Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine.

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.

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 Ͼ10-fold increase in CYP3A5 protein expression observed in 10 -30% of livers of Caucasian origin (8,11). In some persons, CYP3A5 can contribute to Ͼ50% 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 Ͼ60-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 ϳ11% 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.

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 Ϫ80°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 (Ϫ426 to ϩ188 with respect to the transcriptional start site) was amplified by PCR using oligonucleotide primers 5Ј-GGC TCT GTC TGG CTG GGT ATG A-3Ј (bases 110480 -110501 of GenBank TM /EBI accession number AF280107) and 5Ј-GAA CAG TTA CTC ACA GAT AGA GGA GTA TC-3Ј (bases 111093 to 111065). The standard PCR consisted of 1ϫ PCR buffer with 1. , and 20 ng of genomic DNA in a total volume of 50 l. 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 5Ј-ATG ACC TAA GAA GAT GGA GTG-3Ј (bases 110153-110173 of GenBank TM /EBI accession number AF280107) and 5Ј-GAA GGG CAT GGT CTA CAC TAT-3Ј (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, 1ϫ OptiPerform buffer III (PAN Biotech, Aidenbach, Germany), 1ϫ OptiZyme Enhancer (PAN Biotech), 1.5 mM MgCl 2 , 200 M dATP, 200 M dCTP, 200 M dGTP, 200 M dTTP, 1 M each primer, and 1 unit of PowerScript (PAN Biotech) in a volume of 50 l. 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. Firststrand cDNA was generated from 0.5 to 1 g 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 5Ј-reporter dye 6-carboxyfluorescein and the 3Ј-quencher dye 6-carboxytetramethylrhodamine. Oligonucleotides used for CYP3A7 were as follows: primers 5Ј-AAG GGC TAT TGG ACG TTT GAC A-3Ј and 5Ј-ATC CCA CTG GCC CGA AAG-3Ј and probe 5Ј-TAT TTA TGA CTG TCA ACA GCC TAT GCT GGC TAT CA-3Ј. 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 5Ј-TCA GCC TGG TGC TCC TCT ATC TAT-3Ј and 5Ј-AAG CCC TTA TGG TAG GAC AAA ATA TTT-3Ј and probe 5Ј-TCC AGG GCC CAC ACC TCT GCC T-3Ј. 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 5Ј-CTG GCA ACC TTA GGG ACT GG-3Ј and 5Ј-GTT AGC ATT GAA CAC GTC CAC TTT-3Ј and probe 5Ј-CCA GAT CAC TGC TGA GGT CAC AAG C-3Ј. 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 twotailed 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 5Ј-ATG AAT TCC ACC ATG GCC AGT AGG GAA GAT GAG CTG-3Ј, which introduced an EcoRI site and an optimized Kozak consensus sequence, and 5Ј-CGT CTA GAT TAG CTG CAG ATC TCC TGG AGC AG-3Ј, 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 (Ϫ869 to ϩ50) was amplified from human genomic DNA by PCR with oligonucleotides 5Ј-GCG GTA CCA TCT CAT CCA TGC CAT GTC TCT TT-3Ј and 5Ј-ACA AGC TTG CTG TTT GCT GGG CTG TGT GTG-3Ј. The oligonucleotides introduced a KpnI and a HindIII restriction site on the 5Ј-and 3Ј-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(Ϫ869). 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(Ϫ350), henceforth referred to as the CYP3A7 construct, contains the region between positions -350 and ϩ50. 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 5Ј-CAT AGG TAA AGA TCT GTA GGC A-3Ј and 5Ј-ACA AGC TTG CTG TTT GCT GGG CTG TGT GTG-3Ј encompassed the BglII site at position -351 and introduced a HindIII site at position ϩ50, 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 se-quencing 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:Ϫ188,ER6). Second, the mutation at position -129 of CYP3A7*1C was introduced, creating CYP3A7(A4:ER6,Ϫ129). Third, the mutations at positions -181, Ϫ179, and Ϫ178 of CYP3A7*1C were introduced, creating CYP3A7(A4: 3B,ER6). The CYP3A7(A4:Ϫ188,ER6) construct was further mutated by introducing the mutations at positions -181, Ϫ179, and Ϫ178 of CYP3A7*1C, thus creating CYP3A7*1C(A7:Ϫ129). 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(Ϫ1105) (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 ϩ51, pGL3-CYP3A4(Ϫ374), 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 (TTAACTCAATG-GAGGTCA, 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.
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 ϫ 10 5 cells/well. The plasmid DNA was transfected using the Effectene TM transfection reagent (QIA-GEN 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 phosphatebuffered saline to remove Effectene TM -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 M rifampin dissolved in Me 2 SO or with an equivalent amount (0.1%) of Me 2 SO 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 Effectene TM -DNA complexes and lysed with 150 l of 1ϫ passive lysis buffer (Promega). After centrifugation, cleared lysates were used for reporter gene assays, which were done in duplicate. For luciferase measurements, 300 l of an assay solution (25 mM glycylglycine (pH 7.8), 50 M luciferin, 2 mM ATP, 10 mM MgCl 2 , 27 M coenzyme A, and 30 mM dithiothreitol) were automatically injected into 20 l 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.

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 Ͼ700-fold difference (Table I). Statistical analysis demonstrated a clear deviation from gaussian distribution, with a distinct subgroup of 11% of livers that expressed Ͼ25,000 (log 10 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 Ͼ25,000 CYP3A7 transcripts/ng of total RNA, its contribution was 7.7 Ϯ 5.1% (median of 6.4% and range of 2.4 -20.7%). In contrast, the corresponding value was 1.2 Ϯ 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 Ͻ 0.001).
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 Ͻ 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 Ͻ 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 ex- pressing Ͼ25,000 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).
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 Ϯ 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 Ͻ 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.    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 (Ͼ25,000 transcripts/ng of total RNA) are depicted individually as open circles. Differences were tested for statistical significance using the Mann-Whitney U test (*, p Ͻ 0.05; ***, p Ͻ 0.001). Non-CYP3A7*1B and non-CYP3A7*1C subjects with increased CYP3A7 expression (Ͼ25,000 transcripts/ng of total RNA) were included in the Mann-Whitney U test.

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 (Ϫ188 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 PXRbinding 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.
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.
We therefore analyzed the contribution of the additional mutations present in CYP3A7*1C to CAR-mediated activation. CYP3A7 promoter and their identity in CYP3A7 versus CYP3A7*1C and CYP3A4. The G Ͼ 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 Ͼ C or -181T Ͼ A/Ϫ179T Ͼ C/Ϫ178A Ͼ 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 Ͼ T, mutations -181T Ͼ A/Ϫ179T Ͼ C/Ϫ178A Ͼ 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 Ͼ A/Ϫ179T Ͼ C/Ϫ178A Ͼ T, also contributed to CARmediated activation, but only in the presence of -188G Ͼ 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 Ͼ G in the CYP3A4 promoter reduced CAR-dependent activation by ϳ80%. 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.

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 Ͼ25,000 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 Ͼ25,000 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 ϳ11% 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, Ϫ179, and -178 were involved in the activation by CAR. The -188GϾT mutation in CYP3A7*1C creates a putative HNF-3-binding site, whereas mutations of positions -181, Ϫ179, and Ϫ178 are not located within any of the known transcription factor-binding sites (34). The -129AϾC 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 CARmediated activation on position -188 and, to a more limited extent, on at least one of positions -181, Ϫ179, and Ϫ178 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 theconstitutiveexpressionofCYP3A.ThedescribedCYP3A7*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.