Bile acids are the major determinant and driving force for the generation of bile flow (1). Bile formation at the canalicular level results from the active transport of bile acids and other solutes, followed by the passive movement of water (2). The capacity for bile acid uptake greatly exceeds the maximum capacity for excretion, indicating that canalicular bile acid excretion is rate limiting in the vectorial transport by bile acids by the hepatocyte. Owing to an efficient enterohepatic circulation, 20-30 g of bile acids is secreted daily against a steep concentration gradient of 100- to 1000-fold between portal blood and canalicular bile.
Mechanisms of Canalicular Bile Acid Transport
Bile acid transport across the canalicular membrane is primarily an ATP-dependent process (3,4). The predominant transporter has been identified and found to be a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transporters. It is referred to as the bile salt export pump (BSEP, ABCB11). BSEP is a 160-kD protein and is localized exclusively to the canalicular domain of the hepatocyte plasma membrane. BSEP belongs to the multidrug resistance (MDR) gene family of ABC transporters with orthologous proteins expressed in many species including rat, mouse, rabbit, pig and the skate (5). As is typical for an ABC transporter, BSEP consists of 12 membrane-spanning domains that determine the substrate specificity and 2 intracellular nucleotide-binding loops that are highly conserved with Walker A and B motifs for binding and hydrolysis of ATP. Figure 1 shows transcellular transport of taurocholate in MDCK (Madin-Darby canine kidney) cells stably transfected with expression vectors for the rat sodium taurocholate cotransporting polypeptide, NTCP and BSEP. Accumulation of radiolabeled bile acid occurs much more rapidly and to a greater extent in cells expressing NTCP and BSEP than in cells expressing NTCP alone. BSEP expressed at high levels in baculovirus-infected Sf9 insect cells exhibited a high affinity for bile salts with a Michaelis constant (Km) for taurocholate of 4.25-7.9 μmol/L (6). The relative affinity for bile salts is taurochenodeoxycholate > taurocholate > tauroursodeoxycholate glycocholate (7). BSEP is also competitively inhibited by many therapeutic drugs including cyclosporin A, rifampicin, glibenclamide, cloxacillin, troglitazone, midecamycin and the estrogen metabolite, estradiol-17b glucuronide (8,9).
Transcriptional Regulation of BSEP
Because intracellular accumulation of bile salts can lead to cholestasis, hepatocyte apoptosis and progressive liver damage, regulatory mechanisms that can coordinate the expression of genes encoding bile acid transport proteins and the enzymes involved in bile acid biosynthesis are critically important. Bile salts are natural ligands for several nuclear hormone receptors expressed in liver and intestine (10). Nuclear receptors are transcription factors that bind specific ligands and regulate gene expression according to the metabolic requirements of the cell. Nuclear receptors generally bind to AG(G/T)TCA-like hexameric repeat motifs in the promoters of target genes as heterodimers with the retinoid X receptor alpha (RXRα) (11).
In cloning of the BSEP gene, we found that the sequence of the promoter contained an inverted repeat (IR)-1 element (5′-GGGACA T TGATCCT-3′) at base pairs -63/-50 consisting of 2 nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide (12). This binding site has been shown in several recent studies to serve as a binding site for the farnesoid X receptor (FXR), a nuclear receptor for bile acids (10). FXR activity requires heterodimerization with a 9-cis retinoid receptor (RXR alpha), and when bound by bile acids and retinoic acid, the complex effectively regulates the transcription of several genes involved in bile acid transport and synthesis. FXR/RXR alpha heterodimers bound specifically to the IR-1 element in the BSEP promoter on gel mobility shift assays. Cotransfection of FXR and RXR alpha into HepG2 cells led to full transactivation of the BSEP promoter by bile acids. Two FXR transactivation-deficient mutants failed to transactivate, indicating that the effect of bile acids is FXR-dependent. Further, mutational analysis confirms that the FXR/RXR alpha heterodimer activates transcription through the IR-1 site in the human BSEP promoter. The activity of the promoter was modest in the absence of FXR, suggesting that this nuclear orphan receptor may be critical for even basal activity of the BSEP promoter. This notion was supported by experiments in CV-1 cells in which BSEP promoter activity was observed only when FXR/RXRα heterodimer was expressed and activated by the addition of bile acids (Fig. 2). Moreover, mice with targeted disruption of the FXR gene have extremely low levels of BSEP expression, even when fed cholic acid (13). These results demonstrate a mechanism by which bile acids transcriptionally regulate the activity of the BSEP, a critical component involved in the enterohepatic circulation of bile acids.
FXR also activates the transcription of the genes encoding ileal bile acid-binding protein (IBABP) and short heterodimer partner (SHP), while inhibiting the transcription of the genes encoding cholesterol 7α hydroxylase (CYP7A1), the ileal sodium-dependent bile acid transporter (ASBT) and NTCP; also known as SLC10A1 (2,4,11). Further studies have shown that the repressive effect of FXR is predominantly mediated through its activation of SHP, although SHP-independent mechanisms are also involved (10).
Despite the capacity of FXR function to regulate BSEP and other aspects of bile acid homeostasis through transcriptional activation of a number of target genes, it was unknown how FXR achieves a transcriptionally active state and what nuclear protein components are associated with the FXR/RXRα heterodimer as a higher-order ternary complex. Recent studies have indicated that a code is imprinted within the enzymatic posttranslational conjugation of histones that provides a template for many nuclear processes including the regulation of transcription (14). There is a growing body of evidence for the activation of nuclear hormone receptors through the remodeling of chromatin by histone modification involving acetylation, in concert with methylation of H3 and H4 histones. Different proteins or protein complexes are recruited as coactivators such as p160 and CBP/p300 that regulate chromatin function (15). The process of ligand-induced nuclear receptor activation is incompletely understood and involves the cyclical assembly and disassembly of these transcriptional complexes on DNA promoter elements (16).
The mono- and dimethylation of a discrete arginine within the NH2-terminal end of histone H3 positioned at Arg-17 are involved in ligand-dependent nuclear hormone receptor activation of target genes (15). Several enzymes that can catalyze either arginine or lysine methylation have been recently identified. Despite the ability of protein-arginine methyltransferases to methylate the NH2-terminal regions of the various core histones, it is now clear that these proteins exist as components of larger complexes within the active transcriptional apparatus (15). Of those known to target the methylation of arginine at the NH2-terminal end of histones, coactivator-associated arginine methyltransferase 1 (CARM1) has been shown to work with the glucocorticoid receptor-interacting protein 1 to activate transcription through ligand-dependent regulation by estrogen receptors (16). CARM1 preferentially methylates histone H3 at the arginine residue at position 17 from the NH2 terminus, a modification associated with active transcription.
Previous studies on the role of histone methylation in regulating the expression of nuclear receptors led us to ask whether FXR can associate with histone methyltransferase activity in vivo. We found FXR incorporates histone methyltransferase activity within the gene locus for bile salt export pump (BSEP), a well established FXR target gene (17). This methyltransferase activity is directed specifically to arginine 17 of histone H3. FXR is directly associated with CARM1 activity. Chromatin immunoprecipitation showed that the ligand (bile acid)-dependent activation of the human BSEP locus is associated with a simultaneous increase of FXR and CARM1 occupation. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP (17). Consistent with these findings, CARM1 led to increased BSEP promoter activity with an intact FXR regulatory element, whereas CARM1 failed to transactivate the BSEP promoter with a mutated FXR binding site (FXRE). Induction of endogenous BSEP messenger RNA (mRNA) in HepG2 cells and Arg-17 methylation by an FXR regulatory element ligand, chenodeoxycholic acid, require CARM1 activity. These data indicate that histone methylation at Arg-17 by CARM1 is a downstream target of signaling through ligand-mediated activation of FXR. FXR directly recruits specific chromatin modifying activity of CARM1 necessary for full potentiation of the BSEP locus in vivo.
Recent studies by Rizzo and associates have demonstrated a role for another protein arginine methyltransferase, PRMT1, as a coactivator of the FXR/RXR receptor and regulator of FXR responsive genes (18). PRMT1 is the predominant H4 arginine-methyltransferase in mammalian cells and functions as a coactivator of several nuclear receptors, including the thyroid, estrogen, and androgen receptors. Chromatin immunoprecipitation assays indicated that natural and synthetic bile acids induced both the recruitment of PRMT1 and the H4 methylation to the promoter of BSEP and SHP genes. Similar to the studies with CARM1, 2 FXR-responsive genes, BSEP and SHP, were induced in liver cell lines by transient transfection of PRMT1 in the presence of FXR ligands.
The involvement of CARM1 and PRMT1 in modifying histones and acting as coactivators of FXR has now been documented for regulation of BSEP. Several studies have now shown that CARM1 and PRMT1 may act synergistically to enhance gene activation by other nuclear receptors (19,20). Although CARM1 and PRMT1 both transfer methyl groups from S-adenosylmethionine to specific arginine residues of specific target proteins, their substrate specificities differ significantly and so they are unlikely to be redundant in their functions and may rely on different mechanisms for their coactivator activities (20). Methylation of histones H3 and H4 may cooperate with acetylation and other types of covalent histone modifications to remodel chromatin structure and/or provide a signal to facilitate assembly of the active transcription initiation complex (15). Further studies are necessary to determine how these PRMTs cooperate to regulate the expression of BSEP.
Development of Canalicular Bile Acid Transport
Studies in neonatal and to a lesser extent fetal animals indicate that rates of bile flow and bile acid excretion in response to an exogenous infusion of bile acids are decreased in comparison with the adult (21). In unpublished studies, our laboratory has defined developmental changes in bile acid transport across the canalicular plasma membrane (22). Taurocholate uptake by rat liver canalicular membrane vesicles isolated from 7-day-old rat was ATP-dependent with properties virtually identical to that described in the adult. However, the Vmax for ATP-dependent transport was approximately 60% of that determined in membrane vesicles from adult liver; the Km was similar at both ages. These findings indicate that an ATP-dependent system is functional in neonatal liver and may be sufficient for biliary secretion of bile acids at a time at when other components of the enterohepatic circulation of bile acids (including synthesis, pool size, ileal absorption and basolateral transport) are immature.
Several groups have studied the development of the rat BSEP by quantitation of BSEP mRNA and protein in fetal and neonatal rats. In our laboratory, BSEP mRNA measured by Northern blotting and RT-PCR was expressed minimally in the near term fetus and increased abruptly to 50% of adult levels on postnatal day 1 and further to 90% of adult values by 1 week of life (23). On western blotting, there was minimal expression of BSEP protein before birth, with an increase to 40% of the adult on the first day of life. BSEP protein levels increased to 90% of the adult by 1 week of life and further increased to adult values by 4 week of life. The developmental patterns of mRNA and protein expression were quite similar in 2 other studies (24,25). There was minimal transcription of BSEP gene assessed by nuclear run-on assays at the fetal age with an abrupt increase in transcription on the first day of life. Transcription rates in adult nuclei were not significantly different from 1-week-old nuclei, indicating that there was no further increase in transcription between 1 week of age and adults (23).
Chen et al. recently described the development of BSEP in human fetal liver samples at gestational age 14-20 weeks (26). The mean expression levels of BSEP mRNA in the fetus using real-time RT-PCR were 30% of adult values. The mean expression level of FXR was surprisingly at 75% of adult levels. The immunohistochemical localization of BSEP in fetal liver was distinctly different from the adult liver. In the adult liver, there was sharp-linear staining of bile canaliculi for BSEP. However, in fetal liver, BSEP partially showed an intracellular and partially showed a canalicular pattern. These findings suggested decreased expression of BSEP and inefficient targeting of BSEP to the canalicular membrane in human fetal liver (26).
Because of the central role of several nuclear receptors in regulating bile acid homeostasis and other transport systems that contribute to bile formation, we recently completed a study on the development of the most important nuclear receptors (27). For the purposes of this review only the data related to BSEP will be discussed.
Real-time PCR analysis of hepatic nuclear receptor (NR) expression revealed that steady-state mRNA levels for all NRs were low in the rat fetus (27). FXR mRNA was barely detected at 1.5-5.9% of adult levels between fetal days 17 and 21. However, on postnatal day 1, mRNA rose to 13.6%, and by day 7 there was a further rise to 101% of adult values. The level remained between 104% and 117% for the next 14 days but increased to 144% by day 21 of life. In contrast to FXR, the RXRα mRNA level remained relatively low between 4.4% and 35% between fetal day 17 to postnatal day 7 and reached 69.9% of the adult level during the postnatal week 4. These data suggest that different mechanisms of transcriptional regulation are operative for FXR and RXRα.
SHP is a non-DNA binding protein of the nuclear receptor family that acts as a strong repressor of many genes including cyp7α1 and NTCP. The expression of SHP mRNA is low in the fetus between days 17 and 20 remaining at 0.25-0.35% of the adult. On day 21, SHP mRNA was 8.3% of the adult but increased and was maintained at 60% of adult values between postnatal days 1 and 14. The amount of mRNA reached adult levels by day 21.
The amount of FXR protein was at 16.8% of adult values on fetal day 20 and postnatal day 7. However, because FXR mRNA was at 100% of adult level by day postnatal 7, posttranscriptional regulatory mechanisms are likely involved in determining FXR expression in the postnatal period. At 4 week of age, protein levels were 75.2% of the adult level.
There was also significant disparity between mRNA and protein levels with regard to RXRα in the developing liver. Whereas the mRNA levels remained at 32.8-69.9% at postnatal days 7-28, protein levels were at 113.6% and 96.5% at 7 and 28 days, respectively. Because RXRα heterodimerizes with all of the type II NRs, we speculate that its availability, possibly enhanced by a long half-life, is not limiting the activity of these receptors in the postnatal period.
Despite the extremely low levels for SHP mRNA during fetal days 17-20, we observed 8.4% of adult SHP protein level at fetal day 20. However, during the postnatal period, there was good correlation between the mRNA and protein levels for this potent repressor of cyp7α1 and NTCP. The SHP protein level was 50% of the adult by day 7 and at 120% of the adult by 28 days. On the basis of these data, it is likely that the repressive effect of SHP on transporter gene expression is significant only during the postnatal period.
The functional expression of FXR/RXRα was also assessed by electromobility shift assays (EMSA). The complex formed by binding of FXR to its IR-1 element was 32% of the adult amount on fetal day 20 and reached adult levels by postnatal day 28. Comparison of the degree of FXR binding in the EMSA to the amount of its mRNA demonstrates more activity than can be accounted for by the level of mRNA, implying additional levels of posttranscriptional control. However, in the postnatal period, the temporal pattern of the EMSA compares well with mRNA levels (full activity at day 28), indicating transcriptional regulation as the major mechanism of control.
Bile acid synthesis and metabolism in the liver of human fetus and neonate are significantly different from that occurring in the liver of adults (21,28,29). The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1, C-4 and C-6 hydroxylation are important pathways in bile acid synthesis during development. Relatively large amounts of unusual bile acids are detected during infancy, especially during the period up to 1 month of age. At that time, 1beta, 3alpha, 7alpha, 12alpha-tetrahydroxy-5beta-cholan-24-oic, 7alpha, 12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic and 7alpha, 12alpha-dihydroxy-3-oxo-4-cholen-24-oic acids are predominant among the unusual urinary bile acids present (30). These bile acids are unlikely to be good substrates for transport by BSEP and may not be ligands for the bile acid receptor, FXR,that is critical for transactivation of the BSEP gene.
BSEP in Progressive Familial Intrahepatic Cholestasis
Targeted inactivation of the BSEP gene in mice impairs the canalicular secretion of bile salts (31). Because secretion of bile acids is impaired, BSEP-/- mice are cholestatic as evidenced by the accumulation of taurocholate in their plasma. Secretion of cholic acid is greatly reduced to 6% of wild-type mice, whereas total bile salt output in mutant mice is about 30% of the level in wild-type mice (31). Large amounts of tetra-hydroxylated bile acids (not detected in wild-type) are secreted into the bile of mutant mice. Hydroxylation and an alternative mechanism for canalicular transport of more hydrophilic bile salts may protect the mutant mice from severe cholestatic damage in the absence of BSEP. When BSEP-/- mice are fed a cholic acid-supplemented diet, they became severely cholestatic with jaundice, weight loss and elevated serum bile acid and aminotransaminase levels (32). The mutant mice also developed a cholangiopathy (proliferation of bile ductules and cholangitis), liver necrosis and high mortality.
In contrast to BSEP-/- mice which are mildly cholestatic and do not develop severe liver disease unless treated with hydrophobic bile salts, patients with mutations in BSEP develop progressive cholestasis with onset in the neonatal period (33,34). These infants have severe jaundice, hepatomegaly, failure to thrive and later pruritus. Laboratory studies typically show direct hyperbilirubinemia, elevated serum aminotransferases and paradoxically normal serum gamma glutamyl transpeptidase and cholesterol concentrations. Biliary bile acid concentrations are less than 1% of normal (35). On liver biopsy, there is prominent giant cell transformation of hepatocytes, chronic inflammation and fibrosis. Numerous mutations in BSEP have been detected in patients with progressive familial intrahepatic cholestasis type 2 (PFIC2). The bile of PFIC2 patients is amorphous or filamentous on transmission electron microscopy. There is a rapidly progressive course to cirrhosis and liver failure (36). Because BSEP is expressed exclusively in the liver, the disease is cured by liver transplantation.
Many mutations in patients with PFIC2 are sporadic without a family history of the disease (37). Some PFIC2 affected individuals are compound heterozygotes. Several mutations have been studied in vitro (38,39). Mutations may lead to impaired function (e.g., loss of substrate binding) or impaired trafficking. Other missense mutations abolished both the protein's function and apical expression, possibly by misfolding (39). In all cases of PFIC2 in which mutations have been detected, BSEP has been absent on immunohistochemical staining of the liver (35,36,40). This finding may prove to be a useful diagnostic tool when sequencing of the BSEP gene is not readily available.
An unexpected turn has recently come about in our understanding of the pathogenesis of the inherited disorder (PFIC1) or Byler disease. Patients with this disorder usually present in infancy with jaundice, hepatomegaly, diarrhea, failure to thrive and later pruritus (34,41). Liver biochemical tests show elevated serum aminotransferases, direct bilirubin, alkaline phosphatase and bile acids. Biliary bile acid concentrations are markedly reduced. However, similar to PFIC2, the serum γ-glutamyltransferase level is low. Initial liver biopsy shows a bland cholestasis with variable fibrosis. Dilated bile canaliculi containing coarse granular bile, so-called Byler bile, are seen on electron microscopy. The disease is progressive with evolution to cirrhosis and liver failure occurring usually within the first decade of life.
The gene mutated in PFIC1 has been identified and is called FIC1. The FIC1 gene codes for a type IV P-type ATPase whose function is unknown, but that resembles other transporters that function as ATP-dependent aminophospholipid transporters or flippases that move aminophospholipids from the outer to inner leaflets of membranes (42). There is in vitro experimental evidence for phosphatidylserine translocation in canalicular membranes and in cells transiently transfected with FIC1 cDNA (43). Because biliary bile acid concentrations are markedly reduced in PFIC1, it had long been thought that FIC1 might be involved in bile acid transport. However, studies by Harris et al. have shown that FIC1 does not transport taurocholate and, when overexpressed in MDCK cells, had no effect on the function of BSEP or the ileal apical sodium-dependent bile acid transporter (ABST) (44). As expected, FIC1 is detected in hepatocytes and cholangiocytes, but is expressed at much higher levels in the intestine (43). Although patients with PFIC1 suffer from diarrhea and malabsorption that persists even after liver transplantation, it has been puzzling why a disorder with predominantly a liver phenotype should be caused by a defective gene most highly expressed in the intestine.
To gain further insight into the pathophysiology of PFIC1, we recently studied factors involved in the transcriptional regulation of bile acid homeostasis in three affected children (45). In tissue obtained at the time of ileal bypass surgery for treatment of intractable pruritus, ileal apical sodium-dependent bile acid transporter mRNA expression was increased in 3 patients with PFIC1 as compared with the expected decrease in a severely cholestatic patient. Ileal lipid-binding protein mRNA expression that should be induced in this setting was paradoxically repressed, suggesting a central defect in bile acid homeostasis. Ileal FXR and SHP mRNA levels were reduced in the same 3 patients. In the intestinal cell line Caco-2, antisense-mediated knock-down of endogenous FIC1 led to upregulation of apical sodium-dependent bile acid transporter and downregulation of FXR, ileal lipid-binding protein, and SHP mRNA. In FIC1-negative Caco-2 cells, the human apical sodium-dependent bile acid transporter promoter activity was markedly enhanced, whereas the human FXR and BSEP promoter activities were reduced. FXR cis-element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from FIC1-negative Caco-2 cells. These studies indicate that FIC1 is necessary for posttranslational modifications associated with or necessary for the nuclear translocation of FXR.
In another recent report, Alvarez and associates confirmed that the expression of 2 main FXR isoforms was specifically decreased in the liver of a FIC1 disease patient (46). There was also reduction in mRNA levels of FXR targets, BSEP and SHP.
The molecular mechanisms by which FIC1 modulates FXR activity are not yet known. FIC1 may influence signal transduction pathways that lead to changes in the functional activity of key target proteins, including transcription factors such as FXR. One of the proposed functions for FIC1 is that of an aminophospholipid flippase. Indeed, a number of potential signal transduction pathways, such as protein kinase C activity, may be influenced by cell membrane aminophospholipid asymmetry associated with FIC1 activity. Thus, in PFIC1 patients, bile salt uptake from the ileum is enhanced and canalicular bile salt secretion from the liver by BSEP is diminished. An increased load of bile acids is retained in the liver leading to cholestasis and progressive liver injury.
This work was supported by R37 HD20632 to FJS from NICHD.
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