From Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received for publication, January 7, 2003, and in revised form, February 20, 2003
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ABSTRACT |
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The orphan nuclear constitutive androstane
receptor (CAR) is proposed to play a central role in the response to
xenochemical stress. Identification of CAR target genes in humans has
been limited by the lack of a selective CAR agonist. We report the identification of
6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel
human CAR agonist with the following characteristics: (a)
potent activity in an in vitro fluorescence-based CAR
activation assay; (b) selectivity for CAR over other
nuclear receptors, including the xenobiotic pregnane X receptor (PXR);
(c) the ability to induce human CAR nuclear translocation;
and (d) the ability to induce the prototypical CAR target
gene CYP2B6 in primary human hepatocytes. Using
primary cultures of human hepatocytes, the effects of CITCO on gene
expression were compared with those of the PXR ligand rifampicin. The
relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.
The nuclear receptors
CAR1 (NR1I3) and PXR (NR1I2)
play key roles in the response to chemical stress (1-4). Both CAR and
PXR have been shown to bind to a wide range of structurally unrelated ligands (5-7) and to regulate genes involved in the humoral response to both endobiotic and xenobiotic stress (2-4). Global gene expression profiling has shown that PXR and CAR regulate an overlapping set of
genes that encode proteins involved in the detoxification of potentially harmful xenobiotics and endobiotics (8, 9). For example,
CAR- and PXR-dependent signaling pathways converge on
common response elements in the regulatory regions of a number of
genes, notably members of the CYP3A and CYP2B
subfamilies of xenobiotic-inducible cytochromes P450 (10-12).
Current studies are aimed at broadening our understanding of the
biology of these receptors and the genes that they regulate. An
important goal is to delineate CAR- and PXR-specific target genes to
define their distinct physiological roles.
The identification of target genes for each receptor is facilitated by
the availability of potent and selective ligands. PXR has been shown to
be activated by a structurally and chemically diverse set of ligands
(13). Examples of human PXR activators include the xenobiotics
rifampicin and SR12813 (14-17), the endobiotics lithocholic acid (4,
18) and 5 In contrast to PXR, a selective chemical tool has not been available to
study the function of CAR in humans. The hepatomitogen TCPOBOP
is a potent murine CAR ligand that has been used to delineate CAR
target genes in mice, but does not activate human CAR (5, 7, 9).
The barbiturate phenobarbital activates both human and mouse CARs;
however, it does so though an indirect mechanism (7, 20). Thus,
although phenobarbital does not bind to the receptor (7), it causes CAR
to be translocated from the cytoplasm to the nucleus (20-22). Because
CAR exhibits an intrinsically high transcriptional activity, nuclear
localization of the receptor results in the activation of target gene
expression in the absence of ligand binding (20, 23). The induction of
CAR translocation by phenobarbital can be blocked by the phosphatase
inhibitor okadaic acid, suggesting that translocation involves a
dephosphorylation event (21). Importantly, phenobarbital has been shown
to induce large numbers of genes in a CAR-independent fashion, which
may be due to its effects on the phosphorylation status of the cell (24); and moreover, in humans, phenobarbital also activates PXR,
further complicating the interpretation of its effects (7). Similarly,
the human CAR ligands 5 Given the species selectivity with respect to activators of human and
mouse CARs and the unusual divergence of their respective ligand-binding domains (LBDs), it is possible that these two receptors perform different roles in mice and humans and in other species (25).
The availability of potent and selective CAR ligands for both the
murine and human receptors will enable direct comparison of their
species-specific roles.
Through a combination of in vitro and cell-based screening,
we have identified an imidazothiazole derivative that is a selective human CAR agonist. This chemical tool has allowed us to unambiguously define CAR target genes. This compound should be a powerful tool in
differentiating the role of human CAR and PXR. We have demonstrated the
utility of this compound in human hepatocyte studies.
Chemicals--
6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-
carbaldehyde
O-(3,4-dichlorobenzyl)oxime (CITCO) was
purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA).
Unless otherwise stated, cell culture reagents were obtained from
Invitrogen. Dexamethasone and rifampicin were acquired from Sigma.
Fluorescence Resonance Energy Transfer (FRET) Ligand Sensing
Assay--
The FRET ligand sensing assay was performed by modification
of a previously published procedure (26) and is described in Ref. 7.
Polyhistidine-tagged human CAR LBD was purified from Escherichia
coli as previously described (7).
Cotransfection Assays--
CV-1 cells were cotransfected with a
CAR expression plasmid in combination with the XREM-CYP3A4-LUC reporter
(27) as described previously (7).
Construction of a Green Fluorescent Protein (GFP)-CAR Expression
Plasmid--
Full-length human CAR cDNA (GenBankTM/EBI
accession number Z30425) was amplified by PCR using primers with
flanking EcoRI and BamHI sites and subsequently
inserted into the EcoRI and BamHI sites of
pEGFP-C1 (Clontech, Palo Alto, CA), producing
pGFP-hCAR. The sequence of the human CAR cDNA was confirmed by
sequence analysis.
Nuclear Translocation Assay--
Primary rat hepatocytes were
prepared by perfusion as previously described (28) and plated at a
density of ~3 × 106 cells/well of a six-well dish
in Williams' E medium containing 10% fetal bovine serum, 100 nM dexamethasone, and 1% ITS-G
(insulin/transferrin/selenium; Invitrogen). After overnight incubation,
cells were transferred to 2 ml of Williams' E medium as described
above, but without serum. For each well, 1.6 µg of pGFP-hCAR
expression plasmid in 100 µl of Opti-MEM was mixed with 3 µl of
LipofectAMINE 2000 (Invitrogen) in 100 µl of Opti-MEM according to
the manufacturer's directions and subsequently added directly to the
hepatocytes in serum-free medium. After a 4-h incubation, cells were
incubated in Williams' E medium containing 10% serum and incubated
overnight prior to the addition of compounds in fresh medium. The
intracellular localization of the GFP-CAR fusion protein was determined
by fluorescence microscopy ~4 h after the addition of compound.
Treatment of Primary Human Hepatocytes--
Primary human
hepatocytes were obtained from BioWhittaker, Inc. (Walkersville, MD)
and plated at an approximate density of 3 × 106
cells/well in a six-well plate. Cells were maintained in Williams' E
medium supplemented with 100 nM dexamethasone, 2 mM L-glutamine, and 1% ITS-G. Cells were
treated with vehicle (0.1% Me2SO), rifampicin (10 µM), or CITCO (100 nM). Fresh compound and
medium were added after 24 h, and cells were harvested after
48 h.
RNA Preparation and Expression Analysis--
Total RNA from
human hepatocyte cultures was isolated using TRIzol reagent
(Invitrogen) according to the manufacturer's instructions. Real-time
quantitative (RTQ) PCR was performed using an ABI PRISM 7700 sequence
detection system instrument and software (Applied Biosystems, Inc.,
Foster City, CA). RNA samples were prepared for RTQ-PCR as described
(9). Gene-specific primers and probes were designed using Primer
Express Version 2.0.0 (Applied Biosystems, Inc.) and synthesized
by Keystone Laboratories (Camarillo, CA). All primers and probes were
entered into the NCBI BLAST program to ensure specificity. -Fold
induction values were calculated by subtracting the mean threshold
cycle number for each treatment group from the mean threshold cycle
number from the vehicle group and raising 2 to the power of this difference.
For Northern blot analysis, total RNA (10 µg) was resolved on a 2.2 M formaldehyde-containing 1% agarose denaturing gel and transferred to a nylon membrane (Hybond N+, Amersham
Biosciences). Blots were hybridized to 32P-labeled
cDNAs corresponding to CYP2B6 (bases 3-659;
GenBankTM/EBI accession number AF182277), CYP3A4 (bases
790-1322; accession number M18907), human UGT1A1 (bases 75-766;
accession number NM_000463), CYP2A6 (bases 121-1200; accession number
X13930), SULT1A1 (bases 561-1300; accession number NM_001055), and rat 18 S rRNA (bases 293-970; accession number X01117). Signal intensity
was quantitated using ImageQuant software (Amersham Biosciences).
Identification of a Selective Human CAR Agonist
Several criteria were used to identify a potent and selective CAR
agonist. These included (a) activity in an in
vitro FRET-based assay, (b) >50-fold selectivity over
PXR in a transient transfection assay, and (c) the ability
to induce CAR translocation from the cytoplasm to the nucleus in
primary hepatocytes. We then sought to use this chemical tool to
identify genes that are regulated by CAR in primary cultures of human hepatocytes.
Identification of a CAR Agonist in a FRET-based Screen--
A
nuclear receptor-biased chemical library was screened in a CAR
FRET-based assay using human CAR LBD and a peptide containing the
second LXXLL motif of SRC-1 (steroid
receptor coactivator-1; amino acids
676-700). Compounds that induced increased interaction between these
partners with potencies of >100 nM were chosen for further analysis. One of the compounds, the imidazothiazole derivative CITCO (Fig. 1A), displayed a
half-maximal effective concentration (EC50) of 49 nM in the CAR/SRC-1 FRET assay (Fig. 1B). CITCO
was 50-fold more potent than the human CAR agonist
5 Selectivity for CAR Versus PXR of >50-fold--
CAR agonists
derived from the in vitro assay were tested for CAR
selectivity. Using the CAR- and PXR-responsive XREM-CYP3A4-LUC reporter
gene construct in CV-1 transient transfection assays, the selectivity
of the compounds for human CAR over human PXR was assessed. The
majority of compounds that were active in CAR/SRC-1 FRET assays were
<50-fold selective for CAR in comparison with human PXR (data not
shown). CITCO was one of the few compounds that displayed >50-fold
selectivity for CAR over PXR in the transient transfection assay (Fig.
2, A and B). CITCO
displayed calculated EC50 values of 25 nM in
the CAR transient transfection assay and ~3 µM in the
PXR transient transfection assay (>100-fold selectivity for CAR). We
also carried out transient transfection studies to show that CITCO and
the human CAR antagonist clotrimazole are competitive in their effects
on CAR transcriptional activity (Fig. 2A). In the presence
of 1.5 µM clotrimazole (an approximate EC70 in this assay), the EC50 of CITCO increased by >10-fold
(EC50 = 304 nM), indicating competition between
the two compounds for the receptor.
We also tested CITCO for selectivity across a panel of 15 nuclear
receptors for which we had available validated transient transfection
activation assays, viz. estrogen receptor- CAR Translocation Assay--
Although CITCO was a potent activator
of human CAR, the efficacy of this compound and other CAR activators
was relatively weak in assays performed in immortalized cell lines,
possibly because CAR is constitutively present in the nucleus in these assays. Thus, the results of the transfection assays do not accurately predict the overall efficacy of the compound in hepatocytes because inactive CAR is restricted to the cytoplasm in these cells. Thus, to
assess the ability of CAR ligands to induce translocation of CAR from
the cytoplasm to the nucleus, we developed a translocation assay in
primary cultures of hepatocytes. The full-length human CAR coding
region was fused in-frame to the GFP coding region and transfected into
rat hepatocytes. The effects of various compounds on CAR translocation
were visualized by fluorescence microscopy (Fig.
3). Although endogenous CAR has
previously been shown to be predominantly localized in the cytoplasm in
the absence of stimulation, GFP-CAR was present in both the
cytoplasm and nucleus (Fig. 3, panels 1 and 2).
This is likely due to the relatively high concentration of the GFP-CAR
chimera expressed under these assay conditions. In addition to a
widespread GFP-CAR distribution, control cells showed a somewhat
crenulated pattern of fluorescence, indicating that CAR may be attached
to a subcellular structure. Further studies are needed to determine
whether this is true for native CAR as well or whether this property is
unique to the GFP-CAR chimera.
When hepatocytes expressing GFP-CAR were treated with phenobarbital,
the GFP signal was localized predominantly in the nucleus (Fig. 3,
panels 3 and 4), as expected. Notably, in cells
treated with CITCO, the pattern of GFP localization was similar to that seen after phenobarbital treatment (Fig. 3, panels 5 and
6), indicating that CITCO causes efficient nuclear
translocation of CAR in hepatocytes. In summary, CITCO fulfills
our criteria for a useful human CAR chemical tool: it is a potent and
selective human CAR ligand that activates the receptor in a
transfection assay and promotes its translocation into the nucleus of hepatocytes.
Comparison of the Effects of CITCO and Rifampicin on Gene
Expression in Primary Human Hepatocytes
In the absence of a selective human CAR agonist, it has been
difficult to ascertain which genes are regulated by this receptor in
human hepatocytes. We used CITCO to study the effects of CAR activation
on gene expression in primary cultures of human hepatocytes. Hepatocytes derived from three separate donors were treated for 48 h with either 1 µM CITCO or 10 µM
rifampicin, a selective PXR agonist. The comparative effects of the
agonists on the expression of eight genes involved in a variety of
aspects of xenobiotic metabolism were quantitated by RTQ-PCR
(Table I). RNA in sufficient quantity was
available from two donors (Donors 1 and 2) to further evaluate selected
gene expression changes by Northern blot analysis (Fig.
4). The mRNAs evaluated included
those encoding multiple cytochrome P450 enzymes (CYP2A6, CYP2B6, and
CYP3A4); enzymes involved in supporting phase I metabolism (aldehyde
dehydrogenase (ALDH1A4) and aminolevulinate synthase); and enzymes
involved in phase II (conjugation) metabolism, including glutathione
S-transferase A2 (GSTA2) and sulfotransferase (SULT1A1). The
mRNA encoding the conjugation enzyme UDP-glucuronosyltransferase
(UGT1A1) was examined exclusively by Northern analysis due to
difficulties in generating functional RTQ-PCR primers for this
mRNA. We also examined the effects of CITCO and rifampicin on the
expression of the MDR1 (multidrug
resistance-1) transporter mRNA.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pregnane-3,20-dione (15), and the botanical hyperforin
(19). In human studies, rifampicin has been shown to be a useful
chemical tool to define PXR target genes in human hepatocytes (9).
Expression studies using rifampicin have shown that PXR activates the
expression of a battery of genes involved in the response to
xenochemical and endobiotic stress, most notably those genes involved
in oxidation (phase I enzymes), conjugation (phase II enzymes), and
transport (9).
-pregnane-3,20-dione (agonist) and
clotrimazole (antagonist) are both effective activators of human PXR
(7), which limits their utility in the identification of CAR target genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pregnane-3,20-dione (EC50 = 3000 nM) (Fig.
1B). The human CAR antagonist clotrimazole (7) was also
evaluated in this assay and demonstrated an IC50 of 58 nM.
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Fig. 1.
Identification of CITCO as a human CAR
agonist. A, shown is the structure of CITCO.
B, the FRET ligand sensing assay was performed with
polyhistidine-tagged human CAR LBD and 5 -pregnane-3,20-dione (
),
CITCO (
), or clotrimazole (
) as described under "Experimental
Procedures." Data are expressed as relative fluorescence
relative to binding reactions that received vehicle (0.1%
Me2SO) alone. The EC50/IC50 values
calculated from this assays are as follows: clotrimazole,
IC50 = 58 nM; CITCO, EC50 = 49 nM; and 5
-pregnane-3,20-dione, EC50 = 3000 nM.
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Fig. 2.
Effects of compounds on CAR and PXR
activities in transient transfection. CV-1 cells were transfected
with an expression plasmid for human CAR (A) or PXR
(B) and the XREM-CYP3A4-LUC reporter. Dose-response curves
are shown for the compound CITCO alone or in combination with the human
CAR antagonist clotrimazole. CITCO displayed EC50 values of
25 nM in the CAR assay (A, ) and ~3
µM in the PXR assay (B). CITCO was also tested
in a dose-response format in the presence of 1.5 µM
clotrimazole, an approximate IC70 in this assay
(A). Under these conditions, the EC50 of CITCO
was increased by over an order of magnitude (EC50 = 304 nM) (A,
), indicating competition between the
two compounds. Data represent the mean of assays performed in
duplicate. In A, data are expressed as luciferase activity
normalized by subtraction of base-line luciferase activity (base-line
values for CAR activity in the presence of clotrimazole were
~2 times the values in the absence of clotrimazole). In B,
data are expressed as a percentage of luciferase activity obtained from
transfected cells treated with 10 µM rifampicin.
and -
; hepatocyte nuclear factor-4
; LRH-1 (liver
receptor homolog-1); liver X
receptor-
and -
; peroxisome proliferator-activated receptor-
, -
, and -
; retinoic acid receptor-
; farnesoid X
receptor; SHP (small heterodimer
partner); thyroid hormone receptor-
; vitamin D receptor
(VDR); and glucocorticoid receptor. CITCO did not have any detectable
activity on any of these additional receptors at a dose of 10 µM (data not shown).
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Fig. 3.
CITCO promotes nuclear translocation of human
CAR. Primary rat hepatocytes were transfected with an
expression vector containing GFP fused to human CAR. After
transfection, cells were treated with vehicle (0.1% Me2SO)
(panels 1 and 2), phenobarbital (1 mM) (panels 3 and 4), or CITCO (100 nM) (panels 5 and
6).
Effects of CITCO and rifampicin on expression of genes in primary human
hepatocytes
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Fig. 4.
Effects of CITCO and rifampicin on gene
expression in primary cultures of human hepatocytes. Human
hepatocytes were treated with vehicle (0.1% Me2SO), CITCO
(100 nM), or rifampicin (10 µM) for 48 h
prior to isolation of total RNA. Northern blots were prepared and
probed with 32P-labeled cDNA probes corresponding to
CYP3A4, CYP2B6, GSTA2, UGT1A1, CYP2A6, and SULT1A1. Signal intensity
was quantitated by densitometry, and data are expressed as -fold
increases relative to their vehicle controls. A probe corresponding to
the 18 S rRNA was used to verify equal RNA loading.
Phase I Enzyme Genes-- CAR regulates both Cyp2b10 and Cyp3a11 in mouse liver (8-10, 12) and has also been implicated in the regulation of CYP2B6 and CYP3A4 in human hepatocytes (11, 12, 29). However, the role of CAR in the regulation of these genes has not been examined rigorously in human hepatocytes using a selective CAR chemical tool. Both CITCO and rifampicin induced CYP2B6 and CYP3A4 in human hepatocytes from Donors 1 and 2 as measured by RTQ-PCR (Table I). Rifampicin was not effective at inducing CYP2B6 in Donor 3, whereas CITCO was not effective at inducing CYP3A4 in this donor. This likely reflects the relative efficacy of rifampicin and CITCO in induction of CYP3A4 and CYP2B6, respectively, coupled with higher basal levels of expression of these genes in Donor 3. When the effects of CITCO were evaluated by Northern blot analysis, CYP2B6 mRNA was found to be induced by 4.4- and 20-fold in Donors 1 and 2, respectively (Fig. 4). Rifampicin also induced CYP2B6 expression, although to a lesser extent than CITCO (Fig. 4 and Table I). In all three donors, CYP2B6 was induced more efficiently by CITCO than by rifampicin, indicating that this gene is more responsive to CAR than to PXR.
In contrast to CYP2B6, CYP3A4 mRNA displayed a more robust response to rifampicin than to CITCO. When assessed by RTQ-PCR, rifampicin induced CYP3A4 mRNA by 15-fold in Donor 1 and by 78-fold in Donor 2, whereas CITCO induced CYP3A4 mRNA by 7.0- and 46-fold in the same donors. When evaluated by Northern analysis, rifampicin induced CYP3A4 expression by 30- and 5.9-fold, respectively, whereas CITCO induced CYP3A4 by 11- and 6.2-fold, respectively (Fig. 4). These data indicate that both PXR and CAR regulate CYP3A4 in human hepatocytes.
Although technically not a phase I enzyme, the CYP2A6 gene was also examined for PXR and CAR regulation. The mouse homolog (Cyp2a4) has previously been shown to be regulated by mouse CAR (8, 9). Analysis of the three donors by RTQ-PCR showed that CYP2A6 mRNA was induced selectively by CITCO (Table I). Analysis of Donors 1 and 2 by Northern blotting was consistent with these results (Fig. 4). In this analysis, CYP2A6 mRNA was induced by 5.4- and 9.6-fold in Donors 1 and 2, respectively, whereas rifampicin did not cause changes in the expression of this mRNA. Thus, CYP2A6 is selectively induced by CAR, but not by PXR.
Other genes involved in phase I metabolism were also evaluated by RTQ-PCR. Aminolevulinate synthase mRNA was induced by both CITCO and rifampicin in all of the donors as assessed by RTQ-PCR (Table I). Notably, ALDH1A4 showed a highly variable response to CITCO and rifampicin. Induction of ALDH1A4 varied from 1.8-fold (Donor 1) to 60-fold (Donor 3) in response to CITCO and from no induction (Donor 1) to a 160-fold induction (Donor 3) in response to rifampicin. Thus, certain genes appear to display a high degree of inter-individual variability in terms of their response to selective CAR and PXR agonists.
Phase II Conjugation Genes-- The effects of CITCO and rifampicin on the expression of the mRNAs encoding the conjugation enzymes GSTA2, SULT1A1, and UGT1A1 were also examined. Both CITCO and rifampicin were able to induce the expression of GSTA2 when assessed by RTQ-PCR and Northern analysis (Fig. 4 and Table I). When mRNAs from Donors 1 and 2 were evaluated by Northern analysis, CITCO induced GSTA2 mRNA by 3.4-fold in Donor 1 and by 19-fold in Donor 2 (Fig. 4). Rifampicin induced GSTA2 mRNA by 3.8- and 3.5-fold in these same donors (Fig. 4).
SULT1A1 mRNA was robustly induced only by CITCO in one of the three donors. In Donor 1, induction by either compound was <2-fold as measured by Northern blotting or RTQ-PCR; and in Donor 3, no induction was seen by either compound by RTQ-PCR. In contrast, CITCO strongly induced SULT1A1 in Donor 2 (11-fold increase as measured by Northern blot analysis and 7.5-fold increase as measured by RTQ-PCR). The variation in donor response may again be attributable to inter-individual heterogeneity in basal levels of gene expression. Northern blot analysis showed that Donor 1 had relatively high basal levels of expression of SULT1A1 (Fig. 4).
In Northern analysis, CITCO induced UGT1A1 mRNA by 1.8- and 3.7-fold in Donors 1 and 2, respectively (Fig. 4). In contrast, rifampicin did not induce expression of UGT1A1 significantly in Donor 2, but induced expression of UGT1A1 mRNA by 2.5-fold in Donor 1. Again, Donor 1 had a higher basal level of activity.
Transporter Expression-- The multidrug resistance genes, including MDR1, function as broad-specificity transporters in the liver. We examined the response of MDR1 to CITCO and rifampicin by RTQ-PCR (Table I). MDR1 expression was induced by both CITCO and rifampicin in Donor 2, but no induction by either compound was seen in Donors 1 and 3. Thus, for MDR1, a significant inter-individual response is seen. In certain individuals, induction of MDR1 gene expression occurs in response to both PXR and CAR agonists.
Model of CITCO Binding in the Ligand-binding Pocket of
CAR--
Although no x-ray structure is available for CAR, x-ray
structures have been done for the closely related receptors PXR (30) and VDR (31). The x-ray structure of PXR revealed a large and practically spherical ligand-binding pocket that can bind a wide range
of lipophilic ligands, whereas VDR has a smaller pocket with polar side
chains positioned to recognize specific ligands (31). In PXR, the
pocket expansion is due primarily to a 50-60-residue insert between
helixes 1 and 3. This helix 1-3 insert displaces helix 6, thereby
opening the pocket. VDR also has a helix 1-3 insert; there is no
evidence that it displaces helix 6. Although residues in the "core"
of CAR LBD have greater identity to PXR (50%) than to VDR (40%), CAR
lacks the helix 1-3 insert, and its helix 6 should have a geometry
more similar to that in VDR than in PXR (6). Consequently, we chose to
use VDR as the template in building a model for CAR. The MVP program
(32) was used to build the model for CAR and to dock CITCO into the
model. A number of different binding modes were obtained for CITCO, one
of which is shown in Fig. 5.
Asn165 lies near the oxime linkage and might possibly
donate a hydrogen bond to the oxime. This particular binding mode has
the para-chlorophenyl ring directed downwards and the
imidazothiazole group directed upwards, but the calculations also gave
binding modes where the positions of these groups were interchanged,
with the oxime linker still located near Asn165. The
modeling and docking calculations are not accurate enough to
distinguish among the possible binding orientations, but it is clear
that the CAR binding pocket can accommodate CITCO. The model CAR pocket
is smaller than that in PXR and somewhat more lipophilic than that in
VDR, suggesting that CAR should be intermediate between VDR and PXR in
terms of ligand promiscuity.
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DISCUSSION |
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To date, little is known about the function of CAR in humans. Attempts to delineate CAR biology in man have been hindered by the significant overlap in the pharmacology of human CAR and PXR and the lack of a selective CAR activator. CITCO is a potent and, importantly, highly selective human CAR agonist that should prove to be a useful tool in dissecting the structure and function of this receptor.
Direct comparison of human CAR and PXR target genes is now possible, as
is direct comparison of CAR target genes in mouse versus
human cells. In mice, PXR and CAR differential gene expression studies
have previously been carried out using the selective tools pregnenolone
16-carbonitrile (selective PXR agonist in mouse) and TCPOBOP (9).
Gene expression studies with these compounds provide the framework to
begin comparative analysis. For the majority of the genes that
overlapped between the mouse and human studies, similar profiles were
seen using PXR- and CAR-specific compounds. For example, similar to
mice, both selective CAR and PXR agonists regulated CYP2B
and CYP3A expression, consistent with previous studies
suggesting that PXR and CAR cross-regulate these genes (10-12, 22).
Interestingly, CITCO had more robust effects on CYP2B
expression, whereas rifampicin had more robust effects on CYP3A4, indicating that the receptors have different
quantitative effects depending on the specific gene (7).
In our comparison of CITCO with the human PXR ligand rifampicin in three separate sets of human hepatocytes, we observed remarkable inter-donor heterogeneity. This observation is consistent with previous studies showing that cytochrome P450 expression is quite variable in primary human hepatocyte preparations (33-35). For example, inter-individual variations in CYP3A4 protein levels ranging up to 40-fold have been reported (36, 37). Moreover, ethnic differences in CYP3A4-mediated drug metabolism have been reported (38), and it is estimated that ~90% of the inter-individual variability in CYP3A4 expression is due to genetic factors (39). In the case of the hepatocytes used in these studies, the inter-individual variability would be expected to be even higher due to the fact that the donors were typically undergoing drug therapy just prior to the harvest of the hepatocytes. The relatively high basal levels of multiple genes seen in Donor 1 versus Donor 2 might reflect genetic differences, differences in drug exposure, or both.
When comparing CAR target genes in mice versus humans, we found that, generally, genes regulated by only selective CAR ligands in mice were also regulated by the selective human CAR ligand. Notably, the phase II conjugation enzyme sulfotransferase gene (mouse SULTN/human SULT1A1) was more responsive to 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in mouse cells and to CITCO in human cells. Also, the mouse Cyp2a4 and human CYP2A6 genes were typically more responsive to CAR compounds than to PXR compounds. These genes are particularly interesting because, in addition to hydroxylating xenobiotics, the Cyp2a4 and CYP2A6 gene products hydroxylate a variety of steroid hormones, including androgens and estrogens (40). It is possible that CAR plays a different role from PXR in metabolizing endogenous steroids. Other similarities in gene response to CAR and PXR activators were also noted. For example, the phase II genes Gstm2 (mouse) and GSTA2 (human) and the transporter genes Mdr1a (mouse) and MDR1 (human) were induced by both PXR and CAR activators.
Differences between the human and mouse studies were also observed. For example, the UGT1A1 gene has been shown to be regulated by CAR in humans, and a CAR response element has been identified in its promoter (41). Interestingly, we found previously that the mouse CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene does not induce mouse UGT1A1 mRNA (9). In contrast and consistent with the findings of Sugatani et al. (41), we found that a human CAR agonist induces human UGT1A1 mRNA expression. These interspecies differences are intriguing, but, at this point, must be interpreted with caution because the mouse liver studies were carried out in an in vivo setting, whereas the human compounds were assessed in primary hepatocyte cultures. Despite dramatic differences in the amino acid sequences of their respective LBDs, we have demonstrated that human and mouse CARs regulate overlapping sets of genes. These studies have also begun to differentiate some of the distinct physiological pathways regulated by CAR versus PXR.
Finally, the majority of the compounds we identified in in
vitro binding assays were not able to induce CYP2B6 mRNA in
primary human hepatocytes (data not shown), suggesting that ligand
binding alone is not sufficient to induce CAR translocation to the
nucleus. Thus, CITCO, in conjunction with these compounds, should be a useful comparative tool to define the key determinants for
ligand-induced CAR nuclear translocation. We have presented a model
that supports direct binding of CITCO within the ligand-binding pocket
of human CAR. Future crystallography studies of human CAR complexed
with CITCO and CITCO analogs will further improve our understanding of
the structural features required for CAR nuclear translocation and activation.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: GlaxoSmithKline, 5 Moore Dr., V-116B, Research Triangle Park, NC 27709. Tel.: 919-483-3936; Fax: 919-315-6720; E-mail: john.t.moore@gsk.com.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300138200
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ABBREVIATIONS |
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The abbreviations used are: CAR, constitutive androstane receptor; PXR, pregnane X receptor; LBD, ligand-binding domain; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; RTQ, real-time quantitative; VDR, vitamin D receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.
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