FXR, a multipurpose nuclear receptor

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FXR, a multipurpose nuclear receptor Florence Y. Lee1*, Hans Lee1*, Melissa L. Hubbert1, Peter A. Edwards1,2,3 and Yanqiao Zhang1,2 1

Department of Biological Chemistry, University of California at Los Angeles, CA 90095, USA Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles, CA 90095, USA 3 The Molecular Biology Institute, University of California at Los Angeles, CA 90095, USA 2

The farnesoid X receptor (FXR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. In the past six years, remarkable inroads have been made into determining the functional importance of FXR. This receptor has been shown to have crucial roles in controlling bile acid homeostasis, lipoprotein and glucose metabolism, hepatic regeneration, intestinal bacterial growth and the response to hepatotoxins. Thus, the development of FXR agonists might prove useful for the treatment of diabetes, cholesterol gallstones, and hepatic and intestinal toxicity. Introduction In 1995, Forman et al. [1] and Seol et al. [2] isolated a novel cDNA that encoded an ‘orphan’ nuclear receptor. At the time it was named the farnesoid X receptor (FXR) on the basis of its weak activation by farnesol and juvenile hormone III [1], and it has been subsequently classified as NR1H4. There are two known FXR genes, which are commonly referred to as Fxra and Fxrb. Fxra is conserved from humans to fish (teleost fish, Fugu rubripes) [3]. The single Fxra gene in humans and mice encodes four FXRa isoforms (FXRa1, FXRa2, FXRa3 and FXRa4) as a result of the use of different promoters and alternative splicing of the RNA [4,5] (Figure 1a,b). FXRa3 and FXRa4 possess an extended N terminus, which encompasses the poorly defined ‘activation function 1 domain’. In addition, FXRa1 and FXRa3 have an insert of four amino acids (MYTG) immediately adjacent to the DNA-binding domain in a region referred to as the ‘hinge domain’ (Figure 1b). Many FXR target genes are regulated in an isoform-independent manner; however, a few genes, including those encoding intestinal bile acid binding protein (IBABP), syndecan-1, aA-crystallin and fibroblast growth factor 19 (FGF19), are more responsive to the FXRa2 and FXRa4 isoforms lacking the MYTG motif than to FXRa1 and FXRa3 [5–7] (Table 1). Nonetheless, the physiological importance of gene activation by specific FXR isoforms remains to be established. FXRa is expressed mainly in the liver, intestine, kidney and adrenal gland, with much lower levels in adipose tissue [1,4,5]. Like many other non-steroid hormone nuclear receptors, FXRa binds to specific DNA response elements as a heterodimeric complex with the retinoid X receptor [8,9] (Figure 1c). Corresponding authors: Edwards, P.A. ([email protected]); Zhang, Y. ([email protected]). * Authors contributed equally to this review. Available online 14 August 2006. www.sciencedirect.com

The second FXR gene, Fxrb, encodes a functional member of the nuclear receptor family in rodents, rabbits and dogs, but is a pseudogene in human and primates [8]. FXRb has been proposed to be a lanosterol sensor, although its physiological function remains unclear. In 1999, specific bile acids were identified that both bind to the ligand-binding domain of FXRa and potently activate the transcription of FXRa target genes [10–12]. These effects were noted at micromolar concentrations of bile acids. Because serum contains similar concentrations of bile acids, these studies demonstrated for the first time that bile acids function as hormones. Subsequent studies led to the identification of potent synthetic FXRa agonists including GW4064 [13] and fexaramine [14], compounds such as AGN34 that function as gene-selective agonists or antagonists depending on the target gene [15], and natural compounds such as guggulsterone that function as FXRa antagonists [16]. The mechanism of gene activation that follows binding of these agonists to the ligand-binding domain of FXRa is beyond the scope of this review. In addition to activating FXR, bile acids have several other functions including (i) facilitation of lipid and fatsoluble vitamin absorption; (ii) activation of three other nuclear receptors, the pregnane X receptor (PXR) [17], the vitamin D receptor [18] and the constitutive androstane receptor (CAR) [19,20]; (iii) activation of the c-Jun N-terminal kinase (JNK) cascade [21,22]; (iv) regulation of the mitogen-activated protein kinase pathway [23]; and (v) activation of TGR5, a G-protein-coupled receptor [24]. Watanabe et al. [25] recently discovered a particularly exciting connection between bile acids, TGR5 and obesity. They demonstrated that administration of cholic acid to mice results in resistance to diet-induced obesity owing to activation of TGR5 in brown adipose tissue, induction of uncoupling protein 1, and enhanced energy expenditure. Despite these many intriguing properties of bile acids, in this review we focus on FXRa, a nuclear receptor that is sometimes called the ‘bile acid receptor’. The generation of mice deficient in Fxra (hereafter referred to as Fxr) [26], the identification of FXR target genes, and the availability of synthetic FXR-specific agonists [13–15] have provided important insights into the mechanisms by which this nuclear receptor controls many diverse metabolic pathways. There are several excellent reviews on bile acid synthesis and metabolism and on FXR [9,27–29]. Here, we focus specifically on recent developments in our understanding of the regulatory role of FXR in bile acid synthesis, lipoprotein metabolism, liver

0968-0004/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2006.08.002

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Figure 1. Genomic organization and protein isoforms of FXRa. (a) Structure of the Fxra gene. The 11 exons of the mouse and human Fxra genes are indicated, along with the two functional promoters that initiate transcription at exons 1 or 3. Alternative splicing of the initial RNAs produces four mRNAs. The alternative splicing of the 12 bp at the 30 end of exon 5, which encode the MYTG motif, is indicated by the dark blue box. Asterisks indicate the translational start sites (ATG). (b) The four FXR protein isoforms with the different domains color-coded. Abbreviations: AF, activation function; DBD, DNA-binding domain; LBD, ligand-binding domain. (c) FXR binds to FXR response elements, such as the inverted repeat separated by one nucleotide (IR-1), as a heterodimer with the retinoid X receptor (RXR). Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; FXRE, FXR response element; RA, retinoic acid.

regeneration, glucose metabolism, protection from hepatotoxic agents, and repression of bacterial overgrowth in the intestine. FXR and bile acid metabolism Catabolism of cholesterol to bile acids and their subsequent excretion in the feces is the body’s principal means of eliminating cholesterol. Bile acid synthesis is restricted to hepatocytes and occurs via two distinct pathways: the ‘classic’ or neutral pathway, and the ‘alternative’ or acidic pathway [29]. Once synthesized, bile acids are conjugated to amino acids (taurine or glycine) before being secreted www.sciencedirect.com

into the bile canaliculi (Figure 2). Bile acids, cholesterol, phospholipids and small amounts of bile pigments and proteins constitute bile, which is stored in the gall bladder until it is secreted into the small intestine in response to dietary fat. Bile is involved in the emulsification and absorption of dietary lipids and fat-soluble vitamins from the small intestine. As bile acids travel down the small intestine and reach the distal ileum,
95% are reabsorbed and transported back to the liver via the enterohepatic circulation (Figure 2). Because
5% of the bile acids are eliminated in each enterohepatic cycle, the liver must synthesize an equivalent amount to maintain a constant

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Table 1. Genes regulated by activated FXRaa Organ Liver

Function Bile acid and lipid metabolism

Lipoprotein metabolism

Hepatoprotection c

Glucose metabolism

Small intestine

Bile acid resorption

Antibacterial

Kidney

Adrenal gland

Bile acid transport

Unknown

Gene product BSEP

Regulatory effect Induced

Refs [39]

MRP2 MDR3 (human), Mdr2 (mouse) SHP BAT BACS NTCP CYP7A1 CYP8B1 SREBP-1c ApoC-II

Induced Induced

[40] [41,42]

Induced Induced Induced Repressed Regulated Repressed Repressed Induced

[31,33] [50] [50] [43] [21,22,31,33–38] [35] [59,60] [56]

PLTP SDC-1 b VLDLR HL ApoC-III ApoA-I Pon1 CYP3A4 PXR aA-crystallin b SULT2A1 (human), Std (rat) UGT2B4 C3 PEPCK

Induced Induced Induced Repressed Repressed Repressed Repressed Induced Induced Induced Induced

Reviewed [88] Reviewed Reviewed Reviewed Reviewed [57,58] Reviewed [75] [6] [51]

Induced Induced Induced and Repressed Repressed

[49] Reviewed in Ref. [28] [67,69,70]

Induced

[44]

OSTa, OSTb FGF15, FGF19 b ASBT Antiogenin Carbonic anhydrase 12 Inducible nitric oxide synthase OSTa, OSTb

Induced Induced Repressed Induced Induced

[7,46,47] [7,37] [45] [74] [74]

Induced

[74]

Induced

[7,46,47]

MRP2 ASBT OSTa, OSTb

Induced Repressed Induced

[40] [45] [7,46,47]

SHP

Induced

[31,33]

Glucose-6phosphatase IBABP b

in Ref. [9] in in in in

Ref. Ref. Ref. Ref.

[28] [28] [28] [28]

in Ref. [28]

[69,70]

a

Many of the genes are regulated by FXRa in a tissue-specific manner. The induced genes have been identified as direct FXRa target genes as determined by the identification and characterization of FXR response elements. The mechanisms involved in repression have not been identified for many of the genes listed. Other FXR-regulated genes include those encoding kinninogen, apoE and fibrinogen. b The gene indicated is preferentially regulated by FXRa2 and FXRa4. c Including bile acid detoxification and xenobiotic clearance.

pool of bile acids. Thus, bile acid metabolism is a tightly regulated process, and its perturbation affects cholesterol homeostasis across the whole body [29]. Research in the past ten years has established that FXR has a crucial role in regulating all aspects of bile acid metabolism. Although it has long been known that bile acids can inhibit their own synthesis via a negative feedback pathway (reviewed in Refs [22,29]), the mechanism involved in this feedback inhibition was not understood until the discovery of FXR and the subsequent identification of its specific target genes. Cholesterol 7a-hydroxylase, the rate-limiting enzyme in the classic pathway of bile acid synthesis, is encoded by www.sciencedirect.com

CYP7A1 [22,29]. In rodents, basal expression and induction of Cyp7a1 are dependent on, respectively, the transcription factor liver receptor homolog 1 (LRH1; NR5A2) and the oxysterol-activated liver X receptor a (LXRa; NR1H3) [22]. By contrast, the human CYP7A1 gene lacks a LXR response element and thus is unresponsive to oxysterols [30]. A rise in intracellular bile acid levels results in an increase in binding of bile acids to the ligand-binding domain of FXR and in transcriptional activation of its target genes (Figure 2). One such hepatic FXR target gene is the small heterodimer partner (SHP; NR0B2) [31,33], an atypical member of the nuclear receptor superfamily that lacks a DNA-binding domain [32].

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Figure 2. Overview of the enterohepatic circulation of bile acids. Shown is the hepatic synthesis of cholesterol (C) and its subsequent conversion to bile acids (BAs). Bile acids directly bind and activate both hepatic and intestinal FXRa, resulting in the transcription of target genes. Genes that are induced or repressed after activation of FXRa are shown in yellow boxes or green ovals, respectively. Bile acids are synthesized in hepatocytes, conjugated with taurine or glycine via BACS and BAT, and secreted across the bile canalicular membrane by two ABC transporters (BSEP and MRP2). They are stored in the gall bladder before being excreted into the intestinal lumen, where they function to emulsify dietary lipids. Roughly 95% of the BAs in the intestinal lumen are reabsorbed via ASBT. Bile acids in the enterocytes bind FXRa and increase the expression of IBABP and two transporters, OSTa and OSTb, that facilitate BA transport into the portal vein. Activation of intestinal FXRa also increases the expression and secretion of mouse FGF15 into the portal vein. The subsequent binding of FGF15 to the hepatic cell-surface receptor FGFR4 activates the JNK pathway, leading to repression of Cyp7a1 and Cyp8b1. Repression of hepatic Cyp7a1 and Cyp8b1 is also mediated by SHP, another FXR target gene (see text). Also highlighted is the increased expression of angiogenin, carbonic anhydrase 12 and inducible nitric oxide synthase (iNOS) in the enterocytes. These proteins are not involved in enterohepatic circulation but are discussed in the text. Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; lithcholic acid; PL, phospholipids.

SHP can dimerize with and inactivate both LRH1 and LXRa, resulting in a decrease in Cyp7a1 expression [31,33,34] (Figure 2). Support for this model comes from studies showing that treatment of Shp/ mice with a potent, synthetic FXR agonist (GW4064) fails to repress Cyp7a1 mRNA levels [35,36]. Another pathway that regulates bile acid production is initiated after activation of FXR in enterocytes; this activation results in enhanced transcription and secretion of fibroblast growth factor 15 (FGF15) [37]. Subsequent binding of FGF15 to fibroblast growth factor receptor 4 (FGFR4), a transmembrane tyrosine kinase receptor localized on the hepatocyte cell surface, results in activation of the JNK pathway and repression of Cyp7a1 and Cyp8b1 [37] (Figure 1 and Table 1). A clue to the importance of FGF15 in this pathway came from the earlier observation that both Cyp7a1 expression and the bile acid pool are increased in Fgfr4/ mice [38]. www.sciencedirect.com

Importantly, hepatic Cyp7a1 mRNA levels are repressed after administration of bile acids to Shp/ mice [35,36]. The relationship between SHP and FGF15 repression of Cyp7a1 is unclear. In addition, alternative pathways of repression of Cyp7a1 have been described. One such alternative pathway might involve the release of cytokines from Kupffer cells, the resident macrophages in the liver, in response to bile acids; the cytokines subsequently activate JNK in hepatocytes, thereby leading to repression of Cyp7a1 [22]. Indeed, earlier studies have shown that addition of bile acids to hepatocytes represses CYP7A1 transcription as a result of activation of the JNK–c-Jun pathway and that this repression is independent of FXR [21]. Taken together, these data indicate that expression of Cyp7a1 is tightly regulated by multiple pathways to ensure that bile acid, and thus cholesterol, homeostasis is maintained. In addition to Cyp7a1 and Cyp8b1, FXR also regulates genes involved in (i) bile acid (e.g. BSEP, MRP2) [39,40]

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and phospholipid (e.g. human MDR3 or mouse Mdr2) [41,42] secretion across the bile canalicular membrane; (ii) bile acid transport (ASBT, NTCP, IBABP, OSTa– OSTb) [7,43–48]; and (iii) bile acid conjugation and detoxification (e.g. SULT2A1, UGT2B4, BACS, BAT) [49–51] (Figure 2; Table 1). These findings suggest that FXR is the primary bile acid sensor that coordinately regulates bile acid metabolism. FXR, lipoprotein metabolism and atherosclerosis In the early 1970s, individuals affected with gallstones were orally treated with chenodeoxycholic acid, which it was hoped would enhance the concentration of bile acids in the bile and thus slowly ‘solubilize’ the cholesterol-rich stones. Unexpectedly, this treatment led to a reduction in plasma triglyceride levels both in individuals suffering from gallstones and in those with hypertriglyceridemia [52,53]. The molecular mechanism underlying the hypotriglyceridemic effect of orally administered bile acids has now been elucidated and found, at least in rodents, to be linked to FXR. Sinal et al. [26] originally proposed that FXR might control plasma lipid levels on the basis of their observation that Fxr/ mice show an increase in plasma triglycerides and cholesterol. Fxr/ mice have also been shown to have higher plasma levels of high-density lipoprotein (HDL) cholesterol, consistent with reduced hepatic expression of SR-B1 [54,55], the scavenger receptor that facilitates clearance of HDL cholesterol from blood. Notably, administration of FXR agonists to normal rats and mice reduces plasma triglyceride levels [13,56]. Furthermore, activated FXR alters the transcription, either directly or indirectly, of several genes involved in fatty acid and triglyceride synthesis and lipoprotein metabolism [9,57–60] (Table 1). Metabolic studies have also demonstrated that activation of FXR leads to a decrease in the secretion of very-low-density lipoprotein (VLDL) from the liver [59,61,62]. These results – together with the finding that activation of FXR leads to repression of SREBP-1c [59,60], a transcription factor that controls genes involved in fatty acid and triglyceride synthesis – provide a mechanism to account for the triglyceride-lowering effects of bile acids and synthetic FXR agonists. Two recent studies have reported the effect of loss of FXR on atherosclerosis. Hanniman et al. [63] have demonstrated that mice lacking both FXR and apolipoprotein E (Fxr/apoE/ mice) have increased levels of atherosclerosis compared with apoE/ mice, consistent with their hyperlipidemia [63]. By contrast, a recent study by Zhang et al. [55] has shown that male mice lacking the low-density lipoprotein receptor (LDLR) and FXR (Fxr/Ldlr/ mice) have less atherosclerosis than do Ldlr/ mice [55]. This decrease in atherosclerosis correlates with a reduction in plasma LDL levels in the double knockout mice. Thus, the effect of loss of FXR on atherosclerosis is dependent on the genetic background of the mice. FXR and glucose homeostasis Several studies have linked the regulation of carbohydrate metabolism to FXR [64–68]. Indeed, one report concluded that activation of FXR results in the induction of www.sciencedirect.com

phosphoenolpyruvate carboxykinase (PEPCK) expression and an increase in glucose output from primary hepatocytes, but does not affect plasma glucose levels in wild-type mice [67]. However, three recent reports have now provided direct evidence that activation of FXR in wild-type or diabetic db/db or KKA-(y) mice promotes hypoglycemia and increases insulin sensitivity [69–71]. Consistent with these data, Fxr/ mice show impaired glucose tolerance and insulin resistance [69–71]. Because the expression of constitutively active FXR in the livers of either wild-type or Fxr/ mice also results in hypoglycemia [70], it seems that hepatic FXR has an important role in glucose homeostasis. Activation of FXR results not only in hypoglycemia, but also in altered hepatic expression of PEPCK and glucose-6phosphatase [69,70] (Table 1), raised hepatic glycogen levels, and increased hepatic signaling downstream of the insulin receptor [70]. Fxr/ mice show impaired signaling in response to insulin in muscle [69,71] and white adipose tissue [71]. FXR is not expressed in muscle, however, and has only very low expression in white adipose tissue. In addition, no FXR target gene has been identified in white adipose tissue in mice after the oral administration of FXR agonists. One possibility is that the changes in lipids (especially free fatty acids) in blood or muscle might affect insulin sensitivity in all three tissues. Regardless, the relative importance of the liver, muscle and adipose tissue in FXR-dependent hypoglycemia remains to be determined. The observations that glucose levels in Fxr/ mice are unchanged [70], increased [69] or repressed [71] as compared with wild-type littermates indicate that other crucial factors have yet to be identified. It is possible that differences in experimental procedures and/or the genetic backgrounds of the mice account for at least some of these variations. Nonetheless, the finding that activation of FXR in diabetic mice results in significant reductions in plasma cholesterol, triglyceride and/or glucose levels [70,71] seems particularly intriguing. These effects warrant further investigation in order to elucidate the underlying molecular mechanisms and to assess whether they are applicable to individuals with type 2 diabetes. FXR and the control of intestinal bacterial growth Interruption of bile flow by bile duct ligation or disease results in bacterial proliferation in the small intestine and bacterial translocation. Notably, these effects are attenuated in rats after the oral administration of bile acids [72,73]. Recently, Inagaki et al. [74] provided an explanation for this protective effect of bile acids by demonstrating that intestinal FXR has a crucial role in limiting bacterial overgrowth and thus protecting the intestine from bacterial damage. Inagaki et al. [74] studied Cyp27/ mice, which have impaired bile acid synthesis and thus low levels of FXR activation and FXR target gene expression. They found that treatment of Cyp27/, but not Fxr/, mice with a specific FXR agonist (GW4064) repressed bacterial overgrowth and translocation, and attenuated mucosal injury [74]. Activation of intestinal FXR by GW4064 led to the identification of several novel intestinal FXR target genes including those encoding angiogenin, carbonic anhydrase 12 and inducible nitric oxide synthase [74]

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(Figure 2; Table 1). Because these genes have been reported to have antibacterial properties, Inagaki et al. [74] proposed that they might provide protection from bacterial overgrowth after activation of FXR. How these FXR target genes, and possibly others that have not been identified, function to maintain intestinal homeostasis will surely be active areas of future investigation. FXR and hepatoprotection Bile acids are physiologically important owing to their detergent-like properties that facilitate lipid absorption; however, these same properties render bile acids highly cytotoxic when their blood or cellular levels increase as a result of disease. Studies in rat models of intrahepatic and extrahepatic cholestasis have demonstrated that activation of FXR by the synthetic agonist GW4064 provides protection against cholestatic liver damage [42]. This FXRdependent hepatoprotection has been proposed to result from the repression of bile acid synthetic enzymes, such as Cyp7a1, and the induction of proteins involved in transporting bile acids and phospholipids into the bile, including multidrug resistant protein 2 (Mdr2) [42] (Figure 2; Table 1). An increase in hepatic expression of aA-crystallin, which follows activation of FXR [6], might provide additional protection from the deleterious effects of high levels of bile acids in the liver because aA-crystallin is thought to function as a chaperone and to prevent protein denaturation. Activation of the nuclear receptor PXR is known to induce genes that enhance the breakdown of bile acids and thus protect the liver from the toxic effects that result from the intracellular accumulation of excess bile acids. Indeed, Fxr/Pxr/ double knockout mice show enhanced toxicity in response to bile acids [19]. Of note, activation of the xenobiotic receptor CAR by phenobarbitol in Fxr/Pxr/ double knockout mice results in both a marked reduction in serum bile acid and an increase in hepatic genes involved in bile acid metabolism [19]. In addition, data from studies on Car/Pxr/ double knockout mice also suggest that CAR is important in the detoxicification of lithocholic acid, a hepatotoxic bile acid [20]. More recently, FXR has been shown to activate transcription of the Pxr gene, providing insight into one mechanism by which FXR protects the liver from the toxic effects of excess bile acids [75]. Taken together, these data demonstrate that both PXR and CAR, in addition to FXR, are important in controlling bile acid homeostasis, especially during cholestasis. Although FXR has not been directly linked to any human disease, its target genes have been implicated in several inherited cholestatic liver disorders. Mutations in bile salt export pump (BSEP), MDR3 and multidrug resistant related protein 2 (MRP2), all of which are FXR target genes, have been shown to cause, respectively, progressive familial intrahepatic cholestasis type 2 (PFIC2), PFIC3 and Dubin–Johnson syndrome (reviewed in Ref. [76]). Furthermore, individuals affected with PFIC type I have been shown to have decreased expression and activity of FXR [77,78]. These studies suggest that FXR and its target genes are essential for maintaining normal liver function. In addition, FXR has been shown to be involved in liver cirrhosis. Activation of hepatic stellate cells is thought to www.sciencedirect.com

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mediate the process of liver fibrosis through the secretion of extracellular matrix proteins, ultimately leading to scarring of the liver [79]. Recent studies have demonstrated that FXR is expressed in rat and human stellate cells [6,80], and that activation of FXR reduces the expression of extracellular matrix proteins by these cells [81]. FXR activation also prevents and resolves liver fibrosis in rats [80–82]. Lastly, FXR has been shown to be important in preventing gallstone formation in susceptible mice. Using quantitative trait locus analysis, Wittenburg et al. [83] originally suggested that FXR and the heterodimer ABCG5/ABCG8 are possible determinants of cholesterol gallstone formation in mice. In addition, Fxr/ mice are more susceptible to cholesterol gallstone formation than are wild-type mice after administration of a lithogenic diet [84]. Moreover, treatment of C57L mice, which are susceptible to cholesterol gallstones, with a potent FXR agonist prevents gallstone formation [84]. Protection might result from induction of the FXR target gene Bsep and an increase in the transport of bile acids into the lithogenic bile [84] (Figure 2). FXR and liver regeneration Liver, at least in rodents, shows a remarkable ability to regenerate after the removal of up to 75% of the organ. A recent study by Huang et al. [85] has demonstrated that FXR is important in the liver regeneration process. They found that administration of dietary cholic acid to mice that have undergone partial hepatectomy results in accelerated regeneration and this effect is greatly attenuated in Fxr/ mice [85]. Because expression of the transcription factor FoxM1b and its downstream target gene cdc25B is attenuated in Fxr/ mice, Huang et al. [85] have proposed that these proliferative genes might have an important role in regeneration. They suggest that FXR, and possibly other nuclear receptors, regulate the size and/or regeneration of the liver by sensing the levels of metabolites such as bile acids. FXR in the kidney and adrenal gland Although FXR is known to be expressed at high levels in the mouse adrenal cortex [1,4,5], the site of active steroidogenesis, its functional role in the adrenal gland remains an enigma. Unlike their presence in the liver, intestine and kidney, bile acids have not been found to flux through the adrenal gland in a physiologically important way. Thus, an alternative possibility for activation of FXR is that the adrenal gland synthesizes its own unique FXR agonist. Indeed, Howard et al. [86] have used a FXR reporter assay to show that androsterone, a product of androgen catabolism, weakly transactivates an FXR reporter gene, although the physiological relevance of this observation remains to be established. To begin to understand the role of FXR in the adrenal gland, Lee et al. [7] studied Fxr/ mice, mouse adrenal organ cultures and a human adrenal cell line and thereby identified three genes (Osta, Ostb and Shp) that are induced in the adrenal gland in a FXR-dependent manner [7] (Table 1). Osta and Ostb are also induced in the kidney and intestine in response to activation of FXR [7,46,47]. Because OSTa and OSTb transport bile acids [7,46–48]

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(Figure 1), the most likely interpretation is that bile acids regulate adrenal FXR; however, the OSTa–OSTb heterodimer has also been shown to transport dehydroepiandrosterone 3-sulfate out of preloaded oocytes [46]. Because the human adrenal cortex synthesizes and secretes large amounts of dehydroepiandrosterone 3-sulfate, it is possible that OSTa–OSTb functions to export conjugated steroid intermediates from the adrenal gland into the blood, rather than to transport bile acids into the adrenal gland. Clearly, additional studies are necessary to elucidate the physiological role of FXR in the adrenal gland. The role of FXR in the kidney is also poorly understood. Bile acids in the blood are normally filtered through the glomeruli and reabsorbed via renal tubular cells by a process dependent on the Na+ gradient [87]. Identification of the ileal apical sodium and bile acid transporter (ASBT) in the kidney and localization of the FXR target genes OSTa and OSTb on the basolateral surface of renal tubular cells [46] are consistent with the idea that these transporters function in bile acid reabsorption. Concluding remarks The initial cloning of FXR in 1995 and the subsequent demonstration that bile acids function as endogenous agonists in 1999 have resulted in an amazing period of discovery. The findings that bile acids, by activating FXR, regulate many diverse metabolic pathways were unexpected. These pathways affect plasma levels of lipids and glucose, hepatic regeneration, hepatoprotection, gallstone production and bacterial growth in the intestine. Taken together, these findings in animals suggest that the development of improved synthetic FXR agonists might prove to be clinically useful for treating human diseases that arise from defects in these pathways. Acknowledgements Space limitations have precluded the inclusion of many appropriate publications and we apologize to those authors. This work was supported by grants from the National Institutes of Health (grants HL30568 and HL68445) and a grant from Laubisch Fund (P.A.E.), a Beginning Grantin-aid from American Heart Association (0565173Y to Y.Z.).

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