; Nishida et al., 1991; Stieger and Beuers, 2011). Conjugated monovalent BSs are the major substrate for BSEP (Byrne et al., 2002; Gerloff et al., 1998; Hayashi et al., 2005; Noe et al., 2002; Stieger et al., 2000), among which taurochenodeoxycholic acid (TCDCA) is the preferred substrate followed by taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA) and glycocholic acid (GCA) (Noe et al., 2002).

Various BSEP mutations differently impact on transporter activity leading to a wide variety of clinical manifestations in humans (Ho et al., 2010; Kagawa et al., 2008). Complete loss of function BSEP mutations manifest as severe cholestasis in progressive familial intrahepatic cholestasis type 2 (PFIC2) (Jansen et al., 1999; Strautnieks et al., 1998, 2008) and patients carry a considerable risk for development of hepatobiliary malignancies (Knisely and Portmann, 2006; Knisely et al., 2006; Scheimann et al., 2007; Sheridan et al., 2012; Strautnieks et al., 2008) likely due to persistent cell injury by elevated concentrations of intracellular BSs and impairment of cell repair mechanisms (Knisely et al., 2006; Palmeira and Rolo, 2004; Sokol et al., 2006; Souza et al., 2008). In contrast, BSEP variants with mildly impaired transporter function manifest in form of benign recurrent intrahepatic cholestasis type 2 (BRIC2) (Kubitz et al., 2006; van Mil et al., 2004) and may have a pathogenetic role in acquired cholestatic syndromes such as intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI) (Dixon et al., 2009; Eloranta et al., 2003; Keitel et al., 2006; Kubitz et al., 2006; Lang et al., 2007; Meier et al., 2008; Pauli-Magnus et al., 2004; van Mil et al., 2004). Although BSEP is critical for the maintenance of biliary BS excretion and protection against intrahepatic BS accumulation in humans, BSEP deficiency in mice causes only mild and non-progressive cholestasis with 30% preserved biliary BS output (mainly as tetra-hydroxylated BSs). It is important to note that in addition to BSEP, canalicular transporters such as the multidrug resistance protein 1 (MDR1/ABCB1), the multidrug resistance-associated protein 2 (MRP2/ABCC2) and the breast cancer resistance protein (BCRP/ABCG2) may also mediate canalicular export of sulfated bivalent and unusual tetra-hydroxylated BSs (Akita et al., 2001; Janvilisri et al., 2005; Keppler et al., 1997; Lam et al., 2005; Mennone et al., 2010; Wang et al., 2001a) accounting for partially preserved biliary BS elimination in rodents.

In addition to genetic BSEP variants, BSEP represents a vulnerable target for inhibition by various endogenous hormones/metabolites, inflammation or drugs resulting in acquired cholestasis such as ICP, DILI, sepsis/endotoxin-induced cholestasis and cholestasis caused by total parenteral nutrition (TPN). The pathogenesis of ICP is complex, but hypersensitivity to female hormones or their metabolites is likely to be involved (Arrese et al., 2008). Inhibition of BSEP gene transcription by β-estradiol or inhibition of its functional activity through trans-inhibition and internalization by the estrogen metabolite estradiol-17β-glucuronide (Crocenzi et al., 2003; Gerloff et al., 2002; Stieger et al., 2000) are important mechanisms predisposing to estrogen-induced cholestasis (Barth et al., 2003; Yamamoto et al., 2006). Apart from female hormones, drugs such as cyclosporine, glibenclamide, rifamycin SV, rifampicin, indomethacin and bosentan (Byrne et al., 2002; Fattinger et al., 2001; Fouassier et al., 2002; Morgan et al., 2010; Noe et al., 2002; Ogimura et al., 2011; Stieger, 2010; Stieger et al., 2000) or constituents of TPN solutions (Li et al., 2012; Nishimura et al., 2005) may also inhibit BSEP-mediated BS export through either competitive inhibition (Stieger et al., 2000) or through mechanisms involving MRP2-dependent stimulation of BS-independent bile flow (Fouassier et al., 2002).

Notably, genetic or acquired BSEP dysfunction is also likely to predispose to biliary cholesterol precipitation, since biliary BSs are essential for cholesterol solubilization in bile. This may explain the high prevalence of gallstone disease (32%) in PFIC2 patients (Pawlikowska et al., 2010). In support of this hypothesis, patients with cholesterol gallstones show low expression of the BSEP upstream regulator farnesoid X receptor (FXR/NR1H4) (Bertolotti et al., 2006) and its coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α (Zhang et al., 2004), whereas FXR activation prevents gallstone formation in mice (Moschetta et al., 2004). In addition, reduced efflux of biliary BSs may lead to malabsorption of fat and fat-soluble vitamins and subsequently impair whole body energy homeostasis. Indeed, children with PFIC2 developed severe steatorrhoea (Walkowiak et al., 2006), whereas BSEP overexpression increased dietary cholesterol and fatty acid absorption and promoted development of diet-induced obesity in mice (Henkel et al., 2011; Wang et al., 2010). Finally, altered biliary BS elimination may have multiple extrahepatic implications, which may be attributed to the emerging key role of BSs in lipid, glucose and energy homeostasis (Thomas et al., 2009; Thomas et al., 2008; Wang et al., 2003a; Watanabe et al., 2006). Taken together, targeting mechanisms promoting BSEP-mediated BS export can be expected to have beneficial effects in cholestatic liver injury and its extrahepatic complications.

Transcriptional regulation of BSEP

Regulation of BSEP-mediated BS efflux is complex and involves transcriptional as well as posttranscriptional mechanisms (the latter discussed in chapter by J.L. Boyer). The nuclear BS receptor FXR acts as the major stimulator of BSEP transcription in humans, mice and rats through its heterodimerization with the retinoid X receptor alpha (RXRα) and binding to the FXR response element (FXRE), inverse repeat 1 (IR-1), in the BSEP promoter (Ananthanarayanan et al., 2001; Gerloff et al., 2002; Plass et al., 2002). BS-mediated BSEP expression is likely to be influenced by intracellular BS flux rather than steady state concentrations (Wolters et al., 2002) and differs dependent on the ability of endogenous BSs to activate FXR. Notably, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and cholic acid (CA) are strong FXR activators (Makishima et al., 1999; Parks et al., 1999; Wang et al., 1999), whereas the less potent FXR activator litocholic acid (LCA) even inhibited FXR-mediated BSEP transactivation by endogenous and synthetic FXR ligands (CDCA and GW4064 respectively) (Yu et al., 2002). Importantly, murine-specific primary BSs α- and β-tauromuricholic acid (TαMCA and TβMCA) were also identified as FXR antagonists and may be responsible for lower expression of FXR target genes in ileum of germ-free mice (Sayin et al., 2013). Furthermore, TβMCA biotransformation by gut microbiota may significantly influence BS synthesis as well as transport through mitigation of TβMCA-mediated FXR inhibition in gut (Sayin et al., 2013).

Recent studies identified the role of several nuclear receptor co-activating proteins as important mediators of FXR-mediated BSEP transcription. In particular, the key regulator of energy homeostasis PGC-1α and components of the activating signal cointegrator-2-containing complex (ASCOM) interact with FXR to enhance FXR-dependent BSEP expression in a ligand-dependent manner (Ananthanarayanan et al., 2011; Kanaya et al., 2004; Savkur et al., 2005). Importantly, recruitment of the ASCOM complex components to the BSEP promoter was disrupted in cholestasis induced by common bile duct ligation (CBDL) (Ananthanarayanan et al., 2011). This could provide a plausible explanation for paradoxal lack of BSEP induction in cholestasis, despite potential BS-dependent FXR activation by accumulating intrahepatic BSs. In addition, secondary co-activators such as co-activator-associated arginine methyltransferase 1 (CARM1) (Lee et al., 2005) and direct methylation of FXR by Set7/9 methyltransferase facilitate FXR/RXRα-dependent BSEP transcription (Ananthanarayanan et al., 2004; Balasubramaniyan et al., 2012). The recent discovery of FXR-mediated BSEP regulation through recruitment of a transcription co-activator, steroid receptor co-activator 2 (SRC2), provides a novel mechanistic link between intracellular energy depletion and fat absorption. Hepatocyte-specific SRC2−/− mice showed reduced BSEP gene expression and developed signs of cholestasis such as increased intrahepatic and serum BS levels and impaired dietary fat absorption, while dietary CA supplementation or restoration of hepatic BSEP expression by adenoviral vector rescued cholestatic phenotype in these mice (Chopra et al., 2011). Since SRC2 is phosphorylated and activated by the intracellular energy sensor AMP-activated protein kinase (AMPK), this study provided a novel functional crosstalk between hepatic energy depletion and FXR-mediated BS secretion to promote dietary fat absorption and further emphasizes the key role of BSEP in lipid metabolism (Chopra et al., 2011). In line with these findings, AMPK activation is directly involved in formation and maintenance of the hepatocyte canalicular network (Fu et al., 2010). Thus, AMPK activators such as the antidiabetic drug metformin and the antiischemic drug 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) may – apart from their direct action on intracellular metabolism – also have indirect beneficial effects through regulation of biliary BS secretion and promotion of cellular energy supplementation by BSs. In addition, AMPK activators are likely to have cytoprotective effects through promotion of AMPK–SRC2–BSEP-mediated elimination of BSs.

Apart from FXR, several other transcriptional factors also target BSEP gene expression. The human and mouse BSEP promoters contain a response element for the liver receptor homologue 1 (LRH1/NR5A2) (Song et al., 2008), an essential regulator of development, metabolism and BS synthesis (Fayard et al., 2004). LRH-1 mediates CDCA- or FXR-dependent maximal BSEP transcription, whereas its loss markedly reduces expression of hepatobiliary transporters BSEP, NTCP, MRP2 and multidrug resistance-associated protein 3 (MRP3/ABCC3) as well as nuclear receptors FXR and short heterodimer partner (SHP/NR0B2) (Lee et al., 2008; Mataki et al., 2007). Interestingly, the FXR target SHP negatively regulates LRH-1-mediated BSEP transactivation (Kerr et al., 2002), a finding that appears inconsistent with BS-FXR-dependent induction of BSEP. However, BSEP is a direct target of FXR and LRH-1, and therefore, FXR- or LRH-1-mediated induction is likely to dominate over BS-SHP-mediated repression (Song et al., 2008). Since phospholipids (PLs) bind LRH-1 (Krylova et al., 2005; Ortlund et al., 2005), they could in addition to their protective role against biliary BS toxicity (Cohen et al., 1994; Smit et al., 1993) also reduce intracellular BS toxicity through promotion of LRH-1-mediated canalicular export. In support of this assumption, dietary lecithin was protective in mice lacking the canalicular PL exporter multidrug resistance protein 2 (MDR2/ABCB4, human orthologue of MDR3) after dietary CA supplementation (Lamireau et al., 2007), but had no effects on development of spontaneous liver injury without BS challenge in this model (Baghdasaryan et al., 2008). Another important protective mechanism against BS toxicity is the upregulation of BSEP as part of the primary cell response to oxidative stress. Nuclear factor erythroid 2-related factor 2 (NRF2) mediates antioxidative defense through heterodimer formation with musculo-aponeurotic fibrosacroma (MAF) protein and binding to the MAF recognition element (MARE) in the promoter of target genes (Itoh et al., 1997). Two MAREs were identified in the human BSEP promoter and mutations of the predicted MARE1 region abolished BSEP induction by a potent NRF2 activator oltipraz (Weerachayaphorn et al., 2009). Thus, in addition to direct anti-oxidative effects, NRF2 activators may diminish liver injury through canalicular elimination of potentially toxic BSs. Apart from the above mentioned mechanisms, BSEP transcription is also induced by ligands of the pregnane X receptor (PXR/NR1I2) (Teng and Piquette-Miller, 2005), whereas several in vitro studies reported controversial effects on the glucocorticoid receptor (GR/NR3C1). Although the GR agonist dexamethasone was essential for BSEP expression in cultured rat primary hepatocytes (Warskulat et al., 1999), it failed to induce BSEP expression in sandwich cultured cells forming functional canalicular units (Turncliff et al., 2006).

Importantly, BSEP transcription is negatively regulated in pathological conditions such as inflammation or drug toxicity. A clear decrease of peri-portal BSEP was detected in CBDL- and lipopolysaccharide (LPS)-induced liver injury, whereas peri-central expression remained unchanged (Donner et al., 2007). BSEP restoration after blockade of tumor necrosis factor alpha (TNF-α or interleukin-1 beta (IL-1β (by etanercept or anakinra respectively) in models of CBDL-, LPS- and TPN-induced liver injury established a pathogenetic role of inflammatory cell-derived cytokines for BSEP inhibition (Carter and Shulman, 2007; Donner et al., 2007; Li et al., 2012). In support of this hypothesis, TNF-α, IL-1β and interleukin-6 (IL-6) reduced BSEP gene expression in human sandwich-cultured hepatocytes (Diao et al., 2010). Reduced binding of FXR/RXRα heterodimer to IR-1 (Geier et al., 2005a) and direct interaction of the nuclear factor kappa-B (NF-κB) subunits with FXR (Gadaleta et al., 2011) are the main mechanisms mediating cytokine-dependent BSEP inhibition. In addition, cytokines caused down-regulation of RXRα, an obligate partner for heterodimer formation with the major BSEP stimulator FXR and PXR (Beigneux et al., 2000, 2002; Pascussi et al., 2000c; Teng and Piquette-Miller, 2005). Notably, hepatotoxic effects of the antidiabetic drug troglitazone (agonist of peroxisome proliferator-activated receptor gamma (PPARγ)) are mediated through stable complex formation with the FXR ligand binding domain and suppression of FXR-mediated BSEP transactivation (Kaimal et al., 2009). However, this effect of troglitazone was PPARγ-independent as other PPARγ activators did not interfere with FXR activity.

Functional role of OSTα/OSTβ in health and disease

In addition to canalicular BS export, retrograde elimination/overflow of BSs into sinusoidal blood through basolateral efflux transporters represents an alternative rescue mechanism to reduce intracellular BS load. This process is mediated by several transport proteins with wide substrate specificity, such as the organic solute transporter (OST/SLC51), MRP3 and multidrug resistance-associated protein 4 (MRP4/ABCC4). Although MRP3 and especially MRP4 are induced and play a protective role in cholestasis (Mennone et al., 2006; Zelcer et al., 2006), MRP4 is likely to be regulated mainly at the posttranscriptional level (Denk et al., 2004). In contrast, transcriptional upregulation of OST is even more pronounced (Wagner et al., 2003; Zollner et al., 2003b), indicating that adaptive upregulation of OST may be the predominant mechanism of basolateral BS elimination. OST is composed of 2 proteins – OSTα and OSTβ – (Wang et al., 2001b) and is expressed on the basolateral surface of cell populations critically involved in BS transport/reabsorption: enterocytes, hepatocytes, bile duct lining cells – cholangiocytes and tubular renal cells (Ballatori et al., 2005; Dawson et al., 2005; Rao et al., 2008). OSTα/OSTβ expression in hepatocytes enables BS elimination into blood sinusoids, whereas in enterocytes it is critical for intestinal BS absorption and their enterohepatic circulation. Its expression in cholangiocytes serves cholangiocellular transport and intrahepatic recycling of BSs. Apart from BSs OSTα/OSTβ also mediates transport of molecules such as estrone 3-sulfate, dehydroepiandrosterone, digoxin and prostaglandine E2 (Ballatori, 2011; Ballatori et al., 2005; Dawson et al., 2010; Seward et al., 2003). Importantly, in contrast to humans, OSTα/OSTβ expression is very low in mouse liver (Ballatori et al., 2005; Dawson et al., 2005). Although hepatic OSTα/OSTβ is induced under cholestatic conditions both in patients and rodents (Boyer et al., 2006; Cui et al., 2009) its pathogenetic role in liver diseases remains poorly understood. Notably, OSTα deficiency in mice resulted in a reduced BS pool size and impaired cholesterol absorption without development of cholestatic phenotype or liver damage (Ballatori et al., 2008; Rao et al., 2008). Furthermore, experimental models of obstructive cholestasis and dietary BS supplementation showed attenuated liver injury in OSTα−/− mice through involvement of adaptive mechanisms in liver (phase I and II detoxification, phase III elimination through MRP4) and augmentation of renal BS excretion (Denk et al., 2004; Soroka et al., 2010, 2011). In addition to the above mentioned mechanisms, OSTα deficiency reduced BS pool size through retention of BSs in enterocytes and BS-FXR-fibroblast growth factor 15 (FGF15)-mediated inhibition of hepatic CYP7A1 (the rate-limiting enzyme in BS synthesis) (Lan et al., 2011, 2012; Soroka et al., 2011). Whether OSTα/OSTβ dysfunction may cause elevation of serum cholesterol levels and promote development of gallstone disease due to FGF19 (human orthologue of FGF15)-mediated inhibition of cholesterol utilization and decreased biliary excretion remains to be explored in detail. However, non-obese female gallstone patients had reduced intestinal OSTα/OSTβ mRNA as well as protein levels, which correlated with lower expression of FXR and its other targets apical sodium-dependent bile acid transporter (ASBT) and FGF19 (Renner et al., 2008). Given the fact that OSTα/OSTβ induction in liver will promote basolateral BS elimination, whereas its inhibition in gut will reduce BS pool size (through decreased absorption and synthesis), organ-specific modulation of OST-mediated BS transport appears to be an attractive therapeutic concept in cholestasis.

Transcriptional regulation of OSTα/OSTβ

OST-mediated BS transport requires co-expression of both subunits by 2 distinct genes: OSTα and OSTβ (Dawson et al., 2005; Seward et al., 2003; Wang et al., 2001b). Importantly, coexpression and heterodimerization of OSTα and OSTβ subunits and their mutual interaction enables the proper function of the transporter (Dawson et al., 2005; Seward et al., 2003). Specifically, normal OSTα gene and protein expression are critical for the proper function of OSTβ (Li et al., 2007; Rao et al., 2008; Sun et al., 2007), whereas OSTβ promotes trafficking of OSTα to the cell membrane and its functional activity (Christian et al., 2012; Wang et al., 2001b). Since OSTα and OSTβ protein levels correlate with respective transporter mRNA levels in human ileal biopsies, OSTα/OSTβ is believed to be regulated mainly at the transcriptional level (Renner et al., 2008). The key role of BS-FXR-mediated mechanism of OSTα/OSTβ gene regulation was uncovered in mice deficient in the ileal BS uptake transporter ASBT (ASBT−/− mice) (Dawson et al., 2005). Interestingly, blockade of BS uptake in ileum repressed OSTα/OSTβ expression in this part of the gut, whereas its expression in colon was induced due to high transporter-independent intracellular BS flux (Dawson et al., 2005). Indeed, OSTα/OSTβ gene transcription is directly controlled by FXR in human and mouse tissues (Landrier et al., 2006; Lee et al., 2006) and loss of FXR abolished OSTα/OSTβ induction by CA feeding (Zollner et al., 2006b). Two functional FXR-binding sites have been identified in the human OSTα and one in the OSTβ gene promoter (Landrier et al., 2006). Moreover, human OSTα/OSTβ is also induced by GR, whereas vitamin D receptor (VDR/NR1I1) and PXR are unlikely to play a key regulatory role for the human transporter (Khan et al., 2009). Of note, mouse OSTα/OSTβ gene expression may be induced by the liver X receptor (LXR/NR1H3) through binding to the elements of FXRE IR-1 (Okuwaki et al., 2007). In contrast, LRH-1 is responsible for negative regulation of mouse OSTα/OSTβ expression in response to BSs (Frankenberg et al., 2006). However, positive OSTα/OSTβ regulation by FXR appears to be dominant and LRH-1 is unlikely to play a critical role in baseline expression of OST transporters. Collectively, these findings suggest that FXR and GR ligands could have therapeutic potential to induce OSTα/OSTβ-mediated basolateral BS export from hepatocytes in humans.

Functional role of NTCP in health and disease

During their enterohepatic circulation, approximately 95% of biliary BSs are efficiently reabsorbed in intestine and return to liver where they are taken up by hepatocytes and undergo a further cycle of biliary excretion and subsequent enterohepatic circulation (Hofmann, 1999). Hepatic uptake of BSs takes place against a 5–10-fold concentration gradient at the basolateral surface of hepatocytes and is mediated through the Na+-taurocholate co-transporting polypeptide (NTCP/SLC10A1) (Ananthanarayanan et al., 1994; Hagenbuch et al., 1991; Stieger et al., 1994) and a group of Na+-independent organic anion transporting polypeptides (OATPs) (Nathanson and Boyer, 1991). NTCP mediates Na+-dependent uptake of all physiological BSs in their conjugated form (Stieger et al., 1994), estrone-3-sulphate, thyroid hormones (Meier and Stieger, 2002) and drugs bound to TCA, e.g., chlorambucil-taurocholate (Kullak-Ublick et al., 1997). In contrast to NTCP OATPs have a lower affinity for BSs (Na+-independent transport) and broader substrate specificity (Konig et al., 2000; Kullak-Ublick et al., 2001).

Although no mutations of NTCP have been identified as primary causative factor for human cholestasis, the NTCP variant Ser267Phe in Chinese-Americans showed a near complete loss of function of BS transport with preserved uptake of other substrates, thereby uncovering the critical role of this region for BS recognition (Ho et al., 2004). Furthermore, dependent on substrate specificity, NTCP variants may significantly impair the uptake of TCA and drugs such as statins, thereby limiting their clinical effectiveness (Choi et al., 2011; Pan et al., 2011). NTCP gene expression is reduced in human cholestatic conditions such as biliary atresia, advanced primary biliary cirrhosis (PBC) and cholestatic alcoholic liver injury (Shneider et al., 1997; Zollner et al., 2003a, 2001) as well as in several cholestatic animal models (Gartung et al., 1996; Lee and Boyer, 2000; Trauner et al., 1998; Zollner et al., 2003b). However, the pathophysiological role of altered NTCP expression in cholestasis remains unclear. Nevertheless, NTCP repression may contribute to impairment of bile formation, although this mechanism appears to reflect hepatocyte adaptation to increased intracellular BS levels.

Transcriptional regulation of NTCP

Repression of NTCP in response to BS challenge in normal/pathologic conditions is an important step in protection against intracellular BS overload since the majority of BSs excreted into bile are derived from the enterohepatic circulation. NTCP regulation differs considerably between species. Studies in knock-out or overexpression animal models identified the key transcriptional regulators of rodent NTCP such as homeodomain protein hepatocyte nuclear factor 1 alpha (HNF-1α), hepatocyte nuclear factor 4 alpha (HNF-4α), RXRα, hepatocyte nuclear factor-3β (HNF-3β) and FXR/SHP (Denson et al., 2001; Hayhurst et al., 2001; Jung and Kullak-Ublick, 2003; Karpen et al., 1996; Lee et al., 2000; Rausa et al., 2000; Shih et al., 2001). Despite virtually absent baseline NTCP expression in models of HNF-1α deficiency and deficiency of the binding site for retinoic acid receptor alpha (RARα/NR1B1/RXRα heterodimer, no respective response element was found in the minimal promoter of mouse NTCP (Geier et al., 2003b; Jung et al., 2004b). In contrast, HNF-4α directly binds to the murine NTCP promoter through functional HNF-4α-response element (Geier et al., 2008) and PGC-1α potentiates NTCP transactivation by HNF-4α. Since HNF-1α controls HNF-4α transcription, repressed NTCP expression in HNF-1α deficiency may reflect potentially HNF-4α-mediated mechanism (Hansen et al., 2002; Odom et al., 2004). Importantly, the human NTCP promoter is devoid of a TATA regulatory box (Shiao et al., 2000), which explains the absence of binding sites for respective transcriptional factors regulating murine NTCP. Among the known binding sites of NTCP promoter only the HNF-3β response element is shared by the mouse, rat and human sequence (Jung et al., 2004b). Human NTCP is also positively regulated by the GR, for which PGC-1α and components of co-activating complex ASCOM act as co-activators (Ananthanarayanan et al., 2011). In addition, cAMP response element members of the CCAAT/enhancer-binding protein (C/EBP) family were able to transactivate human NTCP (Jung et al., 2004b; Shiao et al., 2000), whereas C/EBP mutations reduced NTCP expression by 70% (Shiao et al., 2000).

Negative feedback regulation of murine NTCP by BSs is mediated through FXR-SHP-mediated prevention of its positive regulation by RXRα/RARα (Denson et al., 2001) or competition of SHP with co-activators for binding to HNF-4α and RXRα (Lee et al., 2000), whereas SHP-mediated negative interaction with GR and PGC-1α represent the main molecular mechanisms mediating BS-dependent repression of human NTCP (Eloranta et al., 2006). Notably, CA feeding repressed NTCP expression in SHP−/− mice, indicating that SHP-independent mechanisms may also be involved in mediating NTCP repression in cholestasis (Wang et al., 2003b). Since BSs inhibit human HNF-1α activation by HNF-4α in SHP-independent manner (Jung and Kullak-Ublick, 2003), this mechanism is also likely to be involved in NTCP repression in mice. In addition to BS-mediated mechanisms, endotoxin/cytokine-mediated inhibition of main NTCP regulators RARα/RXRα, HNF-4α and HNF-1α or their binding activity may also contribute to NTCP repression in cholestasis (Beigneux et al., 2000; Geier et al., 2005a, 2003b; Green et al., 1996; Hartmann et al., 2002; Moseley, 1999; Moseley et al., 1996; Trauner et al., 1998). Inhibition of c-Jun N-terminal kinase (JNK)-mediated RXRα phosphorylation is involved in reduced RARα/RXRα binding activity and NTCP downregulation by Il-1β (Li et al., 2002). In line with rodent data, IL-1β also suppressed human NTCP transcription and protein levels (Le Vee et al., 2008). However, cytokine inactivation or Kupffer cell depletion failed to restore inhibition of NTCP gene expression in mice (Geier et al., 2005b). In line with these findings, decreased nuclear HNF-1α and HNF4α protein levels and inhibited HNF-1α binding activity were also not restored after cytokine inactivation in CBDL (Geier et al., 2005b). Furthermore, reduced levels of the key regulators (HNF-1α, HNF-4α, RXR/RARα) did not differ between FXR/ and FXR−/− mice after CBDL or LCA feeding, despite pronounced difference in NTCP expression (Zollner et al., 2005). These findings indicate that BS-mediated mechanisms, but not cytokines play the predominant role in downregulation of NTCP in cholestasis (Geier et al., 2005b).

Finally, sex hormones also play an important role in regulation of NTCP gene expression as demonstrated by 2-fold higher expression in male compared with female rats. NTCP expression was repressed in pregnant female animals or after estradiol administration in males (Arrese et al., 2003; Bossard et al., 1993; Geier et al., 2003a; Simon et al., 1996), while ovarectomy resulted in increased NTCP expression in females (Simon et al., 2004). These data provides a potential molecular mechanism for sex hormone-induced cholestasis. Importantly, models of experimental hypophysectomy provided evidence for the role of the complex interplay of pituitary hormones in regulating NTCP expression by sex hormones (Simon et al., 2004). The pituitary gland hormone prolactin facilitates NTCP transcription through prolactin receptor-mediated binding of signal transducers and activators of transcription (STAT-5) to its two binding sites in the rat NTCP promoter (Ganguly et al., 1997), whereas estradiol repressed PLR-induced NTCP upregulation in human cells (Cao et al., 2004). Similarly, a STAT-5-mediated mechanism is involved in growth hormone-mediated upregulation of rat NTCP (Cao et al., 2001).

Taken together, transcriptional regulation of the main hepatobiliary BS transporters BSEP, OSTα/β and NTCP play an important role in cholestatic liver disease. The key positive and negative transcriptional regulators of human BSEP, OSTα/OSTβ and NTCP are summarized in Table 1.

Table 1

Transcriptional regulators of main bile salt transporters BSEP, OSTα/OSTβ and NTCP in humans.

Transporter	Positive regulators	Negative regulators
BSEP/ABCB11	FXR/NR1H4- Co-activators: - PGC-1α - Components of ASCOM complex - Set7/9 methyltransferase - CARM-1 - SRC2 LRH1/NR5A2- Potentiates FXR-mediated transactivation NRF2PXR/NR1I2	Pro-inflammatory cytokines TNF-α, IL-6 and IL-1β- Inhibition of FXR/RXRα binding activity - Direct interaction of NF-κB with FXR - Repression of RXRα Drugs (e.g., troglitazone)- Stable complex formation with FXR and suppression of FXR-dependent transactivation Female hormones- β-estradiol
OSTα/OSTβSLC51A/SLC51B	FXR/NR1H4GR/NR3C1	Not known
NTCP/SLC10A1	GR/NR3C1Co-activators:- PGC-1α - Components of ASCOM complex RARα/RXRαC/EBP	FXR- SHP-mediated negative interaction with RXRα, GR and PGC-1α IL-1β- Reduction of RARα/RXRα binding activity through inhibition of RXRα phosphorylation

Novel therapeutic strategies targeting transcription factors in cholestasis

Pharmacological stimulation of canalicular BS efflux through BSEP and basolateral elimination through OSTα/OSTβ or inhibition of hepatocellular BS uptake through NTCP via targeted stimulation of the key regulatory transcription factors may have important clinical applications and represents a promising direction for novel drug development in cholestasis. Several traditional drugs widely used to treat cholestatic liver disorders have turned out to act at least in part through activation of transcription factors regulating hepatobiliary transport and metabolism. Moreover, key regulatory transcription factors have also become the focus of targeted therapies in cholestasis.

Ursodeoxycholic acid and derivatives

Currently, pharmacological therapy of cholestatic liver diseases is limited to use of ursodeoxycholic acid (3α,7β-dihydroxy-5β-cholanoic acid or UDCA), which represents 1–3% of total BSs in human bile (Hofmann, 1994). Importantly, UDCA does not activate FXR (Makishima et al., 1999; Parks et al., 1999; Sato et al., 2008), but binds to GR (discussed below) (Miura et al., 2001) and is capable of activating PXR after its transformation to LCA by intestinal flora (Staudinger et al., 2001b; Xie et al., 2001). Anti-cholestatic effects of this drug are mediated through induction of BS-dependent as well as BS-independent bile secretion by several mechanisms (Jazrawi et al., 1994; Paumgartner and Beuers, 2004; Poupon and Poupon, 2000; Trauner and Boyer, 2003). These include induction of BSEP and MRP2 on the transcriptional level in mice (Fickert et al., 2001) as well as posttranscriptional mechanisms influencing vesicular transport, exocytosis and membrane insertion (Beuers et al., 2001; Dombrowski et al., 2006; Kubitz et al., 2004; Kurz et al., 2001; Milkiewicz et al., 2002). Interestingly, despite increased BSEP protein levels BSEP mRNA remained unchanged in non-cholestatic gallstone patients after UDCA administration, indicating that predominantly posttranscriptional mechanisms are involved (Marschall et al., 2005). In addition, UDCA restored pathologically reduced expression of the biliary exchange transporter anion exchanger 2 (AE2/SLC4A2) in PBC patients (Medina et al., 1997; Prieto et al., 1999) and its combination with glucocorticoids synergetically induced AE2 alternate promoter in human cells (Arenas et al., 2008). Furthermore, UDCA also has several immunomodulatory effects in vitro (Lacaille and Paradis, 1993; Nishigaki et al., 1996; Yamazaki et al., 1999; Yoshikawa et al., 1992). These pleiotropic mechanisms may explain the beneficial clinical effects of UDCA in a broad range of cholestatic disorders including PBC (biochemical and histological improvement and prolonged survival without liver transplantation) (Lazaridis et al., 2001; Paumgartner and Beuers, 2004; Poupon, 2011, 2012, 2000) and ICP (improved maternal pruritus, serum transaminases as well as neonatal outcomes) (Bacq et al., 2012; Brites, 2002; Glantz et al., 2005, 2008; Kondrackiene et al., 2005; Mazzella et al., 2001; Palma et al., 1997). However, standard doses of UDCA did not improve survival in primary sclerosing cholangitis (PSC) and high dose UDCA was even associated with higher risk of severe side effects and mortality (Lindor et al., 2009).

A side chain shortened derivative of UDCA, 24-norUDCA reduced serum liver enzymes, induced bile flow and improved hepatic inflammation and fibrosis in MDR2−/− (ABCB4−/−) model of sclerosing cholangitis (Fickert et al., 2006). Beneficial effects of 24-norUDCA in this model of liver injury were associated with coordinated induction of BS detoxification (through CYP2B10, CYP3A11 and SULT2A1) and basolateral export through alternative transporters MRP3 and MRP4 on transcriptional level without significant effects on BSEP or NTCP gene expression (Fickert et al., 2006). Importantly, in comparison with UDCA, 24-norUDCA is more resistant to amidation, which may promote its cholehepatic shunting and thereby contribute to increased secretion of protective . Clinical trials with 24-norUDCA for PSC have been already initiated and the results of a phase II randomized double-blind multi-center placebo-controlled study will provide first answers about clinical efficacy of this promising compound.

Importantly, derivatives of 24-norUDCA tauro-norUDCA and di-norUDCA do not share pharmacological properties of 24-norUDCA and failed to improve liver injury in the MDR2−/− model. Moreover, di-norUDCA even worsened liver injury, underlining the unique beneficial properties of 24-norUDCA due to its resistance to amidation and cholehepatic shunting (Halilbasic et al., 2009).

Therapeutic potential of FXR activators

Since FXR represents a master regulator of BS homeostasis and inflammation by targeting a broad spectrum of molecular mechanisms (Modica et al., 2012; Trauner et al., 2011; Wagner et al., 2011; Wang et al., 2008) its pharmacological activation may exert multiple protective mechanisms in cholestasis. The main mechanisms by which FXR activation may protect against cholestatic liver injury are summarized in Table 2. A significant proportion of FXR’s therapeutic effects in cholestasis may be due to the fact that FXR regulates gene expression of the key BS transporters including uptake transporter NTCP and export transporters BSEP and OSTα/OSTβ in addition to promoting BS detoxification through phase I hydroxylation by CYP3A4 (Gnerre et al., 2004), phase II sulfoconjugation by SULT2A1 (Song et al., 2001) and glucoconjugation by UGT2B4 (Barbier et al., 2003b). Thus, FXR activation by high affinity agonists with no side effects may represent an attractive therapeutic concept in cholestasis. Non-steroidal compounds GW4064, FXR-450 or WAY-362450 and PX20350 are potent FXR activators (Abel et al., 2010; Maloney et al., 2000; Mehlmann et al., 2009; Pellicciari et al., 2002). GW4064 induced BSEP, SHP, MRP2 and MDR2, inhibited NTCP, repressed BS synthesis and showed hepatoprotective effects in rat models of intrahepatic (alpha-naphthylisothiiocyanate administration) and extrahepatic (CBDL) cholestasis (Liu et al., 2003). Furthermore, GW4064 induced BS-independent bile secretion in human gallbladder through FXR-mediated induction of vasoactive intestinal peptide receptor 1 (VPAC1) transcription, the major regulator of bile secretion (Chignard et al., 2005). A recent study showed that GW4064 and PX20350 may also prevent/treat hepatocellular carcinomas through FXR-dependent induction of tumor suppressor gene NMYC downstrean-regulated gene 2 (NDRG2) (Deuschle et al., 2012). The currently available ligands for clinical trials represent chemical modifications of endogenous BSs (Table 3). Specifically, a semi-synthetic obeticholic acid (OCA) also known as 6-ethylchenodeoxycholic acid (6-ECDCA) or INT-747 was synthesized on the basis of CDCA backbone and is an approximately 100-fold more potent FXR activator (Pellicciari et al., 2002). OCA was beneficial in animal models of LCA-, estrogen-, carbone tetrachloride (CCl4)- or CBDL-induced cholestasis (Fiorucci et al., 2005; Pellicciari et al., 2002). It is important to consider that in contrast to these models of liver injury, dietary OCA supplementation deteriorated liver injury in the MDR2−/− model despite its strong FXR activating capacity (Baghdasaryan et al., 2011). Since biliary secretion of protective PLs is absent in MDR2−/− mice (corresponding to liver disease from MDR3 mutations in humans), increased biliary excretion of hydrophobic OCA and concominant reduction of endogenous BSs (in mice mainly in form of more hydrophilic muricholic acid) could result in a net increase of more hydrophobic BS proportion in bile, thereby promoting liver injury in this specific model. Recent clinical trials have shown beneficial effects of OCA treatment in patients with PBC. Short- and long-term combination of OCA with UDCA reduced serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in PBC patients biochemically not responding to UDCA treatment (Hirschfield et al., 2011; Mason et al., 2010). Moreover, OCA monotherapy significantly reduced serum ALT, ALP and gamma-glutamyl transpeptidase (GGT) levels in treatment-naive PBC patients (Kowdley et al., 2011). Itching was the most common adverse effect in patients receiving high dose OCA treatment. A phase III, double blind, placebo controlled trial and long term safety study of low-dose OCA treatment has been initiated in PBC patients.

Table 2

Molecular targets of FXR in liver and potential beneficial effects of FXR activation in cholestasis.

Molecular targets of FXR in liver	Effects in cholestasis
- Canalicular export of BSs through BSEP and MRP2 - Basolateral BS elimination via OSTα/OSTβ - Inhibition of NTCP- and OATP1B1-mediated BS uptake - Inhibition of BS synthesis (inhibition of CYP7A1 through FGF19- and SHP- mediated mechanisms, inhibition of CYP8B1and CYP27A1) - Promotion of BS detoxification (through upregulation of CYP3A4, SULT2A1, UGT2B4)	Reduction of intracellular BS toxicity
- Reduced biliary excretion of BSs through inhibition of synthesis - Canalicular PL secretion through MDR3 and promotion of mixed micelle formation - Promotion of HCO3- -rich bile secretion through carbonic anhydrase 14 - Induction of gallbladder secretion via VPAC-1	Reduction of bile toxicity
- Repression of pro-inflammatory gene expression through interaction with NF-κB	Inhibition of inflammation

Table 3

FXR ligands with potential clinical application.

Agonist	Clinical application	Comments
CDCA (Chenofalk TN)	Gallstone disease, cerebrotendinous xanthomatosis	Hepatotoxicity in rhesus monkey, but not in humans
OCA/6-ECDCA/INT-747 (semisynthetic derivative of CDCA)	PBC, NAFLD/NASH, bile acid diarrhoea, gallstone disease, portal hypertension	Beneficial in combination with UDCA (biochemically non-responders to UDCA) or as monotherapy (treatment-naïve patients) in PBC.Dose-dependent pruritus
INT-767 (synthetic derivative of OCA)	Preclinical	Preclinical: biochemical and histological improvement in MDR2−/− mouse model of sclerosing cholangitis
GW4064 (synthetic agonist)	Preclinical	Not further developed for clinical use
FXR-450/WAY-362450 (synthetic agonist)	NAFLD	Phase 1 trial in healthy Japanese men was terminated due to pharmacokinetic issues
PX-104	NAFLD	Phase 2 trial to assess the safety and tolerability in NAFLD patients

Further modifications of OCA led to synthesis of a semi-synthetic 23-sulfate-derivative 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt also called INT-767 (Rizzo et al., 2010). In contrast to OCA, INT-767 is a hydrophilic compound with higher affinity to FXR. In addition, INT-767 may also activate the membrane BS receptor TGR5/GPBAR1 (Rizzo et al., 2010). Importantly, INT-767 significantly reduced serum ALT levels, decreased portal inflammation and biliary fibrosis in the MDR2−/− animal model of sclerosing cholangitis (Baghdasaryan et al., 2011). These beneficial effects were mediated by several FXR-dependent hepatoprotective mechanisms including induction of BSEP mRNA and protein levels, promotion of BS-independent bile flow through promotion of protective secretion with simultaneous inhibition of BS synthesis and NTCP repression. Altogether, INT-767 treatment resulted in lower BS and higher output into bile. In addition, since INT-767 may also act as a potent activator of the membrane BS receptor TGR5, it may also induce bile secretory function though non-transcriptional cAMP signaling pathways (Kawamata et al., 2003; Maruyama et al., 2002). Thus, INT-767 represents a very promising new drug to treat non-obstructive cholestasis in humans. However, TGR5 expression is induced in cholangiocellular carcinoma, where it may confer resistance to apoptosis (Keitel et al., 2011), indicating that ligands which can also activate TGR5 have to be seen with some caution.

Notably, FXR activation induces hepatocellular proliferation in animal models (Baghdasaryan et al., 2011; Huang et al., 2006) which theoretically may raise concerns in regards to risk for tumor development. However, FXR directly regulates tumor suppressor NDRG2 (Deuschle et al., 2012) and may also prevent tumor development via its anti-inflammatory properties. Conversely, FXR and BSEP expression is reduced in hepatocellular (Chen et al., 2012; Su et al., 2012). Finally, stimulation of bile flow may have critical deleterious effects in obstructive cholestasis (Stedman et al., 2006; Wagner et al., 2003; Zollner et al., 2003a). Therefore, preserved biliary drainage will be necessary to fully achieve beneficial effects of FXR activators. However, since FXR is expressed in almost all tissues and regulates a broad range of metabolic pathways FXR ligands are likely to have several additional metabolic off-target effects. Finally, endogenous BSs as well as hepatobiliary transport systems differ considerably between species, which makes it difficult to predict the undesired effects of FXR activation in humans based on animal studies. Thus, carefully designed clinical studies are required to answer all these open questions.

Since frequent BSEP mutations are associated with impaired trafficking of BSEP from the ER to the plasma membrane, pharmaceutical targeting of post-transcriptional regulatory mechanisms represents another promising approach to reduce cholestasis (discussed in detail in chapter by J.L. Boyer). Interestingly, the natural ligands/substrates of BSEP, BSs, arise as perfect candidates to act as pharmacological chaperones promoting proper folding and trafficking of mutated BSEP (Misawa et al., 2012b). Novel derivatives of GW4064 compound enhanced BSEP transport activity through their function as chaperons and the 7c analog of the reverse-amide GW4064 derivative has been identified as selective BSEP function enhancer (Misawa et al., 2012a,b). Thus, separation of FXR-agonistic activity from BSEP function-enhancing activity will help to selectively target cholestatic liver injury without broad metabolic effects of FXR.

Therapeutic potential of CAR and PXR

In obstructive cholestasis, detoxification and basolateral elimination of BSs without induction of canalicular secretion may be critical. Genes encoding hepatocellular drug metabolizing enzymes and alternative transporters are direct targets of nuclear receptors such as constitutive androstane receptor (CAR/NR1I3) and PXR (NR1I2) (Chen et al., 2003b, 2004; Ferguson et al., 2005; Gerbal-Chaloin et al., 2002; Goodwin et al., 2002, 1999; Nebert and Jones, 1989), establishing a substantial role of these transcriptional factors in defense against toxic liver injury (Zollner et al., 2006a). In addition to stimulating bilirubin clearance (Chen et al., 2003a; Huang et al., 2003, 2004; Kast et al., 2002) and BS detoxification (Honkakoski et al., 1998; Saini et al., 2004; Sueyoshi et al., 1999) CAR activation promotes MRP4-mediated basolateral elimination of BSs (Chai et al., 2011). Importantly, CAR ligands phenobarbital and a component from Artemisia capillaris (dimethylesculetin) which is used in Chinese herbal teas Yin Zhi Huang and Yin Chin efficiently reduced pruritus and neonatal jaundice (Bachs et al., 1989; Bloomer and Boyer, 1975; Huang et al., 2004; Stiehl et al., 1972). However, CAR activation was hepatotoxic in mice despite significant reduction of serum BS and bilirubin levels (Wagner et al., 2005; Yamazaki et al., 2011). Furthermore, CAR ligands promoted hepatic tumor development (Hojo et al., 2012; Huang et al., 2005; Kiyosawa et al., 2008; Yamamoto et al., 2004) through upregulation of the anti-apoptotic effector myeloid cell leukemia factor 1 (MCL-1) gene expression (Baskin-Bey et al., 2006; Morita et al., 2011).

Apart from CAR, PXR may also be beneficial in cholestasis by induction of BS hydroxylation (Marschall et al., 2005; Xie et al., 2000), inhibition of BS synthesis (Staudinger et al., 2001a) and bilirubin clearance (Chen et al., 2003a; Huang et al., 2003, 2004; Kast et al., 2002) as well as inhibition of serum cholestatic enzyme autotaxin activity (Kremer et al., 2010, 2012). PXR ligand rifampicin improved pruritus in cholestatic patients (Bachs et al., 1989; Cancado et al., 1998; Yerushalmi et al., 1999) and reduced serum liver enzymes in PBC and animal models of liver injury (Staudinger et al., 2001a; Teng and Piquette-Miller, 2005, 2007; Xie et al., 2001). However, rifampicin therapy may be associated with development of hepatitis and an increase of serum liver enzymes in 7% of patients (Bachs et al., 1992; Prince et al., 2002). Apart from these undesired effects, induction of drug-metabolizing enzymes by CAR or PXR may induce generation of reactive metabolites and have critical importance for drug interactions (Goldstein and de Morais, 1994; Miners and Birkett, 1998). Together, these findings indicate that despite desired induction of BS detoxification and elimination mechanisms, CAR and PXR activation is associated with the risk of tissue damage and tumor development. Therefore, ligands of these transcriptional regulators, if strongly indicated, may be administered only for a short-term treatment. Furthermore, CAR antagonists may represent an interesting strategy to treat oxidative stress-mediated liver injury in humans (Kublbeck et al., 2011). However, further studies are required to establish the biological role of CAR inhibition in health and disease.

Therapeutic potential of GR, PPARα, PPARγ and VDR

In addition to FXR, CAR and PXR there is arising evidence for the role of additional transcriptional factors such as GR, peroxisome proliferator-activated receptors alpha (PPARα/NR1C1) and gamma (PPARγ/NR1C3) and VDR in cholestasis. Glucocorticoids, which are widely used in clinical practice due to their pronounced immunosuppressive action, but may also play an important role in cholestasis through transcriptional regulation of BS transporters NTCP, ASBT and OSTα/OSTβ (Eloranta et al., 2006; Jung et al., 2004a; Khan et al., 2009). Moreover, GR may interact with other transcription factors such as CAR, PXR and RXRα leading to synergetic stimulation of target gene expression (Pascussi et al., 2000a,b; Sugatani et al., 2005; Zimmermann et al., 2009).

Combination therapy of UDCA with glucocorticoids induced transporter AE2 gene expression in PBC (Arenas et al., 2008; Medina, 2011; Medina et al., 1997; Poupon, 2011; Prieto et al., 1993), which may also explain stimulation of bile secretion in patients with biliary atresia after Kasai operation (Meyers et al., 2003) and -dependent bile flow in rats (Alvaro et al., 2002). Compared with UDCA monotherapy combination of budesonide (a glucocorticoid with low adverse effects) with UDCA was more efficient with respect to reduction of serum liver enzymes and liver histology in early stage PBC (Leuschner et al., 1999; Rautiainen et al., 2005). However, budesonide monotherapy showed severe side effects and increase in Mayo risk score in UDCA non-responders (Angulo et al., 2000). Furthermore, budesonide treatment in stage 4 PBC patients was associated with portal vein thrombosis and death, thereby establishing an absolute contraindication for budesonide use in late-stage PBC (Hempfling et al., 2003). Notably, GR activation inhibited FXR transcriptional activity and led to cholestasis through recruitment of C-terminal binding protein (CtBP) co-repressor complexes and subsequent induction of BS synthesis and repression of hepatocellular transporters in mice (Lu et al., 2012). Furthermore, suppressed expression of FXR and FXR target genes by glucocorticoids (Rosales et al., 2013) may contribute to development of cholestasis as demonsrated by increased serum BS levels in patients with Cushing syndrome (Lu et al., 2012; Yamanishi et al., 1985). Interestingly, UDCA effects are partially mediated through GR activation (Tanaka and Makino, 1992; Tanaka et al., 1996). Interaction of UDCA with a specific part of the GR ligand-binding domain leads to loss of specific co-activator recruitment and subsequently weak induction of glucocorticoid response element (GRE) gene response while strongly inhibiting NF-κB target genes (Miura et al., 2001). Thus, dissociation of GR-dependent transcription of selective targets by UDCA may represent a very promising starting point for development of novel drugs with less undesired effects.

Agonists of PPARα, which are widely used as antihyperlipidemic drugs, can induce hepatic detoxification (Barbier et al., 2003a; Fang et al., 2005; Honda et al., 2013) and promote MDR3-mediated canalicular PL secretion (Ghonem and Boyer, 2013; Matsumoto et al., 2004; Roglans et al., 2004; Shoda et al., 2004), modify intestinal BS uptake (Jung et al., 2002) and inhibit BS synthesis (Marrapodi and Chiang, 2000; Patel et al., 2000; Post et al., 2001). Unlike CAR and PXR, direct induction of mitochondrial uncoupling protein 2 gene (UCP2) expression (Patterson et al., 2012) by PPARα may be protective against reactive metabolites and therefore might have a potential in the treatment of BS- and drug-induced toxicity. Indeed, combination of UDCA with PPARα agonists bezafibrate and fenofibrate improved serum liver tests in PBC patients with incomplete biochemical response to UDCA (Caroli-Bosc et al., 2001; Honda et al., 2013; Itakura et al., 2004; Kanda et al., 2003; Levy et al., 2011; Nakai et al., 2000) and even showed histological improvement as monotherapy (Kurihara et al., 2002, 2000; Yano et al., 2002). Since these studies have been conducted in a small number of patients and mainly after a short-term treatment, additional longer treatment studies with higher patient numbers are required to establish the long-term efficacy and safety of fibrate treatment in PBC.

In contrast to PPARα, PPARγ plays an important role for cholangiocyte injury (Harada et al., 2005) and is critical for the maintenance of a quiescent state of profibrogenic cells (Hazra et al., 2004a,b; She et al., 2005; Zhang et al., 2012). PPARγ activation in cholangiocytes and portal myofibroblasts by curcumin improved liver injury and biliary fibrosis in the MDR2−/− model of sclerosing cholangitis (Baghdasaryan et al., 2010). Interestingly, PPARγ ligand rosiglitazone improved LPS-induced liver injury by attenuating cytokine-mediated nuclear export of RXRα and restoring its binding activity in hepatocytes with subsequent induction of BSEP and NTCP in mice (Ghose et al., 2007). Since PPARγ is not expressed in hepatocytes, beneficial effects of rosiglitazone on hepatocellular transporter gene expression in vivo and in vitro may reflect indirect mechanisms involving potential effects of rosiglitazone metabolites, activation of additional signaling pathways or interaction with cytokines. Importantly, PPARγ agonists showed pronounced side effects and troglitazone was withdrawn from the market due to its hepatotoxic effects, which notably were PPARγ-independent (Funk et al., 2001; Snow and Moseley, 2007). Since PPARγ is unlikely to influence hepatocellular homeostasis, selective targeting of cholangiocytes and fibrogenic cells by PPARγ activators is likely to have major beneficial effects in portal inflammation and biliary fibrosis (Harada and Nakanuma, 2010).

Importantly, VDR variants were identified in PBC and autoimmune hepatitis (Fan et al., 2005; Halmos et al., 2000; Tanaka et al., 2009; Vogel et al., 2002). They reduced VDR protein levels and were shown to be inversely correlated with histological severity of nonalcoholic steatohepatitis and viral hepatitis C (Barchetta et al., 2012). However, CDCA-mediated induction of BSEP expression was impaired by VDR interaction with FXR in vitro, indicating potential undesired effects of VDR activation in BS toxicity (Honjo et al., 2006). Given the fact that VDR directly inhibits activation and proliferation of hepatic stellate cells (Abramovitch et al., 2011) and proinflammatory cytokine expression (Ogura et al., 2009), VDR activation is likely to improve cholestasis via improvement of inflammation and fibrosis but not through direct modulation of hepatic BS transport systems. In addition, induction of antibacterial cathelicidin gene expression by VDR in biliary epithelium may be essential for biliary tract sterility and help to prevent/treat cholangiopathies (D’Aldebert et al., 2009). Since limited absorption of fat-soluble vitamins may affect Vitamin D levels in cholestatic patients, VDR activators may also have a beneficial role to prevent/treat osteoporosis in cholestatic patients.

Conclusions

Transcriptional regulators of hepatocellular transport systems have emerged as promising drug targets with extraordinary potential in the treatment of cholestatic liver disease. Novel and (compared with endogenous BSs) less toxic FXR activators may counteract cholestasis through induction of biliary and sinusoidal BS export, promotion of BS-dependent and BS-independent bile flow, inhibition of BS synthesis and anti-inflammatory effects. Since FXR has very broad tissue and organ distribution and its activation might also impact on multiple mechanisms regulating glucose and lipid homeostasis, selective BSEP or OSTα/OSTβ inducers (via FXR) may represent the future direction for novel FXR-directed drug development. GR activators are predicted to be beneficial to control disease flare and acute inflammation, but long-term glucocorticoid therapy will have critical undesired effects. As an alternative, novel GR agonists with an ability to selectively block the NF-κB signaling and limited or absent side effects may represent a future strategy for GR activation in chronic cholestasis. PXR and CAR agonists, which prevent accumulation of cytotoxic BS by induction of their detoxification and basolateral elimination, represent an attractive treatment strategy in obstructive cholestasis. Nevertheless, clinical use of these ligands might be limited due to potential hepatotoxic and tumor promoting effects. Finally, drug interactions and potential impact of other transcriptional factor/nuclear receptor-directed drugs has to be evaluated in depth to minimize potential adverse effects and improve therapeutic efficiency of novel drugs.

Funding

This work was supported by grant F3517 from the Austrian Science Foundation (to MT) and SFB35 project part 3509 (to PC).