Introduction

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The cytochromes P450 (P450) are a superfamily of heme-containing enzymes that catalyze the metabolism of a wide range of endogenous substrates as well as the detoxification/metabolic activation of exogenous compounds (Guenguerich, 1993). Human CYP3A4 is the primary catalyst of testosterone 6-hydroxylation (Waxman et al., 1991) and is involved in the metabolism of more than 50% of currently used therapeutic drugs (Li, 1995). The major role of CYP3A4 in xenobiotic metabolization and the large intra- and interindividual variability to which it is subjected (Forrester et al., 1992) strongly contribute to the important differences in the therapeutic and toxic effects of many drugs.

As with most xenobiotic-metabolizing P450s, CYP3A4 is highly expressed in liver, where its is one of the most abundant enzymes (Yamashita et al., 2000), but low levels are also found in extrahepatic tissues. Detailed studies of typical hepatic genes have shown that liver-specific gene expression is accomplished by the concerted action of a small number of liver-enriched transcription factors (LETFs) (Cereghini, 1996). Although the mechanisms that control CYP3A4 high and variable basal expression in human hepatocytes are still unknown, it has been shown that the LETFs hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4, and CCAAT/enhancer-binding protein (C/EBP) play important roles in regulating the expression of P450 genes (Gonzalez and Lee, 1996) and that in most cases, two or more LETFs are responsible for the expression of a hepatic gene.

C/EBP is a member of the basic region leucine zipper family of transcription factors (Antonson and Xanthopoulos, 1995) and its expression controls, among others, the terminal differentiation of adipocytes and hepatocytes (Shugart and Umek, 1997). In the liver, C/EBP plays a major role in the maintenance of energy homeostasis by regulation of glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase (Wang et al., 1995), as well as in the inflammatory response (Burgess-Beusse and Darlington, 1998). A direct demonstration of C/EBP implication in P450 expression was first obtained in Hep G2 cells, which showed augmented levels of CYP2B6, -2C9, and -2D6 mRNAs, when they were stably transfected with a C/EBP expression vector (Jover et al., 1998). Although the expression of CYP3A4 in these cells was not investigated in detail, previous preliminary evidence indicating that C/EBP trans-activates CYP3A4 promoter was gained in gene reporter assays (Ourlin et al., 1997).

HNF-3 belongs to a large family of transcription factors that is characterized by the presence of a winged helix/forkhead domain. This domain is similar to the globular domain of linker histone (Clark et al., 1993) and enables HNF-3 to directly control nucleosome position (Shim et al., 1998). The HNF-3 proteins are involved in the regulation of numerous liver-specific genes (Kaestner et al., 1998; Wang et al., 2000). They regulate the expression of human CYP2Cs (R. Bort, R. Jover, C. Rodríguez-Antona, M. J. Gómez-Lechón, and J. V. Castell, manuscript in preparation), and recombinant promoter analysis has demonstrated that HNF-3 trans-activates rat CYP2C6 and CYP2C12 (Shaw et al., 1994; Delesque-Touchard et al., 2000). In addition, footprint analysis revealed HNF-3 binding sites in the rat CYP2C13 promoter (Legraverend et al., 1994). From the three HNF-3 isoforms expressed in liver, , , and , we focused our studies on HNF-3 based on its temporal expression during embryogenesis (Kaestner et al., 1994) and on knock-out mice data: inactivation of HNF-3 resulted on an altered expression of liver specific genes in contrast to the HNF-3 and HNF-3 knock-out mice (Kaestner et al., 1998, 1999; Sund et al., 2001).

In the present study, we establish the role of C/EBP and HNF-3 in the basal expression of human CYP3A4 by assaying the trans-activating ability of C/EBP and HNF-3 on CYP3A4 promoter deletions and identifying the precise location of the binding sites by EMSA analysis. By using adenoviral expression vectors encoding both LETFs, we found that C/EBP up-regulated CYP3A4, whereas HNF-3 had a synergistic effect. This cooperative effect, which was also detected in the CYP3A5 and CYP3A7 genes, was hepatic specific and probably occurs via chromatin remodeling.

	Materials and Methods

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Construction of Plasmids. Putative binding sites for the transcription factors C/EBP and HNF-3 were identified within the 1843, +6 region of the human CYP3A4 promoter using computer programs (positions are relative to the transcription start site, +1). The MatInspector software (Wingender et al., 2000) was used to identify HNF-3 putative binding sites using search conditions of 100% similarity in core and 82.5% in matrix. Because C/EBP can bind as an - homodimer or an - heterodimer, C/EBP putative binding sites were selected using TFSearch software (Heinemeyer et al., 1998) with search conditions of 80% similarity for C/EBP sites and 82.5% similarity for C/EBP sites. Six C/EBP and eight HNF-3 putative binding sites were identified in this search (Fig. 1). Based on this data and using human genomic DNA isolated from human liver, we generated by PCR different deletion fragments of the CYP3A4 promoter containing different putative binding sites. The amplified fragments were: 1843, 1365, 956, 163, and 104 to +6 (the PCR primers used had restriction enzymes sites for KpnI or XhoI at the 5' end and are described in Table 1). After the PCR reaction, the fragments were double-digested with KpnI and XhoI and ligated to the pGL3-Basic vector (Promega) that had previously been digested with the same enzymes. Plasmids isolated from transformed bacterial colonies were sequenced to confirm the inserted sequence. The complete cDNA of rat C/EBP (a kind gift of Dr. J. Patrick Condreay) was cloned by sticky-blunt ligation of a XbaI-KpnI fragment into the pAC/CMVpLpA vector (Gómez-Foix et al., 1992) predigested with XbaI-HindIII, generating an expression vector for C/EBP (pAC-C/EBP). The expression plasmid for HNF-3 (pAC-HNF-3) was constructed by PCR amplification of the complete human HNF-3 cDNA and ligation into the pAC/CMVpLpA (R. Bort, R. Jover, C. Rodríguez-Antona, M. J. Gómez-Lechón, and J. V. Castell, manuscript in preparation).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   CYP3A4 promoter constructs and putative binding sites for C/EBP and HNF-3. Schematic nucleotide sequences of the CYP3A4 promoter constructs cloned in pGL3-Basic, showing putative binding sites for C/EBP () and HNF-3 (). The positions are relative to the transcriptional start site + 1 and the location of the putative binding sites for C/EBP and HNF-3 in the CYP3A4 promoter are shown in the table.

View this table: [in this window] [in a new window]   TABLE 1 Oligonucleotide PCR primers for cloning CYP3A4 promoter fragments and for PCR mutagenesis of the C/EBP DNA-binding site at 121/130 in CYP3A4 promoter

PCR Mutagenesis of the C/EBP DNA-Binding Site at 121/130 in CYP3A4 Promoter. The CTTTGCCAAC wild-type C/EBP DNA binding site at 121/130 in the CYP3A4 promoter was mutated to CTAGAGAGAC. Two separate PCR reactions were set up to amplify 56- and 152-bp fragments with mutations within the C/EBP binding site using 163/+6 pGL3-Basic plasmid as a template. The C/EBP binding site in the 56- and 152-bp fragments is within 25 overlapping nucleotides that can subsequently be annealed together to serve as templates for further amplification of a full-length 183-bp fragment containing selective point mutations in the C/EBP binding site. The 56- and 152-bp fragments were amplified in independent reactions containing 1 ng of 163/+6 pGL3-Basic, 0.2 µM of sense and antisense oligonucleotide primers, 200 µM of each nucleotide, Expand High Fidelity buffer with 1.5 mM MgCl₂ (Roche Applied Science, Indianapolis, IN), and 2 units of Expand high-fidelity Taq polymerase (Roche Applied Science) in a total volume of 50 µl. DNA was amplified for 30 cycles (denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s). The following specific primers were used for the 56-bp PCR fragment: 163-FP and C/EBPmut-RP and the 152-bp PCR fragment: C/EBPmut-FP and +6-RP (primer sequences are shown in Table 1). The DNA fragments of expected mobility were excised from 2% agarose gels and purified with the UltraClean DNA purification kit (Mo Bio Laboratories, Inc., Carlsbad, CA). To generate a full-length 163/+6 promoter fragment with mutations within the C/EBP binding site, 3 ng of each of the purified 56- and 152-bp DNA fragments were annealed in a reaction mixture containing 200 µM of each nucleotide and Expand high-fidelity buffer with 1.5 mM MgCl₂ at 94°C for 2 min and 55°C for 5 min. Two units of Expand high-fidelity Taq polymerase (Roche Applied Science) were added to the reaction mixture in a total volume of 50 µl. DNA was amplified for 10 cycles (denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min). Sense 163-FP and antisense +6-RP oligonucleotide primers (0.2 µM) were subsequently added to the reaction mixture, and DNA was amplified for an additional 30 cycles (1 cycle = 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s) with a final extension at 72°C for 6 min. The PCR products were precipitated, washed with 70% ethanol, and digested with KpnI and XhoI. The digestion product was electrophoretically fractioned in a 1.5% agarose gel, purified as described above, and cloned into the pGL3-Basic vector. The mutation was confirmed by DNA sequencing.

Cell Culture and Transfection Assays. Hep G2 cells were plated in Ham's F-12/Leibovitz L-15 media [1:1 (v/v)], supplemented with 7% newborn calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and cultured to 70% confluence. HeLa and human embryonic kidney 293 cells were maintained as monolayer cultures and grown in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, 50 U/ml penicillin and 50 mg/ml streptomycin; 293 cell medium was supplemented with 3.5 g/liter of glucose.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay. Nuclear extracts from Hep G2 cells infected with C/EBP or HNF-3 adenovirus were prepared as described previously (Schreiber et al., 1989). Briefly, cells were scraped, washed with ice-cold phosphate-buffered saline, homogenized, and centrifuged to pellet nuclei. The nuclei were incubated with a high-salt buffer at 4°C for 15 min. After centrifugation, the supernatant was stored at 70°C. For EMSA, 12 µg of nuclear extract were preincubated at 37°C for 20 min with 1.5 µg of poly(dI/dC), 100 mM of NaCl, 15 mM HEPES, pH 7.9, 0.25 mM EDTA, 0.25 mM EGTA, 0.25 mM dithiothreitol, and 5% glycerol. The double-stranded oligonucleotide was radiolabeled (50,000 to 100,000 cpm) using [³²P]dATP and T4 polynucleotide kinase (Roche Applied Science), added to the reaction mixture, and incubated for 40 min at 37°C. The binding of proteins to the oligonucleotides was determined by fractionating the reaction mixture by electrophoresis through a nondenaturing 4% polyacrylamide gel at 150 V for 3 to 4 h at 4°C, using a Tris-glycine-EDTA buffer (50 mM Tris, 375 mM glycine, 2 mM EDTA, pH 8.5). Where appropriate, a 50-fold excess (unless another amount is indicated) of competitor DNA was included in the preincubation, before the addition of the ³²P labeled DNA. For antibody supershifts, 1 µg of C/EBP and HNF-3 or 4 µg of C/EBP antiserums (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were added after the incubation with the labeled probe and incubated for 45 min on ice. Gels were dried and exposed at 70°C to an X-ray film with intensifying screens.

Development of Adenoviral Vectors Encoding C/EBP and HNF-3. Recombinant adenovirus encoding C/EBP and HNF-3 were prepared as described elsewhere (Gómez-Foix et al., 1992). Briefly, pAC-C/EBP was cotransfected with pJM17 into 293 cells (AdE1A-transformed human embryonic kidney cells) by calcium phosphate/DNA precipitation. The CMV-driven cassette of pAC/CMVpLpA is located between the sequences representing 0 to 1.3 map units and 9.2 to 16 map units of the adenovirus type 5, whereas pJM17 encodes a full-length adenovirus-5 genome (dl309) interrupted by the insertion of the bacterial plasmid pBRX at position 3.7 map units, thereby exceeding the packaging limit. Homologous recombination between adenovirus sequences in the transfer plasmid (recombinant pAC/CMVpLpA) and in the pJM17 plasmid results in the substitution of the pBRX sequences in pJM17 by the chimeric gene. This generates a genome of packageable size in which most of the adenovirus early region 1 is lacking, thus rendering the replication defective recombinant virus. The resulting virus named Ad-C/EBP was plaque-purified, expanded into a high-concentration stock, and titrated by plaque assay as described previously (Castell et al., 1997). The preparation of Ad-HNF-3 was performed in a similar way (R. Bort, R. Jover, C. Rodríguez-Antona, M. J. Gómez-Lechón, and J. V. Castell, manuscript in preparation). To confirm that the C/EBP protein expressed with the adenoviral vector was functional, we measured albumin synthesis and mRNA contents of C-reactive protein in Hep G2 cells infected with the C/EBP virus, finding that both were increased. A recombinant adenovirus encoding the insertless vector pAC/CMVpLpA (Ad-pAC) was used as control.

RNA Purification and Semiquantitative RT-PCR Analysis. Cellular RNA was extracted with RNeasy Total RNA Kit (QIAGEN), contaminating genomic DNA was removed by incubation with DNase I Amplification Grade (Invitrogen), and 1 µg of total RNA was reverse transcribed. cDNA fragments of C/EBP and HNF-3 were amplified by PCR using 3 µl of diluted cDNA in 40 µl of 20 mM Tris-HCl, pH 8.4, containing 50 mM KCl, 1.5 mM MgCl₂, 50 µM concentrations of each deoxynucleotide triphosphate, 1 unit of Taq DNA polymerase (Invitrogen), 0.2 µM concentrations of each specific primer (Table 2), and, in the case of amplifying C/EBP cDNA, 2.8 µl of glycerol. After denaturing for 4 min at 94°C, amplification was performed by 30 to 35 cycles (94°C for 35 s; 60°C or 57°C for C/EBP or HNF-3, respectively, for 30 s; and 72°C for 45 s) and a final extension of 72°C for 7 min. mRNA levels of CYP3A4, CYP3A5, CYP3A7, and -actin were quantified by RT-PCR with the LightCycler Instrument (Roche Applied Science) using the LightCycler-DNA Master SYBR Green I (Roche Applied Science). Aliquots (15 µl) of the PCR reactions were subjected to electrophoresis on 1.5% agarose gel, for size and purity confirmation. Sample-to-sample variations were normalized by analysis of -actin content in the same cDNA series. Primers used for PCR amplification are shown in Table 2.

Immunoblot Analysis. Protein extracts were electrophoresed in a SDS-polyacrylamide gel (20 µg protein/lane). Proteins were transferred to Immobilon-P membranes (Millipore) and incubated with appropriate polyclonal antibodies (Santa Cruz Biotechnology). After washing, blots were developed with horseradish peroxidase-labeled IgG using an enhanced chemiluminescence kit (Amersham Biosciences).

Statistical Analysis. Statistical analysis was done by Student's t test. A P value less than 0.05 was considered significant.

	Results

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C/EBP but Not HNF-3 trans-Activates CYP3A4 Promoter Constructs. Computer analysis of CYP3A4 promoter revealed the existence of several putative binding sites for C/EBP and HNF-3 (Fig. 1). Their biological relevance was examined by reporter gene assays using progressive 5' deletions of the CYP3A4 promoter fused upstream of the firefly luciferase gene in the pGL3-Basic plasmid. The transfection experiments were carried out in a human cervix carcinoma cell line (HeLa) and in a human hepatic cell line (Hep G2), to determine possible differences in trans-activation depending on cell/tissue specific factors.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Basal activity and trans-activation by C/EBPa and HNF-3 of CYP3A4 promoter constructs in HeLa and Hep G2 cells. Deletions of the 5' flanking region of CYP3A4 promoter were cloned in the firefly luciferase reporter plasmid pGL3-Basic. The numbers given indicate the 5' end of the promoter fragment. Forty-eight hours after transfection, cells were scraped, lysed, and both firefly and R. reniformis luciferase activities were measured. A, basal activity of CYP3A4 promoter deletion constructs. These constructs (1 µg) and 0.1 µg of the plasmid pRL-CMV, as a transfection efficiency control, were transfected with calcium phosphate in HeLa and Hep G2 cells. B, trans-activation by C/EBP and HNF-3 of CYP3A4 promoter constructs. The CYP3A4 promoter constructs (1 µg) were cotransfected with pAC-C/EBP (1 µg) or/and pAC-HNF3 (1 µg) expression vectors into HeLa and Hep G2 cells. The insertless plasmid pAC/CMVpLpA was added, to have a constant amount of total expression vector, and 0.1 µg of the plasmid pRL-CMV was used to control transfection efficiency. Values represent firefly/R. reniformis luciferase activity ratios divided by protein content. Bars represent the mean ± S.D. of four independent experiments.

Functional C/EBP Binding Sites Are Present in the Proximal CYP3A4 Promoter at 121/130 and in the Distal CYP3A4 Promoter at 1393/1402 and 1659/1668. In the 163/+6 region, where the maximal C/EBP trans-activation was detected, sequence analysis located at positions 121/130 the motif CTTTGCCAAC, which shows the features of a consensus C/EBP binding site (Osada et al., 1996). To investigate whether C/EBP could bind to this site, we performed EMSA analysis with nuclear extracts from Hep G2 cells overexpressing C/EBP.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Binding of C/EBP to CYP3A4 promoter and functionality of the sites. For EMSAs, double-stranded DNAs matching CYP3A4 promoter sequence were labeled and incubated with nuclear extracts of Hep G2 cells overexpressing C/EBP. Unlabeled oligonucleotides were incubated at 50-fold molar excess for competition. A, binding of C/EBP to the proximal CYP3A4 promoter. EMSAs were performed using labeled P1 (163 to +6) in the left or P2 (115 to 139) in the right. Specific C/EBP () and C/EBP () antibodies were used and gels were exposed to X-ray films for different times to see C/EBP supershift. B, functionality of the C/EBP binding site located at 121/130. Different CYP3A4 promoter fragments: 163 to +6 (163); 104 to +6 (104); and 163 to +6 with 121/130 C/EBP binding site mutated (163 Mut) were cloned in pGL3-Basic. These constructs (0.5 µg) were cotransfected with increasing amounts of pAC-C/EBP expression plasmid. The insertless plasmid pAC/CMVpLpA was added to have a constant amount of total expression vector and 0.1 µg of the plasmid pRL-CMV was used to control transfection efficiency. Forty-eight hours after transfection, cells were scraped, lysed, and both firefly and R. reniformis luciferase activities were measured. Values represent firefly/R. reniformis luciferase activity ratios divided by protein content. Bars represent the mean ± S.D. of four independent experiments. Significantly different (*, p < 0.05; ***, p < 0.005) from cells transfected with the same promoter construct and no pAC-C/EBP expression plasmid. C, binding of C/EBP to the distal CYP3A4 Promoter. Double-stranded oligonucleotides matching the CYP3A4 promoter sequence between positions 1384/1408 and 1652/1676 that contained the putative C/EBP binding sites at 1393/1402 and 1659/1668, were used as labeled probes. N.E., nuclear extract; IS, itself; U, oligonucleotide with an unrelated sequence; P2m, 115 to 139 oligonucleotide with the central nucleotides of the putative C/EBP binding site at 121/130 mutated; FP, free probe; NS, nonspecific complex; C1 and C2, specific complexes; C/EBP, DNA/C/EBP complex; SS, supershifted complex.

Expression of P450s in Cells Transfected with C/EBP and HNF-3 Adenoviral Vectors. The results obtained with the reporter assays need further confirmation in a more complex system because in plasmid constructs, the DNA lacks the native chromatin structure, which is an important feature for gene expression (van Holde, 1997). To investigate the regulation of the CYP3A4 gene with its native structure, we constructed adenoviral vectors encoding C/EBP (Ad-C/EBP) and HNF-3 (Ad-HNF-3) as tools to overexpress these transcription factors in cells. In these experiments, we used hepatic Hep G2 cells, which have lost the expression of CYP3A4 and other hepatic-specific genes (Fig. 4A), and HeLa cells, which are derived from cervix carcinoma cells and have no CYP3A4 expression (Fig. 4C). In both cases, the cells were infected with Ad-C/EBP, Ad-HNF-3, or Ad-pAC, and 48 h after infection, CYP3A4 mRNA content was analyzed by RT-PCR. The expression of C/EBP and HNF-3 was also measured by RT-PCR (data not shown) and Western blot to examine the efficiency of the infection; in all cases, a dose-proportional expression of the corresponding transcription factor was obtained (Fig. 4A).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Effect of C/EBP and HNF-3 adenoviral vectors on P450 expression. Cells were infected with Ad-C/EBP and/or Ad-HNF-3l; 48 h after infection, cells were harvested. Total RNA was isolated and P450 and -actin mRNA contents were measured by RT-PCR as described under Materials and Methods. For quantitative measurements, the P450 mRNA contents were normalized dividing by their respective -actin mRNA contents and were expressed as fold induction relative to the levels of control cells infected with Ad-pAC. Bars are the mean of four independent experiments ± S.D. A, effect of C/EBP and HNF-3 adenoviral vectors on Hep G2 CYP3A4 expression. Representative PCR reactions for CYP3A4 and -actin are depicted after ethidium bromide staining. C/EBP and HNF-3 protein levels were analyzed by immunoblotting using 20 µg of total protein extracts. A representative Western blot is depicted after detection with the specific antibodies. The doses of Ad-C/EBP and Ad-HNF-3 used in the experiments are indicated. B, effect of C/EBP and HNF-3 adenoviral vectors on Hep G2 CYP3As expression. Hep G2 cells were infected with different amounts of adenoviral vectors (Ad-C/EBP, Ad-HNF-3, and Ad-pAC), total RNA was isolated and CYP3A4, CYP3A5, CYP3A7, and -actin mRNAs were measured by RT-PCR. Control, adenoviral-untreated cells; Ad-pAC, Ad-pAC treated cells. C, P450 expression in HeLa cells infected with C/EBP adenoviral vector. Total RNA was isolated from HeLa cells infected with Ad-C/EBP or Ad-pAC adenoviral vectors (7.5 MOI). After reverse transcription, cDNA fragments of CYP1A1, -1A2, -2B6, -2D6, -2E1, -3A4, -3A5, and -3A7 were amplified by 35 PCR cycles using specific primers. Two identical RT-PCR reactions were carried out using the RNA of Ad-pAC infected cells (control) or Ad-C/EBP-infected cells (C/EBP). After gel electrophoresis of the PCR products and staining with ethidium bromide, the fluorescent bands were recorded with video camera. Marker, 100-bp DNA ladder.

HNF-3 Binds CYP3A4 Distal Promoter. To determine whether a direct effect of HNF-3 in CYP3A4 promoter was responsible for the cooperativity with C/EBP, EMSAs were performed with seven labeled oligonucleotides containing the eight different HNF-3 putative binding sites predicted by sequence analysis (Fig. 1) (the two more distal HNF-3 sites overlap, and both were contained in one single probe) and nuclear extracts from Hep G2 cells infected with HNF-3 adenovirus. When a oligonucleotide containing the consensus binding sequence for HNF-3 [T(A/G)TTTNNTT] was used for competition, only the 1710/1738 probe, which contains the two overlapping HNF-3 sites (TGTTTATTTGTCT), showed competed complexes (data not shown and Fig. 5). In agreement with this, when a HNF-3-specific antibody was added to the EMSA binding reaction, supershifted complexes could only be detected with the 1710/1738 labeled probe (Fig. 5A, lane 14). As shown in Fig. 5B, the specific complex of the 1710/1738 probe had a relatively high affinity (100-fold excess of unlabeled probe was required for complete competition) (Fig. 5B, lanes 3 and 4). HNF-3 was identified as the protein present in this complex by competition with a consensus HNF-3 binding sequence and by supershift with a specific HNF-3 antibody. These data support a direct effect of HNF-3 in CYP3A4 promoter.

View larger version (73K): [in this window] [in a new window]   Fig. 5.   Binding of HNF-3 to CYP3A4 promoter. A, HNF-3 binds only one of the eight putative HNF-3 sites identified in CYP3A4 promoter. Double-stranded oligonucleotides matching CYP3A4 promoter sequence between positions: 13/37, 178/202, 557/581, 983/1007, 1424/1448, 1436/1460, and 1710/1738 and those that contained the eight putative HNF-3 sites shown in Fig. 1 were labeled and incubated with nuclear extracts of Hep G2 cells overexpressing HNF-3. During the binding reaction, a specific HNF-3 antibody was added in the indicated samples. B, HNF-3 binds CYP3A4 distal promoter with high affinity. The oligonucleotide 1710/1738, which contains the overlapping 1718/1726 and 1722/1730 putative HNF-3 binding sites, was labeled, and 50- or 100-fold molar excess of unlabeled probes was used for competition: IS, itself; U, oligonucleotide with an unrelated sequence; HNF-3c, consensus HNF-3 binding sequence; FP, free Probe; NS, nonspecific complex; HNF-3, DNA/HNF-3 complex; SS, supershifted complex.

HNF-3 Cooperative Effect Is Prevented by a Deacetylase Inhibitor. HNF-3 proteins can modify nucleosome positioning, disrupt the local chromatin structure, and in this way facilitate the accession of other transcription factors to their binding sites (Crowe et al., 1999; Cirillo et al., 2002). To investigate whether this mechanism could be responsible for the cooperative effect observed between C/EBP and HNF-3, we treated Hep G2 cells overexpressing C/EBP and/or HNF-3 with trichostatin A (TSA), a compound that remodels the chromatin to a transcriptional competent state by inhibiting histone deacetylases (Yoshida et al., 1995). TSA alone had no effect on CYP3A4 expression (Fig. 6), but it increased by 13-fold the C/EBP activatory effect (Fig. 6, compare bars 2 and 6) and clearly abolished HNF-3 cooperative effect (Fig. 6, bars 6 and 8 are not significantly different, whereas bars 2 and 4 are statistically different). These results suggest an important role of chromatin structure in the cooperativity between C/EBP and HNF-3 on the expression of CYP3A4.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of trichostatin A on C/EBP- and HNF-3-dependent activation of CYP3A4. Hep G2 cells were infected with 7.5 MOI Ad-C/EBP and/or 4.5 MOI Ad-HNF-3, adjusting the final adenoviral dose to 12 MOI with Ad-pAC. Twenty-four hours after infection, cells were treated with the deacetylase inhibitor trichostatin A at 3 µM for 24 h. Total RNA was isolated, mRNA levels of CYP3A4 and -actin were determined by RT-PCR, and CYP3A4 mRNA values were normalized, dividing by their respective -actin mRNA values. The results were expressed as fold induction relative to the levels in cells infected with Ad-pAC. Data are the mean of four independent experiments ± S.D. Significantly different (*, p < 0.05) from cells treated with Ad-C/EBP.

	Discussion

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The LETFs are trans-activating factors that control the expression of hepatic genes acting within a network of cooperative and synergistic effects. C/EBP and HNF-3 have been identified as key signals in the regulation of many liver-specific genes, including several P450s (Ourlin et al., 1997; Jover et al., 1998; Delesque-Touchard et al., 2000). However, their role in the regulation of the constitutive expression of CYP3A4 in hepatocytes, which is much higher than in nonhepatic cells, has not been investigated. Among the different C/EBP consensus binding sequences found by computer analysis in CYP3A4 promoter (Fig. 1), C/EBP trans-activated a luciferase reporter gene specifically binding the 121/130 site (Fig. 3, A and B). The similar results obtained in hepatic and nonhepatic cell lines transfected with the proximal promoter constructs, suggested that the mechanisms mediating C/EBP action at the 121/130 site did not depend on specific hepatic factors (Fig. 2B). In addition to the proximal site, two other C/EBP binding sites were located at distal positions in the promoter (1393/1402 and 1659/1668, Fig. 3C). In contrast to the proximal site, the luciferase reporter gene constructs revealed that the distal sites were functional in hepatic cells but not in nonhepatic cells (Fig. 2B), which may lack hepatic-specific activators or express inhibitors that avoid C/EBP action. On the other hand, HNF-3 neither had any trans-activatory effect by itself nor modified the C/EBP-dependent trans-activation.

Because the reporter plasmids are not organized into the nucleosome array characteristic of cellular chromatin (Smith and Hager, 1997), we tested whether the results found with the gene reporter assays could be extrapolated to the endogenous CYP3A4 gene. For this purpose, we developed replicant-defective recombinant adenoviral vectors encoding C/EBP or HNF-3. These expression vectors allow transfection of foreign genes into cells with almost 100% efficiency in a rather nondisturbing manner for the cells (Castell et al., 1997) and were an excellent tool for the expression of different levels of the transcription factors (Fig. 4). As predicted by the reporter assays, C/EBP increased the CYP3A4 mRNA content of Hep G2 cells (4-fold for 7.5 MOI), whereas HNF-3 did not modify CYP3A4 expression. In contrast, unpredicted by the reported assays, when both factors were expressed simultaneously, the CYP3A4 mRNA levels were increased 45-fold, evidencing a cooperative effect between C/EBP and HNF-3. The lack of effect of HNF-3 when C/EBP was not coexpressed provides evidence that the intrinsic levels of C/EBP in Hep G2 cells were insufficient to bring about the HNF-3 cooperative effect (Fig. 4A). Low levels of C/EBP in Hep G2 cells have been described previously (Jover et al., 1998).

The observed HNF-3 action could occur through a direct binding of HNF-3 to CYP3A4 promoter or by a HNF-3-mediated increase of another transcription factor that would bind CYP3A4 promoter and cooperate with C/EBP. EMSA analysis revealed that HNF-3 binds the CYP3A4 promoter at a distal site (1718/1730), supporting the idea that HNF-3 exerts its cooperative effect through a direct mechanism. The similarity of the DNA binding domain of HNF-3 with that of linker histones (Clark et al., 1993) enables HNF-3 proteins to modify nucleosome positioning and facilitate the binding of other transcription factors (Crowe et al., 1999). The HNF-3 site is located 50 nucleotides upstream of a C/EBP binding site (1659/1668), and it is likely that HNF-3 could affect C/EBP binding. This effect cannot occur in the luciferase reporter plasmids lacking the characteristic chromatin structure of genomic DNA (Smith and Hager, 1997), which explains that the cooperative effect was not detected in these assays. Supporting the notion of the direct effect of HNF-3, the overexpression of HNF-3 did not enhance the expression of other hepatic transcription factors such as HNF-4, pregnane X receptor, constitutive androstane receptor, and retinoid X receptor-, which could be indirect mediators.

In the nonhepatic HeLa cells, the adenoviral overexpression of C/EBP increased the CYP3A4 mRNA content to detectable levels, but HNF-3 showed no effect, either alone or in combination with C/EBP. The latter was in contrast with the findings in the hepatic Hep G2 cells but was consistent with the lack of C/EBP effect in the distal binding sites of the CYP3A4 promoter when the luciferase reporter assays were carried out in HeLa cells (Fig. 2B). We have shown that the HNF-3 cooperative effect occurs through a distal site that is located near C/EBP sites that are not active in HeLa cells.

The up-regulation of CYP3A4 expression by the cooperation of C/EBP and HNF-3 was also detected in CYP3A5 and CYP3A7 genes (Fig. 4, B and C), indicating that similar binding sites for C/EBP and HNF-3 should be found in their promoters. In the case of CYP3A7, the proximal C/EBP site had one nucleotide change with respect to CYP3A4, and the distal C/EBP and HNF-3 sites were identical. In the case of CYP3A5 (which shows the lowest response), the proximal C/EBP site had a lower similarity with the consensus sequence than those of CYP3A4 and CYP3A7. The promoter of CYP3A5 could not be successfully aligned with CYP3A4 at distal positions because of a drastic decrease in similarity. However, sequence analysis of the CYP3A5 distal promoter, with conditions identical to those described for CYP3A4 under Materials and Methods, located a C/EBP site at positions 1621/1630 and two overlapping HNF-3 sites between positions 1740/1755, similar to CYP3A4.

The results obtained with TSA (Fig. 6), an inhibitor of histone deacetylases able to change chromatin conformation to a more relaxed state and more accessible to transcription factors, is consistent with the proposed model for the cooperative effect between C/EBP and HNF-3. It is known that TSA can alter the expression of some genes (Yoshida et al., 1995), but TSA treatment by itself did not modify the levels of CYP3A4 in Hep G2 cells (Fig. 6, compare bars 1 and 5). The relevant results are that cells overexpressing C/EBP increased the CYP3A4 mRNA levels 13-fold when treated with TSA, but cells treated with TSA had lost the response to the cooperative effect of HNF-3. This is in agreement with the requirement of cellular chromatin structure to detect HNF-3 effect and suggests that the modification of chromatin structure is a common mechanism for TSA and HNF-3. However, further studies are required to fully understand the molecular mechanism involved.

C/EBP and HNF-3 play important roles in the constitutive expression of human P450s. C/EBP regulates the expressions of CYP2B6, CYP2D6, and CYP2C9 (Jover et al., 1998), and the expression of several CYP2Cs are regulated by HNF-3 (Shaw et al., 1994; Delesque-Touchard et al., 2000). We now have found that the highest expression of CYP3A4, CYP3A5, and CYP3A7 was obtained in hepatic cells expressing a combination of C/EBP and HNF-3, a mechanism that may also operate in other P450s. Because of the important roles played by C/EBP and HNF-3 in the constitutive expression of human CYP3A4, variations in the expression of C/EBP and HNF-3 could ultimately be responsible of the different expression levels of CYP3A4 found in humans. In this context, the levels of C/EBP and HNF-3 proteins are known to change in the liver under several pathophysiological situations. For example, during inflammatory processes, C/EBP and CYP3A4 expression decrease (Donato et al., 1998; Welm et al., 2000). Diet and hormonal status have also been described to greatly alter HNF-3 expression in liver (Imae et al., 2000). Further studies could determine whether variations in C/EBP and HNF-3 expression could be involved in CYP3A4 intra- and interindividual variability.

In conclusion, we have localized binding sites for C/EBP and HNF-3 in CYP3A4 promoter, and by reporter assays we have shown their relevance for gene expression. By use of adenoviral expression vectors, we have found a synergistic effect between C/EBP and HNF-3 in the expression of hepatic CYP3A genes. Finally, the proximity of C/EBP and HNF-3 distal sites and the abolishment of HNF-3 action by a deacetylase inhibitor suggest that HNF-3 facilitates C/EBP action by modification of the chromatin structure of CYP3A4 promoter.