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J. Biol. Chem., Vol. 281, Issue 10, 6734-6741, March 10, 2006

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CCAAT Enhancer-binding Protein Suppresses the Rat Placental Glutathione S-Transferase Gene in Normal Liver^*

Hiromi Ikeda, Kazuki Omoteyama, Kazuhiko Yoshida, Shinzo Nishi, and Masaharu Sakai^¶1

From the Departments of Biochemistry and Ophthalmology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-Ku, Sapporo 060-8638, Japan and the ^¶Department of Health Sciences, Hokkaido University School of Medicine, N12, W5, Kita-Ku, Sapporo 060-0812, Japan

Received for publication, December 6, 2005 , and in revised form, January 5, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The rat placental glutathione S-transferase (GST-P), an isozyme of glutathione S-transferase, is not expressed in normal liver but is highly induced at an early stage of chemical hepatocarcinogenesis and in hepatomas. Recently, we reported that the NF-E2 p45-related factor 2 (Nrf2)/MafK heterodimer binds to GST-P enhancer 1 (GPE1), a strong enhancer of the GST-P gene, and activates this gene in preneoplastic lesions and hepatomas. In addition to the positive regulation during hepatocarcinogenesis, negative regulatory mechanisms might work to repress GST-P in normal liver, but this remains to be clarified. In this work, we identify the CCAAT enhancer-binding protein (C/EBP) as a negative regulator that binds to GPE1 and suppresses GST-P expression in normal liver. C/EBP binds to part of the GPE1 sequence, and the binding of Nrf2/MafK and C/EBP to GPE1 is mutually exclusive. In a transient-transfection analysis, C/EBP activated GPE1 in F9 embryonal carcinoma cells but strongly inhibited GPE1 activity in hepatoma cells. The expression of C/EBP was specifically suppressed in GST-P-positive preneoplastic foci in the livers of carcinogentreated rats. A chromatin immunoprecipitation analysis showed that C/EBP bound to GPE1 in the normal liver in vivo but did not bind in preneoplastic hepatocytes. Introduction of the C/EBP gene fused with the estrogen receptor ligand-binding domain into hepatoma cells, and subsequent activation by -estradiol led to the suppression of endogenous GST-P expression. These results indicate that C/EBP is a negative regulator of GST-P gene expression in normal liver.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The placental form of glutathione S-transferase (GST-P)² is one of a group of enzymes that catalyzes the conjugation of glutathione to xenobiotics (1). GST-P belongs to the Pi class of GST and is weakly expressed in rat tissues, including lung, kidney, spleen, and placenta. GST-P is not detected in normal liver, but it is markedly and specifically increased during early stages of hepatocarcinogenesis. It can be detected in single liver cells as early as 2-3 days after the administration of a chemical carcinogen (2-7). Therefore, this enzyme has been used as a reliable tumor marker for both chemically induced and spontaneously arising precancerous lesions and hepatomas in experimental carcinogenesis studies. The induction of GST-P occurs primarily at the transcriptional level (8), and we have been studying the transcriptional mechanisms involved in the specific activation of the GST-P gene (9-12). The GST-P gene has a strong enhancer element, GPE1, 2.5-kb upstream from the cap site (9-11). This enhancer is a major control element for the expression of GST-P in preneoplastic lesions and hepatomas. Muramatsu and co-workers (13, 14) established a GPE1-transgenic rat line and showed that GPE1 is essential and nearly sufficient for the expression of GST-P during hepatocarcinogenesis in vivo. GPE1 consists of two phorbol-12-O-tetradecanoate-13-acetate-responsive element (TGAGTCA)-like elements with palindromic orientations (5'-TAGTCAGTCACTATGATTCAGCAA-3'; the phorbol-12-O-tetradecanoate-13-acetate-responsive element-like sequences are underlined) (9). We have recently shown that the NF-E2 p45-related factor 2 (Nrf2) and MafK heterodimer interact with GPE1 and activate the GST-P gene early in hepatocarcinogenesis and in rat hepatomas (15). Although the expression of Nrf2 increases moderately during carcinogenesis, Nrf2 is also expressed at considerable levels in the normal liver, in which GST-P is completely repressed. Therefore, there may be other mechanisms that suppress GST-P expression in normal liver. Circumstantial evidence suggests that GPE1 suppresses GST-P expression in the normal liver, in addition to acting as a positive enhancer in preneoplastic lesions and hepatomas. First, the transgenic rat studies by Muramatsu and co-workers (13, 14) showed that the rats expressed the GPE1 transgene when they were treated with a carcinogen. In the normal liver, however, none of the GPE1 transgenic rats expressed the transgene. Second, the mouse Pi class GST gene (a homologue of rat GST-P), which does not contain GPE1 or any related element in the regulatory region, is expressed in normal liver (16, 17). Although hepatocarcinogenesis does increase the expression of the mouse Pi class GST, the induction is much lower than that of rat GST-P (18). These circumstances suggest that GPE1 may repress GST-P in the normal liver.

In this study, we have conducted a computer-mediated search for transcription factors that might interact with the flanking region of the GST-P gene and found that the CCAAT enhancer-binding protein (C/EBP) could potentially bind to the GPE1 sequence. The C/EBP family of basic leucine zipper transcription factors is comprised of six members, to . The various isoforms show different patterns of expression, and they regulate a wide variety of essential differentiation programs, cellular proliferation, and numerous cellular processes (19). In the liver, C/EBP is highly expressed and maintains liver quiescence (20-22). Livers of newborn C/EBP knock-out mice have an increased rate of proliferation (21). C/EBP gene expression is very low or completely repressed in preneoplastic nodules and hepatomas (23, 24). Thus, the expression of C/EBP is negatively correlated with that of GST-P.

To clarify the possible suppressive effect of C/EBP on the transcription of the GST-P gene in normal liver, we have analyzed the interaction of this factor with GPE1 in vitro and in vivo and used transient-transfection analysis of hepatoma and F9 cells to examine the effect of C/EBP on GPE1. We also have analyzed endogenous GST-P expression in C/EBP overexpressing hepatoma cells. From these analyses, we conclude that C/EBP suppresses the expression of GST-P in the normal liver.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of Plasmids—To construct the luciferase reporter genes, the -2.9-kb/+37-bp fragment relative to the cap site of the rat GST-P gene (GST/ELuc) or the -50/+37-bp fragment, the minimal promoter region containing the GC and TATA boxes (50Luc) (9) was inserted into a promoter-less luciferase plasmid vector, pGVB2 (Nippon Gene, Toyama, Japan). The constructs containing internal deletions or synthetic oligonucleotide elements were made by inserting the following sequences into 50Luc: a 0.3-kb SacI-AccI fragment (-2.5/-2.2-kb) containing GPE1 (SA/50Luc), GPE1 (5'-GTAGTCAGTCACTATGATTCAGCAACA-3', GPE1/50Luc), or mutated forms of GPE1 (M11/50Luc: 5'-GTAGTCAGTCACTATGAaTCAGCAtCA-3'; M12/50Luc: 5'-GTAGTCAGTCACTATGAgTCAGCAACA-3'; and M13/50Luc: 5'-GTAGTCAGTCACTATGAcTCAGCAACA-3'; with the mutated positions indicated by lowercase letters). Three copies of the C/EBP-binding consensus sequence (C/EBP-RE) from the mouse transthyretin gene enhancer (5'-TTTTCCATCTTACTCAACATCCTA-3') were joined in tandem and cloned into 50Luc (C/EBP-RE/50Luc) (25). The C/EBP and C/EBP expression plasmids were kindly provided by Dr. Takiguchi (University of Chiba). The Nrf2 expression vector was described previously (15).

Cell Culture, Reporter Transfection Analysis, and Retroviral Transduction—H4IIE and RL34 rat hepatoma and F9 mouse embryonal carcinoma cell lines were maintained in Dulbecco's modified minimal essential medium (Nissui, Tokyo) with 8% (v/v) fetal bovine serum. For transfection analysis, the cells were plated at a density of 5 x 10⁵ cells (H4IIE and RL34) or 2 x 10⁵ cells (F9)/60-mm plate. A total of 4 µg of DNA was transfected into the cells, including 1 µg of the reporter plasmid and 0.5 µg of the -galactosidase expression plasmid (pSVgal; Promega, Madison WI) with or without an effector-gene expression plasmid (C/EBP, C/EBP, or Nrf2) and pUC18 DNA, according to the procedure of Chen and Okayama (26). The cells were harvested 45 h after transfection and assayed for luciferase activity using a luciferase assay kit (Nippon Gene) and for -galactosidase activity (27). Luciferase activities were normalized to -galactosidase activities. All of the experiments were performed at least twice.

The retroviral construct of C/EBP fused with the estrogen receptor ligand-binding domain (pBabePuro-C/EBP-ER) (23, 28) was kindly provided by Dr. Alan D. Friedman (Johns Hopkins University). The BOSC23 packaging cells (29) were cultured in Dulbecco's modified minimal essential medium with 10% fetal bovine serum and were transfected with pBabePuro-C/EBP-ER by the calcium phosphate method. The supernatant was collected after 40 h and infected into RL34 hepatoma cells with 4 µg/ml polybrene. Stable transformants were cloned after 2 weeks of selection with 3 µg/ml puromycin. C/EBP-ER-expressing cells and parent RL34 cells were cultured in phenol red-free minimal essential medium (Nissui, Tokyo) containing 10% fetal bovine serum. C/EBP-ER was activated by the addition of -estradiol (1 µM; Sigma) and harvested after 48 h for RNA and protein analyses.

Animals and Chemical Hepatocarcinogenesis—Rats with hyperplastic nodules induced by the Solt-Farber protocol (30, 31) were kindly provided by Dr. K. Satoh (Hirosaki University, Hirosaki, Japan). All of the animal experiments were conducted according to the Guidelines for Animal Experiments of Hokkaido University.

EMSA and DNase I Footprinting Analysis—The cDNA fragment encoding the DNA-binding domain of each transcription factor (amino acids 318-598 for Nrf2, 1-156 for MafK, and 282-396 for C/EBP) was fused to the Escherichia coli maltose-binding protein (MBP) gene of the pMAL-c2 vector (New England Biolabs). These fusion proteins were expressed in E. coli cells and purified as previously described (15). To prepare probes for the EMSA, synthetic double-stranded DNA elements were cloned into a pBluescript II vector (Stratagene) and fragmented with BamHI (GPE1) or BamHI and SalI (C/EBP-RE, mutant GPE1). The overhanging ends were labeled with Klenow DNA polymerase I (Takara, Kyoto) and [-³²P]dCTP. A fragment of the multiple cloning site of the pBluescript II vector (HindIII-XbaI) was labeled as a negative control probe. EMSA was performed as described previously (32). The recombinant MBP-fused proteins were preincubated with 0.5 µg of poly(dI-dC) (Amersham Biosciences) for 10 min in 10 µl of binding buffer (20 mM HEPES, pH 7.9, 20 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 4 mM MgCl₂, 15% glycerol, and 100 µg/ml bovine serum albumin. A ³²P-labeled DNA probe (2 x 10⁴ cpm) was added, and the mixture was incubated for another 10 min. Protein-DNA complexes were analyzed by 4% (w/v) polyacrylamide gel electrophoresis in 25 mM Tris borate, 0.5 mM EDTA, pH 8.2.

Nuclear extracts were prepared as described by Dignam et al. (33). The cells were suspended in 5 packed cell volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol), incubated on ice for 10 min, and briefly centrifuged. The cell pellet was resuspended in 2 packed cell volumes of buffer A with 1 mM phenylmethylsulfonyl fluoride (PMSF), homogenized, and centrifuged. The nuclear pellet was suspended in 1 volume of buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF) and gently stirred at 4 °C for 30 min. After centrifugation at 35,000 rpm for 30 min, the supernatant was dialyzed against buffer C (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM PMSF) for 5 h. The nuclear extracts (10 µg/reaction) were incubated with the indicated antibody at 15 °C for 30 min prior to EMSA using the same procedures as for recombinant proteins, except that 2.5 µg of poly(dI-dC) was used.

For the DNase I footprinting analysis, the probe (2 x 10⁵ cpm) was incubated with bovine serum albumin or recombinant protein (0.1 µg) in 50 µl of EMSA binding buffer. Fifty µl of DNase I buffer (5 mM MgCl₂, 5 mM CaCl₂, 200 mM KCl, and 4 µg/ml sheared salmon sperm DNA) and 20 ng of DNase I (Takara) were mixed and incubated for 3 min at room temperature. The reaction was terminated by the addition of 100 µl of stop solution (20 mM EDTA, 1% SDS, and 0.2 M NaCl). DNA was extracted and separated in a 6% (w/v) polyacrylamide gel containing 8 M urea. Guanine and adenine bases of the same probes were modified, digested by the Maxam-Gilbert method, and loaded as a marker ladder.

RNA and Protein Analyses—Total cellular RNAs were isolated from rat tissues and cultured cells using an RNA extraction kit (Isogen, Nippon Gene). Northern blot analysis was performed as described previously (34). The fragments of the C/EBP, C/EBP, and GST-P cDNAs were labeled with a random-primed DNA labeling kit (Takara) using [-³²P]dCTP and used as probes. RNase protection analysis was performed as described previously (34). A DNA fragment of the GST-P gene (-109 to +56-bp, relative to the cap site) was subcloned into pBluescript II and antisense RNA was synthesized by T7-RNA polymerase with [-³²P]UTP for the probe.

For Western blot analysis, the cells were lysed with lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF). The proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted on a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was incubated with anti-C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-GST-P antibody (kindly provided from Dr. K. Satoh, Hirosaki University). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences), and the immunoreactive bands were detected using an ECL system (Amersham Biosciences). Band intensities in the autoradiograms of Northern and Western blotting were estimated by NIH Image.

Immunohistochemistry—Eight weeks after the Solt-Farber protocol was started, the rats were fixed by an intracardiac perfusion of 4% paraformaldehyde in phosphate-buffered saline. The liver tissues were embedded in paraffin and sectioned at 3 µm, and sections were processed for immunofluorescence staining. The slides were incubated with normal goat serum and then with the anti-GST-P or anti-C/EBP antibody. Binding of the primary antibody was localized using a Cy-3-conjugated (GST-P) or FITC-conjugated (C/EBP) goat anti-rabbit IgG (dilution, 1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The slides were examined using laser scanning confocal microscopy (MRC-1024, Bio-Rad; and LSM 510, Carl Zeiss, Oberkochen, Germany).

Chromatin Immunoprecipitation Analysis—Chromatin immunoprecipitation analysis was carried out as described previously (15, 35). Briefly, the cells were fixed in 1% formaldehyde and washed with phosphate-buffered saline containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin). Harvested cells were sonicated in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with an ultrasonic generator (Tomy Seiko, Tokyo, Japan) and centrifuged. The supernatant was diluted 10-fold with immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) and precleared with 30 µl of protein A-Sepharose (50% protein A slurry (Amersham Biosciences) containing 20 µg/ml sheared salmon sperm DNA and 1 mg/ml bovine serum albumin). The precleared chromatin fraction was incubated with anti-C/EBP, anti-C/EBP (Santa Cruz Biotechnology), anti-Nrf2 (15), or anti-MafK (kindly provided by Dr. K. Igarashi, Hiroshima University) antibody or preimmune serum for 16 h at 4 °C. Immune complexes were mixed with 50 µl of protein A-Sepharose and washed sequentially with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1) containing 150 mM NaCl, wash buffer 1 containing 500 mM NaCl, wash buffer 2 (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and twice with 10 mM Tris-HCl, pH 8.1, 1 mM EDTA. The complexes were eluted in 1% SDS, 0.1 M NaHCO₃, and cross-linking was reversed by heating to 65 °C for 4 h. The DNAs were purified, and the GPE1 region of the GST-P gene was amplified by PCR with the primers 5'-TGATTCTGCCATCTTTCTGC-3' and 5'-CCAGCTTCTCTGGACAAACC-3'. Amplified DNAs were analyzed using 3% agarose gel electrophoresis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
C/EBP Suppresses GPE1 Enhancer Activity—We found that the 3'-half of GPE1 (5'-TAGTCAGTCACTATGATTCAGCAAC-3', the C/EBP-binding consensus sequence is underlined) almost completely matches the consensus binding sequence of C/EBP (5'-RTTGCGYAAY-3') (36). Further, C/EBP is markedly down-regulated in preneoplastic foci of the liver, in which the GST-P gene is strongly induced (24). Therefore, we used transient-transfection analysis to examine whether C/EBP influences GST-P expression. Luciferase reporter constructs containing the -2.9-kb/+37-bp region of the GST-P gene (GST/ELuc), and the GPE1 enhancer region (-2.5/-2.2-kb, SA/50Luc) were transfected into cells of the H4IIE rat hepatoma line, with or without a C/EBP or C/EBP expression vector. Fig. 1A shows that both GST/ELuc and SA/50Luc were highly expressed in H4IIE cells but that the expression of both constructs was strongly inhibited by co-transfection with C/EBP. C/EBP also inhibited transcriptional activity, but to a lesser extent than C/EBP. C/EBP-RE/50Luc, which contained three tandem C/EBP binding consensus sequences from the transthyretin gene, was activated by both C/EBP and C/EBP.

Previously, we reported that Nrf2/MafK binds to GPE1 and activates GST-P expression during hepatocarcinogenesis. To determine whether C/EBP inhibits the ability of Nrf2/MafK to activate GPE1, we performed a transient-transfection analysis. The GPE1 core element was fused to a luciferase gene with the minimal promoter sequence of the rat GST-P gene (GPE1/50Luc) and transfected into H4IIE cells, with or without co-transfection with the Nrf2, C/EBP, or C/EBP expression vectors (Fig. 1B). In hepatoma cells, Nrf2 stimulated GPE1/50Luc gene expression 10-fold, as described previously (15), and C/EBP dramatically down-regulated GPE1 activity. C/EBP also suppressed GPE1, but by approximately only one-fifth as much as C/EBP. The expression of 50Luc, which contained only the minimal promoter, did not significantly change with co-transfection by the effector plasmids.

To address why GPE1 was suppressed by C/EBP, we performed a reporter transfection analysis using mouse F9 embryonal carcinoma cells, which do not contain any GPE1-stimulating activity (15). In contrast to the results with hepatoma cells, in F9 cells, C/EBP stimulated GPE1/50Luc by 10-20-fold in a dose-dependent manner (Fig. 1C). However, the stimulation of GPE1/50Luc by Nrf2 was much higher (300-fold) than that by C/EBP. The activation of Nrf2 was strongly inhibited by the co-transfection of C/EBP. These results suggest that C/EBP interacts with GPE1 and inhibits the binding of the strong positive factor, Nrf2/MafK, in hepatoma cells. C/EBP-RE/50Luc was greatly stimulated by C/EBP in F9 cells and was not affected by Nrf2.

C/EBP Binds to GPE1—The nucleotide sequence of GPE1 and the results of the reporter transfection analysis described above suggest that C/EBP binds directly to GPE1. To test whether GPE1 and C/EBP interact directly, we performed EMSA using recombinant C/EBP proteins expressed in E. coli and a GPE1 probe. The multiple cloning site of the pBluescript II vector and the C/EBP-binding consensus sequence (C/EBP-RE) were used as negative and positive control probes, respectively. C/EBP bound strongly to GPE1 (Fig. 2A, lane 8). The probe with a single C/EBP-RE sequence yielded one shifted band, and the probe with two C/EBP-RE sequences yielded double bands, as expected (Fig. 2A, lanes 4 and 6). The binding specificity was confirmed by a competition analysis with an excess amount of unlabeled GPE1 probe (Fig. 2A, lanes 11-13).

To identify the C/EBP-binding site in the GPE1 sequence, we performed a DNase I footprinting analysis. The Nrf2/MafK heterodimer completely protected the entire GPE1 core sequence from DNase I digestion (Fig. 2B), as described previously (15). C/EBP clearly protected the 3'-half of GPE1, the region containing the C/EBP-binding consensus sequence.

The reporter transfection analysis described above suggested that C/EBP binding interfered with Nrf2/MafK activity on GPE1. We investigated whether these factors bind to GPE1 simultaneously or exclusively, using an EMSA with a fixed amount of C/EBP and increasing amounts of Nrf2/MafK and vice versa (Fig. 2C). C/EBP was displaced from GPE1 by increasing amounts of Nrf2/MafK (Fig. 2C, lanes 1-5) and increasing amounts of C/EBP prevented Nrf2/MafK binding (Fig. 2C, lanes 6-10), indicating that these factors bound to GPE1 in a mutually exclusive manner. However, the binding reactions of the mixed factors and GPE1 were not exactly stoichiometric, because the intensities of the shifted bands did not increase with increasing amounts of the competing factors (Fig. 2C, lanes 4, 5, 9, and 10). During the course of these experiments, we found that MafK interacts with C/EBP and inhibits its binding to GPE1 (data not shown). The MafK homodimer also binds weakly to GPE1 (15). These complicated interactions may explain the nonstoichiometric binding to GPE1.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. C/EBP suppresses GST-P expression. Reporter transfection assays of the GST-P promoter are shown. A, the indicated GST-P/luciferase or C/EBP-RE/luciferase constructs were transfected into H4IIE cells, with or without co-transfection with the C/EBP or C/EBP expression vectors (1 µg). C/EBP-RE/50Luc contains three tandem C/EBP binding elements from the mouse transthyretin gene joined to the 50Luc vector. B, a 27-bp GPE1 core sequence was joined to the 50Luc vector (GPE1/50Luc) and transfected into H4IIE cells, with or without co-transfection with the Nrf2, C/EBP, or C/EBP expression vectors, as indicated. C, the GPE1/50Luc construct was transfected into F9 cells, with or without co-transfection with the Nrf2 (1 µg) or C/EBP (1 or 2 µg) expression vectors, as indicated.

In contrast to C/EBP, Nrf2/MafK appeared to bind to all three mutants with higher affinity than to the wild-type GPE1 probe (Fig. 3A). Consistent with the results of the EMSA, the enhancer activities of the mutant GPE1s in H4IIE cells were increased as compared with the wild-type GPE1 in the absence of C/EBP (Fig. 3B). This suggests that the wild-type GPE1 sequence is not optimal for Nrf2/MafK binding. Indeed, Nioi et al. (38) recently reported that the sequence TnACnGTnAGnCnnCA is an optimal binding sequence for Nrf2/MafK, as determined by a point mutation analysis of the antioxidant-responsive element sequence of the mouse NAD(P)H:quinone oxidoreductase 1 gene. Most of the bases in the GPE1 sequence (TCACTATGATTCAGCA) match this sequence; however, the underlined nucleotides at the 6th and 10th positions in the optimal sequence are changed from G to A and T in the GPE1 sequence. The 10th position of the antioxidant-responsive element is critical for enhancer activity, and a G-to-T change at this position greatly reduced NAD(P)H:quinone oxidoreductase 1 expression under constitutive and drug-induced conditions (38). In our results, the enhancer activity of the GPE1-M13 mutant, which had a C residue at the 10th position, was 30 times stronger than that of the wild type (Fig. 3B). This suggests that a C residue, rather than the G of the mouse NAD(P)H:quinone oxidoreductase 1 gene, is the optimal nucleotide at the 10th position of the antioxidant-responsive element sequence.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. C/EBP binds to GPE1. A, EMSA of C/EBP and GPE1. Approximately 50 ng of the C/EBP-MBP fusion protein was incubated with labeled probe (2 x 104 cpm). The probes were the multiple cloning site of pBluescript II vector (MCS), single or double C/EBP binding consensus sequences of the transthyretin gene (C/EBP-RE 1x, 2x), or the GPE1 core sequence (GPE1). In the competition analysis, increasing amounts (10x, 20x, and 50x for lanes 11-13, respectively) of unlabeled GPE1 core sequence were added to the binding mixtures. B, DNase I footprinting analysis of Nrf2/MafK and C/EBP with the GPE1 probe. The indicated proteins (bovine serum albumin, Nrf2/MafK or C/EBP fusion proteins) were incubated with the GPE1 probe and treated with DNase I, as described under "Experimental Procedures." Guanine and adenine residues were modified and digested by the Maxam-Gilbert method (M). The vertical lines and dotted lines indicate the region protected by Nrf2/MafK and C/EBP, respectively. C, the binding of Nrf2/MafK and C/EBP to GPE1 is mutually exclusive. EMSAs were carried out with a fixed amount of C/EBP (10 ng) and increasing amounts of Nrf2/MafK (0-50 ng, lanes 1-5) and a fixed amount of Nrf2/MafK (50 ng) and increasing amounts of C/EBP (0-100 ng, lanes 6-10). GPE1 was used as the probe. The arrows indicate the Nrf2/MafK and C/EBP-GPE1 complexes.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. C/EBP binds to mutated GPE1 and represses its activity. A, EMSA of Nrf2/MafK and C/EBP with wild-type and mutant GPE1s. Recombinant factors were incubated with the indicated probes as described in the legend to Fig. 2. B, reporter transfection analysis of mutated GPE1s. The indicated elements were inserted into the 50Luc vector and transfected into H4IIE cells with (closed bar) or without (open bar) the C/EBP expression vector (upper panel). The ratios of luciferase activity without C/EBP to that with C/EBP are indicated in the lower panel. C, nucleotide sequences of GPE1 and its mutants. Mutated nucleotides are indicated by lowercase letters.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Expression of C/EBP and GST-P in normal liver, preneoplastic liver, and hepatoma cells. A, Northern blotting analysis of C/EBP, C/EBP and GST-P mRNAs. Total RNA (20 µg each) extracted from normal rat liver (Normal), rat liver after 8 weeks of treatment with the Solt-Farber protocol (HN), rat hepatoma cells (H4IIE), or mouse F9 cells (F9) was loaded on an agarose gel containing formaldehyde, electrophoresed, and blotted on to membrane filters. The filters were hybridized with the indicated probes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was analyzed as a control. B, immunofluorescent staining of GST-P and C/EBP in a liver with hyperplastic nodules (x200). Serial liver sections from a rat treated with the Solt-Farber protocol for 8 weeks were stained with an anti-GST-P antibody or an anti-C/EBP antibody.

To further examine the binding of C/EBP to the GPE1 region of the GST-P gene in vivo, we performed a chromatin immunoprecipitation assay using anti-C/EBP, -C/EBP, -Nrf2, and -MafK antibodies (Fig. 5B). We analyzed the chromatin from cells of normal rat livers and livers bearing hyperplastic nodules. The anti-C/EBP antibody precipitated the GST-P GPE1 from the chromatin of normal liver cells but did not precipitate it from the chromatin of cells from liver with hyperplastic nodules. The anti-C/EBP antibody did not precipitate the GST-P GPE1 from the chromatin of normal or hyperplastic cells. In contrast to the anti-C/EBP antibody, the anti-Nrf2 and anti-MafK antibodies precipitated GPE1 from the cells of hyperplastic nodules but not from the normal liver cells (Fig. 5B) (15). The results of chromatin immunoprecipitation analysis suggest that the replacement of the C/EBP transcription factor with Nrf2/MafK on GPE1 is a critical event for the induction of the GST-P gene during hepatocarcinogenesis.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. C/EBP binds to GPE1 in normal liver chromatin. A, GPE1 binds to a nuclear extract from normal rat liver but not from H4IIE hepatoma cells. Approximately 10 µg of protein from nuclear extracts were incubated in the absence (-) or presence of normal rabbit IgG (IgG) or anti-C/EBP (C/EBP) antibodies and subjected to an EMSA as described under "Experimental Procedures." B, chromatin immunoprecipitation analyses of chromatin from normal rat liver (Normal Liver) and from liver bearing hyperplastic nodules (HN) were performed with anti-C/EBP, -C/EBP, -Nrf2, and -MafK, antibodies. The DNAs were extracted from total sonicated nuclei (Input), precleared with protein A-Sepharose (-), and incubated with indicated antibody, or rabbit preimmune serum (Preimmune). The GPE1 region was amplified by PCR (33 cycles) using the GST-P GPE1 primers, as described under "Experimental Procedures."

GST-P expression was analyzed in C/EBP-ER-expressing cells treated with or without -estradiol (Fig. 6, B and C). -Estradiol treatment of the cells decreased GST-P mRNA to 25% of the untreated level in clone 13 transformant and to 50% of the untreated level in the other clones. Western blotting analysis showed that the expression of GST-P was suppressed to 30% (clone 11), 40% (clone 21), 50% (clone 13), and 70% (clone 23) of the levels in untreated cells. Consistent with reporter transfection analysis (Fig. 6A), GST-P expression was not completely inhibited in the -estradiol-treated C/EBP-ER-expressing cells. Although suppression of GST-P expression in the C/EBP-ER expressing hepatoma cells was not as effective as in the normal liver cells, these results clearly indicated that C/EBP suppressed GST-P expression in vivo.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The rat GST-P gene is completely silenced in normal liver but is highly and specifically induced during hepatocarcinogenesis. GPE1 is the major enhancer element essential for the activation of the GST-P gene in early stage hepatocarcinogenesis. We recently found that the Nrf2/MafK heterodimer binds GPE1 and activates GST-P expression during this process (15). In the present study, we identify C/EBP as a suppressor of GPE1 in normal liver. Reporter transfection analysis indicates that C/EBP strongly inhibits GPE1-mediated gene expression in hepatoma cells (Fig. 1). In vitro binding analyses shows that C/EBP strongly binds to the 3'-half of GPE1 and that this region overlaps with the binding site of Nrf2/MafK (Fig. 2). The binding of C/EBP to mutated forms of GPE1 correlates with its suppression of activity in these mutants, suggesting that the binding of C/EBP to GPE1 is necessary for suppression (Fig. 3). Furthermore, the expression of C/EBP is specifically suppressed in GST-P-positive foci (Fig. 4). This result is supported by a chromatin immunoprecipitation analysis that shows that C/EBP is bound to GPE1 in the chromatin of normal liver but not in carcinogen-treated preneoplastic liver (Fig. 5). Furthermore, the C/EBP-ER system clearly shows that the activation of C/EBP activity by -estradiol suppresses endogenous GST-P expression in hepatoma cells (Fig. 6). All of these results indicate that C/EBP acts as a negative regulator of GST-P expression in normal liver through the GPE1 enhancer element.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Activation of C/EBP suppressed GST-P expression in C/EBP-ER-expressing hepatoma cells. A, trans-suppression of GST-P/luciferase reporter gene in C/EBP-ER-expressing cells. RL34 and C/EBP-ER clones (clones 11, 13, 21, and 23) were transfected with the C/EBP-RE/50Luc (left panel) or GST/ELuc reporter genes (right panel) and treated with (closed bars) or without (open bars) -estradiol (Est,1 µM, 24 h), as described under "Experimental Procedures." B, RNase protection analysis of GST-P mRNA in C/EBP-ER-expressing cells. RL-34 parent cells and C/EBP-ER clones were treated with (+) or without (-) -estradiol (1 µM, 48 h) and 10 µg (GST-P) or 2.5 µg (GAPDH) of total RNA were analyzed by RNase protection analysis. C, Western blotting analysis of GST-P and C/EBP-ER. The indicated cells were treated with (+) or without (-) -estradiol for 48 h and 10 µg of total protein were analyzed by Western blotting with anti-GST-P antibody (upper panel). C/EBP-ER was detected by anti-C/EBP antibody in RL34 and C/EBP-ER clones (lower panel).

Previously, it has been reported that the mechanism of transcriptional inhibition by C/EBP does not require direct DNA binding but rather is mediated by protein-protein interactions with positive transcription factors, including E2F (21, 22, 37). Our preliminary EMSA results suggest that C/EBP interacts with MafK, preventing binding to the C/EBP consensus element (data not shown). Thus, the possibility that these protein-protein interactions results in the transcriptional suppression of the GST-P gene cannot be ruled out.

C/EBP is a family of transcription factors that plays an important role in the growth and differentiation of many cell types and tissues (19, 43). C/EBP is expressed at high levels in liver and maintains liver quiescence (20) by direct interaction with cdk2 and cdk4 or by interacting with E2F-DP, which activates several genes required for cell cycle progression (21, 22). These findings are consistent with the induction of GST-P in the preneoplastic foci of the liver. Chemical carcinogens somehow shut down the expression of C/EBP, releasing C/EBP from the GPE1 region of the GST-P gene and enabling Nrf2/MafK to bind to GPE1 and induce GST-P expression. Simultaneously, suppression of C/EBP leads to progression of the cell cycle. On the other hand, Nrf2 is known to be an essential transcription factor for phase II detoxifying enzymes activated by xenobiotics (44). In the steady state, Nrf2 is retained in the cytoplasm by specific binding with Keap1 (Kelch-like ECH-associated protein 1), a key cytoplasmic mediator of Nrf2 activation. Upon induction by xenobiotics such as chemical carcinogens, Nrf2 is released from Keap1 and translocates into the nucleus, where it induces a group of detoxifying enzyme genes (45, 46). It is known that GST-P is not induced by the majority of chemical carcinogens in normal rat liver but that in hepatoma cells xenobiotic drugs markedly stimulate GST-P expression (15). It is possible that GST-P expression cannot be activated by the Keap1-Nrf2 pathway when it is completely repressed by C/EBP and silencer functions in normal liver; however, in hepatoma cells, GST-P expression is derepressed and can be further stimulated by Nrf2 activation. Similarly, in the carcinogen-treated liver foci, the GST-P gene is induced by a dissociation of C/EBP from GPE1, allowing the Keap1-Nrf2 pathway to reinforce strong GST-P expression. Our findings are very interesting because they show that the expression of a tumor maker gene is directly co-regulated with genes that relate to cellular proliferation and transformation.

	FOOTNOTES

 
^* This work was supported by grants-in-aid from the Saitama Medical School Research Center for Genomic Medicine, Ministry of Education, Science, Sports and Culture of Japan, and the YASUDA Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Health Sciences, Hokkaido University School of Medicine, N12, W5, Kita-Ku, Sapporo 060-0812, Japan. Tel.: 81-11-706-3407; Fax: 81-11-706-3407; E-mail: m_sakai{at}med.hokudai.ac.jp.

² The abbreviations used are: GST-P, placental (Pi class) glutathione S-transferase of the rat; GPE1, GST-P enhancer 1; Nrf2, NF-E2 p45-related factor 2; C/EBP, CCAAT enhancer binding protein; EMSA, electrophoretic mobility shift assay; MBP, maltose-binding protein; PMSF, phenylmethylsulfonyl fluoride.

	ACKNOWLEDGMENTS

 
We thank Dr. K. Satoh (Hirosaki University) for providing rats treated with the Solt-Farber protocol and the anti-GST-P antibody. We also thank Drs. A. D. Friedman (Johns Hopkins University), K. Igarashi (Hiroshima University), M. Takiguchi (Chiba University), and K. Uchida for providing us with pBabePuro-C/EBP-ER, the anti-MafK antibody, the C/EBP expression vectors, and RL34 cells, respectively.

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