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J. Biol. Chem., Vol. 276, Issue 42, 39411-39418, October 19, 2001
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From the
Received for publication, July 6, 2001, and in revised form, August 16, 2001
Sister of P-glycoprotein (SPGP) is
the major hepatic bile salt export pump (BSEP). BSEP/SPGP expression
varies dramatically among human livers. The potency and hierarchy of
bile acids as ligands for the farnesyl/bile acid receptor (FXR/BAR)
paralleled their ability to induce BSEP in human hepatocyte cultures.
FXR:RXR heterodimers bound to IR1 elements and enhanced bile acid
transcriptional activation of the mouse and human BSEP/SPGP promoters.
In FXR/BAR nullizygous mice, which have dramatically reduced BSEP/SPGP
levels, hepatic CYP3A11 and CYP2B10 were strongly but unexpectedly
induced. Notably, the rank order of bile acids as CYP3A4 inducers and
activators of pregnane X receptor/steroid and xenobiotic receptor
(PXR/SXR) closely paralleled each other but was markedly different from their hierarchy and potency as inducers of BSEP in human hepatocytes. Moreover, the hepatoprotective bile acid ursodeoxycholic acid, which
reverses hydrophobic bile acid hepatotoxicity, activates PXR and
efficaciously induces CYP3A4 (a bile-metabolizing enzyme) in primary
human hepatocytes thus providing one mechanism for its
hepatoprotection. Because serum and urinary bile acids increased in
FXR/BAR Bile acids are synthesized from cholesterol in the liver and
secreted into the bile duct via an active process in the canaliculus. Canalicular secretion of bile acids from the liver in the form of bile
facilitates the emulsification of dietary lipids and fat-soluble vitamins. Bile acids are found in high concentrations in hepatic nuclei
(1), where they regulate gene expression through the farnesol
X-activated receptor (FXR)1
(also known as bile acid receptor (BAR)) (2, 3). Recent studies in mice
lacking FXR/BAR indicate that fecal excretion of bile acids is markedly
impaired in these mice, but serum concentrations and urinary excretion
of bile acids are increased (4). The decreased hepatic bile acid
secretion correlated with decreased expression of the major canalicular
bile salt transporter, sister of P-glycoprotein (SPGP) (5) also known
as the bile salt export pump (BSEP) (6). This finding is consistent
with results demonstrating genetic mutations in BSEP that are
associated with a severe genetic disease, type 2 progressive familial
intrahepatic cholestasis, in which total bile salt secretion decreases
to about 1% of normal (7, 8). However, despite the decreased
expression of BSEP/SPGP in the absence of FXR, it is unknown if the
murine BSEP/SPGP gene is transcriptionally regulated by bile acids.
Moreover, it is unknown if any other hepatic-ABC transporters
compensate for decreased SPGP, leading to increased serum and urinary
bile acids.
In the absence of appropriate bile acid secretion, activation of other
compensatory metabolic pathways might occur. This possibility was
suggested by the recent studies in the SPGP nullizygous mouse (9). In
the absence of SPGP the liver accumulates large amounts of
tetra-hydroxylated bile acids. Typically, such poly-hydroxylated bile
acids are not seen in the liver, and this suggests an increase in
metabolizing enzymes. Notably, one of the major human cytochromes, CYP3A4, is capable of mono-oxygenation of bile acids leading to formation of more hydrophilic bile acids, such as 6 In this report we demonstrate a critical relationship between bile
acids and the expression of BSEP/SPGP and CYP3A4 in primary human
hepatocytes. Indeed, although recent reports demonstrate that some bile
acids are PXR ligands and that FXR can influence BSEP/SPGP mRNA
expression in mouse liver, there are significant differences between
mice and humans in the composition of the bile salt pool necessitating
studies to determine whether these same mechanisms are operational in
human liver. These current studies demonstrate that both human and
mouse BSEP/SPGP promoters are transcriptionally regulated by FXR and
further imply that loss of BSEP/SPGP-mediated efflux causes
compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters.
In total, this work adds considerably to our understanding of the
regulation of bile acid homeostasis and how variations in the
composition and concentration of bile acid can affect the activity of
two different transcription factors, and how those bile acid activated
transcription factors regulate distinct pathways involved in bile acid elimination.
Plasmids
hPXR excised from pSG5-hPXR Human SPGP Luciferase Reporter Construct--
A 140-bp fragment
of the SPGP promoter was obtained from human genomic DNA by PCR using
primers 5'-ACACTCTGTGTTTGGGGTTATTGC-3' and
5'-TAAAGCACTGAACAGAATTCAA-3'. The resultant PCR product was cloned into
pCR2.1-TOPO and the sequence verified. The insert was then released
from pCR2.1-TOPO (Invitrogen) using KpnI and EcoRV, gel-purified (QiaexTM,
Qiagen), and ligated into pGL3-Basic (Promega) that had been linearized
previously using KpnI and SmaI to create
hBSEP/SPGP-LUC.
Mouse BSEP Luciferase Reporter--
50 ng of BALB/c mouse
liver DNA (CLONTECH) was amplified with 50 pmol of
mouse BSEP/SPGP primers (forward) 5'-CACTCTGGGTTTGGGT-3' and
(reverse) 5'-TAAAGCATTGAACAGAAATCAGGC-3', and high fidelity Taq polymerase and the PCR product were subcloned
into pCR2.1. The resulting construct was digested with EcoRI
and subcloned into pGL2 basic (Promega) to create mBSEP/SPGP-LUC.
CYP3A4-PXRE-LUC containing +53/ Preparation of Primary Cultures of Human Hepatocytes
Human livers were procured from donor organs that were not
suitable for whole organ transplantation or from remaining tissue after
reduced allograft transplantation. Donor livers were flushed, in
situ, and maintained with Belzar's UW solution. Hepatocytes were
isolated within 24 h of cross-clamp. Reasons for not using tissues
for transplantation included traumatic damage, errors in organ harvest,
brief anoxic periods, or macro- or microsteatosis. Human hepatocytes
were isolated essentially as described by Strom et al. (15,
16). Cells were plated on collagen-coated 6-well plates or 60-mm
culture dishes and maintained in Modified Williams E for 48 h and
then treated with bile acids or drugs for 48 h. The activity of
CYP3A4 was determined by measuring the testosterone metabolism in
cultured hepatocytes as described by Kostrubsky et al. (17).
Cells were then scraped from the plates and proteins analyzed on immunoblots.
Immunoblot Analysis
For membrane transport proteins, crude membranes were prepared
from human liver or mouse as described previously (18). Protein was
estimated by the Bio-Rad protein assay using bovine serum albumin as
standard. The crude membrane proteins (300 µg) were analyzed on 7.5%
polyacrylamide gels followed by immunoblotting with rabbit anti-SPGP
IgG (19) appropriate secondary antibody and developed with the Amersham
ECL detection system (Amersham Pharmacia Biotech). The same blot was
stripped of antibodies and redeveloped with polyclonal rabbit
anti-P-glycoprotein (18). Proteins on the blot were stained with
Ponceau Reagent (Sigma), and the SPGP signal was normalized to Ponceau
staining signal in a region of comparable molecular weight. Mouse liver
microsomes were prepared (20), and 10 or 20 µg of protein was
analyzed on 10% slab polyacrylamide gels electrophoresed and
immunoblotted using the following antibodies: polyclonal goat anti-rat
CYP3A1 antibodies (21) or a monoclonal antibody against rat CYP2B1 (mAb
BE28.2) from Dr. Paul Thomas (Rutgers University, Piscataway, NJ). All
primary antibodies were followed by appropriate secondary antibodies
coupled with peroxidase and developed with the ECL detection system
(Amersham Pharmacia Biotech) (22).
Northern Blots
Total RNA was extracted and 10 µg analyzed by Northern blot
with probes to CYP3A11 and Reverse Transcription-PCR
5 µg of total RNA from mouse liver was reverse-transcribed
according to the manufacturer's instructions (Life Technologies, Inc.). Transporter cDNAs were amplified from first-strand cDNA by using the following oligonucleotides: MDR1a sense, aac agc ggt ttc cag gag ctg ctg g, and antisense, cat tgc ctg gaa gaa cat tcc
gat t; MDR1b sense, cag tgt ttg cca tag tat ttt caa gga ttg, and
antisense, ccc ttt aac act aga agc atc ac; MDR 2 sense, tat ccg cta tgg
ccg tgg gaa, and antisense, atc ggt gag cta tca caa tgg; MRP 1 (bp
2072-2573 in AF022908) sense, 5'-TGCTGGCTGAGATGGACAAG-3', and
antisense, 5'-CGGTCTAGCAGCTCCTGATA-3'; MRP 2 (bp 112-446 in AA027420)
sense, 5'-GTCATCACTATCGCACACAG-3', and antisense, 5'-TTCTACAGGGTGGTTGAGAC-3'; MRP 3 (bp 42-352 in AI391398)
sense, 5'-CGCTCTCAGCTCACCATCAT-3', and antisense,
5'-GGTCATCCGTCTCCAAGTCA-3'; MBCRP (bp 47-373 in AF103875) sense,
5'-CCATAGCCACAGGCCAAAGT-3', and antisense, 5'-GGGCCACATGATTCTTCCAC-3';
MRP4 (bp 161-382 in W54702) sense, 5'-GGTTGGAATTGTGGGCAGAA-3', and
antisense, 5'-TCGTCCGTGTGCTCATTGAA-3'.
The control GAPDH cDNA was amplified with the following: sense,
5'-acc aca gtc cat gcc atc ac-3', and antisense, 5'-tcc acc acc ctg ttg
ctg ta-3' primers.
PCRs were performed with 1-5 µl of cDNA, 5 µl of buffer, 3 µl of 25 mM MgCl2, 1 µl of each 10 mM dNTP, 1-2 µl of 10 µM primers, 0.4 µl
of high fidelity Taq polymerase, and 50 µl of water. The reactions were initially denatured at 95 °C (5 min) and then cycled in a MJ Research Tetrad (Waltham, MA) at 95 °C for 30 min; 55, 58, or 60 °C for 30 min; and 68 °C for 1 min for 23 cycles (GAPDH) or
35 cycles (ABC transporters) with a final 68 °C extension step performed for 10 min at the end of all the cycles. PCR products were
analyzed on 0.5% NuSieve, 0.5% agarose (w/v) containing ethidium bromide for visualization.
Electrophoretic Mobility Shift Assay
Human FXR and RXR- Transient Transfection Assays
HepG2 cells were plated in 24-well dishes at 0.3 × 106 cells per well. Twenty four hours later cells were
transfected with 1000 ng of human or mouse Sister-P-glycoprotein-LUC,
200 ng of mFXR, and 500 ng of Animals
Mice were housed in a pathogen-free animal facility under
standard 12 h light/12 h dark cycle. Prior to the administration of special diets, mice were fed standard rodent chow and water ad
libitum. All diets were prepared by Bioserv (Frenchtown, NJ) and
were based upon a standard AIN-93G rodent diet containing 58.6%
carbohydrate, 18.1% protein, 7.2% fat, 5.1% fiber, 3.4% ash, 10%
moisture (control diet). The cholic acid diet was identical to the
control diet but supplemented with 1% (w/w) cholic acid. 8-12-Week-old male mice were used for all experiments and were allowed
water ad libitum. After 5 days of feeding the indicated diets, animals were were euthanized by carbon dioxide asphyxiation at
the mid-light phase period. Tissues were weighed, sectioned, and
snap-frozen in liquid nitrogen and stored at Expression of BSEP in Human Liver and in Primary Human
Hepatocytes--
We found over 7-fold variation in BSEP/SPGP
expression in normal human liver (Fig.
1a). Because some of the
normal human liver samples were from donors who received drugs that can
induce drug detoxification genes due to activation of PXR/SXR and CAR
nuclear hormone receptors, we reasoned that some of the interindividual variation in BSEP/SPGP expression was due to drug induction of this
protein. We compared the expression of BSEP/SPGP in primary human
hepatocytes treated with prototypical ligands for PXR and CAR receptors
(24, 26, 27). However, treatment with rifampin or phenobarbital or
troglitazone (Fig. 1, b and c) caused no
induction of BSEP expression.
Because the hepatic concentration of bile acids varies among
individuals, we tested whether any bile acids affected BSEP expression in primary human hepatocytes. The major human bile acids
chenodeoxycholic acid (CDCA; all bile acids are according to accepted
nomenclature (47)) and deoxycholic acid (DCA) (1) (Fig. 1, b
and c) each induced BSEP/SPGP at 10 µM and
further induced it at 50 µM. Although there was
interindividual variation in BSEP/SPGP induction between preparations
of human hepatocytes, the bile acids consistently induced BSEP/SPGP
with an apparent rank order of potency of CDCA > DCA with LCA the
least potent (Fig. 1c). The hydrophilic DKCA also
modestly induced BSEP/SPGP. This rank order of BSEP/SPGP induction is
similar to the potency of these same bile acids as ligands for the FXR
receptor and is consistent with a role for FXR in the induction of
BSEP/SPGP.
Bile Acids Transcriptionally Activate the Human and Mouse BSEP
Promoters and Require FXR--
To determine whether bile acids utilize
FXR to activate transcriptionally the murine and human BSEP promoters,
we isolated the murine BSEP gene and determined its CAP site by primer
extension analysis (not shown) (Fig.
2a). Comparison of ~2
kilobase pairs upstream of the murine (AY039785) and human BSEP
(AC008177) revealed that the genes were about 68% identical over the
entire 2 kilobase pairs, with the region of greatest sequence identity (83%) between bp
Electrophoretic mobility shift assay analysis was performed with
in vitro translated FXR, RXR, and PXR on segments of both the mouse and human BSEP promoters that contained putative FXR response
elements (Fig. 2d). Both human and mouse BSEP promoters contained regions that specifically bound FXR·RXR complexes,
and FXR·RXR binding was competed dose-dependently from
murine and human BSEP promoter fragments by the unlabeled region of the
human BSEP that bound FXR·RXR. PXR·RXR failed to form any complex
with either the mouse or human BSEP/SPGP promoter fragments (not
shown). In total, these studies indicate that the human and mouse
BSEP/SPGP genes are transcriptionally activated by FXR and that their
minimal promoters specifically bind FXR.
BSEP/SPGP Expression Requires FXR in Vivo, whereas CYP3A, CYP2B10,
MRP3, and MRP4 Are Induced in the Absence of FXR--
BSEP mRNA
decreases in the absence of FXR (4). We compared expression of BSEP
protein in normal mice and mice nullizygous for FXR, and in those same
mice fed either normal chow or 1% cholic acid, a concentration that
increases the amount of total hepatic nuclear bile acids (4) (Fig.
3a). Consistent with the
expression of BSEP mRNA, basal levels of BSEP were dramatically
reduced in the absence of FXR, and dietary cholic acid increased BSEP
expression only in the wild type mice (Fig. 3a).
Mice lacking BSEP/SPGP (9, 29) showed elevated concentrations of
hydroxylated bile acids in the liver and serum suggesting compensatory
changes in expression of bile acid-metabolizing enzymes and alternative
transporters. Moreover, a recent report (11) found that although
treatment with lithocholic acid induced CYP3A in mouse liver, this
up-regulation occurred even in mice lacking the pregnane X receptor
(PXR), demonstrating that some other receptor, such as FXR, must also
mediate CYP3A induction by bile acids. However, FXR is clearly not a
positive regulator of CYP3A expression because CYP3A11 protein and
mRNA were dramatically increased in the absence of FXR (Fig. 3,
a and b). Concurrent analysis of CYP2B10 protein
revealed a pattern of regulation identical to CYP3A11. These studies
indicate that a strong decrease in BSEP/SPGP in the absence of FXR and
an increase in hepatic bile acid concentration correlate with enhanced
CYP3A11 and CYP2B10 levels.
Mice lacking BSEP also demonstrated (a) increased bile acids
in the serum, (b) an increase in biliary phospholipids, and
(c) enhanced secretion of hydroxylated bile salts into bile.
These findings suggest that in the absence of BSEP compensatory
induction of hydroxylated bile acid and phospholipid efflux
transporters occurs. Induction of mdr2 (Fig. 3c), a drug and
phospholipid transporter at the bile canaliculus, observed in the FXR
( Regulation of CYP3A by Bile Acids in Primary Human
Hepatocytes--
To determine if bile acids directly up-regulated the
human orthologue, CYP3A4, we utilized human hepatocyte cultures and
treated them with bile acids (Fig.
4A). In four separate human
hepatocyte preparations we observed CYP3A4 induction by bile acids.
Notably, the hierarchy of bile acids as CYP3A inducers (Fig. 4) was
dramatically different than the rank order of these same bile acids as
inducers of BSEP or as ligands of FXR (Fig. 1). Whereas CDCA was the
most efficacious inducer of BSEP, UDCA and DKCA were the most effective bile acids inducing CYP3A protein (Fig. 4A) and associated
testosterone 6
To confirm that each of the bile acids inducing CYP3A4 was a PXR/SXR
ligand, we compared activation of a CYP3A4 PXRE3-LUC reporter in HepG2 cells with and without stable expression of PXR. All
of the cells were co-transfected with an NTCP expression plasmid to
permit uptake of the conjugated bile acids. Robust transcriptional
activation of PXRE3-LUC by rifampin and
dose-dependent induction by bile acids was observed in
HepG2-PXR cells (Fig. 5) but not in cells not co-transfected with PXR
(not shown). To determine if UDCA and the taurine-conjugated bile acids
directly activated PXR, we utilized a chimeric receptor system
employing (a) the ligand binding domain of the human SXR
fused to the DNA binding domain of the yeast transcription factor,
GAL4, co-transfected into cells with (b) a reporter
containing the GAL4-DNA binding domain. The majority of bile acids
activating PXRE3-LUC were also good activators of the
SXR-Gal4 fusion (Fig. 5). However, 50 and 100 µM LCA and
100 µM DCA and CDCA caused a decrease of 25-60% in
total cellular protein in COS-7 cells but not HepG2 cells, resulting in
a significant decrease in GAL-SXR activation in COS-7 cells and likely
accounting for the discrepancy in their ability to activate SXR in the
two assays. The data also shows that in the presence of NTCP, the more
physiologically relevant taurine-conjugated bile acids can also
activate PXR.
These studies were initiated by our observation that human liver
BSEP/SPGP is subject to considerable interindividual variability. We
reasoned that differences in hepatic BSEP/SPGP might be attributed, in
part, to induction of BSEP/SPGP by co-administered drugs or variable
activation of FXR/BAR by endogenous ligands such as bile acids. The
current studies in human hepatocytes demonstrate that representative
PXR/SXR and CAR ligands (rifampin and phenobarbital) did not increase
BSEP/SPGP expression or activate its promoter. Thus, our studies
indicate that the enhanced bile acid elimination in patients treated
with rifampin (13) is unlikely due to transcriptional up-regulation of
BSEP/SPGP. Nonetheless, we show that FXR/BAR transcriptionally
activates both murine and human BSEP/SPGP promoters. Moreover, bile
acids increased BSEP/SPGP expression in primary human hepatocytes with
the potency being CDCA > DCA > LCA, a finding closely
agreeing with their ability to activate the nuclear farnesyl/bile acid-receptor, FXR/BAR (2, 3). FXR and RXR heterodimers bind to
specific DNA motifs (e.g. consensus = AGGTCA (3))
arranged as an inverted repeat with a single nucleotide spacer (IR1).
Such an element exists in a conserved region of both mouse and human BSEP/SPGP promoters (Fig. 2), and these DNA motifs specifically bound
authentic FXR/RXR heterodimers and were activated in transient transfections by the same bile acids that increased expression of the
endogenous BSEP/SPGP in human hepatocytes. It appears that coordinated
induction of BSEP/SPGP is part of a pleiotropic response to regulate
bile acids because hepatic canalicular bile output decreases in the
absence of FXR as does expression of BSEP/SPGP (4), and because
BSEP/SPGP is positively up-regulated by the major bile acids.
Therefore, interindividual differences in the expression of hepatic
BSEP/SPGP could arise from varying levels of hepatic bile acids as well
as functional differences in either BSEP/SPGP or FXR, perhaps due to
mutations of other alterations. Hepatic variation in BSEP/SPGP
expression could contribute to individual differences in bile flow,
levels of bile acids, hepatic clearance of drugs (31), and also to
gallstone susceptibility. Indeed, hepatic BSEP/SPGP expression is
dramatically elevated in the mouse strain C57L, a strain highly
susceptible to gallstones after ingesting a high fat diet, but BSEP
expression is much lower in AKR-resistant mice that do not develop
gallstones (32).2 Therefore,
we imagine that high SPGP/BSEP expression could increase gallstone
susceptibility in humans.
Our studies further demonstrate an intriguing relationship between the
absence of the nuclear receptor, FXR, loss of SPGP expression, and
up-regulation of CYP3A, CYP2B, and ABC transporters such as MRP3 and
MRP4. These observations suggest the following sequelae: absence of FXR
leads to strong SPGP/BSEP down-regulation that causes elevated
hepatocellular bile acids, particularly SPGP/BSEP substrates
(e.g. CDCA and DCA (9)), and these bile acids then bind PXR
(this work and Ref. 10) and up-regulate CYP3A and CYP2B. This scenario
is consistent with CYPs likely participating in the enhanced formation
of tetrahydroxylated bile acids in mice lacking BSEP/SPGP (9). Notably,
several bile acids, but particularly the more hydrophilic bile acids
such as DKCA and UDCA, significantly up-regulated in primary human
hepatocytes CYP3A4 protein and catalytic activity (testosterone
6 Our results reveal that expression of ABC transporters compensatorily
increase in FXR nullizygous mice. Although MRP3 transports bile acids
(12), MRP4 has not been linked to bile acid transport and has only been
demonstrated to transport nucleotide monophosphate derivatives (33).
Thus, it is a formal possibility that MRP4 plays a role in hepatic bile
acid homeostasis. The induction of these ABC transporters in the
absence of FXR and a further increase after cholic acid feeding
indicate regulation independent of FXR but nonetheless dependent upon
hepatic bile acid accumulation. Such compensatory increases in ABC
transporters are not without precedent as the absence of MRP2 due to a
genetic deficiency leads to a compensatory increase in the expression
of MRP1 to facilitate hepatic removal of anionic compounds by transport
into the sinusoidal blood. Notably, MRP3 up-regulation by bile acids is
also consistent with the recent report showing a 30-fold increase in
hepatic MRP3 after ligation of the common bile duct, a model of
cholestasis (34, 35). Finally, the pattern of MRP3 and MRP4
up-regulation in the FXR-null animals is remarkably similar to CYP3A
and CYP2B and suggests a common regulator, such as PXR, in these animals.
Because rifampin can activate PXR/SXR to induce CYP3A which metabolizes
lithocholic acid (10, 11), and because rifampin has been shown to
induce remission of cholestasis in some patients (13), rifampin therapy
has been proposed as a treatment for cholestatic liver disease (10,
11). Intriguingly, rifampin increases bile salt concentrations in serum
(36) suggesting rifampin-activated PXR up-regulates alternative ABC
transporters (perhaps such as MRP4 or MRP3) that contribute to the
reported decrease in bile acid toxicity. However, it should also be
noted that rifampin has been reported to decrease biliary secretion of
bile acids (36), and high doses of rifampin have also been shown to
induce reversible cholestasis in some patients (37), presumably
mediated through inhibition of BSEP/SPGP (36) and canalicular secretion
of bile acids. Therefore, alternative PXR agonists that do not inhibit
BSEP/SPGP may be preferable to rifampin for treatment of human cholestasis.
Although a good correspondence exists between bile acids as inducers of
either BSEP or CYP3A4 proteins in primary human hepatocytes and
activation of the BSEP promoter and PXR, respectively, in HepG2 cells,
some discrepancies exist. For instance, LCA is an efficacious PXR and
FXR activator in HepG2 and CV1 cells. In contrast, in human
hepatocytes, LCA less effectively increases SPGP/BSEP and CYP3A4, a
finding suggesting hepatic bile acid metabolism (e.g.
CYP3A-mediated (10)) plays a role in the amount of ligand available for
FXR and PXR in the liver. Discrepancies between LCA activation of FXR
and PXR in CV1 cells and induction of target genes in human hepatocytes
may also due to the much greater toxicity of LCA at 50 and 100 µM to primary hepatocytes. Another possibility is that
LCA is differentially conjugated (e.g. sulfation) in primary cells compared with replicating cell lines, and this conjugation alters
its efficacy as a nuclear receptor ligand.
Paradoxically, some bile acids can decrease liver toxicity. Leuschner
et al. (38) found that ursodeoxycholic acid (UDCA) administration reversed hepatotoxicity, and UDCA treatment is used to
ameliorate primary biliary cirrhosis (39, 40). Similarly, chenodeoxycholic acid- or deoxycholic acid-induced rodent liver hepatotoxicity can be reversed by co-administration of less detergent bile acids such as UDCA or tauroursodeoxycholic acid (TUDCA) (41). How
does administration of one bile acid reverse the toxicity of another in
rodents or humans? Whereas a number of mechanisms have been proposed
(39, 42), one possibility was that hydroxylated bile acids such as UDCA
(as opposed to the major hydrophobic bile acids) preferentially
activated alternative bile acid signaling pathways. UDCA does not
activate FXR (28), which is consistent with its poor induction of
BSEP/SPGP in primary human hepatocytes. In contrast, UDCA activated PXR
and was one of the most effective bile acids tested at inducing CYP3A4
in human hepatocytes. Thus the reversal of cholestasis in humans by
UDCA may include PXR-mediated activation of CYP3A4 and perhaps drug
transporter targets that lead to enhanced metabolism and efflux of
hepatotoxic bile acids.
We have shown previously a functional relationship between the MDR1
transporter, P-glycoprotein, CYP3A, and the nuclear hormone receptor
PXR (18, 22, 43-45). Our results now extend this paradigm to include
FXR, BSEP/SPGP, and CYP2B as well as MRP3 and MRP4 transporters. This
interactive network of transporters and cytochromes P450 participate in
hepatic bile acid homeostasis. Normal physiological concentrations of
bile acids feed forward to activate FXR and induce BSEP/SPGP. In turn,
BSEP/SPGP efflux of bile acids modulates the strength and duration of
FXR activation. The intact FXR/BSEP pathway appears essential to keep
the bile acid-mediated activation of PXR in check. Thus one can
envision during abnormal physiological conditions where FXR signaling
or BSEP/SPGP function is impaired or ablated, elevated concentrations
of bile acids can activate nuclear hormone receptors such as PXR to
induce CYPs and alternative ABC transporters to accelerate removal of
bile acids. Although our studies reveal strong parallels between mouse
and man and the genes regulating bile acid homeostasis, it should be
noted that marked differences exist between the bile acid pool
composition in mouse and man (29, 40). In man the primary bile acids
are less hydrophilic (e.g. cholic and chenodeoxycholic
acid), whereas in mouse the major and more hydrophilic bile acids are
cholic and muricholic acids. Thus, accumulation of hepatic bile acids due to alteration or loss of BSEP/SPGP expression could lead to different phenotypes between mouse and man, and these may depend, in
part, upon the ability to activate their respective nuclear receptors.
This speculation seems likely when we consider that LCA is a much more
potent activator of the human PXR/SXR compared with the murine form
(11). Furthermore, it is tempting to postulate that species differences
in bile acid composition are one physiological selective pressure
driving the unexpectedly large differences in ligand binding pocket
between rodent and human PXR/SXR (46).
We thank the members of the Schuetz
laboratory for their thoughtful comments and Dr. Rommel Tirona for
construction of human BSEP-LUC.
*
This work was supported by National Institutes of Health
Research Grants GM60346, ES08648, ES058571, ES08648, ES05780, and P30
CA21745, Cancer Center support grant, the American Lebanese Syrian
Associated Charities, and Grant DK92310.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 16, 2001, DOI 10.1074/jbc.M106340200
2
E. G. Schuetz, S. Strom, K. Yasuda,
V. Lecureur, M. Assem, C. Brimer, J. Lamba, R. B. Kim, V. Ramachandran, B. J. Komoroski, R. Venkataramanan, H. Cai,
C. J. Sinal, F. J. Gonzalez, and J. D. Schuetz,
unpublished data.
The abbreviations used are:
FXR, farnesol
X-activated receptor;
ABC, ATP-binding cassette;
BAR, bile acid
receptor;
PXR, pregnane X receptor;
SXR, steroid and xenobiotic
receptor;
RXR, retinoid X receptor;
BSEP, bile salt export pump;
SPGP, sister of P-glycoprotein;
PCR, polymerase chain reaction;
NTCP, sodium taurocholate cotransporter protein;
bp, base pair;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CA, cholic acid
(3
Disrupted Bile Acid Homeostasis Reveals an Unexpected Interaction
among Nuclear Hormone Receptors, Transporters, and Cytochrome P450*
,,,,,,
,,,
Department of Pharmaceutical
Sciences, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, the Departments of § Pathology
and Pharmaceutical Sciences, the University of Pittsburgh,
Pittsburgh, Pennsylvania 15261, the ¶ Department of
Pharmacology, Vanderbilt University, Nashville, Tennessee 37203, and
the ** Laboratory of Medicine, National Institutes of Health,
Bethesda, Maryland 20892
ABSTRACT
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ABSTRACT
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/ mice, we evaluated hepatic transporters for compensatory changes that might circumvent the profound decrease in BSEP/SPGP. We
found weak MRP3 up-regulation. In contrast, MRP4 was substantially increased in the FXR/BAR nullizygous mice and was further elevated by
cholic acid. Thus, enhanced hepatocellular concentrations of bile
acids, due to the down-regulation of BSEP/SPGP-mediated efflux in FXR
nullizygous mice, result in an alternate but apparent compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters that is
consistent with activation of PXR/SXR by bile acids.
INTRODUCTION
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-hydroxylated lithocholic acid (10). Indeed, recent studies demonstrated that one
nuclear hormone receptor that regulates CYP3A, the pregnane X
receptor/steroid and xenobiotic receptor (PXR/SXR), was unexpectedly bound and activated by some bile acids (e.g. LCA (10, 11)). Thus, CYP3A metabolism of bile acids such as lithocholic acid (10) may
protect from bile acid-mediated hepatotoxicity. In addition, it is
possible that transporters that efflux bile acids (e.g.
BSEP/SPGP or MRP3 (12)) compensatorily increase. Such an alternate
route of bile acid disposal could also explain how rifampin decreases
hepatic cholestasis (13) and complements the conventional idea that
this occurs primarily by enhancement of bile acid metabolism secondary
to PXR activation (11). Currently, it is unknown how bile acid
alterations affect expression of ABC transporters.
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ATG (kindly provided by Dr. S. Kliewer) was subcloned into pcDNA3. CMX-FXR was kindly provided by
Dr. Barry Forman. Gal4-PM2-SXR and TK-(MH100)4-LUC
(tkUAS-LUC) were kindly provided by Dr. Bruce Blumberg. The human
hepatic sodium taurocholate cotransporter protein (NTCP) open reading frame was subcloned into the EcoRI site in the pTM1 vector.
362 and 7208 to 7836 of
the CYP3A4 gene in pGL3B (Promega) was prepared by Dr.
Rommel Tirona as described previously (14).
-actin as described previously
(23).
were each in vitro transcribed
and translated using a TNT Kit (Promega, Madison, WI) according to the manufacturer's instructions. Double-stranded, 32P-labeled
oligonucleotides representing the human SPGP FXR DNA-binding sequence,
containing a potential inverted repeat with 1-base pair spacer (IR-1,
underlined) (hereafter referred to as hBSEP)
5'-GCTGCCCTTAGGGACATTGATCCTTAGGCAAATAGATAAT-3' or an oligonucleotide of the mouse BSEP promoter (mBSEP) containing an
IR-1 (underlined)
5'-TCTGGACTTTAGGCCATTGACCTATAAGCAAATAGATAGT-3', were incubated with 10 mM Tris (pH 8.0), 40 mM
KCl, 0.05% Nonidet P-40, 6% glycerol, 1 mM
dithiothreitol, 0.2 µg of poly(dI-dC), and 2.5 µl of RXR and FXR
in vitro transcribed and translated protein (24). Reactions
were set up in the absence or presence of 50-, 100-, or 200-fold molar
excess unlabeled hBSEP, 200 × excess mBSEP or hBSEP-S for
competition. Complexes were resolved by electrophoresis through a
nondenaturing 4% polyacrylamide gel and analyzed on PhosphorImager.
-galactosidase plasmid
(pSV--galactosidase; Promega Corp., Madison, WI) by calcium
phosphate overnight. HepG2-PXR cells stably expressing PXR (created by
calcium phosphate co-precipitation of hPXR-pcDNA3 and clonal
selection in G418) were co-transfected with 300 ng of CYP3A4-PXRE-LUC
and 30 ng of an NTCP expression plasmid. COS-7 cells were plated in
12-well dishes at 0.3 × 106 cells per well. Twenty
four hours later, cells transfected with 400 ng of Gal4-PM2-SXR, 600 ng
of TK-(MH100)4-LUC (tkUAS-LUC), and 60 ng of NTCP
expression plasmid by LipofectAMINE (Life Technologies, Inc.). The next
day, all cells were washed once with medium and fresh medium containing
10% charcoal-stripped dilipidated calf serum (Sigma) with or
without xenobiotics or bile acids. Twenty four hours later, cells were
harvested, lysed, and centrifuged at 10,000 × g, and
luciferase activities were determined on an aliquot of supernatant (25)
according to the manufacturer's instructions (Luciferase Assay System,
Promega Corp., Madison, WI) using an automated luminometer (model
OPTOCOMP 1, MGM Instruments, Hamden, CT). An aliquot (50 µl) of
supernatant was assayed for -galactosidase using a spectrophotometer
(model 3550, Bio-Rad) set at a wavelength of 415 nm. Luciferase
activities were normalized to -galactosidase activity or cellular
protein. All experimental values were averaged from triplicate
determinations in individual experiments, and the experiment was
repeated at least three times.
80 °C until use. All
protocols and procedures were approved by the NCI Division of Basic
Sciences Animal Care and Use Committee and are in accordance with the
National Institutes of Health guidelines.
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View larger version (43K):
[in a new window]
Fig. 1.
BSEP/SPGP expression in human liver and
up-regulation by bile acids in human hepatocyte culture.
a, 300 µg of crude membranes from human livers were
analyzed by immunoblot for BSEP/SPGP and P-glycoprotein. The signal for
BSEP was normalized to the amount of Ponceau staining. b and
c, primary human hepatocytes from representative persons
(HH786 and HH867) were cultured for 48 h and
then treated from 48 to 96 h with CDCA, DCA, DKCA, and LCA at 10 or 50 µM (50 µM bile acids b,
HH 786), 10 µM rifampin (RIF) or
troglitazone (TGZ), or 2 mM phenobarbital
(PB), and lysates were prepared and 100 µg examined on
immunoblots developed with anti-BSEP/SPGP IgG. 20 µg of LLC-PK1 cells
stably expressing SPGP/BSEP served as a positive control.
48 and +96 (relative to the murine BSEP CAP site).
We amplified this region from both mouse and human BSEP and subcloned
them into promoterless luciferase vectors. The human and mouse
BSEP/SPGP promoters were co-transfected with an FXR expression vector
into HepG2 cells (Fig. 2, b and c). The BSEP promoters were activated by FXR even without addition of exogenous bile
acids, and this is likely due to the fact that HepG2 cells have the
enzymatic machinery, i.e. CYP7A, to synthesize bile acids (2). Addition of bile acids strongly activated both the human and mouse
BSEP promoters with the strongest activation by the two primary bile
acids CDCA and DCA (Fig. 2, b and c), bile acids that are also effective activators of FXR (28) and inducers of BSEP in
primary human hepatocytes (Fig. 1). LCA was also a good activator of
the BSEP promoters. The BSEP promoter was not activated by rifampin
treatment regardless of whether FXR (Fig. 2, b and
c) or PXR (not shown) was co-transfected.
View larger version (35K):
[in a new window]
Fig. 2.
The human and mouse BSEP/SPGP promoters and
their regulation by FXR/BAR. a, alignment of mouse and
human BSEP/SPGP 5'-untranslated regions and promoter regions cloned
into luciferase reporter constructs. b and c,
HepG2 cells were co-transfected with SV40- -galactosidase. The
human (hBSEP/SPGP) promoter-luciferase reporter was
co-transfected with the FXR expression plasmid (open bars)
or not (closed bars) and treated with the indicated bile
acids or rifampicin (RIF). (b) The mouse
BSEP/SPGP promoter-luciferase promoter with or without the FXR
expression plasmid were untreated controls (CT) or were
treated with 20 µM rifampin (RIF) or 50 µM bile acids as indicated (c). Luciferase
activities were normalized to -galactosidase activity, and the fold
increases in treated groups over untreated controls are shown. Values
represent the mean ± S.D. from triplicate determinations of a
single experiment that was performed at least three times.
d, electrophoretic mobility shift assay for
FXR·RXR-BSEP promoter complexes. 32P-Labeled
oligonucleotides containing the conserved FXR binding sequence in mBSEP
or hBSEP were incubated with in vitro transcribed and
translated FXR and RXR. Reactions were incubated in the absence (no
competitor) or presence of 50-200-fold molar excess of the indicated
unlabeled oligonucleotides and electrophoresed and complex formation
was assessed by a PhosphorImager.
View larger version (31K):
[in a new window]
Fig. 3.
Absence of FXR profoundly decreases BSEP/SPGP
but induces CYPs and other ABC transporters. Livers of FXR (+/+)
or ( /) mice (half of each genotype ate a normal diet and half ate a
diet containing 1% cholic) were analyzed. a, crude liver
membranes or microsomes were analyzed on immunoblots developed with
antibody to BSEP/SPGP, CYP3A, or CYP2B. b, Northern blot
analysis of CYP3A11 and -actin in total liver RNA.
c, total liver RNA was analyzed by reverse transcription-PCR
analysis using primers specific for the indicated mouse ABC
transporters or GAPDH.
/) mice fed cholic acid could be responsible for enhanced efflux
of phospholipids. It should be noted that although mdr2 has been
reported as up-regulated by bile acids (30), no FXR-binding motifs were
found in the first 1380 bp of the mdr2 promoter. The
ABC transporters MRP3 and MRP4 were induced in FXR null animals and
further increased in FXR null mice fed cholic acid (Fig.
3c). These efflux transporters are located on the lateral
membrane of the hepatocyte and possibly participate in enhanced
secretion of bile acids into the serum. The pattern of induction of
these transporters in these mice was strikingly similar to that of
CYP3A11 and CYP2B10 (Fig. 3, a and b). Thus, it
appears that in the absence of FXR and the concurrent loss of the bile
acid exporter, alternative mechanisms of metabolism and efflux
transporters step up to handle the toxic build up of bile acids in the liver.
-hydroxylase activity (Fig. 4B). Although
two recent reports in CV1 cells (10, 11) and our own studies in COS-7
cells (Fig. 5) found that 100 µM lithocholic acid, CDCA, and DCA could activate PXR/SXR
and transcriptionally activate the CYP3A4 promoter, this concentration
of bile acids was visibly toxic to the primary human hepatocytes. Total
hepatic protein was decreased 50, 23, and 12% by 100 µM
LCA, UDCA, and CDCA, respectively, and 38% by 50 µM LCA.
In hepatocyte preparations from two other persons, only 50 µM DKCA (as directly compared with 50 µM
LCA, DCA, or CDCA) induced CYP3A4 protein expression and associated
testosterone 6-hydroxylase activity (2.5- and 3.4-fold). Of the bile
acids tested, only 100 µM LCA and taurohydrodeoxycholic
acid failed to induce testosterone 6-hydroxylase activity (Fig.
4B). We also tested UDCA, TUDCA, TLCA, and TLDCA, because
these more hydrophilic bile acids have been shown to decrease the
toxicity of more hydrophobic bile acids in humans or rodents, a
protective mechanism that potentially involved induction of CYP3A4.
Only UDCA and TUDCA were effective inducers of CYP3A4 protein in
primary human hepatocytes.
View larger version (25K):
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Fig. 4.
Bile acids induce CYP3A in primary human
hepatocytes. A, primary hepatocytes were cultured for
48 h and then treated with the indicated µM
concentrations of bile acids, 10 µM rifampin, or were
untreated controls (CT). 100 or 50 µg of total cell lysate
was analyzed on immunoblots with antibodies to BSEP/SPGP and CYP3A
(18), respectively. B, immediately before harvest, the
hepatocytes were incubated with testosterone and the medium later
analyzed for the formation of 6 -hydroxytestosterone.
View larger version (14K):
[in a new window]
Fig. 5.
Solid bars, HepG2 cells stably
expressing hPXR were co-transfected with CYP3A4-PXRE-LUC, an NTCP
expression plasmid, and a -galactosidase construct. Gray
bars, COS-7 cells were co-transfected with an NTCP expression
plasmid, TK-(MH100)4-Luc (tkUAS-LUC) reporter and a
chimeric GAL4-SXR plasmid containing the SXR/PXR ligand binding domain.
The transfected cells were subsequently treated for 24 h with 50 or 100 µM bile acids, 10 µM rifampin, or
were untreated controls (CT). Luciferase activities were
normalized to -galactosidase activity (HepG2) or cell protein
(COS-7), and fold increases in treated group over untreated controls
are shown. Values represent the mean ± S.D. from triplicate
determinations of a single experiment that was performed at least three
times.
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-hydroxylase). Furthermore, we provide evidence that these bile
acids were potent ligands for SXR/PXR and provide a mechanistic basis
for their induction of CYP3A4. In contrast, we unequivocally
demonstrate that FXR does not mediate CYP3A induction by bile acids
that seemed a formal possibility based upon a previous report (10).
This finding coupled with a previous report demonstrating that
lithocholic acid induces CYP3A in PXR nullizygous mice, and our finding
of the parallel induction of CYP2B in FXR nullizygous mice, suggest
that either CAR or another nuclear hormone receptor mediates this response.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Pharmaceutical Sciences, St. Jude Children's Research Hospital, 332 N. Lauderdale Ave., Memphis, TN 38105. Tel.: 901-495-2174; Fax:
901-525-6869; E-mail: John.schuetz@stjude.org.
ABBREVIATIONS
,7,12-trihydroxy-5-cholan-24-oic acid);
CDCA, chenodeoxycholic acid (5-cholanic acid-3,7-diol);
DKCA, 5-cholanic acid 3,7-dion (3,7-diketo-5-cholan-24-oleic acid);
DCA, deoxycholic acid (3,12-dihydroxy-5-cholan-24-oic acid);
LCA, lithocholic acid (5-cholan-24-oic acid-3-ol);
UDCA, ursodeoxycholic acid;
TUDCA, tauroursodeoxycholic acid.
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DISCUSSION
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Hofmann, A. F.,
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33,
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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