Transcriptional Regulation of Human CYP3A4 Basal Expression by CCAAT Enhancer-Binding Protein and Hepatocyte Nuclear Factor-3

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/ enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 was overexpressed. In contrast, overexpression of HNF-3 simultaneously with C/EBP resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP binding site. Trichostatin A increased C/EBP effect but abolished HNF-3 cooperative action. These findings revealed that C/EBP and HNF-3 cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling. The cytochromes P450 (P450) are a superfamily of hemecontaining 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 intraand 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 This work was supported by the European Union, BIOTECH contract BIO4-CT96-0052 and BIOMED contract BMH4-CT86-0254 (Eurocyp). C. R.-A. was the recipient of a fellowship of Generalitat Valenciana. ABBREVIATIONS: P450, cytochrome P450; LETF, liver-enriched transcription factor; HNF, hepatocyte nuclear factor; C/EBP, CCAAT enhancerbinding protein; MOI, multiplicity of infection; EMSA, electrophoretic mobility shift assay; Ad-C/EBP , recombinant adenovirus encoding C/EBP ; Ad-HNF-3 , recombinant adenovirus encoding HNF-3 ; Ad-pAC, recombinant adenovirus encoding pAC/CMVpLpA; TSA, trichostatin A; PCR, polymerase chain reaction; bp, base pair(s); CMV, cytomegalovirus; RT, reverse transcription. 0026-895X/03/6305-1180–1189$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 5 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2198/1065374 Mol Pharmacol 63:1180–1189, 2003 Printed in U.S.A. 1180 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from 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 glucose6-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. Rodrı́guez-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; DelesqueTouchard 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 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 (pACHNF-3 ) was constructed by PCR amplification of the complete human HNF-3 cDNA and ligation into the pAC/CMVpLpA (R. Bort, R. Jover, C. Rodrı́guez-Antona, M. J. Gómez-Lechón, and J. V. Castell, manuscript in preparation). 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 56and 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 56and 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 56and 152-bp fragments were amplified in independent reactions containing 1 ng of 163/ 6 pGL3-Basic, 0.2 M of sense and antisense oligonucleotide primers, 200 M 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 l. 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., 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 (u) and HNF-3 (f). 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. Regulation of Human CYP3A4 by C/EBP and HNF-3 1181 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from 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 56and 152-bp DNA fragments were annealed in a reaction mixture containing 200 M 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 l. 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 M) 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. Plasmid DNAs were purified with QIAGEN Maxiprep kit columns (QIAGEN, Valencia, CA) and quantified by absorbance at 260 nm and fluorescence using PicoGreen (Molecular Probes, Eugene, OR). The day before transfection, cells were plated in 35-mm diameter dishes with 1.5 ml of medium. Two hours before transfection, medium was changed to Dulbecco’s modified Eagle’s medium/Nut F12 (Invitrogen, Carlsbad, CA) supplemented with 10% newborn calf serum, 50 U/ml penicillin, and 50 mg/ml streptomycin. Firefly luciferase pGL3-Basic constructs (0.5 to 1 g) were transfected with or without pAC-C/EBP and pAC-HNF-3 (0.5 to 1 g) by calcium phosphate precipitation. 0.1 g of pRL-CMV (a plasmid expressing Renilla reniformis luciferase under the CMV immediate early enhancer/promoter) was cotransfected to correct for variation in transfection efficiency. Calcium phosphate/DNA coprecipitates were added directly to each culture and incubated for 6 (Hep G2) or 20 h (HeLa). Then, the medium was replaced; 48 h after transfection, firefly and R. reniformis luciferase activities were determined using the dual-luciferase reporter assay system (Promega, Madison, WI). In all experiments, luciferase activity was normalized to transfection efficiency (R. reniformis luciferase activity by pRL-CMV) and protein