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Correspondence to:
John Y. L. Chiang
Fibrates are widely used hypolipidemic drugs that regulate the expression of many genes involved in lipid metabolism by activating the peroxisome proliferator-activated receptor
These results suggest that PPAR
Supplementary key words:
bile acid synthesis, gallstones, peroxisome proliferators, hypolipidemic drugs, cytochrome P450, HNF-4
Fibrates are extensively used drugs in the treatment of hyperlipidemia, a risk factor for the development of coronary heart diseases (1). This class of compounds exerts its effect on plasma lipids by altering the expression of genes involved in lipid metabolism through activation of PPAR (2). Upon activation by ligands, such as fatty acids, eicosanoids, and hypolipidemic drugs, PPAR forms a heterodimer with RXR
Of clinical importance is the fact that treatment with fibric acid derivatives leads to cholesterol saturation of the bile resulting in an increased risk of gallstone formation (8). Fibrates reduce bile acid synthesis, presumably by inhibition of cholesterol 7
The CYP7A1 expression is restricted to the liver and its liver-specific promoter contains functional binding sites for the liver-enriched transcription factors, HNF-3, HNF-4, and COUP-TFII (16) (17). The orphan nuclear receptor HNF-4 binds as a homodimer to the DR-1 without ligand binding (18). Previous study has shown that HNF-4 regulates CYP7A1 transcription through a completely conserved DR-1 at nt -146 to -130 of the rat CYP7A1 promoter, which was also mapped as a bile acid response element (BARE) (16) (19). We have proposed a receptor;–mediated mechanism for down-regulation of CYP7A1 transcription by bile acids (9). Recently, orphan nuclear receptor FXR has been identified as a bile acid-activated receptor, which mediates the repression of CYP7A1 transcription by bile acids (20) (21) (22) (23). However, the mechanism by which FXR down-regulates CYP7A1 transcription is not known. Interaction of transcription factors bound to the BARE may explain the negative regulation of CYP7A1 transcription by physiological regulators such as bile acids, fibrates, insulin, and phorbol esters. The goal of this study was to identify the cis-acting elements conferring the inhibitory effect of PPAR
Plasmids
Transfection assays
Electrophoretic Mobility Shift Assays (EMSA)
Quantitative Western immunoblot analysis
Mapping of PPAR
PPAR
HNF-4 protein expression level is reduced by Wy14,643 treatment in HepG2 cells
Suppression of CYP7A1 promoter activity by Wy14,643 is a liver-specific effect
In this study, we showed that the potent peroxisome proliferator Wy14,643 suppressed CYP7A1 expression in HepG2 cells. The inhibition of CYP7A1 transcription by Wy14,643 explains the reduced cholesterol 7
Our results show that the suppression of CYP7A1 promoter activity by Wy14,643 and PPAR
Screening of fibrates for their minimal effects on cholesterol 7
This research project was supported by National Institutes of Health grants GM-31584 and DK-44442.
Manuscript received June 17, 1999; and in revised form December 23, 1999
Abbreviations:
CYP7A1, cholesterol 7
Copyright © 2000 by Lipid Research, Inc.
Original Article
Peroxisome proliferator-activated receptor
Maria Marrapodia and
John Y. L. Chianga
(PPAR) and agonist inhibit cholesterol 7-hydroxylase gene (CYP7A1) transcription
a Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, P.O. Box 95, Rootstown, OH 44272-0095
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PPAR). The objective of this study was to investigate the mechanism of action of peroxisome proliferators and PPAR on the transcription of cholesterol 7-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. When cotransfected with the expression vectors for PPAR and RXR, Wy14,643 reduced human and rat cholesterol 7-hydroxylase gene (CYP7A1)/luciferase reporter activities by 88% and 43%, respectively, in HepG2 cells, but not in CV-1 or CHO cells. We have mapped the peroxisome proliferator response element (PPRE) to a conserved sequence containing the canonical AGGTCA direct repeats separated by one nucleotide (DR1). This DR1 sequence was mapped previously as a binding site for the hepatocyte nuclear factor 4 (HNF-4) which stimulates CYP7A1 transcription. Electrophoretic mobility shift assay (EMSA) showed no direct binding of in vitro synthesized PPAR/RXR heterodimer to the DR1 sequence. PPAR and Wy14,643 did not affect HNF-4 binding to the DR1. However, Wy14,643 and PPAR/RXR significantly reduced HNF-4 expression in HepG2 cells. and agonist repress cholesterol 7-hydroxylase activity by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore attenuates the transactivation of CYP7A1 by HNF- 4.—Marrapodi, M., and J. Y. L. Chiang. Peroxisome proliferator-activated receptor (PPAR) and agonist inhibit cholesterol 7-hydroxylase gene (CYP7A1) transcription. J. Lipid Res. 2000. 41: 514;–520.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which binds to the peroxisome proliferator response element (PPRE) and modulates gene transcription (3). The PPRE consists of a direct repeat of the canonical AGGTCA sequence separated by one base pair (bp) (2). Three PPAR isoforms, , 
, and 
have been identified (4). PPAR is the predominant form in the liver and plays a pivotal role in regulation of lipid metabolism. Disruption of PPAR in mice caused a defect in lipid and lipoprotein metabolisms (5) (6) (7). -hydroxylase, the rate-limiting enzyme in the catabolic pathway of cholesterol to bile acids in the liver (9). Cholesterol 7-hydroxylase (CYP7A1) is regulated mainly at the transcriptional level by factors including negative feedback by bile acids, insulin, and phorbol esters (10) (11). Bezafibrate has been shown to reduce the activity of cholesterol 7-hydroxylase in rodents (12) (13), as well as in gallstone (14) and hyperlipoproteinemia patients (15). Interestingly, development of supersaturated bile is prevented when fibrate therapy is combined with the bile acid sequestrant, cholestyramine (15). This suggests that increased expression of CYP7A1 activity by cholestyramine treatment may increase bile acids or decrease cholesterol in bile. and agonist on CYP7A1 transcription. We show that peroxisome proliferators regulate CYP7A1 at the transcriptional level by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore interfering with transactivation of CYP7A1 by HNF4.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Construction of human CYP7A1/Luc reporter genes has been described previously (11). The rat CYP7A1/Luc mutant plasmids and the HNF-4 expression vector pCMV-HNF-4, were generated as described previously (16). The rat acyl-CoA oxidase gene (AOX) from nucleotides -1198 to -463 contains a well-characterized PPRE and was obtained by polymerase chain reaction (PCR) amplification of rat genomic DNA (24). PCR primer pair, 5'-ATAG GTACCATCCCCAGTAGAACCTTGTTCAGG-3' and 5'-ATGAG ATCTTTCAGGGTCTCGGGCGGAGTGAAG-3', was used to generate a 736 bp fragment. The reporter gene plasmid AOX-1198/-463 was constructed by fusing the PCR fragment upstream to the SV40 promoter in pGL3-Promoter (Promega, Madison, WI). The expression plasmid for human PPAR, pcDNA3-hPPAR, was constructed by subcloning the human PPAR cDNA (Dr. F. J. Gonzalez, NCI, Bethesda, MD) into pcDNA3 vector (Invitrogen, Carlsbad, CA). The expression plasmid for the retinoid X receptor, pCMX-RXR, was obtained from Dr. R. Evans (Salk Institute, La Jolla, CA).
HepG2, CHO, and CV-1 cells, grown in 12-well tissue culture plates to 100% and 70% and 70% confluence, respectively, were transfected with the chimeric gene constructs by the calcium phosphate co-precipitation method as described previously (25). In cotransfection experiments, expression plasmids for PPAR and RXR (0.5 µg each) or an equal amount of the empty vector were co-transfected with 2.5 µg of the test plasmid. After transfection, cells were treated with 100 µM Wy14, 643 or an equal volume of ethanol as the vehicle. Cells were harvested 42 h after treatment and luciferase activity was determined with the luciferase assay kit (Promega) using a Lumat LB9501 luminometer (Berthold System, Inc., Pittsburgh, PA). Luciferase activities were normalized for transfection efficiency by dividing relative light units by ß-galactosidase activity expressed from cotransfected pCMVß plasmid. Transfection assays were performed in triplicate and the results were analyzed for statistical significance by Student's t-test using Sigma plot software (Jandel Scientific, San Rafael, CA). Each experiment was repeated at least twice.
PPAR, RXR, and HNF-4 were synthesized in vitro by programming transcription/translation (TNT) system (Promega) with the expression vectors pcDNA3-hPPAR, pCMX-RXR, and pCMV-HNF4, respectively. Synthetic oligonucleotides carrying the 5' overhang GATC were annealed by heating at 100°C in 2x SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) and slowly cooling to room temperature. The double-stranded oligonucleotides were labeled by filling-in with [ 32P]dCTP using the Klenow fragment of DNA polymerase I. In vitro translated proteins were pre-incubated for 15 min on ice in a buffer containing 12 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 15% glycerol, and 1 µg poly (dI-dC). After incubation for 20 min at room temperature with 75,000 cpm of labeled probe, the DNA/protein complexes were separated on a 4% native polyacrylamide gel in 0.5 x TBE running buffer, at 200 V for 2 h at room temperature. One µl of an antibody directed against PPAR (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture. Results were quantified with IP Lab Gel software (Signal Analytics, Corp., Vienna, VA) in conjunction with a Molecular Dynamics PhosphoImager 445Si (Sunnyvale, CA).
Confluent HepG2 cells were transfected with the expression vectors for PPAR/RXR or the empty vector pcDNA3 as a control. These cells were then treated with 100 µM Wy14,643 or the vehicle ethanol. Nuclear extracts were isolated from HepG2 cell preparations according to Dent and Latchman (26). Ten µg of protein was resolved by 10% SDS-PAGE and electroblotted onto nitrocellulose filters. The membranes were blocked in non-fat dry milk at 4°C overnight and then incubated with anti-HNF4 antisera (Dr. F. Sladek, University of California, Riverside, CA) for 6 h at 4°C. After washing with PBS and 0.05% Tween-20, the blot was incubated with the horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, washed again, and HNF-4 was detected with enhanced chemiluminacent ECL kit (Amersham Life Science, Arlington Heights, IL). Filters were then washed and blocked again. Antibody against Sp1 was used to detect Sp1 protein as an internal standard. X-ray films were analyzed with the Kodak digital science 1D 2.0.2 software and the intensity of HNF-4 band was normalized by dividing the Sp1 band intensity. Data from three independent nuclear extract preparations were analyzed. Statistical significance between two treatments of three independent experiments was analyzed using ANOVA procedure (SAS software).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
response element in CYP7A1 promoter activity
It has been established that fibrate treatment in rodents (12) (13) and humans (14) (15) results in a decrease of cholesterol 7-hydroxylase activity by 65%. To study the effect of PPAR and agonist Wy14,643 on CYP7A1 transcription, transient transfection assays in HepG2 cells were performed. Wy14,643 (100 µM) alone and over-expression of PPAR/RXR alone reduced human CYP7A1/luciferase reporter (ph-3025luc) activity by about 40% and 60%, respectively ( Fig 1). The combination of both Wy14,643 and PPAR/RXR reduced the promoter activity by 90%. These results suggested that endogenous PPAR might be activated by Wy14,643 and PPAR/RXR suppressed CYP7A1 transcription. As expected, the reporter activity of the positive control plasmid AOX-1198/463 was stimulated 2-fold by Wy14,643. PPAR and Wy14,643 strongly stimulated this reporter activity by about 11-fold (Fig 1). The 5' deletion mutants of human CYP7A1/Luc were transfected in HepG2 cells to map the PPRE. As shown in Fig 1, deletion of nucleotides between -150 and -136 significantly reduced the inhibitory effect of Wy14,643 and PPAR. This result suggest that a negative PPRE is localized between -150 and -136. This region includes the DR1, TGGACTtAGTTCA ( Fig 2). Fig 2 shows the alignment of rat and corresponding human nucleotide sequences in this region. Three AGGTCA-like sequences form overlapping DR1 and DR5 motifs, which have been mapped as an HNF-4 and RXR/RAR binding site in the rat CYP7A1 promoter, respectively (17). As the DR-1 motif is perfectly conserved in the rat and human CYP7A1 promoter (Fig 2), we also tested the effect of Wy14,643 on the wild-type rat CYP7A1/luciferase chimeric plasmid, p-376luc, and mutant plasmids with mutations in AGGTCA-like sequences (Fig 2 and Fig 3). Overexpression of PPAR/RXR in the presence of Wy14,643 suppressed the activity of the wild-type rat p-376luc construct by 50%. Mutations in the 5' half-site of the DR1 (m-146/-141) reduced the basal reporter activity (1.7 x 106 RLU/ß-gal vs. 4.8 x 106 of the wild-type reporter). The T at position 1 of the 5' half-site TGGACT has been shown to be more specific for HNF-4 than for PPAR binding (27). This mutant reporter did not respond to Wy14,643. On the other hand, the plasmid m-139/-132, which has mutations in the 3' half-site of the DR1 or 5' half-site of the DR5, had the lowest basal activity (0.43 x 106 RLU/ß-gal) among wild-type and three mutant reporters, but was responsive to Wy14,643. Previously, we showed that mutations in the 3' half-site of the DR-1 allowed some, although weakened, interaction with HNF-4 (28). This may explain the observed inhibitory effect of Wy14,643 on m-139/-132. The RXR/RAR heterodimer only binds to the rat CYP7A1 promoter and competes with HNF-4 for binding to a partially overlapped consensus sequence (16). As previously reported, plasmid m-129/-123, which has mutations in the 3' half-site of the DR5, showed higher basal activity when compared with the wild-type plasmid (8.8 x 106 RLU/ß-gal vs. 4.8 x 106). This is expected as the mutation disrupted RXR/RAR binding site but allowed HNF-4 to bind to the intact DR-1. Interestingly, abolishing the RXR/RAR binding site in plasmid m-129/-123 enhanced the down-regulation by Wy14,643. This mimics the stronger inhibitory effect of Wy14,643 on the human CYP7A1 promoter, which lacks the RXR/RAR binding site. These data provided strong evidence for mapping the PPRE to the DR1 sequence of the CYP7A1 promoter.
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Figure 1.
Effects of Wy14,643 and PPAR /RXR on reporter activities of human CYP7A1/luciferase chimeric constructs in transient transfection assays in HepG2 cells. Confluent HepG2 cells were transfected with the human CYP7A1/Luc reporter genes and the expression vectors for PPAR and RXR as described under Experimental Procedures. The AOX-1198/-463 plasmid was used as a positive control. Results are expressed as the percent of reporter activity in HepG2 cells cotransfected with reporter plasmid and the empty vector pcDNA3 and incubated with the vehicle ethanol. Data presented are representative of three independent experiments each performed in triplicate. Symbols (*, #) indicate statistically significant difference in reporter activity between two constructs. Basal reporter activities (RLU/ß-gal) for the chimeric constructs were 2.2, 7.9, 3.0, 3.7, 6.0, 1.1, 0.49, 0.34, and 0.21 x 106 for ph-3025Luc, ph-1887Luc, ph-785Luc, ph-372Luc, ph-298Luc, ph-150Luc, ph-135Luc, ph-80Luc, and pGL3basic, respectively. Error bar indicates standard error of the mean (SEM) from three determinations.
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Figure 2.
Alignment of the rat and human CYP7A1 promoter in the region under study. The canonical AGGTCA sequences are represented by bold characters and half-sites of direct repeats are underlined. X indicates a non-conserved nucleotide between the rat and human CYP7A1 sequence. Arrows point to nuclear receptors binding sites. Relevant sequences in the rat CYP7A1/luciferase mutants used in transfection assays in Fig 3 are also shown (mutations indicated in lower case).
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Figure 3.
Effect of Wy14,643 and PPAR /RXR on reporter activities of the wild-type and mutant rat CYP7A1/luciferase chimeric gene constructs. The wild-type rat CYP7A1/luciferase chimeric reporter plasmid p-376Luc and its mutant constructs were transfected in confluent HepG2 cells. Results (filled bars) are expressed as percent of the reporter activity in HepG2 cells cotransfected with PPAR/RXR plasmids versus that with the empty vector pcDNA3 and treated with vehicle ethanol. Data are representative of three independent experiments each performed in triplicate. Basal promoter activities (RLU/ß-gal) for the chimeric constructs were 4.8, 1.7, 0.43, 8.8, and 0.53 x 106 for p-376luc, m-146/-141luc, m-139/-132luc, m-129/-123luc, and pGL3basic, respectively. On the left, AGGTCA half-sites are represented by a box. The shaded boxes represent DR1 motif. A box with a line crossed indicates a half-site with mutations as indicated in Fig 2. The (*) indicates reporter activity significantly different from the control (100%). Error bar indicates SEM./RXR does not bind to the human and rat DR-1
Electrophoretic mobility shift assay (EMSA) was used to determine whether the effect of PPAR on CYP7A1 was due to direct binding of PPAR/RXR to the DR1 region, which is the preferred binding site for PPAR. As a positive control, the AOX-PPRE probe bound in vitro synthesized the PPAR/RXR heterodimer but not PPAR or RXR alone ( Fig 4 A). An antibody against PPAR was able to super-shift the PPAR/RXR/DNA complex (Fig 4a). PPAR/RXR did not bind to the human hCYP7A1 -157/-127 or the rat rCYP7A1 -149/-128 probe (Fig 4a and Fig b). Addition of Wy14,643 did not have any effect on EMSA. The hCYP7A1 -157/-127 probe bound the in vitro synthesized HNF-4 (Fig 4a). Incubation of the hCYP7A1 -157/-127 probe with the in vitro synthesized PPAR/RXR did not affect HNF-4 bound to the probe, both in the presence and in absence of the ligand Wy14,643 (Fig 4a). These results clearly demonstrated that PPAR/RXR did not bind to the functional PPRE in the CYP7A1 promoter and did not compete with HNF-4 for the same binding site.
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Figure 4.
EMSA of PPAR /RXR binding to the human and rat CYP7A1 probes. A: EMSA using 32P-labeled oligonucleotide probe of the human CYP7A1 sequences from -157 to -127 (hCYP7A1-157/-127) and in vitro synthesized PPAR, RXR, and HNF-4. Where indicated Wy14,643 (100 µM) was added to the binding reaction. The AOX-PPRE probe containing a known PPRE was the positive control for binding of PPAR/RXR. DNA/protein complexes are indicated by an arrow. B: EMSA using 32P-labeled oligonucleotide probe of the rat CYP7A1 sequences from -149 to -128 (rCYP7A1-149/-128), and PPAR and RXR. An antibody against PPAR was used to super-shift the AOX-PPRE/protein complex. Nucleotide sequences of oligonucleotide probes used are shown on the bottom. Overhanging sequences are in lower case. DR-1 sequence is represented by bold characters and half-sites are underlined.
To further investigate the mechanism by which PPAR represses CYP7A1 transcription, we studied the effect of Wy14,643 and PPAR on the expression of HNF-4 in HepG2 cells. Western immunoblot assay was used to measure HNF-4 expression levels in HepG2 cells. Fig 5 shows that treatment with Wy14,643 and overexpression of PPAR/RXR reduced HNF-4 levels in nuclear extracts isolated from HepG2 cells. Wy14,643 also reduced HNF-4 levels in control cells transfected with the pcDNA3 empty vector. Table 1 summarizes the quantitative analysis of three independent experiments. Wy14,643 or PPAR/RXR overexpression reduced HNF-4 levels to 59% and 69% of the control, respectively. Addition of Wy14,643 significantly repressed the HNF-4 levels to 42% (P < 0.0085) in HepG2 cells overexpressed with PPAR/RXR. PPAR overexpression also reduced HNF-4 levels in HepG2 cells treated with Wy14,643 (P < 0.0528). These results clearly demonstrated that modulation of HNF-4 protein expression might be a mechanism for the suppression of CYP7A1 transcription by PPAR and Wy14,643.
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Figure 5.
Western immunoblot analysis of the effect of PPAR on HNF-4 protein levels in HepG2 cells. A representative autoradiograph of Western immunoblot performed with nuclear extracts from confluent HepG2 cells transfected with the expression vectors for PPAR/RXR or the empty vector pcDNA3 and incubated with Wy14,643 (100 µM) or the vehicle ethanol is shown. Arrows indicate the HNF-4 and the internal standard Sp1 detected with antibodies. Quantitative analysis of the band intensity is shown in Table 1.
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Table 1.
Quantitative analysis of HNF-4 protein levels in HepG2 cells
In order to determine whether the down-regulation of CYP7A1 by fibrates is a phenomenon restricted to the liver, we tested the effect of Wy14,643 on several non-hepatic cell lines. As shown in Fig 6 A, Wy14,643 (100 µM) stimulated human p-372/Luc reporter activity by 20% in CHO and did not have any effect in CV-1 cells. Cotransfection with increasing amounts of PPAR/RXR heterodimer (0.1 to 0.5 µg) and Wy14,643 (100 µM) stimulated reporter activity by 20 to 40% in CHO cells (Fig 6b). Therefore, liver-specific factors important for regulation of CYP7A1 transcription must be present to mediate the negative effect of Wy14,643 and PPAR/RXR.
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Figure 6.
Effect of Wy14,643 and PPAR /RXR on CYP7A1 promoter activity in transient transfection assays using HepG2 and non-hepatic cells. A: HepG2, CHO, and CV-1 cells were transfected with the human reporter construct ph-372Luc and incubated for 42 h with Wy14,643 or the vehicle ethanol. B: CHO cells were cotransfected with ph-372Luc and increasing amounts of the expression vectors for PPAR and RXR (0.1 µg, 0.25 µg, and 0.5 µg each vector, respectively) and treated with Wy14.643 or the vehicle ethanol. The (*) indicates statistically significant difference from the control (100%).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase activity in patients treated with fibrates, resulting in diminished excretion of bile acids and consequently increased incidence of cholesterol gallstones formation. Peroxisome proliferators have been shown to activate many genes involved in lipid metabolism through transcriptional activation mediated by binding of PPAR to PPREs in the gene promoter. This mechanism involves heterodimerization of PPAR with RXR and ligand-dependent recruitment of co-activators (29). However, the mechanism for suppression of genes by peroxisome proliferators is less understood. Several mechanisms have been proposed which includes competition with HNF-4 for binding site, suppression of HNF-4 expression (30) (31), and up-regulation of the repressor Rev-erb (32). Our mutagenesis analysis of the human and rat CYP7A1 promoter in transfection assays showed that the PPRE co-localizes with a functional HNF-4 binding site (DR1). Mutations in this conserved sequence reduced the basal promoter activity and abolished the inhibitory effect of not only PPAR but also bile acids and phorbol esters (25). This indicates that HNF-4 is required in maintaining basal level expression of the CYP7A1 as well as in mediating the response to physiological regulators. The DR-1 elements are quite promiscuous in allowing the binding of RXR homodimer, PPAR/RXR and RAR/RXR heterodimers, HNF-4, and COUP-TFII (27). However, PPAR/RXR did not bind to the DR-1 sequence of the rat and human CYP7A1 promoter and did not interfere with the binding of HNF-4. A reduction of HNF-4 protein levels in HepG2 cell nuclear extracts upon treatment with Wy14,643 suggests that one possible mechanism for down-regulation of CYP7A1 transcription by fibrates is through reduced availability of HNF-4 for binding to the DR1, hence reduced CYP7A1 expression. This mechanism has been suggested for the repression of HNF-4-activated liver genes, apolipoprotein C-III, and transferrin by PPAR (30) (31). Although functional PPRE has not been identified in the promoter of HNF-4, it is known that HNF-4 regulates its own synthesis (33). It is possible that the ligand-activated PPAR may compete with HNF-4 for the same binding site on the HNF-4 promoter and that may result in the reduced synthesis of HNF4 protein in liver cells. Thus, reduction of HNF-4 expression and interference with HNF-4 function by PPAR explains the pleiotropic effect exerted on liver gene expression by hypolipidemic peroxisome proliferators. is restricted to HepG2 cells. Overexpression of PPAR/RXR caused a small but significant increase of CYP7A1 reporter activity in CHO cells. Therefore, liver-enriched transcription factors must be involved in the suppression of CYP7A1 transcription by peroxisome proliferators. This again supports our conclusion that HNF4 is involved in PPAR-mediated repression of CYP7A1 transcription. -hydroxylase gene expression may be considered for reducing the risk of developing cholesterol gallstone disease in hyperlipidemic patients under fibrates therapy. A better understanding of the basic mechanisms by which fibrates affect the expression of genes involved in lipid metabolism will allow the development of more rational lipid-lowering strategies with enhanced specificity and reduced side effects.
ACKNOWLEDGMENTS -hydroxylase; CYP7A1, cholesterol 7-hydroxylase gene; PPAR, peroxisome proliferator activated receptor ; PPRE, peroxisome proliferator response element; HNF-4, hepatocyte nuclear factor 4; DR; direct repeat; bp, base pair; nt, nucleotide; Luc, luciferase; AOX, acyl-CoA oxidase; EMSA, electrophoretic mobility shift assay, BARE, bile acid response element; PCR, polymerase chain reaction
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and . Proc. Natl. Acad. Sci. USA. 94:4312-4317-deficient mice. J. Biol. Chem. 272:27307-27312-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat. J. Biol. Chem. 266:3416-3421-hydroxylase gene. CYP7A in(HepG2 cells. J. Lipid Res. 37):1831-1841.-hydroxylase gene (CYP7A) by nuclear hormone receptors. J. Lipid Res. 39:2192-2200-hydroxylase gene. J. Biol. Chem. 272:3444-3452-hydroxylase gene (CYP7A). Am. J. Physiol. 273:G508-G517-hydroxylase gene (CYP7A) by nuclear hormone receptor bound to the bile acid response elements (BARE). In Bile Acids and Cholestasis. G. Paumgartner, A. Stiehl, W. Gerok, D. Keppler, and U. Leuschner, editors. Kluwer Academic Publishers, Norwell, MA. 51;–58.-hydroxylase gene. CYP7A J.(Lipid Res. 36):2419-2432.-hydroxylase gene (CYP7A). J. Lipid Res. 41:1-11
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 G. F. Gbaguidi and L. B. Agellon The inhibition of the human cholesterol 7{alpha}-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor {alpha} and peroxisome proliferator-activated receptor {alpha} heterodimer Nucleic Acids Res., February 11, 2004; 32(3): 1113 - 1121. [Abstract] [Full Text] [PDF]
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O. Barbier, D. Duran-Sandoval, I. Pineda-Torra, V. Kosykh, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-activated Receptor {alpha} Induces Hepatic Expression of the Human Bile Acid Glucuronidating UDP-glucuronosyltransferase 2B4 Enzyme J. Biol. Chem., August 29, 2003; 278(35): 32852 - 32860. [Abstract] [Full Text] [PDF]
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S. L. Ripp, K. C. Falkner, M. L. Pendleton, V. Tamasi, and R. A. Prough Regulation of CYP2C11 by Dehydroepiandrosterone and Peroxisome Proliferators: Identification of the Negative Regulatory Region of the Gene Mol. Pharmacol., July 1, 2003; 64(1): 113 - 122. [Abstract] [Full Text] [PDF]
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D. Jung, M. Fried, and G. A. Kullak-Ublick Human Apical Sodium-dependent Bile Salt Transporter Gene (SLC10A2) Is Regulated by the Peroxisome Proliferator-activated Receptor alpha J. Biol. Chem., August 16, 2002; 277(34): 30559 - 30566. [Abstract] [Full Text] [PDF]
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 S. M. Post, H. Duez, P. P. Gervois, B. Staels, F. Kuipers, and H. M.G. Princen Fibrates Suppress Bile Acid Synthesis via Peroxisome Proliferator-Activated Receptor-{alpha}-Mediated Downregulation of Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase Expression Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1840 - 1845. [Abstract] [Full Text] [PDF]
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R. A. Memon, A. H. Moser, J. K. Shigenaga, C. Grunfeld, and K. R. Feingold In Vivo and in Vitro Regulation of Sterol 27-Hydroxylase in the Liver during the Acute Phase Response. POTENTIAL ROLE OF HEPATOCYTE NUCLEAR FACTOR-1 J. Biol. Chem., August 3, 2001; 276(32): 30118 - 30126. [Abstract] [Full Text] [PDF]
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