Molecular Mechanisms of Polymorphic CYP3A7 Expression
in Adult Human Liver and Intestine*
Oliver
Burk
§,
Heike
Tegude,
Ina
Koch¶,
Elisabeth
Hustert¶,
Renzo
Wolbold,
Hartmut
Glaeser,
Kathrin
Klein,
Martin F.
Fromm,
Andreas K.
Nuessler
,
Peter
Neuhaus,
Ulrich M.
Zanger,
Michel
Eichelbaum**, and
Leszek
Wojnowski¶
From the Dr. Margarete Fischer-Bosch Institute of
Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany,
¶ Epidauros Biotechnologie AG, Am Neuland 1, D-82347 Bernried,
Germany, the Department of Surgery, Charité, Campus
Virchow-Clinic, Humboldt University, D-13353 Berlin, Germany,
and the ** Division of Clinical Pharmacology, Eberhard Karls
University, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany
Received for publication, March 11, 2002
 |
ABSTRACT |
Human CYP3A enzymes play a pivotal role in the
metabolism of many drugs, and the variability of their expression among
individuals may have a strong impact on the efficacy of drug treatment.
However, the individual contributions of the four CYP3A
genes to total CYP3A activity remain unclear. To elucidate the role of
CYP3A7, we have studied its expression in human liver and
intestine. In both organs, expression of CYP3A7 mRNA
was polymorphic. The recently identified CYP3A7*1C allele
was a consistent marker of increased CYP3A7 expression both
in liver and intestine, whereas the CYP3A7*1B allele was
associated with increased CYP3A7 expression only in liver.
Because of the replacement of part of the CYP3A7 promoter by the corresponding region of CYP3A4, the
CYP3A7*1C allele contains the proximal ER6 motif of
CYP3A4. The pregnane X and constitutively activated
receptors were shown to bind with higher affinity to CYP3A4-ER6 than to
CYP3A7-ER6 motifs and transactivated only promoter constructs
containing CYP3A4-ER6. Furthermore, we identified mutations in
CYP3A7*1C in addition to the ER6 motif that were necessary only for activation by the constitutively activated receptor. We
conclude that the presence of the ER6 motif of CYP3A4
mediates the high expression of CYP3A7 in subjects carrying
CYP3A7*1C.
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INTRODUCTION |
Cytochrome P450 enzymes play a pivotal role in the oxidative,
peroxidative, and reductive metabolism of many endogenous compounds, procarcinogens, and drugs. The CYP3A subfamily composed of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 in humans is of special importance because
it accounts for as much as 30% of total liver cytochrome P450 content
(1). At least 50% of all medicines are metabolized by enzymes of the
CYP3A subfamily (2). The most abundant CYP3A isoform in liver and
intestine is CYP3A4. Its interindividual hepatic expression varies
60-fold (3), and the in vivo function as assessed by
clearance displays at least a 20-fold difference (4). Induction by
xenobiotics (e.g. rifampin) and endogenous compounds
(e.g. steroid hormones) further modulates the variability of
CYP3A4 expression among individuals. The induction of CYP3A4 and most likely that of other CYP3A genes is mediated by the
nuclear receptor NR1I2 (pregnane X receptor
(PXR)1) (reviewed in Ref. 5).
CYP3A4-inducing compounds bind to PXR and stimulate the
transcriptional activity of the receptor. Additional nuclear receptors
such as NR1I3 (constitutively activated receptor (CAR)) and NR1I1
(vitamin D receptor) have also been implicated in the transcriptional
regulation of CYP3A4 (6, 7). Although the substrate
specificity of CYP3A5 is similar to that of CYP3A4, CYP3A5 has been
regarded to be less important for drug elimination because it is
expressed at much lower levels than CYP3A4 in most livers of Caucasian
origin (8). CYP3A43 is expressed at very low levels in adult
human livers, accounting for only 0.1-0.2% of CYP3A4
transcripts (9, 10). Therefore, its contribution to the elimination of
CYP3A substrates is regarded to be negligible (10). This variability in
CYP3A expression and function explains why the intensity and duration
of drug action and the occurrence of side effects show large
patient-to-patient variability. Although a recent analysis suggests
that, depending on the drug, 60-90% of patient-to-patient variability
in CYP3A function is caused by genetic factors (3), the sources for
variability in constitutive CYP3A expression remain largely
unknown. However, a common genetic polymorphism in intron 3 of
CYP3A5 was recently identified. It results in high
expression genotypes and explains the >10-fold increase in CYP3A5
protein expression observed in 10-30% of livers of Caucasian origin
(8, 11). In some persons, CYP3A5 can contribute to >50% of total
CYP3A content, thus exceeding CYP3A4 levels. Correspondingly, CYP3A5
has been proposed to contribute substantially to the elimination of
CYP3A substrates (11). These findings could explain why there is
less variability in the in vivo clearance than one would
predict based on the >60-fold variability in CYP3A4 expression.
The role of CYP3A7 in the biotransformation of CYP3A substrates in
adult liver and intestine is not known. CYP3A7 accounts for 30-50% of
total cytochrome P450 in fetal liver (12) and was at first regarded to
be exclusively expressed there (13). Since then, CYP3A7
expression was detected in 54-88% of adult livers (14, 15). However,
quantitative data on CYP3A7 expression in adult livers are
missing. Moreover, the mechanisms responsible for expression of
CYP3A7 in adult livers remain unknown.
Using a large collection of human livers, we report here that
CYP3A7 mRNA is polymorphically expressed in both liver
and intestine, with ~11% of subjects belonging to a distinct
subgroup of high expression phenotype. Two-thirds of the subjects in
this group carry the CYP3A7*1C or (less frequently) the
CYP3A7*1B promoter allele. The CYP3A7*1C allele
is the exclusive marker of high CYP3A7 expression in the
intestine. Functional differences between CYP3A7 and
CYP3A7*1C proximal promoter ER6 (everted
repeat separated by 6 base pairs) motifs in
binding and activation by the nuclear receptors PXR and CAR were
identified as the mechanisms responsible for the high CYP3A7
expression in CYP3A7*1C carriers.
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EXPERIMENTAL PROCEDURES |
Human Tissue Samples--
Normal human liver tissue samples were
obtained from patients of Caucasian origin undergoing liver resection
at the Department of Surgery, Campus Virchow-Clinic, Humboldt
University (Berlin, Germany) (16). Liver pieces were immediately placed
into RNAlater (Ambion Inc.), incubated for 24 h at 4 °C, and
then stored frozen at 
80 °C until RNA isolation. Human intestine
samples were obtained from patients of Caucasian origin undergoing
either gastrectomy or pancreatoduodenectomy at the Department of
Surgery, Robert Bosch Hospital (Stuttgart, Germany). In addition, a
liver wedge biopsy was obtained from a subset of these patients. Normal
intestine tissue (duodenum or proximal jejunum) was dissected and
either immediately frozen in liquid nitrogen or incubated for 24 h
at 4 °C in RNAlater and then stored at -80 °C until RNA
preparation. The matching liver biopsies were processed as the
intestine samples.
Genotyping by Sequencing--
Genomic DNA was isolated from
blood samples of the patients using the QIAamp DNA blood kit (QIAGEN
Inc.). To determine the CYP3A7 genotypes, a 614-bp fragment
of the CYP3A7 proximal promoter and exon 1 (426 to +188
with respect to the transcriptional start site) was amplified by
PCR using oligonucleotide primers 5'-GGC TCT GTC TGG CTG GGT ATG
A-3' (bases 110480-110501 of GenBankTM/EBI accession
number AF280107) and 5'-GAA CAG TTA CTC ACA GAT AGA GGA GTA TC-3'
(bases 111093 to 111065). The standard PCR consisted of 1× PCR
buffer with 1.5 mM MgCl2 (QIAGEN Inc.), 200 µM dATP, 200 µM dCTP, 200 µM
dGTP, 200 µM dTTP, 0.5 µM each primer, 1.25 units Taq polymerase (QIAGEN Inc.), and 20 ng of genomic DNA
in a total volume of 50 µl. Cycling conditions were one cycle at
94 °C for 2 min and 34 cycles at 94 °C for 45 s, 62 °C
for 45 s, and 72 °C for 1 min, followed by a final cycle at
72 °C for 5 min. PCR products were purified using the QIAquick PCR
purification kit (QIAGEN Inc.), directly sequenced using the ABI BigDye
terminator cycle sequencing kit (Applied Biosystems) and an ABI 3700 DNA analyzer (Applied Biosystems), and analyzed using the
PHRED/PHRAP/POLYPHRED/CONSED software package (University
of Washington, Seattle, WA).
Restriction Fragment Length Polymorphism Assay for
CYP3A7*1C--
A restriction fragment length polymorphism assay
was developed for the CYP3A7*1C allele. Oligonucleotide
primers 5'-ATG ACC TAA GAA GAT GGA GTG-3' (bases 110153-110173 of
GenBankTM/EBI accession number AF280107) and 5'-GAA GGG CAT
GGT CTA CAC TAT-3' (bases 112620 to 112600) were used to amplify a
2468-bp fragment of the CYP3A7 gene. PCR was performed with
100-200 ng of genomic DNA, 1× OptiPerform buffer III (PAN
Biotech, Aidenbach, Germany), 1× OptiZyme Enhancer (PAN
Biotech), 1.5 mM MgCl2, 200 µM
dATP, 200 µM dCTP, 200 µM dGTP, 200 µM dTTP, 1 µM each primer, and 1 unit of
PowerScript (PAN Biotech) in a volume of 50 µl. Cycling conditions
comprised an initial denaturing cycle at 94 °C for 2 min, followed
by 32 cycles of 94 °C for 30 s, 55 °C for 30 s, and
72 °C for 3 min and by a final extension step at 72 °C for 5 min.
PCR products were analyzed on an agarose gel, and the 2468-bp
fragment was purified using the QIAquick gel extraction kit (QIAGEN
Inc.) and digested with the restriction endonuclease SspI.
The wild-type allele fragment has two SspI sites (positions 110734/110739 and 112353/112358), resulting in three fragments of 1617, 585, and 266 bp. In contrast, in the CYP3A7*1C allele, the
site at position 110734/110739 is destroyed through mutation of the T
at position 110739 to G (corresponding to position -167 of the
CYP3A7 promoter). The resulting two fragments have
sizes of 2202 and 266 bp, respectively (see also Fig. 2).
RNA Analysis and Real-time PCR--
Total RNA was prepared from
human liver and intestine samples using the RNeasy kit (QIAGEN Inc.)
according to the recommendations of the manufacturer. RNA was treated
with DNase I to remove contaminating genomic DNA. First-strand cDNA
was generated from 0.5 to 1 µg of total RNA using random hexamer
primers and TaqMan reverse transcription reagents (Applied Biosystems)
according to the standard protocol of the manufacturer. PCRs were set
up with cDNA corresponding to 35 or 40 ng of total RNA for liver or
intestine samples, respectively, and the TaqMan Universal PCR Mastermix
(Applied Biosystems). Primers were used at a final concentration of 400 nM, and probes at 200 nM. Expression levels of
CYP3A7, CYP3A4, villin, and 18 S rRNA were
quantified by TaqMan real-time quantitative PCR using the ABI 7700 sequence detection system (Applied Biosystems). The experiments were
performed according to a standard protocol for the ABI 7700 sequence
detection system. Assays were done in triplicate. Oligonucleotide primers and probes were designed with the PrimerExpress software (Applied Biosystems). Probes were labeled with the 5'-reporter dye
6-carboxyfluorescein and the 3'-quencher dye
6-carboxytetramethylrhodamine. Oligonucleotides used for
CYP3A7 were as follows: primers 5'-AAG GGC TAT TGG ACG TTT
GAC A-3' and 5'-ATC CCA CTG GCC CGA AAG-3' and probe 5'-TAT TTA TGA CTG
TCA ACA GCC TAT GCT GGC TAT CA-3'. The specificity of the assay was
determined using in vitro transcribed CYP3A4,
CYP3A5, and CYP3A43 RNAs. Serial dilutions of
in vitro transcribed CYP3A7 or of a plasmid
containing CYP3A7 cDNA were used to create the
calibration curve. Oligonucleotides used for CYP3A4 were as
follows: primers 5'-TCA GCC TGG TGC TCC TCT ATC TAT-3' and 5'-AAG CCC
TTA TGG TAG GAC AAA ATA TTT-3' and probe 5'-TCC AGG GCC CAC ACC TCT GCC
T-3'. The specificity of the CYP3A4 assay was determined
using in vitro transcribed CYP3A5,
CYP3A7, and CYP3A43. Serial dilutions of in
vitro transcribed CYP3A4 were used to create the
calibration curve. Oligonucleotides used for villin were as follows:
primers 5'-CTG GCA ACC TTA GGG ACT GG-3' and 5'-GTT AGC ATT GAA CAC GTC
CAC TTT-3' and probe 5'-CCA GAT CAC TGC TGA GGT CAC AAG C-3'. To create
the calibration curve for the villin assay, serial dilutions of Caco-2
TC7 cDNA were used. The expression levels of CYP3A7 and
CYP3A4 in liver samples were normalized with respect to the
expression of 18 S rRNA, as determined using the TaqMan ribosomal RNA
control reagents (Applied Biosystems) and serial dilutions of HepG2
cDNA for the calibration curve. CYP3A7 and
CYP3A4 expression levels in intestine samples were
normalized with respect to the expression levels of villin.
Statistical Analysis--
CYP3A7 expression data were
tested for gaussian distribution by calculating a probability plot with
Statgraphics Plus Version 2.0 for Windows (Statistical Graphics Corp.).
The two-tailed Mann-Whitney U test was used to analyze for
statistically significant associations between CYP3A7
expression and CYP3A7 genotype. These analyses were
performed using GraphPAD InStat Version 3.05 for Windows 95.
Plasmid Constructs--
The eukaryotic expression plasmid for
human PXR has been described previously (17). To construct a eukaryotic
expression plasmid for human CAR, the open reading frame of human CAR
was amplified by PCR from human liver cDNA using primers 5'-ATG AAT TCC ACC ATG GCC AGT AGG GAA GAT GAG CTG-3', which introduced an EcoRI site and an optimized Kozak consensus sequence, and
5'-CGT CTA GAT TAG CTG CAG ATC TCC TGG AGC AG-3', which introduced an XbaI site. The EcoRI/XbaI-digested PCR
fragment was cloned into appropriately digested vector pcDNA3
(Invitrogen), creating pcDhCAR1, and verified by sequencing.
The CYP3A7 proximal promoter (869 to +50) was amplified
from human genomic DNA by PCR with oligonucleotides 5'-GCG GTA CCA TCT
CAT CCA TGC CAT GTC TCT TT-3' and 5'-ACA AGC TTG CTG TTT GCT GGG CTG
TGT GTG-3'. The oligonucleotides introduced a KpnI and a
HindIII restriction site on the 5'- and 3'-ends of the
amplified fragment, respectively. The PCR product was digested with
KpnI and HindIII and cloned into appropriately
digested luciferase reporter gene vector pGL3-Basic (Promega). The
identity of the cloned DNA fragment with the CYP3A7 promoter
was verified by sequencing of the resulting plasmid,
pGL3-CYP3A7(869). This plasmid was then digested with KpnI
and BglII, leading to deletion of the region between
positions -869 and -350; and the overhanging ends were converted to
blunt ends by treatment with T4 DNA polymerase and religated. The
resulting plasmid, pGL3-CYP3A7(350), henceforth referred to as the
CYP3A7 construct, contains the region between positions -350 and +50.
A reporter gene construct containing the CYP3A7*1C variant
of the CYP3A7 promoter was obtained by amplification and
cloning of the appropriate portion of genomic DNA from a subject heterozygous for CYP3A7*1C. Oligonucleotide primers 5'-CAT
AGG TAA AGA TCT GTA GGC A-3' and 5'-ACA AGC TTG CTG TTT GCT GGG CTG TGT
GTG-3' encompassed the BglII site at position -351 and
introduced a HindIII site at position +50, respectively. The
PCR product was digested with BglII and HindIII
and cloned into appropriately digested pGL3-Basic. A plasmid clone
containing the CYP3A7*1C variant was selected by sequencing
and is referred to as CYP3A7(*1C). Mutagenesis of the CYP3A7
promoter was done using the QuikChange site-directed mutagenesis kit
(Stratagene) according to the recommendations of the manufacturer. The
single base pair mutations constituting the CYP3A7*1C allele
were introduced either alone or in combinations. Thus, mutations at
positions -167 and -159, which convert the ER6 motif of
CYP3A7 into the corresponding ER6 motif of CYP3A4 (TGAACTCAAAGGAGGTCA, mutated bases in boldface
italics), were introduced into the CYP3A7 construct, creating
CYP3A7(ER6A4). This construct was further mutated. First, the mutation
at position -188 of CYP3A7*1C was introduced, creating
CYP3A7(A4:188,ER6). Second, the mutation at position -129 of
CYP3A7*1C was introduced, creating CYP3A7(A4:ER6,129).
Third, the mutations at positions -181, 179, and 178 of
CYP3A7*1C were introduced, creating CYP3A7(A4:3B,ER6). The
CYP3A7(A4:188,ER6) construct was further mutated by introducing the
mutations at positions -181, 179, and 178 of CYP3A7*1C, thus creating CYP3A7*1C(A7:129). The introduction of all the mutations and the absence of other, undesired mutations were verified by sequencing.
To create a CYP3A4 promoter reporter gene comparable in size
to the CYP3A7 constructs, unidirectional deletion of
pGL3-CYP3A4(1105) (18) was performed with the double-stranded nested
deletion kit (Amersham Biosciences). Sequencing identified a clone
encompassing the CYP3A4 promoter region from positions -374
to +51, pGL3-CYP3A4(374), further referred to as the CYP3A4
construct. Mutation of the ER6 element of the CYP3A4
promoter was done by sequential PCR steps according to standard
procedures and converting the ER6 motif into the corresponding ER6
motif of CYP3A7 (TTAACTCAATGGAGGTCA, mutated bases in boldface italics), thus creating CYP3A4(ER6A7). The
CYP3A4 construct was further mutated using the QuikChange site-directed
mutagenesis kit: the T at position -188 was converted to G, creating
CYP3A4(A7:188). The introduction of all the mutations and
the absence of other undesired mutations were verified by sequencing.
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were performed as previously described (17). The
human PXR, CAR, and RXR
proteins were synthesized using the using
expression plasmids pcDhPXR (17), pcDhCAR1, and pCMX-hRXR (kindly
provided by R. Schüle, Klinik fuer Tumorbiologie, University of
Freiburg, Freiburg, Germany) and the TNT T7 Quick Coupled
transcription/translation system (Promega). Oligonucleotides for the
MDR1 -7.8-kb enhancer nuclear receptor response element
DR4(I) (wild-type) were as described (17). Oligonucleotides for the
CYP3A4 and CYP3A7 proximal ER6 PXR-binding sites
were as follows: ER6-A4 sense, 5'-GAT CCA ATA TGA ACT CAA AGG AGG TCA
GTG A-3'; ER6-A4 antisense, 5'-GAT CTC ACT GAC CTC CTT TGA GTT CAT ATT
G-3'; ER6-3A7 sense, 5'-GAT CCA ATA TTA ACT CAA TGG AGG TCA GTG A-3';
and ER6-3A7 antisense, 5'-GAT CTC ACT GAC CTC CAT TGA GTT AAT ATT
G-3'. Retarded complexes were quantified with the BAS1800 II
phosphor-storage scanner (Fuji) and AIDA software (Raytest,
Straubenhardt, Germany).
Cell Culture, Transient Transfections, and Reporter Gene
Assays--
The human colon carcinoma cell line LS174T (19) was
obtained from American Type Culture Collection and cultivated as
described previously (17). One day before transfection, LS174T cells
were plated in 24-well plates (Falcon) at a density of 1.5 × 105 cells/well. The plasmid DNA was transfected using the
EffecteneTM transfection reagent (QIAGEN Inc.) according to
the manufacturer's recommendations. Usually, 150 ng of reporter gene
plasmid, 20 ng of 
-galactosidase reference plasmid pCMV
(CLONTECH), and 0.1 ng of human PXR or 10 ng of
human CAR expression plasmid were used per well. pUC18 plasmid DNA was
used to fill up to a total amount of 200 ng of DNA/well. Six to seven
hours after transfection, the cells were washed with phosphate-buffered
saline to remove EffecteneTM-DNA complexes and supplied
with fresh medium (without phenol red and supplemented with fetal calf
serum that was pretreated with dextran-coated charcoal). Cells
cotransfected with the PXR expression plasmid were treated with 10 µM rifampin dissolved in Me2SO or with an
equivalent amount (0.1%) of Me2SO only. Transfections were
done in triplicate. At least three independent experiments were
performed, using at least two different plasmid DNA preparations of
each construct. Cells were harvested 40 h after the removal of the
EffecteneTM-DNA complexes and lysed with 150 µl of 1×
passive lysis buffer (Promega). After centrifugation, cleared lysates
were used for reporter gene assays, which were 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, and 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,
Bad Wildbad, Germany). -Galactosidase assays were done
according to Jain and Magrath (20), and the enzyme activity was
measured as described above. Luciferase activity was normalized with
respect to transfection efficiencies using the corresponding
-galactosidase activity. To identify statistically significant
differences, one-way analysis of variance with the Student-Newman-Keuls
post-test was performed with mean values of at least three independent
experiments done in triplicates using GraphPAD InStat Version 3.05 for
Windows 95.
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RESULTS |
CYP3A7 Is Polymorphically Expressed in Adult Livers and
Significantly Contributes to Hepatic CYP3A
Expression--
CYP3A7 mRNA expression was analyzed in
a total of 127 liver samples obtained from adult individuals.
CYP3A7 was expressed in every liver. However,
interindividual variability of CYP3A7 expression was very
pronounced, showing a >700-fold difference (Table
I). Statistical analysis demonstrated a
clear deviation from gaussian distribution, with a distinct subgroup of
11% of livers that expressed >25,000 (log10 4.4)
transcripts/ng of total RNA (Fig. 1,
A and B). To compare the expression of
CYP3A7 with that of CYP3A4, we analyzed
CYP3A4 expression in the same samples (Table I). On average,
CYP3A7 transcripts accounted for only 1.5% of combined
CYP3A4 and CYP3A7 transcripts. However, in the 11% of livers expressing >25,000 CYP3A7 transcripts/ng of
total RNA, its contribution was 7.7 ± 5.1% (median of 6.4% and
range of 2.4-20.7%). In contrast, the corresponding value was
1.2 ± 2.0% (median of 0.46% and range of 0.01-14.5%) in
the 89% of livers expressing lower levels of CYP3A7. The
difference between the two groups was statistically significant
(Mann-Whitney U test, p < 0.001).
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Fig. 1.
A distinct subset of adult human livers
exhibit high CYP3A7 expression. CYP3A7
expression was analyzed in adult human livers (n = 127). The CYP3A7 expression data were normalized with
respect to the expression of 18 S rRNA and
log10-transformed. A, probability plot for
CYP3A7 expression data. The frequencies of
log10-transformed CYP3A7 expression data were
transformed to probits. The plot includes a least square regression
line for comparison. B, frequency distribution of
log10-transformed CYP3A7 expression.
Closed arrows indicate subjects homozygous or heterozygous
for CYP3A7*1C; open arrows denote
subjects heterozygous for CYP3A7*1B.
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CYP3A7*1C and CYP3A7*1B Are Associated with High Hepatic CYP3A7
Expression--
Recently, several single nucleotide polymorphisms have
been described in the CYP3A7 proximal promoter region, and
three alleles (CYP3A7*1B, CYP3A7*1C, and
CYP3A7*1D) were defined in Caucasians (11). To investigate
the role of these alleles in CYP3A7 expression, we sequenced
the proximal promoter region of CYP3A7, encompassing these
alleles, in the subjects of the liver bank. The results, presented in
Table II, were in accordance with the
previously published allele frequencies (11). A restriction fragment
length polymorphism assay (Fig. 2) was
used to confirm the sequencing data regarding the CYP3A7*1C
allele. Subsequently, this assay was used to screen for the presence of
this allele. CYP3A7 expression was significantly increased
in the livers of all subjects heterozygous or homozygous for the
CYP3A7*1C allele (Mann-Whitney U test,
p < 0.001) (Fig. 3). The
CYP3A7*1B allele was detected in two subjects, and it was
also significantly associated with high CYP3A7 expression (Mann-Whitney U test, p < 0.05) (Fig. 3). A
third CYP3A7*1B heterozygote was present in the study group
from which we obtained intestine and liver samples. This subject also
showed strongly increased CYP3A7 expression in the liver
(Fig. 4B). In contrast,
CYP3A7*1D was not associated with high CYP3A7
expression (data not shown). CYP3A7*1B,
CYP3A7*1C, and CYP3A7*1D were never found
together in any subject investigated. With the exception of one
CYP3A7*1C heterozygote, all subjects with the
CYP3A7*1C or CYP3A7*1B allele belonged to the
group expressing >25,000 transcripts/ng of total RNA. Taken together,
CYP3A7*1C and CYP3A7*1B alleles were specifically associated with approximately two-thirds of the cases of increased CYP3A7 expression in this group (Fig. 1B).
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Fig. 2.
Restriction fragment length polymorphism
assay for detection of the CYP3A7*1C allele. The
scheme shows the genomic region of CYP3A7 amplified by PCR.
The sizes (in bp) of fragments derived by digestion with
SspI (S) are shown for the CYP3A7
wild-type allele (wt) and the CYP3A7*1C allele
(*1C). The gel shows an electrophoretic separation of
SspI digests of DNA samples from subjects homozygous for the
CYP3A7 wild-type allele (wt/wt), heterozygous for
the CYP3A7*1C allele (wt/*1C), and homozygous for
the CYP3A7*1C allele (*1C/*1C). The flanking
lanes show size markers; the numbers indicated are sizes in
bp.
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Fig. 3.
CYP3A7*1C and CYP3A7*1B
are associated with high CYP3A7 expression in
adult human livers. Genotypes are indicated as described in the
legend to Fig. 2. wt/*1B indicates subjects heterozygous for
CYP3A7*1B. CYP3A7 expression is presented in box
and whisker diagrams. Minimum, maximum, and median values (in the
box) are shown as lines; the box shows
the 25-75th percentile. "Non-CYP3A7*1B" and
"non-CYP3A7*1C" subjects with increased
CYP3A7 expression (>25,000 transcripts/ng of total
RNA) are depicted individually as open circles. Differences
were tested for statistical significance using the Mann-Whitney
U test (*, p < 0.05; ***, p < 0.001). Non-CYP3A7*1B and non-CYP3A7*1C
subjects with increased CYP3A7 expression (>25,000
transcripts/ng of total RNA) were included in the Mann-Whitney
U test.
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Fig. 4.
Impact of CYP3A7 promoter
alleles on intestinal versus hepatic CYP3A7
expression. A, the distribution of intestinal
CYP3A7 expression is presented in box and whisker diagrams
as described in the legend to Fig. 3. CYP3A7 expression
values were normalized with respect to the expression of villin. The
statistical significance of the association between CYP3A7
expression and the presence of the CYP3A7*1C allele was
analyzed by the Mann-Whitney U test (**, p < 0.01). wt/wt, homozygous for the CYP3A7
wild-type allele; wt/*1C, heterozygous for the
CYP3A7*1C allele. B, shown is CYP3A7
expression in matched liver and intestine samples. CYP3A7
expression values are given as copies of CYP3A7 mRNA/ng
of total RNA. Liver expression data (gray bars,
left y axis) were normalized with respect to the
expression of 18 S rRNA; expression values given for intestines
(black bars, right y axis) were
normalized with respect to the expression of villin. Letters
indicate the donors of the sample pairs. CYP3A7*1B and
CYP3A7*1C heterozygotes are marked with *1B and
*1C, respectively.
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CYP3A7*1C Is a Predictor of High CYP3A7 Expression in Adult
Intestine--
The expression of CYP3A7 was studied in 23 human intestine samples. Because the samples were derived from surgical
specimens containing variable amounts of epithelial enterocytes,
CYP3A7 expression was normalized using the expression of the
epithelium-specific gene villin (21). Most intestine samples expressed
very low amounts of CYP3A7 (71 ± 178, range of 3-679
copies/ng of total RNA). However, two intestine specimens showed 34- and 38-fold more CYP3A7 transcripts, respectively, than the
mean of all the other samples (p < 0.01) (Fig.
4A). They were obtained from the two subjects heterozygous
for the CYP3A7*1C allele in this study group. To elucidate
whether CYP3A7 was co-regulated in liver and intestine, we
analyzed CYP3A7 expression in the liver samples matching 15 of these intestine samples. The expression of hepatic CYP3A7
was increased in the two subjects who were heterozygous for either
CYP3A7*1B or CYP3A7*1C as well as in one
additional subject (Fig. 4B). No matching liver sample was
available from the second CYP3A7*1C heterozygote. Thus, in
contrast to liver, high intestinal CYP3A7 expression was
exclusively associated with the presence of the CYP3A7*1C allele.
Mutation of the ER6 Motif Is Responsible for
PXR-dependent Transcriptional Activation of
CYP3A7*1C--
Because CYP3A7*1C was detected in half of
the subjects highly expressing CYP3A7 and was the only
allele associated with high CYP3A7 expression in both liver
and intestine, we attempted to elucidate the mechanism underlying its
effect. In the CYP3A7*1C allele, 60 bp of the
CYP3A7 promoter (188 to -129) are replaced by the
corresponding region of CYP3A4 (11). This replacement results in a difference of 7 bases between the CYP3A7
wild-type and CYP3A7*1C alleles. Two bases alter the
proximal ER6 element found in all CYP3A gene promoters (Fig.
5). The nuclear receptor PXR binds to the
proximal ER6 motif (22). Consequently, in CYP3A7*1C, the
proximal ER6 PXR-binding site of CYP3A7 is replaced by the corresponding ER6 motif of CYP3A4 (Fig. 5).
CYP3A4 is highly expressed in adult liver and small
intestine, and PXR has been described to be a key regulator for
CYP3A4 expression. Therefore, we hypothesized that the
presence of CYP3A4-ER6 in CYP3A7*1C could be responsible for
the increased expression of CYP3A7 in individuals carrying the CYP3A7*1C allele. Fig.
6A demonstrates a markedly
stronger binding of PXR/RXR heterodimers to the ER6 motif of
CYP3A4 than to the ER6 motif of CYP3A7. This
result suggested that the high CYP3A7 expression, associated
with the CYP3A7*1C allele, may indeed be caused by the
higher affinity of binding of PXR for CYP3A4-ER6 compared with
CYP3A7-ER6 and consequently by a higher transactivation potential of
PXR at the CYP3A7*1C promoter. Reporter gene analysis performed in LS174T cells, which are capable of PXR-mediated
CYP3A4 induction (18), provided further support for this
hypothesis (Fig. 6B). A CYP3A7 promoter construct
was not activated by PXR, whereas a CYP3A4 construct was. In
accordance with the presence of CYP3A4-ER6 in CYP3A7*1C, a
CYP3A7*1C reporter gene construct was activated by PXR.
Mutation of the ER6 motif in the CYP3A7 promoter to
CYP3A4-ER6 had the same effect as the entire set of mutations
constituting CYP3A7*1C. Conversion of the ER6 motif in the
CYP3A4 promoter construct into CYP3A7-ER6 abolished the activation by PXR. These results demonstrated that the presence of a
CYP3A4-ER6 element was necessary and sufficient for
PXR-dependent activation of CYP3A7*1C. Thus, it
is very likely that differences in binding and activation of the
proximal ER6 motifs of CYP3A4 and CYP3A7 by PXR
are the mechanism underlying the observed effect of
CYP3A7*1C on CYP3A7 expression.
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Fig. 5.
Proximal promoter region mutated in the
CYP3A7*1C allele and hypothesized consequences for
gene expression. In the CYP3A7*1C promoter allele, the
region between positions -188 and -129 is replaced by the
corresponding region of CYP3A4. Bases different between
CYP3A7 and CYP3A4 in the region replaced are
depicted as open circles. Numbering is with respect to the
transcriptional start site. Also indicated is the localization of the
ER6 element. The size of the arrows indicates the
hypothesized strength of gene expression in adults, depending on the
presence of CYP3A4-ER6. Open ellipse, the ER6 motif of
CYP3A7; closed ellipses, the ER6 motif of
CYP3A4.
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Fig. 6.
CYP3A4-ER6 is responsible for
PXR-dependent induction of CYP3A7*1C.
A, electrophoretic mobility shift assays were carried out
using in vitro translated proteins bound to radiolabeled
oligonucleotides corresponding to proximal ER6 motifs of
CYP3A4 (ER6 A4) and CYP3A7 (ER6
A7). Binding reactions contained (+) or lacked () the indicated
proteins. Complexes of PXR/RXR heterodimers with the
oligonucleotides are marked by an arrow. Equal amounts of
ER6 motifs radiolabeled to the same specific activity were used in the
assays. B, the CYP3A promoter reporter genes
indicated on the left are described under "Experimental
Procedures." The results of cotransfection experiments with the PXR
expression plasmid in LS174T cells are illustrated on the right. The
bars show the mean induction factors (±S.D.) of the
reporter genes by 10 µM rifampin. The activity of each
reporter in the presence of Me2SO only was designated as 1. Statistically significant differences are indicated by
asterisks (***, p < 0.001).
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|
Mutations in CYP3A7*1C in Addition to the ER6 Motif Are Required
for CAR-dependent Transcriptional
Activation--
Recently, it was shown that CYP3A4
transcription is also regulated by the nuclear receptor CAR, which
binds with equal affinity as PXR to the same ER6 element (23). Fig.
7A shows that CAR/RXR heterodimers bound to the ER6 motif of CYP3A4 with
5-10-fold higher affinity than to CYP3A7-ER6. The CYP3A7*1C
promoter reporter gene was strongly activated by CAR, in contrast to
the lack of activation of the CYP3A7 reporter (Fig.
7B). Mutation of only the ER6 motif in the CYP3A7
promoter to CYP3A4-ER6 was not, however, sufficient to achieve a
significant increase in activation by CAR, whereas the reciprocal
mutation in the CYP3A4 promoter abolished
CAR-dependent activation completely. Thus, the presence of
a CYP3A4-ER6 motif is necessary but not sufficient for CAR-mediated
activation of CYP3A7*1C.
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Fig. 7.
CYP3A4-ER6 is necessary, but not sufficient,
for CAR-dependent activation of
CYP3A7*1C. A, electrophoretic mobility
shift assays were performed using in vitro translated
proteins bound to a radiolabeled oligonucleotide corresponding to the
DR4(I) motif of the MDR1 regulatory cluster (17)
(upper panel). Binding reactions contained (+) or lacked
() the indicated proteins. Complexes of CAR/RXR heterodimers with
the oligonucleotide are marked by an arrow. Competition was
performed with the unlabeled ER6 motif of CYP3A4 (ER6
A4) or the ER6 motif of CYP3A7 (ER6 A7). The
numbers indicate the n-fold molar excess of the
competing oligonucleotide. Retarded complexes were quantified and are
expressed as percent of the initial complex (lower panel).
Open circles denote the CYP3A7 ER6 motif
competitor; closed squares indicate the CYP3A4
ER6 motif competitor. B, LS174T cells were cotransfected
with the indicated CYP3A promoter reporter genes and the CAR
expression plasmid. The bars show the mean activation
factors (±S.D.) of the reporter genes by CAR. The activity of
each reporter in the presence of empty expression vector pcDNA3 was
designated as 1. Statistically significant differences are indicated by
asterisks (***, p < 0.001).
|
|
We therefore analyzed the contribution of the additional mutations
present in CYP3A7*1C to CAR-mediated activation. Fig.
8A shows the positions of the
mutated bases in the CYP3A7 promoter and their identity in
CYP3A7 versus CYP3A7*1C and
CYP3A4. The G > T mutation at position -188 significantly
contributed to CAR-dependent activation of
CYP3A7*1C. Combining this mutation with
CYP3A4-ER6 resulted in a significant increase in
CAR-dependent transactivation compared with the construct
containing only CYP3A4-ER6 (Fig. 8B, constructs 1 and 2). However, the full extent of CAR-mediated activation
of a CYP3A7*1C reporter (Fig. 8B, construct
6) was not yet achieved. Mutation of -129A > C or -181T > A/179T > C/178A > T, each combined with CYP3A4-ER6, did not show
any significant increase in activation by CAR compared with the
CYP3A7 reporter gene containing only CYP3A4-ER6. In
contrast, combining mutation -188G > T, mutations -181T > A/179T > C/178A > T, and CYP3A4-ER6 conferred a CAR-dependent
activation on the CYP3A7 reporter, which was almost
identical to that observed with the CYP3A7*1C reporter (Fig.
8B, constructs 5 and 6). Consequently,
activation by CAR depended not only on the ER6 CAR-binding site, but
also on neighboring nucleotides. Nucleotide T at position -188 seemed
to be of special importance, as it had the strongest effect on
CAR-mediated activation of all mutations tested together with
CYP3A4-ER6. At least one of the mutations, -181T > A/179T > C/178A > T, also contributed to CAR-mediated activation, but only in
the presence of -188G > T and CYP3A4-ER6. The importance of
nucleotide T at position -188 is further highlighted by the results
presented the lower part of Fig. 8B. Mutation of
-188T > G in the CYP3A4 promoter reduced
CAR-dependent activation by ~80%. In conclusion, we have identified specific sequence features responsible for differences in
the activation potential by PXR and CAR. In contrast to PXR, several
additional sites in the promoter sequence, besides the ER6 motif, are
required for CAR-mediated activation of CYP3A4 and
CYP3A7*1C.
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Fig. 8.
CAR-dependent activation of
CYP3A promoters requires bases in addition to
CYP3A4-ER6. A, the regions between positions -188 and -129
of the CYP3A7 and CYP3A7*1C promoters (the latter
being identical to the corresponding region of the CYP3A4
promoter) are shown schematically. Differing nucleotides are shown and
marked by open and closed circles for
CYP3A7 and CYP3A7*1C/CYP3A4,
respectively. Numbering is with respect to the transcriptional start
site. Localization of the ER6 motif is shown. B,
CYP3A reporter genes are shown on the left. Upper
part, CYP3A7 promoter constructs: 1,
CYP3A7(ER6A4); 2, CYP3A7(A4:188,ER6); 3,
CYP3A7(A4:ER6,129); 4, CYP3A7(A4:3B,ER6); 5,
CYP3A7*1C(A7:129); and 6, CYP3A7*1C. Lower
part, CYP3A4 promoter constructs: 7, CYP3A4;
and 8, CYP3A4(A7:188). Closed and open
circles symbolize nucleotides as described for A. The
results of cotransfection experiments with the CAR expression plasmid
are illustrated on the right and presented as described in the legend
to Fig. 7B.
|
|
| |
DISCUSSION |
In this study, we have described the polymorphic expression of
CYP3A7 in adult human liver and intestine and analyzed the functional consequences of two promoter alleles for the expression of
the gene. CYP3A7 mRNA was found in all livers, but
showed pronounced interindividual variability. There was a non-gaussian
distribution, with 11% of livers expressing >25,000 transcripts/ng of
total RNA. Because at the present time no CYP3A7-specific antibody is available, expression at the protein level could not be studied. Nearly
two-thirds of the subjects in the subgroup with hepatic expression of
>25,000 transcripts of CYP3A7 mRNA/ng of total RNA carried the CYP3A7*1C allele or, less frequently, the
CYP3A7*1B allele. The mechanism of the increased
CYP3A7 expression in the subjects without these alleles
remains to be elucidated, but it could involve additional genetic
markers or induction by xenobiotics. Only one subject in this group had
been treated with nifedipine, which is a known inducer of
CYP3A4 expression in vitro (24). However, the
expression of CYP3A4 in the corresponding liver was low
(data not shown). Thus, it is very likely that the high expression of
CYP3A7 in subjects without the CYP3A7*1C or
CYP3A7*1B allele is caused by yet unknown genetic variants
rather than by induction, but this remains to be verified
experimentally. Induction can be ruled out as the mechanism of high
CYP3A7 expression in the subjects carrying
CYP3A7*1C or CYP3A7*1B because none of these patients was treated with known CYP3A inducers.
The increased CYP3A7 expression observed in ~11% of adult
human livers could have consequences for drug
biotransformation. For a number of drugs, data on substrate
affinity and specificity using cDNA-expressed CYP3A4, CYP3A5, and
CYP3A7 demonstrate similarity (25). On the other hand, there are also
examples of pronounced differences in substrate affinity among CYP3A
isoforms, including CYP3A7 (26, 27). For example, CYP3A7 metabolizes
retinoic acid 25 times more efficiently than CYP3A4 (26). Based on the expression levels of CYP3A7 in the subjects of the high
expression subgroup, which come up to 20% of the combined
CYP3A4 and CYP3A7 pool, CYP3A7 could contribute
to up to 80% of total biotransformation of retinoic acid. Thus, the
polymorphic expression of CYP3A7 in adult human livers could
be responsible for part of the variability of CYP3A activity among individuals.
In contrast to previous studies that did not detect CYP3A7
expression in fetal and adult human intestine (28-30), we demonstrated CYP3A7 expression in every intestine sample investigated.
This is in all probability due to the more sensitive method used.
However, high amounts of CYP3A7 transcripts were present
only in intestine samples of subjects with the CYP3A7*1C
allele. This observation demonstrates differences in the regulation of
CYP3A7 between liver and intestine. In addition to the
CYP3A7*1C-dependent regulation, further
mechanisms responsible for high CYP3A7 expression (such as
CYP3A7*1B-dependent regulation) obviously
exist in liver. However, half of the subjects from whom we obtained
intestine samples, including the CYP3A7*1C and
CYP3A7*1B heterozygotes, were treated with known inducers of
CYP3A4 (omeprazole, nifedipine, reserpine, or St. John's
wort). Because the CYP3A7*1C heterozygotes showed only low
intestinal CYP3A4 expression, it is unlikely that their high
intestinal CYP3A7 expression was caused by induction. This assumption is further supported by a lack of association between treatment with inducers and CYP3A7 expression (data not
shown) and the tissue-specific effects of the two alleles determining CYP3A7 expression. In addition, reserpine, which was
administered to one CYP3A7*1C heterozygote, did not induce
CYP3A7 expression in an intestinal cell line, whereas
CYP3A4 was induced by reserpine in the same cell line
(31).
Furthermore, we have provided a mechanistic explanation for the
increased expression of CYP3A7 in the individuals carrying the more frequent marker of the CYP3A7 polymorphism, the
CYP3A7*1C allele. The CYP3A7*1C mutation has
arisen through replacement of part of the CYP3A7 promoter by
the corresponding region of CYP3A4. This led to the
substitution of CYP3A7-ER6 for CYP3A4-ER6. The ER6 motif in the
proximal promoter of the CYP3A4 gene is one of the two
elements mediating PXR-dependent activation. The second element is the distal xenobiotic-responsive enhancer module (32). Two
bases are different between the proximal ER6 motifs of
CYP3A4 and CYP3A7 (33). We showed that the
presence of CYP3A4-ER6 was responsible for the high expression of
CYP3A7 in carriers of the CYP3A7*1C allele. PXR
exhibited stronger binding to CYP3A4-ER6 than to CYP3A7-ER6. However,
in contrast to the results presented by Pascussi et al.
(33), this differential binding also resulted in a dramatic difference
in the PXR-mediated transactivation of CYP3A4 and
CYP3A7 promoters. The discrepancy is most likely due to
differences between the natural CYP3A7 promoter used in our study and the reporter gene containing multimerized CYP3A7-ER6 motifs
used by Pascussi et al. (33). Our data clearly demonstrate that the proximal ER6 motifs of CYP3A4 and CYP3A7
are functionally different with respect to binding and activation by
PXR. This difference appears to be the mechanism of the
CYP3A7*1C effect on CYP3A7 expression.
PXR is not the only transcription factor binding to the proximal ER6
motif of CYP3A4 and activating its transcription. Recently, it has been shown that CAR and vitamin D receptor, which belong to the
same subfamily of nuclear receptors (NR1I) as PXR, also regulate
CYP3A4 expression (6, 7, 23). Similarly to PXR, CAR showed
stronger binding to CYP3A4-ER6 than to CYP3A7-ER6 and activated
CYP3A4 and CYP3A7*1C promoters. But in contrast
to PXR, the presence of CYP3A4-ER6, although necessary, was not
sufficient for CAR activation. Additional single base mutations of the
CYP3A7*1C allele proved to be necessary. We identified
position -188 as the most crucial one, but also positions -181,
179, and -178 were involved in the activation by CAR. The -188G>T
mutation in CYP3A7*1C creates a putative
HNF-3-binding site, whereas mutations of positions -181, 179,
and 178 are not located within any of the known transcription
factor-binding sites (34). The -129A>C mutation in
CYP3A7*1C destroys a functional HNF-3-binding site of the
CYP3A7 promoter (35) and creates a putative octamer motif (34). The strong dependence of CAR-mediated activation on position -188 and, to a more limited extent, on at least one of positions -181, 179, and 178 probably reflects functional interactions between CAR and other transcription factors binding to these sequences. The nature of these interactions and the role of HNF-3 have to be
elucidated in future studies.
The involvement of PXR in the induction of CYP3A genes is
established, but data on its role in the regulation of constitutive CYP3A expression are less convincing. A targeted deletion of
PXR did not alter the constitutive expression of CYP3A in
one strain of mice (36), whereas it resulted in a 3-fold reduction of
CYP3A expression in another mouse strain (37). The data
presented in our study support the hypothesis that nuclear receptors
binding to the proximal ER6 motif may play a role in the constitutive expression of CYP3A. The described
CYP3A7*1C-dependent mechanism of high
CYP3A7 expression is based on functional differences between the proximal ER6 motifs of CYP3A4 and CYP3A7. The
proximal ER6 motif of CYP3A4, but not that of
CYP3A7, mediates activation by the nuclear receptors PXR and
CAR. Therefore, the induction of CYP3A7 observed in most,
although not all, primary adult hepatocyte cultures and hepatoma cell
lines after treatment with the PXR ligands rifampin, clotrimazole, and
RU486 (15, 33) could be mediated by the recently described
xenobiotic-responsive enhancer module in the CYP3A7 far
upstream regulatory region that is functionally conserved between
CYP3A4 and CYP3A7 (38).
No clues regarding the mechanism of action of the
CYP3A7*1B allele can be derived from the sequence
context of the single nucleotide polymorphism that constitutes the
allele. The mechanism has to be clarified in future functional studies.
In conclusion, this study provides clear evidence for an association of
the CYP3A7*1B and/or CYP3A7*1C allele with high
expression of CYP3A7 in adult liver and intestine. These
alleles could serve as markers for the variable CYP3A activity.
Furthermore, we identified functional differences between the ER6
motifs of CYP3A4 and CYP3A7 proximal promoters in
binding and activation by the nuclear receptors PXR and CAR. Taken
together, these results provide a mechanistic explanation for the
increased CYP3A7 expression in CYP3A7*1C
carriers. Whether the proximal ER6 motif and these nuclear receptors
participate also in the developmental switch from CYP3A7 to
CYP3A4 expression in liver, occurring immediately after
birth (39), remains to be elucidated in future studies.
| |
ACKNOWLEDGEMENTS |
We are indebted to K. Abuazi de Paulus, R. Weil, and J. Klattig for excellent technical assistance and to P. Fritz
for the histological examination of the intestine samples. K. P. Thon (Department of Surgery, Robert Bosch Hospital) and F. Läpple kindly helped to collect the intestine samples. We thank R. Schüle, T. Kamataki (Hokkaido University, Sapporo,
Japan), F. J. Gonzales (NCI, Bethesda, MD), and P. Beaune
(University René Descartes, Paris, France) for kindly providing
plasmids containing human RXR, CYP3A7,
CYP3A4, and CYP3A5 cDNAs, respectively.
| |
Note Added in Proof |
The high prevalence of CYP3A7*1C alleles
in subjects expressing high levels of CYP3A7 transcripts is in
agreement with expression data presented together with the original
description of this allele (11).
| |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant Bu 1249/1-1, German Federal Ministry for Education and Science Grant 01GG9846/8, and the Robert Bosch Foundation (Germany).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. Tel.: 49-711-8101-3753;
Fax: 49-711-859295; E-mail: oliver.burk@ikp-stuttgart.de.
Present address: Dept. of Clinical Pharmacology, Georg August
University, Robert-Koch-Str. 40, D-37075 Göttingen, Germany.
Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M202345200
| |
ABBREVIATIONS |
The abbreviations used are:
PXR, pregnane X
receptor;
CAR, constitutively activated receptor;
RXR, retinoic X
receptor;
HNF, hepatocyte nuclear factor.
| |
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