The Pregnane X Receptor: A Promiscuous Xenobiotic Receptor That Has Diverged during Evolution
Stacey A. Jones1,
Linda B. Moore1,
Jennifer L. Shenk1,
G. Bruce Wisely,
Geraldine A. Hamilton,
David D. McKee,
Nicholas C. O. Tomkinson,
Edward L. LeCluyse,
Millard H. Lambert,
Timothy M. Willson,
Steven A. Kliewer and
John T. Moore
Departments of Molecular Endocrinology (S.A.J., L.B.M., J.L.S.,
G.B.W., D.D.M., S.A.K., J.T.M.), Medicinal Chemistry (N.C.O.T, T.M.W.),
and Structural Chemistry (M.H.L) Glaxo Wellcome Inc.
Research and Development Research Triangle Park, North Carolina
27709
Department of Drug Delivery and Disposition (G.A.H.,
E.L.L.) School of Pharmacy University of North Carolina at
Chapel Hill Chapel Hill, North Carolina 27599
 |
ABSTRACT
|
---|
Transcription of genes encoding cytochrome P450 3A
(CYP3A) monooxygenases is induced by a variety of xenobiotics and
natural steroids. There are marked differences in the compounds that
induce CYP3A gene expression between species. Recently, the mouse and
human pregnane X receptor (PXR) were shown to be activated by compounds
that induce CYP3A expression. However, most studies of CYP3A regulation
have been performed using rabbit and rat hepatocytes. Here, we report
the cloning and characterization of PXR from these two species. PXR is
remarkably divergent between species, with the rabbit, rat, and human
receptors sharing only approximately 80% amino acid identity in their
ligand-binding domains. This sequence divergence is reflected by marked
pharmacological differences in PXR activation profiles. For
example, the macrolide antibiotic rifampicin, the antidiabetic
drug troglitazone, and the hypocholesterolemic drug SR12813 are
efficacious activators of the human and rabbit PXR but have little
activity on the rat and mouse PXR. Conversely, pregnane
16
-carbonitrile is a more potent activator of the rat and
mouse PXR than the human and rabbit receptor. The activities of
xenobiotics in PXR activation assays correlate well with their ability
to induce CYP3A expression in primary hepatocytes. Through the use of a
novel scintillation proximity binding assay, we demonstrate that many
of the compounds that induce CYP3A expression bind directly to human
PXR. These data establish PXR as a promiscuous xenobiotic receptor that
has diverged during evolution.
 |
INTRODUCTION
|
---|
The liver and intestine are important sites for the metabolism of
both endogenous and exogenous chemicals. Members of the cytochrome P450
(CYP) superfamily of hemoproteins play critical roles in the oxidative
metabolism of compounds in both of these tissues. The CYP3A gene
products are among the most abundant of the monooxygenases in mammalian
liver and intestine. In humans, CYP3A4 is involved in the metabolism of
more than 50% of all drugs as well as a variety of other xenobiotics
and endogenous substances, including steroids (1 ).
Expression of CYP3A genes is induced by a variety of compounds,
including many drugs (1 2 ). The induction of CYP3A transcription
represents the basis for a number of common drug-drug interactions.
Many xenobiotics have been profiled for their effects on CYP3A
expression in primary hepatocytes from rabbits or rats (3 4 5 ). These
studies have revealed marked species differences and called into
question the validity of using animal models or nonhuman hepatocytes
for predicting the effects of xenobiotics on CYP3A transcription in
humans. Transfection studies in which reporter genes driven by CYP3A
promoter sequences were introduced into rabbit or rat hepatocytes
showed these differences were a consequence of host cell factors rather
than differences in cis-acting sequences in the CYP3A gene
promoters (5 ). However, the molecular basis for these species
differences had remained in question.
We and others recently cloned novel mouse and human orphan members of
the nuclear hormone receptor superfamily and showed that they are
activated by a variety of known inducers of CYP3A expression (6 7 8 9 10 ).
We have named these orphan nuclear receptors pregnane X receptors
(PXRs) based upon their efficacious activation by natural C21 steroids
(pregnanes). Mouse and human PXR are predominantly expressed in liver
and intestine and bind to xenobiotic response elements previously
identified in the human and rat CYP3A promoters (6 7 8 9 ). Based upon
these data, PXR has been suggested to serve as a key regulator of CYP3A
expression. The human and mouse PXR share only 76% amino acid identity
in their ligand-binding domains (LBDs) and display markedly different
activation profiles in response to xenobiotics. Thus, it has remained
an open question whether these receptors are bona fide orthologs or
members of a broader subfamily of closely related orphan nuclear
receptors.
We now report the cloning and characterization of PXR from rabbit and
rat, two species that are frequently used for studies of drug
metabolism and CYP3A regulation. Although PXR has diverged
significantly during the course of evolution, our results provide
evidence that it has an important role in CYP3A regulation in multiple
species. In addition, we also report the development of a scintillation
proximity binding assay for human PXR and show for the first time that
structurally diverse compounds bind directly to this orphan nuclear
receptor.
 |
RESULTS
|
---|
Cloning and Characterization of the Rabbit and Rat PXR
Because pharmacological and toxicological studies are often
conducted in rabbits and rats, we sought to clone PXR from these
species. PCR strategies were employed using oligonucleotides derived
from the mouse and human PXR and cDNA prepared from either rat or
rabbit liver. The resulting rat and rabbit PXR clones encode proteins
of 431 and 411 amino acids, respectively (Fig. 1A
). Rat PXR is closely related to its
mouse ortholog, sharing 97% amino acid identity throughout. Alignment
of the PXR sequences revealed interesting differences between species.
Although their DNA-binding domains (DBDs) are approximately 95%
identical, the LBDs of the rabbit, rodent, and human PXR share only
about 80% amino acid identity (Fig. 1
). Notably, the rabbit PXR is
roughly equally divergent from the human and rodent PXR (Fig. 1B
). This
degree of divergence is unprecedented for nuclear receptor orthologs.
Secondary structure prediction within the LBD suggested that several of
the residues that differ between species might affect the
ligand-binding properties of the receptor (Fig. 1A
). This analysis also
revealed the presence of a large insert between the predicted H2 and
H3, similar to that observed in the peroxisome proliferator-activated
receptors (PPARs) but not the classical steroid receptors (11 12 13 ).

View larger version (68K):
[in this window]
[in a new window]
|
Figure 1. Sequence Comparison of PXR among Species
A, Alignment of the rabbit, rat, human, and mouse PXR amino acid
sequences. Residues that differ from the consensus are boxed in
black. The DBD and the predicted -helices in the LBD are
indicated. B, The percent amino acid identities and differences are
indicated for the PXR DBD and LBD. 1, Rabbit; 2 , rat; 3, mouse; 4,
human.
|
|
The tissue expression patterns of rat and rabbit PXR were determined by
Northern analysis using blots containing poly(A)+
RNA from a variety of adult tissues. Rat and rabbit PXR are most
abundantly expressed in the liver, with PXR mRNA also detected in
tissues of the gastrointestinal tract (Fig. 2
). Two distinct transcripts of
approximately 2.5 and 4.0 kb were seen in rat liver. The 2.5-kb
transcript is also observed in rat stomach and small intestine. PXR
transcripts of 2.6, 4.5, and 5.0 kb were detected in rabbit liver,
small intestine, and kidney. We did not detect PXR in rat kidney, even
in longer exposures of the blot. This was surprising since PXR was
previously observed in mouse kidney (6 ). We conclude that PXR is most
abundantly expressed in liver in all four species, but that differences
exist in PXR transcript size and extrahepatic tissue expression.

View larger version (87K):
[in this window]
[in a new window]
|
Figure 2. Northern Blot Analysis of Rat and Rabbit PXR
Expression Patterns in Adult Tissues
RNA size markers (in kilobases) are indicated on the
left.
|
|
Differential Activation of Mouse, Rat, Rabbit, and Human PXR
A variety of different xenobiotics were previously tested for
their ability to induce CYP3A expression in primary hepatocytes from
either rabbits or rats (3 4 5 ). Many of these compounds, including
dexamethasone, phenobarbital, RU486, spironolactone, clotrimazole, and
trans-nonachlor, induced CYP3A expression in hepatocytes
from both species (4 ). However, there were notable differences. For
example, the macrolide antibiotic rifampicin was a much more
efficacious inducer of CYP3A expression in rabbit hepatocytes than rat
hepatocytes. Conversely, pregnenolone 16
-carbonitrile (PCN) induced
CYP3A expression in rat hepatocytes but not rabbit hepatocytes. We
tested this same panel of compounds for activation of the rabbit and
rat PXR. PXR expression plasmids were cotransfected into CV-1 cells
together with a reporter plasmid containing two copies of the CYP3A1
direct repeat 3 (DR-3) PXR response element upstream of the minimal
thymidine kinase promoter and chloramphenicol acetyltransferase (CAT)
gene. The cells were then treated with 10 µM
concentrations of each compound except for phenobarbital, which was
tested at 1 mM. Both the rabbit and rat PXR
responded to xenobiotics. Rifampicin and dexamethasone were the most
efficacious activators of rabbit PXR, inducing reporter levels more
than 15-fold over the basal level (Fig. 3A
). The synthetic steroids PCN, RU486,
cyproterone acetate (CPA), and spironolactone were efficacious
activators of rat PXR, increasing reporter levels more than 7-fold
(Fig. 3A
). In general, activation of the rat and rabbit PXR agreed with
the reported induction of CYP3A expression in primary hepatocytes from
these same species (4 ). Rifampicin was an efficacious activator of
rabbit PXR but had no effect on rat PXR (Fig. 3A
). Although both
receptors were activated by 10 µM
concentrations of PCN, full dose-response analysis revealed that PCN
was roughly 1 order of magnitude more potent on rat PXR than rabbit PXR
(Fig. 3B
). These data suggest that PXR has an important role in the
regulation of CYP3A expression in multiple species.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 3. The Human, Rabbit, Rat, and Mouse PXR Are Activated
by Xenobiotics
A, CV-1 cells were transfected with expression plasmids for PXR from
the different species and the (CYP3A1 DR3)2-tk-CAT
reporter. Cells were treated with 10 µM of each compound,
except for phenobarbital, which was tested at 1 mM. Cell
extracts were subsequently assayed for CAT activity. Data represent the
mean of assays performed in triplicate ± SE. B, CV-1 cells
were transfected with expression plasmids for rabbit or rat PXR and the
reporter (CYP3A1 DR3)2-tk-CAT. Cells were treated with the
indicated concentrations of PCN, and cell extracts were subsequently
assayed for CAT activity. Data points represent the mean
of assays performed in duplicate.
|
|
The same panel of CYP3A inducers was also tested on the human and mouse
PXR (Fig. 3A
). Based on the finding that rifampicin is a much more
efficacious activator of CYP3A expression in hepatocytes from rabbits
and humans than from rats (4 5 ), together with the observation that
rifampicin is an efficacious activator of human PXR (7 8 9 ), it had been
suggested that the activation profiles of the rabbit and human PXR were
likely to be similar. Indeed, the human and rabbit PXR were both
efficiently activated by rifampicin as well as by RU486, clotrimazole,
trans-nonachlor, and phenobarbital (Fig. 3A
). However, the
rabbit PXR was much more sensitive than its human ortholog to
activation by the synthetic steroids dexamethasone, PCN, CPA, and
spironolactone (Fig. 3A
). Thus, there are clear differences in
responsiveness of rabbit and human PXR to xenobiotics. Given their high
degree of sequence identity, it was not surprising that the rat and
mouse PXR had very similar activation profiles, although there were
subtle differences (Fig. 3A
). We conclude from these studies that
although the human, rabbit, rat, and mouse PXR are activated by several
of the same compounds, each is pharmacologically distinct.
Among the established inducers of CYP3A expression that we tested for
PXR activation was troglitazone.
Troglitazone is a member of the thiazolidinedione class of
insulin-sensitizing drugs that lower glucose, lipid, and insulin levels
in patients with type 2 diabetes. Thiazolidinediones mediate their
therapeutic effects by binding and activating the nuclear receptor
PPAR
(14 ). Troglitazone is known to increase CYP3A4
activity and to enhance the metabolism of other drugs in patients (15 ).
Consistent with this, we found that troglitazone activated
both the human and rabbit PXR (Fig. 3A
). Full dose-response analysis
showed that troglitazone activated human PXR with an
EC50 value of approximately 3 µM,
which is comparable to the concentration required to activate PPAR
(data not shown). Thus, the interaction of troglitazone
with other drugs is likely to result from its activation of PXR.
Interestingly, troglitazone had little effect on the rat
and mouse PXR.
Rexinoids Activate PXR
Like many of the other orphan nuclear receptors, PXR binds to its
hormone response elements as a heterodimer with retinoid X receptor
(RXR) (6 7 8 9 ). These heterodimers have been classified as either
permissive or nonpermissive depending on whether they are
activated by RXR ligands (rexinoids) (16 ). To test whether the PXR/RXR
heterodimer is permissive for activation by rexinoids, we performed
cotransfection assays with PXR from the four species in CV-1 cells in
the presence of the natural RXR ligand 9-cis-retinoic acid
and the synthetic, RXR-selective compounds LGD1069
and LG100268 (17 18 ). The RXR ligands did not activate the
PXR/RXR heterodimer at the nanomolar concentrations that are typically
required to activate the RXR homodimer or the permissive RXR
heterodimers. However, micromolar concentrations of the rexinoids did
weakly activate the heterodimers formed between either the human or
rabbit PXR and RXR (Fig. 4
). Transfection
experiments performed with saturating concentrations of a PXR ligand
showed that LG100268 did not further activate the human PXR/RXR
heterodimer (data not shown). Notably, the rexinoids had no effect on
the rat or mouse PXR heterodimers with RXR (data not shown), suggesting
that these compounds might not mediate their effects through RXR but
rather via the rabbit and human PXR. Consistent with this idea, we have
shown that RXR ligands bind directly to human PXR at micromolar
concentrations (see below). Thus, our data indicate that the
heterodimers formed between either the human or rabbit PXR and RXR can
be activated by micromolar concentrations of rexinoids that cross-react
with PXR.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 4. Rexinoids Activate Human and Rabbit PXR. CV-1 Cells
Were Transfected with Expression Plasmids for Human (A) or Rabbit (B)
PXR and the Reporter (CYP3A1 DR3)2-tk-CAT
Cells were treated with the indicated concentrations of
rifampicin (open circles), LGD1069
(closed circles), LG100268 (open
squares), or 9-cis-retinoic acid (closed
squares). Cell extracts were subsequently assayed for CAT
activity. Data points represent the mean of assays
performed in duplicate.
|
|
SR12813 Is a Potent PXR Activator
The bisphosphonate ester SR12813 lowers cholesterol levels in a
range of species including rats, dogs, and primates (19 20 ). The
molecular mechanism for these hypocholesterolemic effects has remained
unclear. Since SR12813 has been reported to increase CYP3A protein
levels in rat hepatocytes (21 ), we tested its ability to activate PXR.
SR12813 was a very potent and efficacious activator of both the human
and rabbit PXR, with EC50 values of approximately
200 nM and 700 nM, respectively (Fig. 5A
). This is the most potent PXR
activator to be identified to date. By contrast, SR12813 was only a
very weak activator of the rat and mouse PXR (Fig. 5A
).

View larger version (25K):
[in this window]
[in a new window]
|
Figure 5. Bisphosphonate Ester SR12813 Is a Potent Activator
of Human and Rabbit PXR 32P-labeled fragments of CYP3A1 (rat), CYP3A6
(rabbit), or CYP3A4 (human). Pictures of the ethidium bromide-stained
total RNA, including the 18S and 28S ribosomal RNA bands, used to
generate each blot are shown.
A, CV-1 cells were transfected with expression plasmids for PXR from
the different species and the (CYP3A1 DR3)2-tk-CAT
reporter. Cells were treated with the indicated concentrations of
SR12813, and cell extracts were subsequently assayed for CAT activity.
Data points represent the mean of assays performed in
duplicate. B, Northern blot analysis was performed with total RNA (10
µg) prepared from primary rat, rabbit, or human hepatocytes that had
been treated for 24 h with vehicle alone (0.1%
dimethylsulfoxide), 10 µM rifampicin, 5 µM
PCN, or 10 µM SR12813. Blots were probed with
|
|
We next tested whether the differences between rat, rabbit, and human
PXR in response to SR12813 would be reflected at the level of
CYP3A induction in primary hepatocytes derived from each of these
species. Hepatocytes were treated with vehicle alone, SR12813, PCN, or
rifampicin, and CYP3A mRNA levels were determined by Northern blot
analysis using probes for CYP3A1, CYP3A4,and CYP3A6, major inducible
CYP3A family members in rat, human, and rabbit, respectively. As
expected, rifampicin was an efficacious inducer of CYP3A expression in
human and rabbit hepatocytes, but not rat hepatocytes (Fig. 5B
).
Conversely, PCN induced CYP3A expression in rat but not human or rabbit
hepatocytes. In agreement with the results from the transfection
studies, SR12813 induced CYP3A expression in human and rabbit
hepatocytes but only weakly in rat hepatocytes (Fig. 5B
). These results
demonstrate that the PXR activation profile is predictive of the
effects of SR12813 on CYP3A expression in primary hepatocytes from
different species.
Structurally Diverse Xenobiotics Are PXR Ligands
The structural diversity of the compounds that activate PXR is
unprecedented for a nuclear receptor. Do these xenobiotics, which range
in mol wt from 232 (phenobarbital) to 823 (rifampicin), mediate their
effects through direct interactions with PXR? We set out to address
this issue by establishing a competition binding assay employing
[3H]SR12813 as a radioligand. Initial attempts
to express the LBD of human PXR in Escherichia coli were
unsuccessful due to its lack of solubility. However, coexpression of an
88-amino acid region of the steroid coactivator protein 1 (SRC-1) with
the human PXR LBD resulted in soluble protein that was purified to
homogeneity and biotinylated for use in ligand-binding assays. A
scintillation proximity-binding assay was developed using
streptavidin-coated polyvinyltoluene beads and the biotinylated human
PXR. [3H]SR12813 interacted specifically with
human PXR with a dissociation constant (Kd) of 40
nM (Fig. 6A
). This
value is in good agreement with the EC50 value
for SR12813 for activation of human PXR in the transfection assay (Fig. 5A
). These data demonstrate that [3H]SR12813
binds directly to human PXR.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 6. SR12813 and Other Xenobiotics Bind to Human PXR
A, Purified human PXR LBD immobilized on SPA beads was incubated with
concentrations of [3H]SR12813 ranging from 0.5
nM to 1000 nM in the absence (total binding,
open squares) or presence (specific binding,
closed triangles) of 10 µM clotrimazole to
define nonspecific binding. Data points represent the mean of assays
performed in triplicate. The Kd value for
[3H]SR12813 binding to human PXR was 41 nM as
calculated by nonlinear regression. B, Competition binding assays were
performed with 10 nM [3H]SR12813 and 10
µM of each of the xenobiotics, except phenobarbital,
which was tested at 1 mM. Data represent the mean of assays
performed in duplicate ± SE and are plotted as
percent inhibition of [3H]SR12813 binding where
competition with unlabeled SR12813 is defined as 100%.
|
|
We next tested various PXR activators for their ability to compete with
[3H]SR12813 for binding to human PXR. Each
compound was tested at 10 µM except for phenobarbital,
which was tested at the 1 mM concentration required to
activate human PXR. Notably, all the xenobiotics that activated human
PXR in the transfection assay displaced
[3H]SR12813 from the receptor (Fig. 6B
). The
compounds that interacted with human PXR included the thiazolidinedione
trolitazone and the rexinoids 9-cis retinoic acid,
LGD1069, and LG100268. Consistent with their relative
inactivity in the transfection assay, the synthetic steroids PCN, CPA,
spironolactone, and dexamethasone competed only weakly with
[3H]SR12813 for binding to human PXR (Fig. 6B
).
These data demonstrate that a variety of xenobiotics are capable of
interacting with the PXR LBD at concentrations that are consistent with
those required to activate the receptor in transfection assays. Given
the high concentration of phenobarbital that is required for
competition in the binding assay and the fact that it is known to
mediate effects through other mechanisms (22 ), we cannot rule out the
possibility that this barbiturate activates PXR through other signaling
pathways.
Natural Steroids Are PXR Ligands
Human and mouse PXR are activated by a variety of naturally
occurring steroids, among which C21 steroids (pregnanes) are the most
potent (6 7 8 9 ). We tested various pregnanes and other steroids for their
activities on the human, rabbit, rat, and mouse PXR in the transfection
assay. Each PXR displayed a distinct activation profile (Fig. 7A
). However, in each case, the most
efficacious activator was a pregnane. 5ß-Pregnane-3,20-dione was the
most efficacious natural activator of the human, rat, and mouse PXR
(Fig. 7A
). Although 5ß-pregnane-3,20-dione also activated the rabbit
PXR, the closely related compound 17-OH progesterone was the most
efficacious activator of the rabbit receptor. Weaker activation of PXR
was also seen with other steroids, including corticosterone,
dihydrotestosterone, and estradiol (Fig. 7A
), as previously described
(8 9 ).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 7. Natural Steroids Are PXR Ligands
A, CV-1 cells were transfected with expression plasmids for PXR from
the different species and the (CYP3A1 DR3)2-tk-CAT
reporter. Cells were treated with 10 µM of each steroid.
Cell extracts were subsequently assayed for CAT activity. Data
represent the mean of assays performed in triplicate ±SE.
B, Competition binding assays were performed with purified human PXR
LBD immobilized on SPA beads, 10 nM
[3H]SR12813, and 10 µM of each steroid.
Data represent the mean of assays performed in duplicate ±
SE and are plotted as percent inhibition of
[3H]SR12813 binding where competition with 10
µM unlabeled SR12813 is defined as 100%.
|
|
We examined whether these naturally occurring steroids interacted
directly with human PXR. In agreement with the transfection data,
5ß-pregnane-3,20-dione competed most efficiently with
[3H]SR12813 for binding to human PXR (Fig. 7B
).
Full dose-response analysis showed that 5ß-pregnane-3,20-dione bound
to human PXR with an IC50 value of approximately
400 nM (data not shown). Competition was also seen with 10
µM concentrations of other natural steroids that activate
human PXR including corticosterone and estradiol (Fig. 7B
). These data
demonstrate that natural steroids can bind directly to human PXR and,
furthermore, suggest that the natural PXR ligand is likely to be a
metabolite or close analog of 5ß-pregnane-3,20- dione.
 |
DISCUSSION
|
---|
Studies performed over the past 20 yr have revealed marked species
differences in the induction of CYP3A expression in response to
xenobiotics. These differences have complicated the development of
assays to identify compounds that modulate CYP3A transcription. It was
recently shown that the human and mouse orthologs of PXR are activated
by compounds that induce CYP3A expression (6 7 8 9 ). We have now extended
these earlier analyses to include rabbit and rat PXR. The
characterization of PXR from these two species was of particular
interest since a number of inducers of CYP3A expression have been
studied in rabbit and rat hepatocytes, which are readily available.
Overall, we find that there is a good correlation between PXR
activation and the induction of CYP3A transcription in rat and rabbit
hepatocytes. These data provide strong evidence
that PXR is a key regulator of CYP3A transcription in a range of
species, and that much of the cross-species variability in CYP3A
regulation can be accounted for at the level of PXR activation. We note
that several of the xenobiotics that induce CYP3A expression, including
dexamethasone and phenobarbital, activate other nuclear receptors (22 ).
Thus, other signaling pathways are also likely to contribute to the
regulation of CYP3A expression.
Since rifampicin is an efficacious inducer of CYP3A expression in human
and rabbit but not in rat, it was recently postulated that the rabbit
PXR might be more closely related to its human ortholog than the rat
PXR (9 ). In fact, the human, rabbit, and mouse/rat PXR are all roughly
equally divergent, sharing only approximately 80% amino acid identity
in their LBDs. Although both the rabbit and human PXR are activated by
rifampicin, there are marked differences in their responsiveness to
synthetic steroids such as dexamethasone and CPA. These differences in
PXR activation profiles are likely to be reflected at the level of
CYP3A expression in vivo. Thus, caution must be exercised in
extrapolating CYP3A induction data for a particular compound from
rabbit to man. Despite their divergence, several lines of evidence
suggest that that PXRs are orthologs and not closely related members of
a subfamily of nuclear receptors. First, each PXR is most abundantly
expressed in the liver and tissues of the gastrointestinal tract.
Second, our pharmacological data strongly suggest that each PXR
regulates CYP3A expression in its respective species. Finally, our
searches of the expressed sequence tag (EST) databases have not
revealed any other PXR-like sequences (J. T. Moore, unpublished
results). The divergence in PXR could represent either an adaptive
response to different environmental xenobiotic challenges or
differences in natural PXR ligands between species. Although pregnanes
are the most efficacious natural activators of PXR from each of the
species, we have observed cross-species differences in PXR activation
by natural steroids. However, the concentrations of these steroids
required to activate PXR are superphysiological, and the natural ligand
for PXR remains to be determined.
The known ligands for nuclear receptors are all small molecules with
similar volumes and molecular weights (23 ). Larger molecules, such as
growth factors, can also activate nuclear receptors through binding to
cell surface receptors and activation of their second
messenger-signaling cascades (24 ). The observation that rifampicin, a
macrolide antibiotic with a mol wt of 823, activated human PXR and
promoted its interaction with the coactivator SRC-1 in an in
vitro coprecipitation assay was surprising (7 8 9 ). Could PXR bind
to a ligand as large as rifampicin? The development of a radioligand
competition binding assay for this orphan receptor has allowed us to
address this question. Using [3H]SR12813 in a
scintillation proximity assay, we have demonstrated that many of the
xenobiotics, including rifampicin, that induce CYP3A4 expression bind
directly to PXR. The interaction of rifampicin with PXR suggests that
its ligand-binding pocket must be very large in comparison with other
nuclear receptors. The only other nuclear receptors that are known to
have such large ligand-binding pockets are the PPARs (11 12 13 ). x-Ray
crystallography has established that the large cavities in the PPARs
are due, in part, to the presence of an
-helix termed H2' that is
not present in other nuclear receptors. Sequence alignment suggests
that PXR has an even larger insert in the H2' region than the PPARs
(Fig. 1A
). Thus, it is possible that the promiscuity of PXR is due to
the presence of a ligand-binding pocket that is larger than those of
other nuclear receptors.
SR12813 lowers plasma cholesterol levels in a number of species
including primates (19 20 ). Although SR12813 has been reported to
reduce cholesterol biosynthesis, by increasing the degradation of
hydroxy-methylglutarate-coenzyme A reductase (20 ), the
molecular target for its actions remains unknown. Despite its potency
in activating human and rabbit PXR, we believe it unlikely that SR12813
mediates its hypocholesterolemic effects exclusively through PXR. PCN
is a potent activator of the mouse and rat PXR and has effects on bile
composition in rats (25 ). However, in agreement with previous studies
(25 ), we have found that treatment of wild-type rats with PCN does not
decrease serum cholesterol levels (J. L. Shenk and D. Winegar,
unpublished data). Under these same conditions, SR12813, which is only
a very weak activator of rat PXR, effectively lowers cholesterol
levels. Moreover, rifampicin, which is widely used to treat
tuberculosis at doses that induce CYP3A4 expression, has not been
associated with reductions in cholesterol levels. These results suggest
that PXR activation alone is unlikely to account for the
hypocholesterolemic actions of SR12813. It was recently shown that
micromolar concentrations of SR12813 activate the related nuclear
receptor farnesoid X receptor (FXR) (26 ). FXR is a bile acid
receptor that regulates genes such as cholesterol 7
-hydroxylase that
are involved in cholesterol homeostasis (27 28 29 ). Thus, SR12813 may
exert its hypocholesterolemic effects through FXR or other cellular
receptors.
The thiazolidinedione antidiabetic drugs were developed using rodent
models of insulin resistance, without knowledge of their cellular
target. It is now known that these insulin sensitizers mediate their
effects through activation of the PPAR
/RXR heterodimer (30 31 ).
Troglitazone is the first of these drugs to be marketed
for the treatment of type 2 diabetes. Although the drug is devoid of
serious side effects in rodents, in humans it has been shown to
increase CYP3A4 activity (15 ) and is also associated with an
idiosyncratic hepatotoxicity (32 ). Our data showing that
troglitazone activates human PXR at concentrations similar
to those required to activate PPAR
provides an explanation for its
interactions with other drugs, including oral contraceptives.
Interestingly, the relative lack of activity of
troglitazone on the mouse or rat PXR may explain why these
effects were not reported in animal toxicology studies. Additional
studies will be required to determine whether PXR also plays a role in
the hepatotoxicity observed with troglitazone. In this
regard, it is interesting that the PXR ligand rifampicin has also been
associated with hepatotoxicity in humans (33 34 ). Our data suggest
that certain rexinoids, which have been proposed as diabetes drugs
(35 ), may show side effects similar to troglitazone
through activation of PXR. The availability of PXR screening assays
should allow for the development of new drugs for type 2 diabetes with
increased selectivity for their cellular target, the PPAR
/RXR
heterodimer.
In summary, we have demonstrated that PXR is a promiscuous nuclear
xenobiotic receptor with an LBD that has diverged considerably during
the course of evolution. This divergence in the PXR LBD accounts in
large part for the differential effects of various compounds on CYP3A
expression across species. Comparative functional studies using rabbit
and rat PXR will increase our ability to evaluate metabolism data from
relevant nonhuman pharmacological model systems. Moreover, the
availability of PXR in a high throughput binding format provides a
valuable tool for the rapid identification of compounds that will
induce CYP3A expression and interact with other drugs. Through the
early elimination of these compounds from the drug discovery process,
these assays will aid in the development of safer medicines.
 |
MATERIALS AND METHODS
|
---|
Reagents
ITS+ (insulin, transferrin, selenium, linoleic acid, and BSA
supplement), rat tail collagen, type I, and Matrigel were purchased
from Collaborative Biomedical Research Instruments, Inc.
(Bedford, MA). Collagenase (CLS2) was obtained from
Worthington Biochemical Corp. (Freehold, NJ). Petri
dishes (60 mm, Permanox) were purchased from NUNC (Naperville, IL). All
other media and culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). All solvents and other
chemicals used were of HPLC grade or the highest purity available.
Troglitazone, SR12813, LGD1069, and LG100268
were synthesized in house. RU486 and trans-nonachlor were
purchased from BIOMOL Research Laboratories, Inc.
(Plymouth Meeting, PA) and Velsicol (Chicago, IL), respectively. All
other xenobiotics and steroids used in the transfection and binding
assays were purchased from either Sigma (St. Louis, MO) or
Steraloids, Inc. (Wilton, NH).
Cloning of Rabbit and Rat PXR
The full-length coding region of rabbit PXR was derived from a
gt11 phage rabbit liver cDNA library (CLONTECH Laboratories, Inc. Palo Alto, CA). An internal 861-bp rabbit PXR fragment was
first obtained by PCR using oligonucleotides derived from the mouse PXR
LBD sequence (6 ). The forward oligonucleotide sequence was
5'-AAGTGCCTGGAGAGTGGCATG, and the reverse primer was 5'-
TCGCAGCTCAGTGAGGACGGC. The remainder of the rabbit PXR 5'- and
3'-coding sequence was obtained by nested PCR using oligonucleotides
within the 861-bp internal rabbit PXR sequence and oligonucleotides
within
gt11, which flank the EcoRI cloning site. The
oligonucleotides used for the nesting were: Nest 1: 5'
CTGAGCCTCCATCCGTTCTCTC and 5' AGGTGTGGTGCAGCGTGAAG; Nest 2: 5'
ACGGCCACATCGGACATGATC and 5'GATCATGGCCGTCCTCACTGAG. After obtaining the
full-length rabbit PXR sequence by this method, the entire coding
region was amplified and cloned again from rabbit liver cDNA to confirm
the original sequence. The primers used in this step contained flanking
EcoRI restriction sites for subcloning the PCR product into
the mammalian expression vector pSG5 as well as a consensus Kozak
sequence preceding the ATG to optimize mammalian expression. The
forward primer sequence used was 5'-
TCCACCGAATTCACCACCATGGGTGGAAAGCCCACCATCAGTGCAGAT and the reverse primer
was 5'- TCCACCGAATTCTCAGTCATCTGTGGTGCTGAACAGCTCCCG. The sequences
obtained in both cloning steps agreed over the entire coding region.
The sequence of rabbit PXR has been deposited in GenBank (accession
number AF188476).
The coding region of rat PXR was obtained by PCR amplification using
rat liver cDNA (CLONTECH Laboratories, Inc.) and
oligonucleotides derived from the 5'- and 3'-flanking regions of the
mouse PXR. The forward primer sequence was 5'-TGATTCTTCAAGGTGGACCCC,
and the reverse primer was 5'-GCAATTCAGAATGTCTGGGTCTAGC. Clones derived
from three independent PCR reactions were sequenced. We note that while
this work was in progress, the rat PXR clone was reported by another
group (36 ).
Sequence Alignment
The program MVP (37 ) was used to align the PXR sequences
with the human PPAR
sequence. Most of the secondary structure was
identified from the x-ray structure of the PPAR
LBD (11 ). The region
between helix-1 and helix-3 failed to give a clear alignment, so the
PRISM secondary structure prediction procedure, as implemented in MVP,
was used to predict helix-2, helix-2', and ß-strands a and b.
Northern Analysis
Primary human, rabbit, and rat hepatocytes were treated for
24 h with the respective compounds, and total RNA was isolated
using Trizol reagent (Life Technologies, Inc.). Rabbit RNA
was isolated from normal rabbit tissues using Trizol reagent. Total RNA
from each sample (10 µg) was resolved on a 2.2 M
formaldehyde denaturing gel. The gels were stained with ethidium
bromide to examine for equal loading. The RNA was subsequently
transferred onto Nytran nylon membrane from Schleicher & Schuell, Inc. (Keene, NH). Rat blots were purchased from OriGene
(Rockville, MD). Blots were hybridized with 32P-labeled
CYP3A1, CYP3A4, CYP3A6, rat PXR, or rabbit PXR cDNA probes.
Cotransfection Assays
CV-1 cells were plated in 96-well plates at a density of 20,000
cells per well in high glucose DMEM supplemented with 10%
charcoal/dextran-treated FBS (HyClone Laboratories, Inc.
Logan, UT). Transfection mixes contained 5 ng of receptor expression
vector, 12 ng of reporter plasmid, 25 ng of ß-galactosidase
expression vector pCH110 (Amersham Pharmacia Biotech,
Piscataway, NJ) as internal control, and 38 ng of carrier plasmid.
Human and mouse PXR expression plasmids and the (CYP3A1
DR3)2-tk-CAT reporter were previously described
(6 7 ). Transfections were performed with Lipofectamine (Life Technologies, Inc.) essentially according to the manufacturers
instructions. Drug dilutions were prepared in phenol red-free DMEM/F-12
with 15 mM HEPES supplemented with 10% charcoal-stripped,
delipidated calf serum (Sigma). Cells were incubated for
24 h in the presence of drugs, and then cell extracts were
prepared and assayed for CAT and ß-galactosidase activities as
previously described (30 ).
Primary Hepatocytes
Hepatocytes were isolated from human liver tissue obtained as
surgical biopsy samples or from rejected donor livers by the two-step
collagenase digestion method (38 ) with minor modifications as
previously described (39 ). In most cases, encapsulated liver tissue
(25100 g) was perfused with calcium-free buffer containing 5.5
mM glucose, 5% BSA, ascorbic acid (50 mg/ml), and 0.5
mM EGTA for 1015 min at a flow rate of 2535 ml/min
followed by perfusion with DMEM containing 1.5 mM calcium,
5% BSA, ascorbic acid (50 mg/ml), and collagenase (0.30.4 mg/ml) for
1520 min. Hepatocytes were dispersed from the digested liver in DMEM
containing 5% FBS, insulin (4 µg/ml), and dexamethasone (1
µM) and washed by low-speed centrifugation (70 x
g, 4 min). Cell pellets were resuspended in supplemented
DMEM and 90% isotonic Percoll (3:1 vol:vol) and centrifuged at
100 x g for 5 min. The resulting pellets were washed
with fresh medium by low-speed centrifugation and resuspended in
supplemented DMEM. Viability was determined by trypan blue exclusion
and was typically between 80 and 90%. Human, rabbit, and rat
hepatocytes were cultured according to the method of LeCluyse et
al. (39 ). Briefly, approximately 4.5 x
106 hepatocytes were added to 60-mm NUNC Permanox
culture dishes coated with a rigid collagen substratum in 3 ml of
supplemented DMEM and allowed to attach for 24 h at 37 C in a
humidified chamber with 95%/5%, air/CO2.
Culture dishes were gently swirled and medium containing unattached
cells was then aspirated. Fresh ice-cold serum-free modified Chees
medium containing 0.1 µM dexamethasone,
6.25 µg/ml insulin, 6.25 µg/ml transferrin, and 6.25 ng/ml selenium
(ITS+) and 0.25 mg/ml Matrigel were added to each dish and cultures
were returned to the humidified chamber (40 ). Medium was changed on a
daily basis thereafter. Unless otherwise specified, primary cultures of
human, rabbit, and rat hepatocytes were maintained for 3648 h before
treatment with test compounds was initiated.
Synthesis of [3H]SR1281
[3H]SR12813 with a specific activity of
23 Ci/mmol was prepared by Amersham International plc
(Cardiff, UK) from
[3H]3,5-ditertbutyl-4-hydroxy benzaldehyde by
modification of the published procedure (41 ).
Expression of Recombinant Human PXR LBD
The LBD of human PXR (amino acids 130434) was expressed as an
amino-terminal polyhistidine-tagged fusion protein. The PXR LBD was
subcloned into the pRSETa bacterial expression vector
(Invitrogen, San Diego, CA). Sequence encoding a
polyhistidine tag derived from an N-terminal PCR primer (MKKGHHHHHHG)
was fused in frame to the PXR LBD. To enhance its solubility, the PXR
LBD was coexpressed with an 88-amino acid fragment of SRC-1 (42 43 ).
The human SRC-1 expression construct was created by insertion of a
fragment of SRC-1 encoding amino acids 623710 (43 ) flanked by
NdeI-BamHI restriction sites into the pACYC184
vector (New England Biolabs, Inc., Beverly, MA) containing
the T7 promoter region of the pRSETA vector (Invitrogen).
PCR primers were designed to amplify an 88-amino acid region of the
human SRC-1 gene (p160), which encodes two of the LXXLL motifs (motifs
1 and 2) along with an N-terminal tag (MKK). The resulting
SRC-1/pACYC184 plasmid was cotransformed with PXRLBD/pRSETa into the
BL21(DE3) E. coli strain. One-liter shake flask liquid
cultures containing standard Luria-Bertani (LB) broth with 0.05 mg/ml
ampicillin and 0.05 mg/ml chloramphenicol were inoculated and grown at
22 C for 24 h. The cells were induced with 0.05
mM isopropyl
ß-D-thiogalactopyranoside for 46 h at 22 C after
which the cells were harvested by centrifugation (20 min, 3,500 x
g, 4 C). The cell pellet was stored at -80 C. The cell
pellet was resuspended in 250 ml buffer A (50 mM
Tris-Cl pH 8.0, 250 mM NaCl, 50
mM Imidazole, pH 7.5). Cells were sonicated for
35 min on ice and the cell debris was removed by centrifugation (45
min, 20,000 x g, 4 C). The cleared supernatant was
filtered through a 0.45 µM filter and loaded on
to a 50 ml Ni++-charged ProBond Chelation
resin (Invitrogen). After washing to baseline with buffer
A, the column was washed with buffer A containing 125
mM Imidazole, pH 7.5. The PXRLBD/SRC-1 complex
was eluted from the column using buffer A with 300
mM Imidazole, pH 7.5. Column fractions were
pooled and concentrated using Centri-prep 30K (Amicon, Beverly, MA)
units. The protein was subjected to size exclusion, using a column (26
mm x 90 cm) packed with Sepharose S-75 resin (Amersham Pharmacia Biotech, Piscataway, NJ) preequilibrated with 20
mM Tris-Cl, pH 8.0, 200 mM
NaCl, 5 mM dithiothreitol, 2.5
mM EDTA, pH 8.0. Column fractions were pooled and
concentrated.
Scintillation Proximity Binding Assay
A scintillation proximity binding assay (44 ) was developed using
purified human PXR/SRC-1. Streptavidin-PVT scintillation proximity
assay (SPA) beads (Amersham Pharmacia Biotech
RPNQ0007) were suspended in assay buffer (50 mM Tris, pH
8.0, 50 mM KCl, 1 mM EDTA, 1 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate,1
mM dithiothreitol, 0.1 mg BSA/ml) at 0.5 mg/ml.
Biotinylated human PXR was added to a final concentration of 75
nM. The receptor/bead mix was allowed to incubate for 30
min at room temperature. Uncoupled receptor was removed by centrifuging
the receptor/bead mixture at 3,000 rpm for 10 min and pouring off the
supernatant. The receptor-coupled beads were resuspended in assay
buffer. All experiments were run in Packard Optiplates (Packard
6005190, Packard Instruments, Meriden, CT) using 25 µg
bead/well and an assay volume of 100 µl. Twelve-point saturation
curves were generated in triplicate using 10 µM
clotrimazole to define nonspecific binding.
[3H]SR12813 was added such that the final
concentration of radioligand ranged from 0.5 to 1,000 nM.
The Kd value was determined by nonlinear
regression. In competition experiments, test compounds were diluted in
100% dimethylsulfoxide and added to the wells in 1 µl-aliquots.
[3H]SR12813 was added to a final concentration
of 10 nM. The plates were shaken momentarily to ensure
complete mixing. After a 2-h incubation at room temperature, the plates
were counted on a Packard TopCount, which was programmed to compensate
for color quenching.
 |
ACKNOWLEDGMENTS
|
---|
We thank Tom Consler for biotinylation and analysis of the
purified human PXR; Ian Fellows for assistance in the preparation of
[3H]SR12813; D. James Coon for assistance with
the perfusion of human liver samples and isolation of hepatocytes; and
Summer Jolley for assistance with the maintenance and treatment of
hepatocyte cultures.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Steven A. Kliewer, Department of Molecular Endocrinology, Room 3.3124, Glaxo Wellcome Inc. Research and Development, Five Moore Drive, Research Triangle Park, North Carolina 27709.
1 Equal contributions were made by these authors. 
Received for publication August 27, 1999.
Revision received September 21, 1999.
Accepted for publication September 28, 1999.
 |
REFERENCES
|
---|
-
Maurel P 1996 The CYP3A family. In: Ioannides C (ed)
Cytochromes P450: Metabolic and Toxicological Aspects. CRC Press, Inc.,
Boca Raton, FL, pp 241270
-
Guzelian PS 1988 Regulation of the glucocorticoid-inducible
cytochromes P450. In: Miners JO, Birkett DJ, Drew R, McManus M (eds)
Microsomes and Drug Oxidations. Taylor and Francis, London, pp 148155
-
Kocarek TA, Schuetz EG, Guzelian PS 1993 Regulation of
phenobarbital-inducible cytochrome P450 2B1/2 mRNA by lovastatin and
oxysterols in primary cultures of adult rat hepatocytes. Toxicol Appl
Pharmacol 120:298307[CrossRef][Medline]
-
Kocarek TA, Schuetz EG, Strom SC, Fisher RA, Guzelian PS 1995 Comparative analysis of cytochrome P4503A induction in primary cultures
of rat, rabbit, and human hepatocyes. Drug Met Dispos 23:415421[Abstract]
-
Barwick JL, Quattrochi LC, Mills AS, Potenza C, Tukey RH,
Guzelian PS 1996 Trans-species gene transfer for analysis of
glucocorticoid-inducible transcriptional activation of transiently
expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult
rat and rabbit hepatocytes. Mol Pharmacol 50:1016[Abstract]
-
Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones
SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T,
Lehmann JM 1998 An orphan nuclear receptor activated by pregnanes
defines a novel steroid signaling pathway. Cell 92:7382[Medline]
-
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT,
Kliewer SA 1998 The human orphan nuclear receptor PXR is activated by
compounds that regulate CYP3A4 gene expression and cause drug
interactions. J Clin Invest 102:10161023[Abstract/Free Full Text]
-
Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L,
Sydowbackman M, Ohlsson R, Postlind H, Blomquist P, Berkenstam A 1998 Identification of a human nuclear receptor defines a new signaling
pathway for CYP3A induction. Proc Natl Acad USA 95:1220812213[Abstract/Free Full Text]
-
Blumberg B, Sabbagh W, Juguilon H, Bolado J, Vanmeter CM, Ong
E, Evans RM 1998 SXR, a novel steroid and xenobiotic sensing nuclear
receptor. Genes Dev 12:31953205[Abstract/Free Full Text]
-
Schuetz EG, Brimer C, Schuetz JD 1998 Environmental
xenobiotics and the antihormones cyproterone acetate and spironolactone
use the nuclear hormone receptor pregnenolone X receptor to activate
the CYP3A23 hormone response element. Mol Pharmacol 54:11131117[Abstract/Free Full Text]
-
Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa
R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding
and co-activator assembly of the peroxisome proliferator-activated
receptor-
. Nature 395:137143[CrossRef][Medline]
-
Uppenberg J, Svensson C, Jaki M, Bertilsson G, Jendeberg L,
Berkenstam A 1998 Crystal structure of the ligand binding domain of the
human nuclear receptor PPAR
. J Biol Chem 273:3110831112[Abstract/Free Full Text]
-
Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown
PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA,
Milburn MV 1999 Molecular recognition of fatty acids by peroxisome
proliferator-activated receptors. Mol Cell 3:397403[Medline]
-
Kliewer SA, Willson TM 1998 The nuclear receptor PPAR
- bigger than fat. Curr Opin Gen Dev 8:576581[CrossRef][Medline]
-
Michalets EL 1998 Update: clinically significant
cytochrome P-450 drug interactions. Pharmacotherapy 18:84112[Medline]
-
Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan
receptors. Cell 83:841850[Medline]
-
Boehm MF, Zhang L, Badea BA, White SK, Mais DE, Berger E, Suto
CM, Goldman ME, Heyman RA 1994 Synthesis and structure-activity
relationships of novel retinoid X receptor-selective retinoids. J
Med Chem 37:29302941[Medline]
-
Boehm MF, Zhang L, Zhi L, McClurg MR, Berger E, Wagoner M,
Mais DE, Suto CM, Davies JA, Heyman RA 1995 Design and synthesis of
potent retinoid X receptor selective ligands that induce apoptosis in
leukemia cells. J Med Chem 38:31463155[Medline]
-
Berkhout TA, Simon HM, Patel DD, Bentzen C, Niesor E, Jackson
B, Suckling KE 1996 The novel cholesterol-lowering drug SR-12813
inhibits cholesterol synthesis via an increased degradation of
3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Biol Chem 271:1437614382[Abstract/Free Full Text]
-
Berkhout TA, Simon HM, Jackson B, Yates J, Pearce N, Groot PH,
Bentzen C, Niesor E, Kerns WD, Suckling KE 1997 SR-12813 lowers plasma
cholesterol in beagle dogs by decreasing cholesterol biosynthesis.
Atherosclerosis 133:203212[CrossRef][Medline]
-
Williams JA, Chenery RJ, Berkhout TA, Hawksworth GM 1997 Induction of cytochrome P4503A by the antiglucocorticoid mifepristone
and a novel hypocholesterolaemic drug. Drug Metab Dispos 25:757761[Abstract/Free Full Text]
-
Honkakoski P, Zelko I, Sueyoshi T, Negishi M 1998 The nuclear
orphan receptor CAR-retinoid X receptor heterodimer activates the
phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell
Biol 18:56525658[Abstract/Free Full Text]
-
Bogan AA, Cohen FE, Scanlan TS 1998 Natural ligands of nuclear
receptors have conserved volumes. Nat Struct Biol 5:679681[CrossRef][Medline]
-
Weigel NL, Zhang Y 1998 Ligand-independent activation of
steroid hormone receptors. J Mol Med 76:469479[CrossRef][Medline]
-
Turley SD, Dietschy JM 1984 Modulation of the stimulatory
effect of pregnenolone-16
-carbonitrile on biliary cholesterol
output in the rat by the manipulation of the rate of hepatic
cholesterol synthesis. Gastroenterology 87:284292[Medline]
-
Niesor EJ, Flach J, Weinberger C, Bentzen CL 1999 Syntheic
farnesoid X receptor (FXR) agonists: a new class of cholesterol
synthesis inhibitors and antiproliferative drugs. Drugs Future 24:431438
-
Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG,
Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM 1999 Bile acids: natural ligands for an orphan nuclear receptor.
Science 284:13651368[Abstract/Free Full Text]
-
Makishima M, Okamoto AY, Repa JJ, Tu H, Learned M, Luk A, Hull
MV, Lustig KD, Mangelsdorf DJ, Shan B 1999 Identification of a nuclear
receptor for bile acids. Science 284:13621365[Abstract/Free Full Text]
-
Wang H, Chen J, Hollister K, Sowers LC, Forman BM 1999 Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol
Cell 3:543553[Medline]
-
Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson
TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high
affinity ligand for peroxisome proliferator-activated receptor
(PPAR
). J Biol Chem 270:1295312956[Abstract/Free Full Text]
-
Willson TM, Cobb JE, Cowan DJ, Wiethe RW, Correa ID, Prakash
SR, Beck KD, Moore LB, Kliewer SA, Lehmann JM 1996 The
structure-activity relationship between peroxisome
proliferator-activated receptor
agonism and the
antihyperglycemic activity of thiazolidinediones. J Med Chem 39:665668[CrossRef][Medline]
-
Watkins PB, Whitcomb RW 1998 Hepatic dysfunction associated
with troglitazone. N Engl J Med 338:916917[Free Full Text]
-
1998 Physicians Desk Reference. Medical Economics Data,
Montvale, NJ
-
Durand F, Jebrak G, Pessayre D, Founier M, Berneau J 1996 Hepatotoxicity of antitubercular treatments. Rationale for monitoring
liver status. Drug Safety 15:394405[Medline]
-
Mukherjee R, Davies PJA, Crombie DL, Bischoff ED, Cesario RM,
Jow L, Hamann LG, Boehm MF, Mondon CE, Nadzan AM, Paterniti Jr JR,
Heyman RA 1997 Sensitization of diabetic and obese mice to insulin by
retinoid X receptor agonists. Nature 386:407410[CrossRef][Medline]
-
Zhang H, LeCluyse E, Liu L, Hu M, Matoney L, Zhu W, Yan B 1999 Rat pregnane X receptor: molecular cloning, tissue distribution, and
xenobiotic regulation. Arch Biochem Biophys 368:1422[CrossRef][Medline]
-
Lambert MH 1997 Docking conformationally flexible molecules
into protein binding sites. In: Charifson PS (ed) Practical Application
of Computer-Aided Drug Design. Marcel-Dekker, New York, pp 243303
-
Li AP, Roque MA, Beck DJ, Kaminski DL 1992 Isolation and
culturing of hepatocytes from human livers. J Tissue Culture Methods 14:139146
-
LeCluyse E, Bullock P, Parkinson A, Hochman JH 1996 Cultured
rat hepatocytes. In: Borchardt RT, Wilson G, Smith P (eds) Model
Systems for Biopharmaceutical Assessment of Drug Absorption and
Metabolism. Plenum, New York, pp 121159
-
Sidhu JS, Farin FM, Omiecinski CJ 1993 Influence of
extracellular matrix overlay on phenobarbital-mediated induction of
CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture.
Arch Biochem Biophys 30:103113
-
Nguyen LM, Niesor E, Phan HT, Maechler P, Bentzen CL Phenol
substituted gem-biphosphonate derivatives, process for their
preparation, pharmaceutical compositions containing them. US Patent
9011247
-
Onate SA, Tsai SY, Tsai M-J, OMalley BM 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Takeshita A, Yen PM, Misiti S, Cardona GR, Liu Y, Chin WW 1996 Molecular cloning and properties of a full-length putative thyroid
hormone receptor coactivator. Endocrinology 137:35943597[Abstract]
-
Nichols JS, Parks DJ, Consler TG, Blanchard SG 1998 Development of a scintillation proximity assay for peroxisome
proliferator-activated receptor gamma ligand binding domain. Anal
Biochem 257:112119[CrossRef][Medline]