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Originally published In Press as doi:10.1074/jbc.M307145200 on August 15, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45062-45071, November 14, 2003
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Complementary Roles of Farnesoid X Receptor, Pregnane X Receptor, and Constitutive Androstane Receptor in Protection against Bile Acid Toxicity*

Grace L. Guo{ddagger}, Gilles Lambert§, Masahiko Negishi¶, Jerrold M. Ward||, H. Bryan Brewer, Jr.**, Steven A. Kliewer{ddagger}{ddagger}, Frank J. Gonzalez{ddagger}§§, and Christopher J. Sinal{ddagger}¶¶

From the {ddagger}Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892, §INSERM U539, Nantes 44035, France, Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, ||Veterinary and Tumor Pathology Section, Center for Cancer Research, NCI, Frederick, Maryland 21702, **Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, and the {ddagger}{ddagger}Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, July 3, 2003 , and in revised form, August 12, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase {alpha}), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acids, the amphipathic end products of cholesterol metabolism, are critical for the absorption of dietary fats and fat-soluble vitamins, as well as regulation of bile flow and biliary lipid secretion that facilitate the excretion of conjugated drugs and endogenous waste products. Bile acid production also represents a major route for the elimination of excess cholesterol (1). Because disturbances of bile acid biosynthesis, metabolism, or transport cause diseases in the liver, biliary tree, and intestine (2), bile acid homeostasis needs to be tightly controlled. The farnesoid X receptor (FXR1; NR1H4; see Refs. 3 and 4), a member of the nuclear receptor superfamily, was identified as a sensor for bile acids (5–7). It is strongly activated by bile acids, such as chenodeoxycholic acid, CA, deoxycholic acid, and lithocholic acid (LCA). Activated FXR forms a heterodimer with retinoid X receptor {alpha} and binds to its response elements found upstream of FXR target genes (6, 8–11). Disruption of the FXR gene in mice clearly demonstrated that FXR serves as a central coordinator for bile acid biosynthesis, metabolism, and transport (12).

Several lines of evidence indicate that bile acids bind and activate nuclear receptors other than FXR. In FXR-null mice, cyp3a11, a target gene of the pregnane X receptor (PXR; NR1I2) encoding a cytochrome P450 enzyme that facilitates bile acid metabolism and elimination, was significantly up-regulated (13). PXR was recently identified as another bile acid sensor and is bound and activated by LCA, deoxycholic acid, and chenodeoxycholic acid (14, 15). LCA and its major metabolites also activate the vitamin D receptor (NR1I1 (see Ref. 16)). Activation of PXR and vitamin D receptor by bile acids leads to the induction of hepatic and/or enteric phase I and phase II metabolizing enzymes, such as CYP3A11 and sulfotransferase (17), indicating the existence of alternative pathways for metabolism and excretion of bile acids.

In order to determine, in vivo, whether there is a combined role for FXR and PXR in maintaining bile acid, cholesterol, and lipid homeostasis, the FXR-null mice were cross-bred with PXR-null mice to create FPXR-null mice. FPXR-null mice exhibited a marked disruption of bile acid and cholesterol homeostasis when fed a 1% CA diet. Moreover, CAR and its target genes were induced in FXR- or FPXR-null mice fed the CA diet, and pretreatment with CAR activators, PB or TCPOBOP, resulted in a marked reduction in serum bile acid and bilirubin levels in these animals. This study therefore demonstrates a critical and combined role for FXR and PXR as bile acid sensors and further revealed that induction of CAR provided an additional hepatic defense to maintain bile acid and cholesterol homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were obtained from Sigma, and all enzymes for molecular biology were purchased from Invitrogen unless otherwise indicated. [32P]CTP was from PerkinElmer Life Sciences.

Generation of FXR and PXR Double Null Mice—B6;129-Fxrtm1Gonz (FXR-null) mice (12) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice (15) were cross-bred to generate wild type (WT), FXR-null, PXR-null, and Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice. The primer sequences and reaction conditions used for genotyping the FXR-null allele were reported previously (12). The primer sets used for genotyping the PXR-null mice were as follows: PXR-F1 (5'-CTG GTC ATC ACT GTT GCT GTA CCA-3'), PXR-R2 (5'-GCA GCA TAG GAC AAG TTA TTC TAG AG-3'), and PXR-R3 (5'-CTA AAG CGC ATG CTC CAG ACT GC-3'). Amplification of the PXR WT allele produced a 348-bp product, whereas amplification of the null allele produced a 265-bp product.

Diets, Sample Collection, and Histological Analysis—Mice were housed in a pathogen-free animal facility under standard 12-h light/ 12-h dark cycle with access to chow and water ad libitum. All protocols and procedures were approved by the NCI Animal Care and Use Committee and are in accordance with the National Institutes of Health and ALAC Guidelines. All diets were prepared by Bioserv (Frenchtown, NJ) and were based on a standard AIN-93G rodent diet (58.6% carbohydrate, 18.1% protein, 7.2% fat, 0.1% cholesterol, 5.1% fiber, 3.4% ash, and 10% moisture) (12). The 1% CA diet consisted of the control diet supplemented with 1% (w/w) CA. Groups of 8–12-week-old males were used for all experiments. At the end of the study, animals were fasted for 4 h in the morning and anesthetized with avertin. Following blood collection from the orbital plexus, animals were euthanized. Tissues were weighed, snap-frozen in liquid nitrogen, and stored at –80 °C until use.

Plasma Chemistry—Serum was prepared from whole blood by centrifugation at 6,000 x g using Microtainer serum separator tubes (BD Biosciences). Serum levels of total bile acids, total and direct bilirubin, were measured colorimetrically using corresponding Sigma diagnostics analysis kits. Total cholesterol and triglyceride (Sigma) as well as free cholesterol and phospholipid (Wako, Osaka, Japan) concentrations were measured from 12-µl aliquots of serum using commercial kits and the Hitachi 911 automated chemistry analyzer (Roche Applied Science). Fast protein liquid chromatography (FPLC) separation of serum lipoproteins from pooled plasma samples (60 µl; n = 4–7) was achieved by gel filtration using two Superose 6HR 10/30 columns (Amersham Biosciences) in series as described previously (12, 18). Mouse apoA-1, apoA-II, apoE, and apoB were identified by Western blot as described previously (12), using polyclonal rabbit anti-mouse IgG raised against the purified apolipoproteins. Plasma lecithin-cholesterol acyltransferase (LCAT) activity was measured as described previously (19).

Biliary Bile Acid and Lipid Secretion—Mice were fasted for 4 h in the morning following a 1% CA diet for 4 days. Animals were weighed, anesthetized by avertin (2.5%), and their abdominal cavities opened under sterile conditions. The cystic duct was ligated, and the common bile duct was cannulated. The hepatic bile was collected by gravity for 45 min. Biliary cholesterol, phospholipids, and bile acids (Sigma diagnostic kit) were measured immediately after bile collection.

Cloning and Analysis of Gene Expression—Total RNA was prepared using Trizol reagent (Amersham Biosciences) and analyzed by electrophoresis in 0.22 M formaldehyde-containing 1% agarose gels. The cDNA probes and detailed Northern blot analysis procedures were described previously (12, 20), except for ALAS1 (composed of 820–1426 bp of ALAS1 cDNA with accession number NM020559 and recognizes ALAS1), UGT1A (composed of 578–947 bp of UGT 1A1 cDNA with accession number L27122 [GenBank] and recognizes the UGT1A family), and GST{alpha} (composed of 203–592 bp of GST{alpha}1 cDNA with accession number NM008181 and recognizes the GST{alpha} family). Probes were 32P-labeled by the random primer method using Ready-to-Go DNA labeling beads (Amersham Biosciences).

Western Blot Analysis—Hepatic microsomal proteins were prepared as described previously (21), and 1 µg of protein was subjected to 10% SDS-PAGE and transferred by electroblotting to GeneScreen Plus membranes (PerkinElmer Life Sciences). The membranes were incubated with monoclonal anti-rat CYP3A1/2 or polyclonal anti-rat CYP2B antibody (N-19, Santa Cruz Biotechnology, Santa Cruz, CA) for 4 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG for CYP3A1/2 or goat anti-rabbit IgG for CYP2B (Sigma). Bands were visualized with ECL Western blotting detection reagents (Amersham Biosciences).

Statistics—Unless otherwise stated, all values were expressed as the mean ± S.E. All data were analyzed by the unpaired Student's t test for statistical significance between each group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of Both FXR and PXR Results in Severe CA-induced Toxicity—The nuclear receptors, FXR and PXR, play important roles in maintaining bile acid homeostasis. However, the combined role of FXR and PXR in vivo has not been studied, thus FXR-PXR double null mice were generated by cross-breeding PXR-null with FXR-null mice to abolish both FXR and PXR expression in vivo. Under standard housing and dietary conditions, there was no external difference among FPXR-, FXR-, PXR-nulls, or WT mice. However, upon feeding a 1% CA diet for 4 days, FPXR-null mice exhibited the most severe body weight loss, followed by FXR-, PXR-nulls, and WTs (Fig. 1A). Serum bile acids and total and direct bilirubin levels were highest in FPXR-followed by FXR-, PXR-null mice, and WTs fed the CA diet. The serum levels of direct bilirubin or conjugated bilirubin, a direct indicator for hepatic function, were statistically higher in FPXR-null mice than those in FXR-null mice, but there was no statistical significance between FPXR- and FXR-nulls for bile acid and total bilirubin levels (Fig. 1, B–D). Histological examination of the livers by hematoxylin and eosin revealed more fatty metamorphosis in FPXR-, FXR-, and PXR-null mice than WT mice fed the CA diet, but the degree was similar (data not shown).



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  		FIG. 1.
Body weight changes, serum bile acid, and bilirubin levels. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a control (CON) or a 1% CA-containing diet for 4 days. A, daily change of body weight. Serum levels of total bile acids (B), total bilirubin (C), and direct bilirubin (D). Black bars represent the control diets and gray bars the CA diets. Data were obtained from four to seven animals per group. A–D, the statistical significance between WT and PXR-, FXR-, or FPXR-null mice fed the CA diet is noted with an asterisk for p < 0.05, and double asterisks for p < 0.001. B–D, the statistical significance between control and CA diet within each strain is expressed as # for p < 0.05, and in D, the statistical significance between FXR- and FPXR-null mice fed the CA diet is expressed as {diamondsuit} for p < 0.05.

 
Disruption of Serum Cholesterol and Lipid Homeostasis in FPXR-null Mice—Because bile acid synthesis and excretion represents the major pathway for elimination of cholesterol, we examined cholesterol and lipid levels in these animals. On a control diet, the plasma levels of cholesterol were highest in FXR-null mice, followed by FPXR- and PXR-null mice, compared with WT mice; and plasma triglycerides (TG) and phospholipids (PL) were elevated in both FXR- and FPXR-null mice followed by PXR-null and WT mice. The CA diet lowered the plasma lipid levels in FXR-null and PXR-null mice but increased those of FPXR-null mice (Fig. 2A). The serum contents of cholesterol and lipid were further analyzed by fast protein liquid chromatography (FPLC) using pooled serum samples. This revealed that the dramatic hyperlipidemia observed in FXR- and FPXR-null mice on a control diet was mostly due to increased quantities of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and large HDL1 particles (Fig. 2B, panels A and B), accompanied by an increase in plasma apoE and apoB100 (Fig. 2B, panel A, inset). The plasma FPLC profiles of WT and PXR-null mice predominantly consisted of HDL particles that were only slightly affected by the CA diet. In contrast, the CA diet resulted in a dramatic shift in the elution of VLDL and IDL/LDL in FPXR-nulls and to a lesser degree in FXR-nulls (Fig. 2B, panels C and D). There was a virtual absence of HDL particles in the plasma of FPXR-null mice fed the CA diet. Consistent with these data, plasma apoA-I and apoA-II levels were reduced, whereas levels of apoB100 were increased in FPXR-null mice fed the CA diet (Fig. 2B, panel C, inset). The PL elution profiles were similar to those of total cholesterol, and the TG profiles were similar to those of free cholesterol in all groups and for both diets (data not shown).



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  		FIG. 2.
Serum lipoprotein profiles. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a control (CON) or a 1% CA-containing diet for 4 days (n = 4 to 7/group). Blood was collected, and serum was prepared by centrifugation at 6,000 x g for 10 min. A, total cholesterol, TG, and PL levels were measured from 12-µl aliquots of serum using commercial kits and a Hitachi 911 automated chemistry analyzer. The statistical significance between WT and PXR-, FXR-, or FPXR-null mice fed the control diet is noted with an asterisk for p < 0.05, and the statistical significance between WT and PXR-, FXR-, or FPXR-null mice fed the CA diet is noted with a {blacktriangleup} for p < 0.05. The statistical significance between control and CA treatment within each strain is expressed as # for p < 0.05. The statistical significance between FXR- and FPXR-null mice is expressed as {diamondsuit} for p < 0.05. B, FPLC separation of serum lipoproteins from pooled serum samples (60 µl) were achieved by gel filtration using 6HR 10/30 columns. The concentration of cholesterol in each eluted fraction is indicated in the y axes. CON stands for the control diet, and 1% CA for the 1% CA diet. Closed circle, WT mice; closed triangle, PXR-null mice; open square, FXR-null mice; and open circle, FPXR-null mice. Inset, Western blot analysis of mouse apoA-1, apoA-II, apoE, and apoB from pooled serum samples from WT, PXR-, FXR-, and FPXR-null mice. FPLC analyses are shown for free cholesterol with the control diet (a), free cholesterol with the 1% CA diet (b), total cholesterol with the control diet (c), and total cholesterol with the 1% CA diet (d).

 
Biliary Excretion of Bile Acids, Cholesterol, and Phospholipids—Because FXR and PXR regulate many genes encoding hepatic canalicular transporters, biliary excretion of bile acids, cholesterol, and phospholipids was determined in WTs, PXR-, FXR-, and FPXR-null mice fed the 1% CA diet for 4 days to saturated their hepatic biliary secretion ability, as described previously (20). Under these experimental conditions, the flow of biliary cholesterol and phospholipid, measured after cannulation of the common bile duct, was dramatically reduced in FPXR-nulls, followed by FXR-nulls but unchanged in PXR-nulls, compared with WTs (Fig. 3, A and B). The bile acid output was similar in WTs, PXR-, and FXR-nulls but dramatically reduced in FPXR-nulls (Fig. 3C), indicating that loss of both PXR and FXR in mice leads to the most severe impairment of biliary excretion.



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  		FIG. 3.
Biliary bile acid, cholesterol, and phospholipid levels. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a 1% CA-containing diet for 4 days. Mice were fasted for 4 h in the morning, weighed, and anesthetized, and their abdominal cavities were opened under sterile conditions. The cystic duct was ligated, and the common bile duct was cannulated. The bile was collected by gravity for 45 min. Biliary cholesterol (A), PLs (B), and bile acids (C) were measured immediately after bile collection. The statistical significance between WT and PXR-, FXR-, or FPXR-null mice is indicated by an asterisk for p < 0.05, and double asterisks for p < 0.001. The statistical significance between FXR- and FPXR-null mice is expressed as {diamondsuit} for p < 0.05.

 
Expression of the Major Genes Involved in Cholesterol and Lipid Metabolism—To understand the molecular mechanisms responsible for the severe disturbances of lipoprotein homeostasis in FPXR-nulls, especially after CA feeding, the hepatic expression levels of the major genes involved in cholesterol and lipid metabolism were determined by Northern blot analysis (Fig. 4). Among the major apolipoproteins, only the expression of apoA-I, apoA-II, apoE, and apoB was increased in FXR- and FPXR-null mice after CA feeding; expression of other apolipoproteins was either not altered (apoC-I, apoC-II) or up-regulated by the CA diet regardless of genotype (apoA-V; data not shown). The levels of ABCA1 and CD36 mRNA were also increased in the livers of FXR- and FPXR-null mice fed the CA diet, whereas those of SRB1 and sterol regulatory element-binding protein-1c were lower in FXR- and FPXR-nulls. In addition, feeding of CA resulted in a dramatic reduction of plasma LCAT activity in FXR- and FPXR-nulls (5.8 ± 2.1 and 3.7 ± 1.4 nmol cholesterol esterification/ml/h, respectively) compared with WTs and PXR-nulls, respectively (21.2 ± 0.9 and 19.4 ± 3.2 nmol CE/ml/h, n = 4, p < 0.05). There was a dose-dependent and positive correlation between the concentration of bile acids and the inhibition of LCAT activity in the plasma of the animals, suggesting a specific inhibition of LCAT activity by bile acids (data not shown).



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  		FIG. 4.
Northern blot analysis of hepatic mRNA levels of the major genes involved in cholesterol and lipid metabolism. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a control (CON) or 1% CA-containing diet for 4 days. Livers were removed, and total RNAs were isolated. Pooled total RNA (10 µg) from 4 to 7 animals per group were separated on a 1% agarose gel, transferred to GeneScreen Plus membranes, and hybridized with the indicated 32P-labeled cDNA probes. The bands were quantified using a PhosphorImager, and the values are expressed as fold of changes after corrected for 18 S RNA levels. CON, control diet group; CA, 1% CA diet group.

 
Hepatic Expression of the Genes Associated with Bile Acid Metabolism and Transport—To understand the molecular mechanisms responsible for defective biliary secretion in FPXR-null mice, the hepatic expression of the major genes involved in bile acid production, metabolism, and transport was determined (Fig. 5). With the control diet, expression levels of the rate-limiting enzyme for bile acid synthesis, CYP7A1, were decreased in PXR-nulls, elevated in FXR-nulls, and markedly reduced in FPXR-nulls. With the CA diet, CYP7A1 mRNA levels were strongly repressed in WTs and PXR-nulls, weakly in FXR-nulls, but not altered in FPXR-nulls. Basal levels of CYP3A11 mRNA were induced in PXR-, FXR-, and FPXR-nulls but were only further elevated in FXR-nulls fed the CA diet. Basal mRNA levels of the nuclear hormone receptor CAR and its classical target gene, CYP2B, were markedly increased in FXR-nulls and to a lesser degree in FPXR-nulls. The CA diet further induced CAR and CYP2B mRNA levels in FXR-nulls, followed by in FPXR- and PXR-nulls. Basal mRNA levels of the bile salt export pump, BSEP, were higher in PXR-nulls, lower in FXR-nulls, and unchanged in FPXR-nulls fed the control diet. Feeding the CA diet led to an induction of BSEP in WTs and PXR-nulls but a further suppression in FXR- and FPXR-nulls. The expression pattern of MDR1a followed that of CYP3A11, whereas the levels of MRP2 and MRP3 mRNA were slightly induced in FPXR-nulls on the CA diet (1.6- and 2-fold, respectively). On the control diet, the mRNA levels of the biliary phospholipid transporter MDR2, the biliary cholesterol half-transporter ABCG8, and the biliary cholesterol half-transporter ABCG5 appeared to be not altered in FXR- or FPXR-nulls on control diet, but all were induced to a similar degree by the CA diet. With the CA diet, the levels of ABCG1 were suppressed in WTs and PXR-nulls but were up-regulated in FXR- and FPXR-nulls. Levels of other nuclear hormone receptors (retinoid X receptor {alpha}, LXR{alpha}, HNF4{alpha}, and PPAR{alpha}) were not altered in these four strains regardless of the diet (data not shown).



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  		FIG. 5.
Northern blot analysis of hepatic mRNA levels of the major FXR and/or PXR target genes. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a control or 1% CA-containing diet for 4 days. Livers were removed, and total RNAs were isolated. Pooled total RNA (10 µg) from 4 to 7 animals per group were separated on a 1% agarose gel, transferred to GeneScreen Plus membranes, and hybridized with the indicated 32P-labeled cDNA probes. The bands were quantified using a PhosphorImager, and the values are expressed as fold of change after normalization for 18 S RNA levels. CON, control diets; CA, for 1% CA diets.

 
Hepatic Protein Levels of CYP3A and CYP2B—Because hepatic mRNA levels of CYP3A11 and CYP2B in FXR- and FPXR-null mice were increased following a 1% CA diet, their protein levels were determined by Western blot analysis (Fig. 6). Similar to their mRNA levels, basal protein levels of CYP3A were increased in PXR-, FXR-, and FPXR-null mice, with the highest elevation in the latter animals. However, feeding the 1% CA diet only resulted in increased CYP3A protein levels in WTs, PXR-, and FXR-nulls but not in FPXR-nulls, indicating that activation of PXR is mainly responsible for induction of CYP3A in FXR-null mice. Basal levels of CYP2B protein in FPXR-null mice were slightly increased compared with WTs (1.5-fold), which was further increased by feeding with the CA diet (1.8-fold). Compared with CYP3A, the degree of CYP2B induction was much lower.



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  		FIG. 6.
Western blot analysis of hepatic levels of CYP3A and CYP2B. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were fed a control or 1% CA-containing diet for 4 days. Livers were removed, and hepatic proteins containing membrane fractions were isolated. Microsomal protein (1 µg) was subjected to 10% SDS-PAGE and transferred by electroblotting to GeneScreen Plus membranes. The membranes were incubated with monoclonal anti-rat CYP3A1/2 or polyclonal anti-rat CYP2B antibody, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG for CYP3A1/2 or goat anti-rabbit IgG for CYP2B. Bands were visualized with ECL Western blotting detection reagents.

 
Protection of CA Toxicity in FXR- and FPXR Null Mice by Pretreatment with PB or TCPOBOP—Because CAR and CYP2B were induced in FXR- and FPXR-null mice, it was hypothesized that activation of both CAR and CYP2B might reduce the overt hepatic toxicity by bile acids. To test this possibility, two CAR activators, PB (100 mg/kg, intraperitoneal, with saline as control) or TCPOBOP (3 mg/kg, intraperitoneal, with corn oil as control), were administered 2 days before the CA diet feeding was initiated, and the treatment was continued for an additional 4 days with coadministration of the CA diet. Cotreatment of PB or TCPOBOP with CA significantly lowered the serum bile acids and total and direct bilirubin levels in FXR- and FPXR-null mice compared with treatment with CA alone, strongly indicating that activation of CAR reduced the overt bile acid toxicity (Fig. 7, A–C).



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  		FIG. 7.
Pre-treatment with the CAR activators, PB or TCPOBOP, reduced CA-elicited toxicity. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were treated for 2 days with PB (100 mg/kg, intraperitoneal), TCPOBOP (3 mg/kg, intraperitoneal), saline, or corn oil, followed by 4 days coadministration of a 1% CA diet. A, serum total bile acids; B, serum direct bilirubin levels; and C, serum total bilirubin levels. Black bars, saline/CA-treated animals; gray bars, corn oil/CA-treated animals; white bars, PB/CA-treated animals; striped bars, TCPOBOP/CA-treated animals. An asterisk indicates the statistical significance for p < 0.05 between saline/CA and PB/CA, or corn oil/CA and TCPOBOP/CA treatment for each strain.

 
Hepatic Expression of the Genes for Bile Acid and Bilirubin Metabolism and Transport following PB or TCPOBOP Treatment—To determine the mechanism by which PB or TCPOBOP treatment effectively prevented the elevation of serum bile acid and bilirubin levels in CA-fed FXR- or FPXR-null mice, mRNA levels of the major genes involved in bile acid and/or bilirubin metabolism and transport were determined (Fig. 8). Treatment with the CAR activators induced the levels of CYP2B, CYP3A11, UGT1a, GST{alpha}, MRP2, MRP3, and organic anion transporting polypeptide 2, leading to increased metabolism and excretion of bile acids and bilirubin. Surprisingly, the mRNA levels of ALAS1, the rate-limiting enzyme for bilirubin synthesis, were induced by the CAR activators as well. Because the net levels of serum bilirubin were decreased following treatment with the CAR activators, the possibility exists that the degree for induction of bilirubin synthesis was lower than that for its metabolism and excretion. Furthermore, PB and TCPOBOP affected the CAR mRNA levels differently; PB induced CAR mRNA in WT and PXR-null mice but not in FXR- and FPXR-null mice; in contrast, TCPOBOP did not affect CAR mRNA levels in WT mice, and it prevented the induction of CAR mRNA by CA in PXR-, FXR-, and FPXR-null mice. PB and TCPOBOP also differentially affected MDR1a; PB induced but TCPOBOP repressed MDR1a mRNA levels. PB and TCPOBOP tended to reduce the CA toxicity not only by inducing metabolism enzymes and export transporters but also by inhibiting the main bile acid uptake transporter, Na+/taurocholate cotransporting polypeptide. The mRNA levels of BSEP were not affected by the pretreatment with CAR activators (data not shown). In summary, pretreatment with the CAR activators reduced bile acid uptake but enhanced their metabolism and excretion, particularly in FXR- and FPXR-null mice fed with a CA diet, thus resulting in a lower bile acid or bilirubin content in the circulation.



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  		FIG. 8.
Northern blot analysis of hepatic mRNA levels for enzymes or transporters involved in bile acid and/or bilirubin metabolism and transport following CAR activator treatment. WT (F+P–), PXR-null (F+P–), FXR-null (F–P+), and FPXR-null (F–P–) mice were treated as in Fig. 7. Livers were removed, and total RNAs were isolated. An aliquot of pooled total RNA (10 µg) from 6 to 7 animals per group were subjected to 1% agarose gel, and electrophoresis was transferred to GeneScreen Plus membranes and hybridized with the indicated 32P-labeled cDNA probes. The bands were quantified using a PhosphorImager, and the values are expressed as fold of change after correction for 18 S levels. TCP, TCPOBOP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Following the discovery that FXR was a key nuclear receptor for regulating bile acid levels (5–7), PXR was identified as another bile acid sensor with relative specificity toward LCA and its metabolites (14, 15). To test the combined role of FXR and PXR in vivo in regulating bile acid synthesis, metabolism, and transport, as well as for maintaining cholesterol and lipid homeostasis, FPXR double null mice were generated by cross-breeding FXR-null with PXR-null mice. An exacerbated disturbance of bile acid and lipid homeostasis exhibited by FPXR-versus FXR- or PXR-nulls fed a CA diet confirmed a cooperative function for these two nuclear receptors in maintaining bile acid homeostasis in vivo, and further provided insight for a possible role of PXR in regulating cholesterol and lipid homeostasis. Deletion of FXR and PXR caused more severe liver damage as evidenced by a more rapid loss of body weight, higher serum levels of bile acids, and total and direct bilirubins. Meanwhile, CAR and its target gene CYP2B were induced in FXR- and FPXR-nulls fed the CA diet, suggesting that activation of CAR might limit the overt hepatic toxicity elicited by excess bile acids. A protective role for CAR activation in bile acid toxicity was confirmed by markedly reduced bile acid and bilirubin levels in FXR- and FPXR-null mice upon pretreatment with CAR activators, PB or TCPOBOP. These data suggested that induction of CAR target genes enhanced bile acid and bilirubin metabolism and excretion and therefore reduced bile acid-associated toxicity.

Disturbances of bile acid metabolism lead to cholestatic liver diseases that are often associated with hypercholesterolemia (22, 23). The current study demonstrated that deletion of FXR and PXR resulted in severe disturbances of cholesterol and lipid homeostasis, especially upon feeding a diet containing CA. Feeding CA decreased levels of serum cholesterol, PL and TG in WT, PXR- and FXR-nulls but increased those in FPXR-nulls, indicating an inability of FPXR-null mice to respond to elevated levels of bile acids. Consistent with this, a study by Wang et al. (24) showed that small heterodimer partner-null mice have decreased plasma cholesterol when fed a 1% CA diet. In addition, PXR expression is increased in small heterodimer partner-null mice, and PXR target genes are strongly activated upon CA feeding in these animals (24). The influence of PXR on cholesterol and lipid metabolism was further revealed in FPXR-null mice fed the CA diet by a significant shift of the cholesterol spectrum from HDL to VLDL/LDL, with the serum apolipoprotein profile demonstrating elevated levels of apoB100 and reduced levels of apoA-I and apoA-II, parameters associated with a proatherogenic cholesterol and lipid profile. Under a CA diet, FXR- as well as FPXR-null mice produced more nascent HDL particles, as revealed by increased hepatic apoA-I, apoA-II, and ABCA1 mRNA levels, but these particles cannot undergo subsequent maturation as a result of the inhibition of LCAT by bile acids in the serum of these animals. Thus, under CA treatment the up-regulation of hepatic apoA-I, apoA-II, and ABCA1 and possibly the down-regulation of SRB1 in FXR- and FPXR-null mice are compensatory mechanisms aimed at restoring normal plasma HDL levels. Increased cholesterol and lipid levels in the FXR-null mice have been further investigated with the results revealing that hepatic SRB1, the HDL receptor for reverse cholesterol transport, was down-regulated following loss of FXR expression (20). The combined results indicate that both FXR and PXR are critical in maintaining cholesterol and lipid homeostasis, suggesting that PXR plays a role in cholesterol and lipid homeostasis primarily through facilitating bile acid metabolism.

The current study also demonstrated that deletion of both FXR and PXR in mice almost totally blocks the normal flow of bile acids and lipids, whereas deletion of FXR alone only decreases the secretion of biliary lipids. In a previous report (20), evidence revealed that upon FXR deficiency, alternative PXR-regulated pathways could mediate the elimination of biliary acids and lipids in excess. Upon administration of the CA diet, hepatic levels of the canalicular transporter MDR1a roughly paralleled those of CYP3A and could also play a role in bile acid elimination from livers in FXR-null mice. Taken together, these data indicate that PXR-mediated activation of CYP3A11 and possibly of MDR1a permits the elimination, at least partially, of toxic bile acids from FXR-null mice livers. Because the expression pattern of BSEP, the bile salt transporter, the ABCG5/8 sterol transporters, and the MDR2 phospholipid transporter cannot account for the differences observed in bile flow between FXR- and FPXR-null mice, the current study supports the contention that bile acids/bile salts are the main driving force for maintaining regular bile formation and bile flow, which is the major route for biliary cholesterol and lipid excretion (25, 26). Consistent with this, type 2 progressive familial intrahepatic cholestasis patients are defective in BSEP and demonstrate a similar bile component profile, with very low levels of biliary bile salts, cholesterol, and phospholipids but high levels of serum bile salts and cholesterol (27, 28).

The other advantage of studying bile acid and cholesterol metabolism using FPXR-null mice is the potential to discover other nuclear receptors that may be involved in the defense against overt liver damage caused by severe cholestasis. In this regard, the current study revealed that mRNAs encoding CAR and CYP2B were elevated in the livers of FXR- and FPXR-null mice, particularly following a challenge with the CA diet. Evidence from the current study suggests that CAR may be activated by an endogenous substance(s) that exists at increased concentrations in FXR- and FPXR-null mice, particularly with the CA diet. The elevated mRNA levels of CAR in FXR- and FPXR-null mice suggest that the expression of CAR was modulated by transcription factors or nuclear receptors other than FXR at the transcriptional level. The degree of CYP2B and CAR induction in FXR-null mice was higher than in FPXR-null mice, suggesting that PXR may be a positive regulator for CAR and CYP2B in FXR-null mice, which is in line with earlier studies suggesting cross-talk between CAR and PXR, where PXR was found to regulate an overlapping but distinct sets of genes with CAR by sharing ligands and binding to similar response elements (29–31). Treatment of FXR- or FPXR-null mice with CA dramatically increased bile acid, bilirubin, and cholesterol levels, which might serve to either activate and/or serve as a mediator to induce CAR. Indeed, the involvement of CAR in protection against bile acid toxicity was further confirmed by the dramatic reduction of serum bile acid and bilirubin levels in FXR- and FPXR-null mice following pretreatment with PB or TCPOBOP, two potent CAR activators that increase target gene transcription by increasing the nuclear translocation of CAR through a ligand-independent mechanism (PB) or by serving as a direct ligand (TCPOBOP; see Ref. 30). Administration of PB to patients with cholestatic diseases enhances bile flow and reduces pruritus (32–34). In the present study, CAR activators decreased the serum bile acid and bilirubin levels, presumably due to the up-regulation of CAR target genes expressed in liver encoding bile acid and bilirubin metabolism enzymes (cyp2b, cyp3a11, ugt1a, and gst{alpha}), bile acid and bilirubin efflux transporters (mrp2, mrp3, and mdr1a), and suppression of the hepatic bile acid influx transporters (such as ntcp). Activation of CAR has also been shown to enhance bilirubin conjugation by induction of UDP-glucuronosyltransferase 1A1 through a CAR-mediated mechanism (35–37). PXR was also identified as a positive regulator for UGT1A1, and thus activation of CAR and/or PXR enhances bilirubin excretion (29, 37).

In summary, the current study revealed the critical and cooperative roles of FXR and PXR as bile acid sensors to maintain bile acid, cholesterol, and lipid homeostasis. Furthermore, evidence is provided suggesting that activation of other nuclear receptors, such as CAR, were involved in the defense against severe cholestasis. Elucidation of this mechanism(s) will aid in the understanding, prevention, and treatment of hepatic damage elicited by pathophysiological factors or toxic xenobiotics.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

¶¶ Present address: Dept. of Pharmacology, Dalhousie University, Halifax, Nova Scotia 33H 1X5, Canada. Back

§§ To whom correspondence should be addressed: Laboratory of Metabolism, Bldg. 37, Rm. 2A19A, NCI, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-9067; Fax: 301-496-8419; E-mail: fjgonz{at}helix.nih.gov.

1 The abbreviations used are: FXR, farnesoid X receptor; PXR, pregnane X receptor; CAR, constitutive androstane receptor; CA, cholic acid; LCA, lithocholic acid; PB, phenobarbital; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; SRB1, scavenger receptor B-I; ABC, ATP-binding cassette; ALAS1, aminolevulinic acid synthase 1; UGT1A1, UDP-glucuronosyltransferase 1A1; GST{alpha}, glutathione S-transferase alpha; CYP3A, cytochrome P450 3A; CYP2B, cytochrome P450 2B; BSEP, bile salt efflux protein; MDR1a, multidrug resistance protein 1a; MRP2, multidrug resistance-related protein 2; MRP3, multidrug resistance-related protein 3; WT, wild type; TG, triglycerides; PL, phospholipids; TCPOBOP, 1,4-bis-[2-(3,5-dichlorpyridyloxy)]benzene. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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