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INTRODUCTION |
The MDR11
gene product P-glycoprotein (P-gp) plays an important role in the
transport of hydrophobic xenobiotics and peptides from the inside to
the outside of cells. Initially discovered in cancer cells as a
mechanism responsible for resistance against certain cytostatic drugs
(reviewed in Ref. 1), it was later shown that P-gp is also expressed in
different nonmalignant cells of various organs. In agreement with its
assumed physiologic role as a defense mechanism against potential toxic
substances present in the diet and from environmental exposure, it is
expressed in the brush border membrane of the mature enterocytes, the
canalicular membrane of hepatocytes, the brush border of proximal renal
tubular cells, and the luminal side of endothelial cells of brain
capillaries (2, 3).
In the gut P-gp functions as an efflux pump, actively transporting
substances back into the intestinal lumen (4) and, hence, has an
important role for the absorption and presystemic elimination of many
chemicals including drugs. The level of intestinal P-gp expression
shows wide interindividual differences (5) controlled by both genetic
and environmental factors. Recently a genetic polymorphism of the
MDR1 gene has been reported that affects the P-gp expression
in the epithelial cell lining of the small intestine (6). In addition
to this genetic polymorphism, environmental factors can affect the
expression of P-gp. A number of drugs and steroid hormones have been
shown to induce P-gp expression (7-11). For instance, the antibiotic
rifampin not only induces intestinal cytochrome P 450 3A4 enzyme but
also elicits a significant increase of intestinal P-gp (11). As a
consequence, the plasma concentrations of orally administered digoxin
are dramatically reduced.
So far the mechanisms by which rifampin and other inducing substances
cause induction are poorly understood. Induction appears to take place
at the transcriptional level, as MDR1 mRNA expression is
elevated after treatment with inducers (8, 10, 12). Recent studies
indicate that the nuclear receptor NR1I2 (pregnane X receptor (PXR))
(13) mediates xenobiotic induction of CYP3A genes by binding
to PXR response elements in the upstream regulatory region of these
genes (14-18). Since CYP3A4 and MDR1 seem to be co-induced at least by some compounds (11, 12), we investigated whether
a similar PXR-dependent mechanism is also involved in MDR1 induction. In this study we used the human colon
carcinoma cell line LS174T as a model to elucidate the molecular
mechanisms of MDR1 induction by rifampin because in these
cells the endogenous MDR1 gene is highly inducible by
rifampin. By DNA binding assays and transfections, we identified a
distinct PXR binding site, a DR4 nuclear receptor response element,
that is essential for MDR1 induction by rifampin.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The reporter gene plasmid containing
10.2 kb of the MDR1 upstream regulatory region (p-10224) was
constructed by ligating the corresponding NcoI fragment of
the BAC clone CTB-60P12 (Research Genetics; GenBankTM
accession number AC002457), which contains the 5' part of the
MDR1 locus, into the NcoI site of pGL3-Basic
(Promega). The correct orientation was verified by sequencing. p-10224
encompasses the MDR1 sequence from bases
10224 to +261.
Unidirectional deletion mutants of p-10224 were constructed using the
double-stranded Nested Deletion kit (Amersham Pharmacia Biotech). The
precise starting point of the deletion mutants was determined by
sequencing. The MDR1 downstream promoter region encompassing
the sequence between bases
1974 and +281 (19) was amplified by PCR
out of human genomic DNA using the primers 5'-ACG GTA CCA AGG ACT GTT GAA AGT-3' and 5'-ATG GAT CCA ACC CCA CTT GGT CCC CAT-3', which introduce KpnI and BamHI sites, respectively. The
PCR fragment digested with KpnI and BamHI was
ligated into KpnI/BglII-digested pGL3-Basic,
creating p-1974, and sequenced. To create p-1803, p-1974 was digested
with KpnI and BglII and the ends filled in and
religated. The MDR1 promoter fragment (
7975 to
7013)
containing the cluster of nuclear receptor response elements was
amplified by PCR out of the above-mentioned BAC clone with primers
5'-TCT GCT AGC AGT GTT TCT TGT-3', containing a natural NheI
site, and 5'-AAT AGA TCT CAT ATA AGG CAA CTG TTT TGT T-3', introducing
a BglII site. The NheI/BglII-digested
PCR fragment was ligated between the NheI/BglII
sites of a modified pGL3-Basic vector, which contains the sequences
from
105 to + 51 of the thymidine kinase (TK) promoter between its
BglII and HindIII sites, creating p-7975/7013/TK, and sequenced. p-7975(
7012-1804) was constructed by ligating a
KpnI/BglII fragment of p-7975/7013/TK, which
contained the MDR1 sequence between
7975 and
7013, into
KpnI/BglII-digested p-1974.
Site-directed mutagenesis of the DR4(I) and ER6/DR4(III) motifs was
performed within p-7975/7013/TK by using primers that contained two
mutated bases in the center of each half-site (mutated bases are
underlined and in italics): DR4(I), 5'-CAT TGT
TCT AAC TTG TTC TTG CTC-3' and 5'-AGC
AAG AAC AAG TTA GAA CAA
TG-3'; ER6/DR4(III), 5'-GAG TTC ATT TGT TAT TAA
ACA AGA ACA AAG TCT ATG-3' and 5'-CAT AGA CTT
TGT TCT TGT TTA ATA ACA
AAT GAA CTC-3'. The DR4(I)-ER6/DR4(III) double mutation was obtained by
site-directed mutagenesis within the DR4(I)-mutated plasmid with the
ER6/DR4(III)-mutated primers. All mutations were verified by
sequencing. Dimers of DR4(I) and ER6/DR4(III) were obtained by
self-ligation of the appropriate double-stranded oligonucleotides via
added BamHI/BglII sites and cloning into the
BglII site of the modified pGL3-Basic vector with the TK
promoter described above. Dimers with the motifs in the same
orientation as in the MDR1 gene were identified by sequencing.
The open reading frame of human PXR was amplified out of LS180 cells
using the primers 5'-TCG AAT TCC ACC ATG GAG GTG AGA CCC AAA GAA
AGC-3', introducing an EcoRI site and an optimized Kozak
consensus sequence and 5'-CGT CTA GAT CAG CTA CCT GTG ATG CCG AAC A-3',
which introduces an XbaI site. The
EcoRI/XbaI-digested PCR fragment was ligated into
appropriately digested pcDNA3 (Invitrogen), creating pcDhPXR and sequenced.
Cell Culture, Transfection, and Reporter Gene Assays--
The
human colon adenocarcinoma cell line LS174T (20) was obtained from
ATCC. Cells were cultivated in Dulbecco's modified Eagle medium (Life
Technologies) buffered with 25 mM HEPES, supplemented with
100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies),
1% nonessential amino acids (Biochrom), 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine
(Biochrom), and 10% fetal calf serum (Biochrom). Cells were grown at
37 °C, 5% CO2 in a humidified incubator. One day before
transfection, 5.0 × 106 cells/dish were plated in
94-mm dishes (Greiner). 5 µg of reporter gene plasmids, 0.5 µg of
pCMV
(CLONTECH), and 5 µg of pUC18/dish were
introduced into the cells by calcium phosphate transfection. 5 h
after transfection, cells were incubated with 15% glycerol, 1×
phosphate-buffered saline for 3 min, washed twice with 1×
phosphate-buffered saline, and treated for 40 h with rifampin
dissolved in Me2SO to a final concentration of 10 µM or 0.1% Me2SO for the controls. Transfected cells were cultivated in Dulbecco's modified Eagle medium
without phenol red and with fetal calf serum that was pretreated with
dextran-coated charcoal. Transfections were done in duplicate, and
experiments were repeated three times and with different DNA preparations. Cells were harvested and lysed in Nonidet P-40 lysis buffer (250 mM KCl, 50 mM HEPES, pH 7.6, 0.1%
(v/v) Nonidet P-40, 10% (v/v) glycerol, 1 mM
dithiothreitol) for 20 min on ice. After centrifugation, cleared
lysates were used for reporter gene assays done in duplicate. For
luciferase measurements, 300 µl of an assay solution (25 mM glycylglycine, pH 7.8, 50 µM luciferin, 2 mM ATP, 10 mM MgCl2, 27 µM coenzyme A, 30 mM dithiothreitol) were
automatically injected into 20 µl of cell lysate, and luminescence
was measured immediately for 4 s with an AutoLumat Plus (Berthold,
Germany).
-Galactosidase assays were done according to Jain and
Magrath (21) and measured as described above. Luciferase activity was normalized with respect to transfection efficiencies using the corresponding
-galactosidase activity.
RNA Preparation and Northern Blotting--
Preparation of
polyadenylated RNA and Northern blot analysis were done as described
previously (22). Radioactive probes were synthesized using the
DECAprime II Kit (Ambion). To synthesize a probe specific for human
MDR1, the MDR1 cDNA fragment of pMDR V15'
(23) was used. The GAPDH-mouse DECAtemplate (Ambion) was used as a
probe specific for glyceraldehyde-3-phosphate dehydrogenase.
Electrophoretic Mobility Shift Assays--
Gel mobility shift
assays were performed as previously described (18) with modifications.
Briefly, hPXR and hRXR
were synthesized from pcDhPXR and
pCMX-hRXR
(generously provided by R. Schüle) expression
vectors, respectively, using the TNT Quick Coupled transcription/translation reticulocyte system (Promega). Nuclear response elements were prepared by annealing 1 µl each of two complementary oligonucleotide stocks (100 µM) in 48 µl
of 10 mM Tris-Cl pH 7.8 and 1 mM EDTA,
300 mM NaCl. For radioactive labeling, 5 µl of the
annealed oligonucleotides, 2 µl of 10× RB100 (200 mM
Tris-Cl, pH 7.5, 100 mM MgCl2, 1 M
NaCl, 10 mM dithiothreitol), 20 µCi of
[
-32P]dCTP, 1 µl of 5 mM dATP, dGTP, and
dTTP, 0.5 µl of Klenow fragment (2.5 units) and
H2O to a final volume of 20 µl were incubated at 30 °C
for 15 min and purified through Sephadex columns (MicroSpinTM G-25
columns, Amersham Pharmacia Biotech).
Binding reactions contained 10 mM HEPES, pH 7.8, 60 mM KCl, 0.2% Nonidet P-40, 6% glycerol, 2 mM
dithiothreitol, 2 µg of poly(dI-dC), 10,000 cpm labeled probe, and 1 µl of synthesized hPXR and/or hRXR
in a final volume of 20 µl.
If necessary, reactions were filled up with unprogrammed lysate. In
competition experiments, unlabeled competitor oligonucleotides were
added before the addition of in vitro translated protein(s).
Samples were incubated on ice for 20 min after the synthesized proteins
had been added, and the protein-DNA complexes were resolved on a
pre-electrophoresed 5% polyacrylamide gel in 44.5 mM Tris,
44.5 mM boric acid, and 1 mM EDTA (pH 8.3)
at 200 V and room temperature. Gels were dried and
autoradiographed at
80 °C overnight.
Oligonucleotides for Electrophoretic Mobility Shift
Assays--
ER6/DR4(III) wild type sense, 5'-GAT CCC ATT TGA GAT TAA
ACA AGT TCA AAG T-3'; ER6/DR4(III) wild type antisense, 5'-GAT CTA CTT
TGA ACT TGT TTA ATC TCA AAT G-3'; ER6/DR4(III) mutated sense, 5'-GAT
CCC ATT TGT TAT TAA ACA AGA
ACA AAG T-3'; ER6/DR4(III) mutated antisense, 5'-GAT CTA CTT
TGT TCT TGT TTA ATA ACA
AAT G-3'; DR4(I) wild type sense, 5'-GAT CCT CAT TGA ACT AAC TTG ACC
TTG CTC CA-3'; DR4(I) wild type antisense, 5'-GAT CTG GAG CAA GGT CAA
GTT AGT TCA ATG AG-3'; DR4(I) mutated sense, 5'-GAT CCT CAT
TGT TCT AAC TTG TTC TTG CTC CA-3'; DR4(I) mutated antisense, 5'-GAT CTG GAG CAA
GAA CAA GTT AGA ACA ATG
AG-3'; DR4(II) wild type sense, 5'-GAT GGA GAG AGT TCA TTT GAG ATT AAA
CAA-3'; DR4(II) wild type antisense, 5'-GAT TTG TTT AAT CTC AAA TGA ACT
CTC TCC-3'; DR4(II) mutated sense, 5'-GAT GGA GAG AGA
ACA TTT GAG CCT AAA CAA-3'; DR4(II)
mutated antisense, 5'-GAT TTG TTT AGG CTC AAA
TGT TCT CTC TCC-3'; DR3 wild type sense, 5'-GAT TGA ACG TTA CCT CAT TGA ACT AAC TTG-3'; DR3 wild type antisense, 5'-GAT
CAA GTT AGT TCA ATG AGG TAA CGT TCA-3'; DR3 mutated sense, 5'-GAT TGA
ACG TTA CCT CAT TGT TCT AAC TTG-3'; DR3 mutated antisense, 5'-GAT CAA GTT AGA ACA ATG AGG TAA
CGT TCA-3'; Cyp3A23-DR3 sense, 5'-GAT CCT AGA TGA ACT TCA TGA ACT GTC
TA-3'; Cyp3A23-DR3 antisense, 5'-GAT CTA GAC AGT TCA TGA AGT TCA TCT AG-3'.
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RESULTS |
Induction of Endogenous MDR1 in LS174T Cells by Rifampin and Other
CYP3A4 Inducers or PXR Activators--
Expression of intestinal
P-glycoprotein had been shown to be induced by rifampin in
vivo (11). To investigate the mechanisms of induction in the
intestine, we used the intestinal cell line LS174T as a model. LS174T
cells showed a time- and concentration-dependent induction
of MDR1 by rifampin (Fig. 1).
When LS174T cells were exposed to rifampin for 3-48 h, a biphasic
induction was observed that peaked at about 8 h, declined
thereafter to a low at 16 h, and increased again (Fig.
1A). When LS174T cells were treated with increasing
concentrations of rifampin ranging from 0.1 µM to 10 µM, MDR1 induction was maximal between 5 and
10 µM rifampin (Fig. 1B). Thus, LS174T cells
are an appropriate model to investigate the mechanisms of intestinal
MDR1 induction because the endogenous MDR1 gene
is highly inducible by rifampin.

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Fig. 1.
Rifampin induces the expression of
MDR1 in LS174T cells in a time- and
concentration-dependent manner. Northern blot analysis
with polyadenylated RNA of LS174T cells treated with 10 µM rifampin for different time periods as indicated (A)
and for 48 h with different concentrations of rifampin as
indicated in the figure (B). Blots were sequentially
hybridized with probes for the genes indicated. Results of
representative experiments are shown. DMSO,
Me2SO; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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Recently, it has been reported that the induction of CYP3A4
by rifampin is mediated through the nuclear receptor PXR (18), to which
rifampin binds directly (24). Because CYP3A4 and
MDR1 are supposed to be co-induced (12), we hypothesized
that a similar mechanism is also involved in MDR1 induction.
Therefore, LS174T cells were incubated with additional drugs already
known to induce CYP3A4 or to be an activator or ligand for
PXR. Fig. 2 shows that apart from
rifampin, which is one of the most potent inducers, reserpine,
nifedipine, clotrimazole, RU486, and corticosterone were also strong
inducers. Weaker induction was seen with carbamazepine, 5
-pregnane-3,20-dione, 6,16
-dimethylpregnenolone, dexamethasone, and pregnenolone-16
-carbonitrile, and no induction of
MDR1 was observed after treatment with coumestrol. With the
exception of 6,16
-dimethylpregnenolone, reserpine, and
carbamazepine, for which no EC50 values have been reported,
concentrations used to induce MDR1 in LS174T cells exceeded
reported EC50 values for ligand activation of hPXR (14, 15,
24). Regarding the nearly identical pharmacological profile of
MDR1 induction in LS174T cells and hPXR activation, we
suppose that MDR1 induction by rifampin is mediated by a
PXR-dependent mechanism.

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Fig. 2.
MDR1 induction by
CYP3A4 inducers and PXR activators. Northern blot
analysis of polyadenylated RNA of LS174T cells treated for 48 h
with different substances as indicated: lanes 1 and
12, negative control with 0.1% Me2SO;
lanes 2 and 15, rifampin; lane 3,
RU486; lane 4, corticosterone; lane 5,
5 -pregnane-3,20-dione; lane 6,
6,16 -dimethylpregnenolone; lane 7, clotrimazole;
lane 8, nifedipine; lane 9, 50 µM
coumestrol; lane 10, 50 µM carbamazepine;
lane 11, reserpine; lane 13, 50 µM
pregnenolone-16 -carbonitrile; lane 14, 100 µM dexamethasone. If not indicated otherwise,
concentrations were 10 µM each. Blots were hybridized
with probes for the genes indicated. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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PXR/RXR
Heterodimers Bind to Three DR4 Motifs in a Cluster of
Nuclear Response Elements--
The previously published proximal
promoter region of MDR1 (up to approximately
2 kb) (19)
was not involved in induction by rifampin in LS174T cells (Fig.
5A). Unidirectional deletion mutants of this region also did
not reveal any induction by rifampin (data not shown). By
computer-aided analysis with the Lasergene program package (DNASTAR),
we looked for potential PXR binding sites in 20 kb of the
MDR1 upstream region (sequence is available in
GenBankTM accession number AC002457). PXR had been shown to
bind to AG(G/T)TCA repeats of DR3, DR4, DR5, and ER6 organization (15). We identified a cluster of potential PXR binding sites at about
8 kb
of the MDR1 promoter (Fig. 3)
comprising three DR4 motifs (numbered I, II, and III, starting from 5')
and one DR3 and ER6 motif, all overlapping one another with at least
one half-site. DR4(I) and DR3 are arranged in the opposite direction
compared with the other motifs. Within the 20-kb upstream region
analyzed, DR4(I) was the only motif for PXR with two consensus
AG(G/T)TCA half-sites; all the others had one or two mismatches in one
half-site.

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Fig. 3.
Position of MDR1 promoter
elements. The figure shows the position of the regulatory cluster
containing several potential nuclear response elements in relation to
the transcription start site of the human MDR1 gene. The 1 kb fragment comprises bases 7975 to 7013. These include the cluster
from 7864 to 7817, which is depicted in a larger magnification. DR4
motifs are numbered serially from the 5' to the 3' direction. Also
shown is the previously published proximal promoter region (19).
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Electrophoretic mobility shift assays (Fig.
4, A and B) with
wild type and mutated versions of each motif revealed that PXR binds
specifically as a heterodimer with RXR
to all three DR4 wild type
motifs (DR4(III) and ER6 are coincident, since the two elements are too
nested) but not to the mutated forms. DR3 was not able to bind
PXR/RXR
heterodimers, even in its wild type form (Fig.
4B). PXR/RXR
heterodimers did not bind to DR4(I) or DR4(II) as strongly as to ER6/DR4(III). Binding to the latter motif is
as strong as to the DR3 motif of CYP3A23 (Fig.
4A). Competition experiments with the corresponding
unlabeled wild type and mutated versions of the motifs further
demonstrated the specificity of binding as exemplified for DR4(I) in
Fig. 4C. Usually, the two centrally located base pairs of
each half site (DR4(I), DR4(II), and ER6/DR4(III)) were mutated for the
shifts. To demonstrate that site-directed mutagenesis of DR4(I) also
inactivates DR3, only the two central base pairs of the 5' half-site
were changed in the DR3 motif, because this is the one it shares with
DR4(I). Since DR4(III) and ER6 overlap almost completely except for two base pairs in one half-site of each motif, it was impossible to rule
out whether PXR/RXR
heterodimers bind to the ER6 or DR4(III) motif.

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Fig. 4.
PXR/RXR heterodimers
bind specifically to DR4 and ER6/DR4 motifs. A and
B, electrophoretic mobility shift assays using in
vitro translated proteins bound to radiolabeled oligonucleotides
corresponding to unmutated or mutated (mut) potential PXR
binding sites of the MDR1 regulatory cluster or the PXR
binding site of CYP3A23 as a positive control. Binding
reactions contained (+) or lacked ( ) the indicated proteins.
Complexes of PXR/RXR heterodimers and the oligonucleotides are
marked by an arrow. The band of lower molecular weight seen
in the lanes with only one of the two proteins derived from the
unprogrammed reticulocyte lysate, as it was also detected when
unprogrammed lysate was used alone (shown in C). The
intensity of this band varied between experiments and probes used.
C, competition experiment with radiolabeled wild type DR4(I)
as probe and unlabeled wild-type (DR4(I)) or mutated DR4(I)
(DR4(I)mut) as competitor. The numbers indicate the
n-fold molar excess to which the competitor was added.
Specific complexes are marked by an arrow.
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Reporter Gene Assays Confirm That the Cluster of Nuclear Response
Elements Is Responsible for MDR1 Induction by Rifampin--
To analyze
the functional role of the identified cluster of PXR binding sites, we
constructed a reporter gene plasmid that contained about 10 kb of the
MDR1 upstream region (p-10224), including the potential
regulatory cluster at about
8 kb. Transient transfection of p-10224
into LS174T cells revealed that this MDR1 upstream fragment
was inducible by rifampin (Fig.
5A). A deletion mutant still
containing the potential regulatory cluster (p-8055) was also
inducible, whereas a deletion mutant without the cluster (p-7771) was
no longer induced by rifampin. Consequently, the cluster of PXR binding
sites is necessary for the induction of MDR1 by rifampin.
That p-7771 was not inducible by rifampin at all indicates no sequences
further downstream are required for induction. To demonstrate this, a
reporter gene plasmid was constructed in which a 1-kb MDR1
upstream sequence encompassing the regulatory cluster was fused to the
proximal promoter region of MDR1. This region by itself is
not inducible by rifampin, as demonstrated by p-1803. The upstream
sequence containing the regulatory cluster conferred inducibility by
rifampin to the proximal MDR1 promoter, thereby also
demonstrating enhancer properties (Fig. 5A). Furthermore, when the fragment containing the regulatory cluster was fused to a
heterologous promoter (minimal TK promoter), it also conferred inducibility by rifampin. Therefore, the cluster of PXR binding sites
constitutes an enhancer element that is inducible by rifampin.

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Fig. 5.
Activation of the MDR1
promoter by rifampin requires a DR4 motif within a regulatory
cluster at about 8 kb. A, MDR1 reporter
genes are shown schematically on the left. Numbers indicate
positions relative to the transcriptional start site. The black
bar corresponds to the regulatory cluster. The results of
transfection experiments in LS174T cells are illustrated on the right.
Cells were harvested and analyzed for luciferase and -galactosidase
activity. The columns show the mean induction factors of the luciferase
reporter genes by rifampin. The activity of each reporter in the
presence of Me2SO only was designated as 1. Thin lines show S.D. B, reporter genes are shown
schematically on the left. Numbers indicate positions
relative to the transcriptional start site of MDR1.
Ellipses denote DR4(I), and bars denote
ER6/DR4(III) motifs. Filled symbols are in wild type
configuration, and open symbols denote mutated motifs. The
results of transfection experiments are shown on the right, as
described in A. C, LS174T cells were
co-transfected with the reporter gene p-7975/7013/TK (illustrated in
the inset), pCMV , and 0.2 µg of an expression plasmid
for hPXR (+PXR) or empty expression vector
( PXR) and treated with rifampin (+) or Me2SO
only ( ). The cells were harvested and analyzed for luciferase and
-galactosidase activity. The columns show the average induction
factors. The activity in the absence of rifampin and exogenous PXR was
designated as 1.
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To further analyze the contribution of the individual PXR binding sites
in the cluster to induction by rifampin, the DR4(I) and/or the
ER6/DR4(III) motifs were mutated by site-directed mutagenesis. Fig.
4A shows that PXR/RXR
heterodimers cannot bind to the
mutated motifs, thereby demonstrating that the mutated motifs are no
longer functional. When DR4(I) is mutated alone or together with
ER6/DR4(III), induction by rifampin was lost (Fig. 5B). In
contrast, mutation of the ER6/DR4(III) motif alone resulted even in an
increased induction. Mutation of DR4(II) did not significantly
influence the induction by rifampin (data not shown). Fig.
5B also shows that dimers of DR4(I) were induced by
rifampin, consistent with the results of the electrophoretic mobility
shift assay and the reporter gene assay with the mutated DR4(I). In
contrast, ER6/DR4(III) dimers did not show a significant induction.
The data presented in Fig. 2 have provided evidence that PXR is likely
to be the endogenous factor present in LS174T cells that mediates
induction. To demonstrate that PXR can activate transcription of
MDR1, an expression plasmid for hPXR was co-transfected with
p-7975/7013TK. As can be seen in Fig. 5C, co-transfection of
PXR activated transcription of the reporter gene even in the absence of
rifampin. Treatment with rifampin led to a further increase in
induction (about 3-fold). Therefore it can be concluded that induction
by rifampin of the MDR1 enhancer can be mediated by PXR.
Altogether these data indicate that PXR is the endogenous factor
present in LS174T cells mediating induction by rifampin.
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DISCUSSION |
Due to the central role that intestinal P-gp plays in the
absorption and presystemic elimination of many chemicals, including medicines, an understanding of the factors regulating its expression is
of importance both from a clinical and toxicological point of view.
Because the mechanisms of induction are poorly understood, we
investigated the molecular mechanisms by which rifampin induces MDR1 gene expression using an intestinal cell line. Rifampin
was selected because it is a powerful inducer of intestinal P-gp both in human duodenal biopsies (11) and in a cell line (12), whereas for
other inducers, only in vitro data are available, thus
raising doubts if results obtained in cell lines with these substances can be extrapolated for the human in vivo situation. Since a
time- and concentration-dependent induction of
MDR1 by rifampin was observed in the colon carcinoma cell
line LS174T, it proved a suitable model for intestinal MDR1
induction. The biphasic induction is indicative of a two-stage
mechanism of induction by rifampin. The first and immediate increase
can be explained by activation of pre-existing factors, probably PXR,
and is therefore supposed to be independent of de novo
protein biosynthesis. The second increase, starting at about 24 h,
however, probably required de novo protein biosynthesis.
This would imply that additional genes are induced by rifampin, which
then participate in MDR1 induction. The reason for the
decrease in MDR1 expression between 8 and 16 h is
unclear and remains to be elucidated.
The reported co-induction of CYP3A4 and
MDR1 (11, 12) and the recently identified involvement of PXR
in xenobiotic induction of CYP3A genes (18) prompted us to
investigate whether PXR is also involved in induction of
MDR1. Treatment of LS174T cells with the known
CYP3A4 inducers reserpine, clotrimazole, RU486, carbamazepine, dexamethasone, and pregnenolone-16
-carbonitrile (12,
25, 26) and the known activators for PXR, nifedipine, corticosterone,
5
-pregnane-3,20-dione, and 6,16
-dimethylpregnenolone (14-17),
induced MDR1. Only the PXR activator coumestrol did not induce MDR1. Induction of MDR1 by other
drugs apart from rifampin, known as CYP3A4 inducers and PXR
activators, provided evidence that PXR is probably the factor
responsible for induction of MDR1 in LS174T cells. In
agreement with this hypothesis, PXR mRNA is expressed in
LS174T cells (data not shown).
We identified an enhancer element at about
8 kb of the
MDR1 upstream region that contains several nuclear receptor
binding motifs and mediates induction by rifampin in LS174T cells.
Within this enhancer element, there are three DR4 motifs (numbered
I-III from 5' to 3') and one DR3 and ER6 motif, respectively, all
overlapping with at least one half-site. A similar complex organization
of nuclear response elements has also been reported in the distal enhancer module (XREM) of CYP3A4, which mediates the
hPXR-dependent induction by xenobiotics in co-operation
with a proximal promoter element (18). In contrast to
CYP3A4, such a proximal promoter element is missing in
MDR1. As shown by us, the proximal promoter region of
MDR1 cannot be induced by rifampin. Similar to the XREM of
CYP3A4, only one out of several motifs to which PXR binds is necessary for induction. In the XREM of CYP3A4, a DR3 motif
mediates induction (18), whereas in MDR1 it is the DR4(I)
motif, which is the only consensus motif present in MDR1.
Mutation of the ER6/DR4(III) motif in the MDR1 enhancer was
associated with an increased induction. A similar observation has been
made with the mutated ER6 motif in the CYP3A4 XREM (18).
Therefore it is quite likely that there are factors in LS174T cells
that bind to the ER6/DR4(III) and probably exert a suppressive effect
on the activity of DR4(I). In agreement with this, dimers of
ER6/DR4(III) cannot be induced, although PXR binds in vitro
to the motif. The ER6 motif in the proximal promoter of
CYP3A23 also showed binding of PXR but did not mediate
PXR-dependent induction (27). It is a common feature of all
these imperfect ER6 motifs, to which PXR binds in vitro but
which do not mediate induction, that a more or less well conserved DR4
motif is contained within the ER6. It is probably due to the imperfect
nature of these ER6 motifs that other factors can bind to them or to
the enclosed degenerate DR4 with higher affinity than PXR. Likely
candidates could be the nuclear receptors COUP-TFI, LXR, and
CAR, which all bind to degenerate DR4 motifs (28-30). LXR and
COUP-TFI are expressed in LS174T cells (data not shown). Competition of
different nuclear receptors, which exert opposing effects on
MDR1 expression, could explain the pronounced
interindividual variability in MDR1 induction by rifampin
observed in humans (9, 11). The magnitude of induction is probably
dependent on the relative amounts of competing factors. Consequently,
the total amount of PXR alone does not determine the extent of
induction. On the other hand, mutations in the PXR response elements
that change the binding affinity of PXR or competing factors can
also cause the observed variability of MDR1 induction in
different individuals.
The DR4(I) motif mediating induction of MDR1 is the first
physiological PXR response element of DR4-type, whereas previously described PXR response elements in CYP3A promoters were all
of ER6 and DR3-type. This confirms results obtained with synthetic AG(G/T)TCA repeats of different spacing, which suggest the existence of
DR4 and DR5 motifs in PXR-regulated gene promoters (15). The degenerate
DR4(II) motif to which PXR binds in vitro is not necessary
for induction. Other factors in addition to PXR probably bind to
DR4(II) in LS174T cells.
Co-transfection of an expression plasmid for hPXR directly demonstrated
that PXR mediates induction of MDR1 through the identified enhancer. Moreover, PXR activates the reporter gene with the
MDR1 enhancer even in the absence of rifampin treatment.
Co-transfection probably changes the relative amounts of PXR and
competing factors, leading to an increased binding of PXR to its
binding sites in the MDR1 enhancer. Transcriptional
activation then might be due to the presence of endogenous ligands for
PXR in LS174T cells. As reported previously, pregnanes are likely
candidates for endogenous ligands (16).
This study provides evidence that a similar mechanism is responsible
for induction of CYP3A4 and MDR1 by xenobiotics.
The induction of both genes requires nuclear receptor response elements in proximal promoter and/or enhancer regions to which PXR binds. Recently it has been shown in knockout mice that PXR is necessary for
induction of CYP3A (31). Whether this is also true for
MDR1 induction remains to be clarified. CYP3A4 and P-gp are
believed to constitute a physiological barrier in the intestine that
prevents the absorption of harmful chemicals contained in the diet
(32). When the organism is exposed to increasing concentrations of
xenobiotics, induction seems to be a mechanism to strengthen the
barrier function in the intestine by coordinated up-regulation of both
the drug metabolizing enzyme and the drug transporter. The question
arises of whether additional drug-metabolizing enzymes or drug
transporters, which can contribute to barrier function, are also
up-regulated by a similar PXR-dependent mechanism.