Interaction of Bile Acids and the Gut Microbiome Dig Dis 2015;33:357–366 DOI: 10.1159/000371688

Nuclear Receptors in Acute and Chronic Cholestasis Ester Gonzalez-Sanchez Delphine Firrincieli Chantal Housset Nicolas Chignard INSERM UMR_S 938, Centre de Recherche Saint-Antoine, and Sorbonne Universités, UPMC Université Paris 06, Paris, France

Abstract Background: Nuclear receptors (NRs) form a family of 48 members. NRs control hepatic processes such as bile acid homeostasis, lipid metabolism and mechanisms involved in fibrosis and inflammation. Due to their central role in the regulation of hepatoprotective mechanisms, NRs are promising therapeutic targets in cholestatic disorders. Key Messages: NRs can be classified into five different physiological clusters. NRs from the ‘bile acids and xenobiotic metabolism’ and from the ‘lipid metabolism and energy homeostasis’ clusters are strongly expressed in the liver. Furthermore, NRs from these clusters, such as farnesoid X receptor α (FXRα), pregnane X receptor (PXR) and peroxisome proliferator-activated receptors (PPARs), have been associated with the pathogenesis and the progression of cholestasis. The latter observation is also true for vitamin D receptor (VDR), which is barely detectable in the whole liver, but has been linked to cholestatic diseases. Involvement of VDR in cholestasis is ascribed to a strong expression in nonparenchymal liver cells, such as biliary epithelial cells, Kupffer cells and hepatic stellate cells. Likewise, NRs from other physiological clusters with low hepatic expression, such as estrogen receptor α

© 2015 S. Karger AG, Basel 0257–2753/15/0333–0357$39.50/0 E-Mail [email protected] www.karger.com/ddi

(ERα) or reverse-Erb α/β (REV-ERB α/β), may also control pathophysiological processes in cholestasis. Conclusions: In this review, we will describe the impact of individual NRs on cholestasis. We will then discuss the potential role of these transcription factors as therapeutic targets. © 2015 S. Karger AG, Basel

Introduction

Cholestasis is defined by an impairment of bile flow leading to the accumulation of toxic compounds that are normally excreted into bile. Retained toxins will induce liver damage, which is then followed by biliary fibrosis, cirrhosis and finally end-stage liver disease. Currently, medical therapy for cholestatic liver diseases is limited to ursodeoxycholic acid (UDCA), and liver transplantation is often required in patients with end-stage disease. Thus, the development of new therapeutic approaches is eagerly awaited. Because alterations in nuclear receptor (NR) signaling may contribute to the pathogenesis and progression of cholestasis, these transcriptions factors have emerged as promising therapeutic targets in cholestatic disorders. In this review, we will summarize the current knowledge regarding the role of NRs in cholestatic liver diseases. Nicolas Chignard, PhD UPMC, CdR Saint-Antoine, UMR_S 938 Faculté de Médecine Pierre et Marie Curie, site Saint Antoine 27 rue Chaligny, FR–75571 Paris cedex 12 (France) E-Mail nicolas.chignard @ upmc.fr

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Key Words Nuclear receptors · Cholestasis · Liver cells

NRs represent the largest family of transcriptional regulators in animals [1]. In humans, the NR superfamily comprises 48 transcription factors that directly modulate the expression of genes involved in reproductive, developmental, metabolic and immune response activities. Members of the NR superfamily present a conserved structure consisting of a NH3-terminal ligand-independent activation domain (AF1 domain), a central DNAbinding domain which allows binding to specific DNA sequences (hormone response elements), a hinge region and a C-terminal ligand-binding domain responsible for receptor dimerization, ligand recognition and cofactor interaction. NRs bind to hormone response elements as monomers, homodimers and heterodimers with retinoid X receptors (RXRs) [1]. To carry out their functions, NRs bind a wide variety of natural and/or synthetic lipophilic ligands that include endogenous compounds, such as steroid hormones, fatty acids, bile acids and vitamins [2, 3]. In the absence of a ligand, NRs are either located in the cytoplasm or in the nucleus. When in the nucleus, unliganded NRs are constitutively bound to their respective hormone response elements, but remain inactive by corepressor complexes. Once bound to a ligand, NR conformational change leads to the dissociation of corepressor complexes and to the recruitment of tissue-specific coactivators, thus eliciting gene transcription. Due to their key role in modulating gene expression, NR signaling dysfunction leads to proliferative, reproductive and metabolic diseases. Consistently, NR agonists or antagonists are used in clinical practice. As an example, thiazolidinediones, agonists of peroxisome proliferatoractivated receptor γ (PPARγ), are used for the treatment of type 2 diabetes, whereas agonists of the glucocorticoid receptor (GR), like dexamethasone, are used to treat inflammatory conditions [4]. Thus, NRs constitute a potential reservoir of therapeutic targets in a wide range of human diseases. Members of the NR superfamily interact with each other to form complex signaling networks that control physiological and pathophysiological processes. In this context, the characterization of tissue and disease-specific NR profile may shed light on important regulatory pathways of biological functions. To address the latter point, the relative expression level of the 49 mouse NRs was determined in 39 tissues [5]. The unsupervised analysis of NR expression by hierarchical clustering led to a classification into a cluster comprising physiological functions of repro358

Dig Dis 2015;33:357–366 DOI: 10.1159/000371688

duction, development and growth, and another cluster including nutrient uptake, metabolism and excretion. In each cluster, NRs have been included in different subordinated clusters according to their specific function: (1) steroidogenesis, (2) reproduction and development, (3) bile acid and xenobiotic metabolism, (4) lipid metabolism and energy homeostasis, and (5) central nervous system (CNS), circadian and basal metabolic functions [5]. These observations indicate that NRs may have redundant general functions and that individual variations may have limited impact. Thus, a complete analysis of NR expression profiles may contribute to better understand the role of these transcription factors in different pathophysiological processes.

NRs in Liver Pathophysiology

NRs play a central role in the regulation of liver metabolic pathways, such as lipid and glucose metabolism, endo- and xenobiotic detoxification, and bile acid homeostasis. NRs also participate in the regulation of key processes involved in liver pathophysiology, including inflammation, cell differentiation, liver regeneration, fibrosis and carcinogenesis [6]. NR Profile in Liver Recently, we have analyzed the complete profile of NRs in C57BL/6J and FVBn mice liver. Our results indicate that despite differences in individual gene expression, both genetic backgrounds present similar hepatic NR expression profile in terms of physiological clusters. In accordance with previous studies, we have shown that in both mice strains the most highly expressed NRs belong to the ‘bile acid and xenobiotic metabolism‘ and ‘lipid metabolism and energy homeostasis’ clusters [5, 7, 8]. Individual members of ‘reproduction and development’ and ‘CNS, circadian and basal metabolic functions’ clusters present medium-low expression, whereas mRNA levels of ‘steroidogenesis’ cluster members ranged from very low to undetectable (table 1). The liver is a complex tissue made of different cell types like hepatocytes, biliary epithelial cells (BECs), Kupffer cells (KCs), endothelial cells and hepatic stellate cells (HSCs). Notably, nonparenchymal cells (i.e. BECs, KCs, endothelial cells and HSCs) represent only 30% of the total cells in the liver, but play critical roles in cholestatic features, such as bile secretion, inflammation and fibrosis. As nonparenchymal cells are less abundant than hepatocytes, NR expression in these cells may be masked when analyzGonzalez-Sanchez/Firrincieli/Housset/ Chignard

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The NR Superfamily

Table 1. Nuclear receptor expression in mouse liver Cluster/ Abreviation

Nomenclature

Hepatic expression

NPCs expression BECs KCs

HSC

LSECs

+++ +++ +++ +++ +++ – ++ ++ –

* * + * * * * * +

– – + – + * – – *

* – + * * * + * –

– – + – + * – + *

Lipid metabolism and energy homeostasis COUP-TFγ NR2F6 Chicken ovalbumin upstream promoter transcription factor γ TRβ NR1A2 Thyroid hormone receptor β PPARα NR1C1 Peroxisome proliferator-activated receptor α PPARβ/δ NR1C2 Peroxisome proliferator-activated receptor β/δ RXRα NR2B1 Retinoic X receptor α ERRα NR3B1 Estrogen related receptor α GCNF NR6A1 Germ cell nuclear factor TR2 NR2C1 Testicular orphan receptor 2 PPARγ NR1C3 Peroxisome proliferator-activated receptor γ LXRα NR1H3 Liver X receptor α GR NR3C1 Glucocorticoid receptor PNR NR2E3 Photoreceptor-cell-specific nuclear receptor

++ ++ +++ +++ +++ ++ + ++ + ++ ++ –

* * * + * * * * + * * *

+ – – * + + * * + + – *

* * * + + * * * + + * *

+ – – * + + * * + + + *

CNS, circadian and basal metabolic functions LXRβ NR1H2 Liver X receptor β RXRβ NR2B2 Rettinoic X receptor β MR NR3C2 Mineralocorticoid receptor TR4 NR2C2 Testicular orphan receptor 4 TLX NR2E1 Tailless homolog orphan receptor NOR-1 NR4A3 Neuron-derived orphan receptor 1 NGF1-B NR4A1 Nerve-growth-factor-induced gene B RXRγ NR2B3 Retinoic X receptor γ RORα NR1F1 RAR-related orphan receptor α REV-ERBα NR1D1 Reverse-Erb α RARβ NR1B2 Retinoic acid receptor β ERRγ NR3B3 Estrogen related receptor γ ERRβ NR3B2 Estrogen related receptor β RORβ NR1F2 RAR-related orphan receptor β REV-ERBβ NR1D2 Reverse-Erb β NURR1 NR4A2 Nur-related factor 1 TRα NR1A1 Thyroid hormone receptor α COUP-TFα NR2F1 Chicken ovalbumin upstream promoter transcription factor α

++ +++ + + – – + + ++ ++ + + – – – – + +

* * * * * * * * * * * * * * * * * *

+ + * + * * + – – + + * * * – * * +

+ * * * * * * * * * * * * * * * * *

+ + * + * * + + + + + * * * + * * +

Reproduction and development AR NR3C4 ERα NR3A1 COUP-TFβ NR2F2 RARα NR1B1 RARγ NR1B3 ERβ NR3A2 PR NR3C3

Androgen Receptor Estrogen receptor α Chicken ovalbumin upstream promoter transcription factor β Retinoic acid receptor α Retinoic acid receptor γ Estrogen receptor β Progesteron receptor

+ ++ ++ ++ + – –

* + * * * + *

+ – + + + * *

* * * + * * *

+ – + + + * *

Farnesoid X receptor β Steroidogenic factor 1 Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1

– – –

* * *

* * *

* * *

* * *

Bile acid and xenobiotic metabolism CAR NR1I3 PXR NR1I2 FXRα NR1H4 HNF4α NR2A1 LRH-1 NR5A2 HNF4γ NR2A2 SHP NR0B2 RORγ NR1F3 VDR NR1I1

Steroidogenesis FXRβ SF-1 DAX-1

NR1H5 NR5A1 NR0B1

Name

Constitutive androsterone receptor Pregnane X receptor Farnesoid X receptor α Hepatocyte nuclear factor α Liver receptor homologe-1 Hepatocyte nuclear factor γ Small heterodimer partner RAR-related orphan receptor γ Vitamin D receptor

NRs in Acute and Chronic Cholestasis

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NRs hepatic expression level: undetectable (–); low (+); medium (++); high (+++). NRs non-parenchymal liver cells expression level: undetectable (–); detectable (+); no data available (*). Non-parenchymal liver cells (NPCs); biliary epithelial cells (BECs); hepatic stellate cells (HSCs); Kupffer cells (KCs) and endothelial cells (LSECs).

NRs in Cholestatic Liver Diseases NRs regulate different aspects of bile acid homeostasis, including bile acid synthesis, import, conjugation and export, as well as phospholipid and sterol transport, sterol metabolism and organic anion efflux [10]. NRs also participate in the regulation of central mechanisms involved in the progression of chronic liver disease, such as inflammation or fibrosis [11]. Therefore, alterations in NR signaling may contribute to the pathogenesis and progression of cholestatic diseases. Thus, the study of NR expression and function in the liver may lead to a better understanding of the pathophysiological features in cholestatic liver diseases. Here, we will review the most relevant data regarding the role of NRs in cholestasis. NRs will be described according to their physiological cluster classification. Because members of the ‘steroidogenesis’ cluster are not expressed in liver, these NRs will not be discussed herein. Bile Acid and Xenobiotic Metabolism Cluster Farnesoid X Receptor. The Farnesoid X receptor (FXR) has been identified as the main NR for bile acids. FXR plays a central role in the regulation of the enterohepatic circulation and intracellular accumulation of bile acids. In the liver, FXR inhibits bile acid synthesis and uptake through short heterodimer partner (SHP) activation and also stimulates bile acid detoxification pathways [12]. FXR expression is decreased in patients with cholestatic diseases, such as progressive familial intrahepatic cholestasis 1 and 2 (PFIC1 and PFIC2), biliary atresia and late-stage primary biliary cirrhosis (PBC) [13–15]. In chronic cholestasis, adaptive mechanisms, such as repression of uptake systems (Na+-taurocholate cotransporting polypeptide, NTCP, and organic anion transporter polypeptide 2, OATP2) and induction of basolateral efflux systems (multidrug resistance-related proteins 3 and 4, MRP3 and MRP4, and organic solute transporters α/β, OSTα/β) are elicited in support of bile acid elimination. Interestingly, FXR was shown to contribute to these adaptive changes in animal models of cholestasis [16], However, reduced liver injury is observed in Fxr–/– mice submitted to experimen360

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tal cholestasis [i.e. bile duct ligation or (BDL)] [17, 18]. These observations suggest that the use of FXR agonists may represent effective therapeutic approaches for the treatment of chronic cholestasis, preferably without biliary obstruction. Furthermore, FXR activation may also limit hepatic inflammation by inhibiting the NF-κΒ-mediated inflammatory response [19], while restricting fibrosis through the modulation of HSC activity and the reduction of profibrotic gene expression [20]. Thus, anticholestatic, anti-inflammatory and antifibrotic properties make FXR an attractive therapeutic target in cholestasis. Consequently, the therapeutic potential of FXR agonists in PBC has been evaluated in clinical trials. In PBC patients not responding to UDCA therapy, the semisynthetic bile acid derivative 6-ethyl chenodeoxycholic acid (6-ECDCA, INT-747 or obeticholic acid) allows for an improvement of biochemical markers of liver damage and cirrhosis in phase II clinical trials [21–23]. Except for dose-dependent itching, no major adverse events have been described in PBC patients under obeticholic acid. Taken together, these observations suggest that FXR is a strong potential therapeutic candidate in cholestasis. Short Heterodimer Partner. The orphan NR SHP is a transcriptional repressor, which mediates FXR inhibitory effects by interfering with other NRs, such as liver X receptor (LXR), liver receptor homolog-1 (LRH-1) and hepatocyte nuclear factor 4α (HNF4α) [12]. Reduced SHP mRNA levels have been found in patients with PBC and PFIC1 [14, 15]. Mice invalidated for Shp expression showed increased sensitivity to cholestatic liver injury [24]. Conversely, SHP overexpression may reduce cholestatic liver fibrosis by inhibiting Egr-1 signaling [25], which is activated by bile acids to induce inflammation and fibrosis. Thus, SHP may represent an interesting therapeutic target in acute or chronic cholestatic diseases. Pregnane X Receptor and Constitutive Androstane Receptor. The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are central in the detoxifying pathways involving phase I and II enzymes and phase III transporters. Both NRs coordinate protective hepatic responses to toxic stimuli induced by a broad range of endogenous compounds (bile acids, bilirubin) and xenobiotics. The protective effect of PXR and CAR in cholestasis is mediated by their ability to inhibit bile acid synthesis [26] and to activate detoxification pathways [27, 28]. Consistently, low PXR and CAR expression in patients with biliary atresia leads to a poorer prognosis [29]. PXR and CAR mRNA levels are also reduced in late-stage PBC patients [15]. Moreover, PXR polymorphisms have been associated with increased susceptibility to intrahepatic Gonzalez-Sanchez/Firrincieli/Housset/ Chignard

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ing whole liver. For example, vitamin D receptor (VDR) expression is generally accepted to be negative in liver, while it is indeed expressed in BECs, KCs, endothelial cells and HSCs [9]. Thus, analysis of the NR profile in each hepatic cell type in different pathophysiological settings would help to better characterize the molecular mechanisms involved in cholestatic diseases and could lead to the identification of new potential therapeutic targets.

NRs in Acute and Chronic Cholestasis

Liver Receptor Homolog-1. LRH-1, which is highly expressed in liver, participates in bile acid homeostasis by reducing the expression of different hepatobiliary transporters and NRs, such as FXR and SHP [47]. In LRH-1-deficient mice, a more hydrophilic bile acid pool composition has been found [48]. Thus, absence of LRH-1 may either sensitize (by reducing bile acid transport) or protect (by modifying the bile acid pool) liver against cholestatic injury. This latter point needs to be clarified in order to determine the potential of LRH-1 in the treatment of cholestatic disorders. Lipid Metabolism and Energy Homeostasis The role of PPARs in cholestasis has been well documented. Moreover, other NRs from this cluster, such as thyroid hormone receptor β (TRβ), GR and LXRα, have also been related to cholestasis. Peroxisome Proliferator-Activated Receptors. The PPARs form a family of three NRs, namely PPARα, PPARβ/δ and PPARγ. PPARs are activated by endogenous ligands, such as fatty acids, phospholipids and eicosanoids. All PPARs are expressed in the liver where they control fatty acid metabolism, cell proliferation and inflammation. Activation of PPARs with fibrates has anticholestatic activity in patients with PBC under UDCA therapy [49]. The anticholestatic effect of fibrates is linked to a direct control of the multidrug resistance protein 3 (MDR3 or ABCB4) gene expression by PPARα [49, 50]. Indeed, activation of PPARα leads to increased phosphatidylcholine excretion in vitro [50], suggesting a protective mechanism against bile toxicity in vivo. Furthermore, PPARα may limit bile acids deleterious effects by decreasing CYP7A1 expression, while increasing CYP3A4 expression [49]. Moreover, PPARα activation has anti-inflammatory effects on liver cells by inhibiting TNFα expression elicited by lipopolysaccharide [51]. Taken together, these observations suggest that PPARα activation could be protective in chronic cholestatic settings. However, activation of PPARα by fibrates in Abcb4–/– mice aggravates liver injury. Furthermore, mice invalidated for both PPARα and Abcb4 are protected from liver injury when compared to Abcb4–/– mice [52]. The latter observations shed light on the diversity of cholestatic pathological entities and suggest that a better definition of the NRs involved in the pathophysiology of cholestatic diseases is needed. PPARβ/δ is expressed in the liver by parenchymal cells but mostly by nonparenchymal cells [53, 54]. During BDL, PPARβ/δ expression increases in BECs [53], but no clear pathophysiological role has been ascribed to such modifications. The expression of PPARβ/δ in KCs and HSCs suggests that activation of PPARβ/δ Dig Dis 2015;33:357–366 DOI: 10.1159/000371688

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cholestasis of pregnancy [30], but also to primary sclerosing cholangitis and PBC progression [31, 32]. In animal models of obstructive cholestasis, both NR expression levels are induced and the activation of their target genes elicits protective pathways in hepatocytes. Consistently, hepatic damage is increased in both Car–/– and Pxr–/– mice after BDL [18]. PXR may also protect liver from cholestasis-induced damage by modulating inflammation and fibrosis. Indeed, PXR inhibits NF-κΒ leading to reduced inflammation in the liver [33]. In addition, PXR activation reduces proliferation and transdifferentiation of HSC and thus inhibits the expression of α-Sma and TGF-β [34]. Ligands of PXR and CAR, such as rifampicin and phenobarbital, have long been used for the treatment of cholestasis and pruritus. However, broader targeting of these NRs may be limited by undesirable side effects. Vitamin D Nuclear Receptor. VDR is involved in the control of ion homeostasis as well as in immune regulation, cell interactions and cell proliferation. Polymorphisms of VDR have been identified in human cholestatic conditions, such as PBC [35–39]. The direct impact of VDR polymorphisms on PBC pathogenesis, however, is unclear, even though VDR may protect the liver from potential pathogenic triggers by controlling innate immune mechanisms [40]. In mice, absence of VDR worsens hepatic damage due to acute obstructive cholestasis (i.e. BDL) by limiting the protection given by the epithelial barrier formed by BECs [41]. Mice submitted to a vitamin D-supplemented diet are protected from hepatic damage after BDL [41]. Furthermore, hepatic fibrosis is inversely correlated with vitamin D intake in mice invalidated for Abcb4, a well-known model of primary sclerosing cholangitis [42]. Consistent with an effect of vitamin D on liver fibrosis, VDR is the most highly expressed NR in HSCs [43]. Furthermore, VDR activation is able to deactivate HSCs through epigenetic modifications [44]. Taken together, these observations suggest that VDR may represent a therapeutic target in acute or chronic cholestatic diseases. Hepatocyte Nuclear Factor 4 α. HNF4α regulates the expression of different genes responsible for bile acid synthesis and may also participate in bile acid homeostasis indirectly by activating other transcriptional factors and NRs such as CAR, PXR, FXR and PPARγ [10]. HNF4α may also modulate biliary fibrosis [45], even though the molecular mechanisms are unclear. In late-stage PBC patients, HNF4α expression is reduced [15]. Furthermore, polymorphisms in the coding sequence of HNF4α have been related to PBC progression [46]. Yet, no pharmacological approaches have been developed to target HNF4α in cholestatic liver diseases.

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models of cholestasis [66], which may explain the deleterious effects observed in PBC patients treated with UDCA and glucocorticoid combination. Liver X Receptor α. LXRα is highly expressed in several tissues including the liver. In mice, LXRα induces the expression of Cyp7a1, but may also stimulate the expression of Sult2a9 and Mrp4 [67]. Thus, LXRα activation was protective in mice submitted to BDL, even though this effect was restricted to females. Because LXRα modulates inflammatory responses in macrophages, LXRα activation reduces liver injury induced by endotoxins by preventing TNFα and prostaglandin E2 release from KCs [68]. Thus, LXRα is a promising target for the treatment of cholestatic diseases through its role on bile acid homeostasis and its anti-inflammatory effects. CNS, Circadian and Basal Metabolic Functions Cluster NRs from the CNS, circadian and basal metabolic functions cluster display highly variable hepatic expression levels. While the expression of tailless homolog orphan receptor (TLX), neuron-derived orphan receptor 1 (NOR-1) and Nur-related factor 1 (NURR1) is undetectable, chicken ovalbumin upstream promoter-transcription factor α (COUP-TFα), estrogen-related receptor β (ERRβ), estrogen-related receptor γ (ERRγ), LXRβ, mineralocorticoid receptor (MR), nerve-growth-factor induced gene B (NGF1-B), TRα, retinoic acid receptor β (RARβ), retinoic acid receptor-related orphan receptor α (RORα), retinoic acid receptor-related orphan receptor β (RORβ), reverse-Erb α (REV-ERBα),reverse-Erb β (REVERBβ), RXRβ, RXRγ, and testicular orphan receptor 4 (TR4) are expressed at low-to-moderate levels in the liver. Currently, the information on the role of NRs from this cluster in cholestasis is still scarce. Estrogen-Related Receptor γ. ERRγ is constitutively activated in the absence of estrogen and competitively inhibits the estrogen receptor (ER)-dependent effects of estrogen. Despite low hepatic expression, ERRγ is expressed in BECs and treatment with estrogen reduces its expression [69]. Notably, ERRγ expression is increased in women suffering from PBC. Enhanced ERRγ expression was shown to increase susceptibility to apoptosis of BECs in patients with PBC via activation of proapoptotic Bcl-2 family members [69]. As ERRγ overexpression in BECs may contribute to PBC progression, therapeutic approaches able to modulate its expression may have beneficial effects in the treatment of PBC. Liver X Receptor β. LXRβ participates in the regulation of bile acid homeostasis [10] and may thus play a protecGonzalez-Sanchez/Firrincieli/Housset/ Chignard

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may be of interest in the context of inflammatory fibrosis. Indeed, PPARβ/δ agonists have hepatoprotective and antifibrotic effects in mice submitted to carbon tetrachloride intoxication [54]. However, the effect of PPARβ/δ activation could not be ascribed to a modulation of KCs or HSCs biology [54]. Thus, the potential to target PPARβ/δ in cholestatic settings remains to be evaluated. Finally, PPARγ expression is decreased in the damaged BECs of PBC livers [55]. Furthermore, PPARγ expression decreases in KCs during BDL, resulting in a limited inhibition of inflammatory cytokine production by PPARγ activation [56]. These observations suggest that PPARγ activation has a protective role against inflammation in cholestasis. This assumption is further supported by the observation that activation of PPARγ leads to a decreased expression of VCAM-1 on BECs of Abcb4–/– mice [57]. Consistently, the use of a PPARγ agonist decreases liver damage, cholestasis and fibrosis in models of acute or chronic cholestasis [57, 58]. Overall, these observations suggest that PPARγ, by anti-inflammatory properties targeting mostly nonparenchymal liver cells (i.e. BECs and KCs), may represent a therapeutic target in cholestatic settings. Thyroid Hormone Receptor. TRs mediate the effect of triiodothyronine (T3). In the liver, TRs controls physiological functions, such as bile acid homeostasis and ammonia metabolism. TRβ is expressed by perivenous hepatocytes [59], but also by BECs [60]. In rats submitted to BDL, T3 limits the ductular reaction by inhibiting BECs proliferation [60]. However, the effect of T3 on the proliferation of BECs is mediated by intracellular pathways linked to calcium [60]. Thus, the exact role of TRβ in a cholestatic setting remains to be elucidated. Glucocorticoid Receptor. GR plays a role in a large number of metabolic pathways, including carbohydrate and protein homeostasis, anti-inflammatory responses and bile acid homeostasis. Indeed, GR regulates the expression of several bile acid transporters, including NTCP, the apical sodium-dependent bile acid transporter and organic solute transporters α/β. GR may also modulate the expression of other NRs, including CAR, PXR and RXRα [61, 62]. Activation of GR by glucocorticoids in patients with PBC under UDCA therapy has led to anti-inflammatory and anticholestatic effects [63]. The anticholestatic effect of GR activation is in part linked to its ability to stimulate the expression of the bicarbonate carrier AE2, thus increasing BECs bicarbonate secretion [64]. The promising clinical benefits of GR ligands are, however, often limited by well-known severe side effects, increased bile acid synthesis and an increased incidence of gallstone disease [65]. Indeed, GR activation represses FXR activation in mouse

Relative mRNA expression

25 20

a

0

Sham BDL

15 10 5

35

WT Abcb4–/–

72 h BDL were used as a model of acute cholestasis. b FVBn-Abcb4–/– male mice were investigated as a model of chronic cholestasis. NR expression was determined by RT-QPCR in whole liver samples. The relative mRNA level in each physiological cluster represents the sum of individual NR expression.

25 20 15 10 5 0

b

Bile acid and xenobiotic metabolism

Lipid metabolism and energy homeostasis

CNR, circadian and basal metabolic functions

Reproduction and development

Steroidogenesis

tive role in cholestasis. Double KO mice deficient in both Lxrα/β isoforms exhibit increased sensitivity to cholestasis-induced liver damage [67]. Moreover, LXRβ also exerts anti-inflammatory effects in KCs by preventing TNFα and prostaglandin E2 release [68], suggesting a potential role in cholestatic settings. Reverse-Erb α and Reverse-Erb β. REV-ERBα/β are involved in lipid and glucose metabolism, adipogenesis and circadian control. Both isoforms may participate in bile acid homeostasis by regulating the expression of Cyp7a1 [70, 71]. Furthermore, REV-ERBα ligand SR6452 decreases expression of fibrogenic markers in HSCs, thus ameliorating fibrosis in rodent models [72]. This data identifies REV-ERBα as a novel regulator of HSC transdifferentiation and suggests this NR as a potential therapeutic target in chronic cholestatic diseases.

Reproduction and Development Cluster Hepatic expression levels of NRs from this cluster are low or undetectable. However, some members of this cluster may play crucial roles in the molecular mechanisms leading to cholestatic liver disease. For example, ERα and ERβ have been described to stimulate BEC proliferation both in vitro and in vivo. Although not expressed in normal liver, ERα and ERβ have increased expression in BECs of patients with PBC [73]. Increased ERs expression was also observed in other hepatic diseases characterized by BEC injury and proliferation [74]. Because PBC typically affects women after menopause, a combination of low estrogen levels and decreased ERs expression may directly lead to a defect in BEC proliferation, setting the pace for ductopenia [73]. Moreover, activation of ERα may inhibit FXR function during pregnancy, leading to intrahepatic cholestasis of

NRs in Acute and Chronic Cholestasis

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Fig. 1. a C57BL/6J male mice subjected to

Relative mRNA expression

30

pregnancy in genetically predisposed individuals [75]. Thus, therapeutic strategies targeting ERs receptors may have the potential to improve the status of patients’ cholestatic diseases.

Perspectives

NRs, by being able to modulate expression of genes involved in a wide variety of biological activities, represent highly valuable potential therapeutic targets. In cholestasis, NRs have been involved in the pathogenesis or progression of the disease. Indeed, NRs from the ‘bile acid and xenobiotic metabolism’ cluster govern adaptive mechanisms that are able to protect the liver from the deleterious effects of cholestasis. In this context, FXR agonists have been successfully transferred to the clinics to serve as a medical therapy for PBC. Other NRs from other physiological clusters may also represent potential therapeutic targets. However, information regarding the involvement of these NRs in cholestasis may be lacking. As an example, ERs are not expressed in the normal liver, but expression arises in cholestatic settings. Consequently, ERs may represent potential therapeutic targets in cholestatic diseases. The latter observation illustrates the need to better define expression levels of NRs in cholestatic settings. We have thus analyzed NR profiles in acute (i.e. BDL) and chronic (i.e. Abcb4–/– mice) cholestasis. As shown in figure 1, our

results indicate specific modifications of NR expression in cholestasis according to both the physiological cluster and the type of cholestatic disease. NR expression may also be restricted to specific cell types in the liver as documented for VDR. Thus, NRs may be of low expression when considering the whole liver, but of significant expression in cell types involved in the pathogenesis or progression of cholestasis. In this context, NR profiling in all liver cells whether parenchymal (i.e. hepatocytes) or nonparenchymal (i.e. KCs, BECs, HSCs) would be of particular interest. In conclusion, NRs represent extremely valuable potential targets in cholestatic diseases. A better definition of the involvement of individual members of this superfamily in cholestatic settings will expand the opportunities to develop new therapeutic strategies.

Financial Support This work was supported by ‘Fond CSP Vaincre la Cholangite Sclérosante Primitive’. Ester Gonzalez-Sanchez is recipient of a postdoctoral fellowship from the Spanish Association for the Study of the Liver (AEEH).

Disclosure Statement The authors disclose no conflicts.

References

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