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J Biol Chem, Vol. 275, Issue 17, 12530-12536, April 28, 2000


The Murine and Human Cholesterol 7alpha -Hydroxylase Gene Promoters Are Differentially Responsive to Regulation by Fatty Acids Mediated via Peroxisome Proliferator-activated Receptor alpha *

Sukhinder K. CheemaDagger and Luis B. Agellon§

From the Medical Research Council Group on Molecular and Cell Biology of Lipids and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We determined if fatty acids can regulate the murine Cyp7a1 and human CYP7A1 gene promoters via peroxisome proliferator-activated receptor alpha  (PPARalpha )/9-cis-retinoic acid receptor alpha  (RXRalpha ). In transfected cells, the murine Cyp7a1 gene promoter displayed markedly lower basal activity, but greater sensitivity to fatty acid- or WY 14,643-activated PPARalpha /RXRalpha when compared with the human CYP7A1 gene promoter. PPARalpha /RXRalpha can bind to a site (Site II) located within the region at nucleotides -158 to -132 of both promoters. Mutagenesis of the human CYP7A1 Site II element abolished the response to activated PPARalpha /RXRalpha . The murine Cyp7a1 gene promoter contains an additional PPARalpha /RXRalpha -binding site (Site I) located within nucleotides -72 to -57. Replacement of a single residue in human CYP7A1 Site I with that found in the murine Cyp7a1 Site I sequence enabled PPARalpha /RXRalpha binding, and this mutation resulted in reduced basal activity, but substantially improved the response to activated PPARalpha /RXRalpha in transfected cells. We conclude that fatty acids can regulate the cyp7a gene promoter via PPARalpha /RXRalpha . The differential response of the murine Cyp7a1 and human CYP7A1 gene promoters to PPARalpha activators is attributable to the additional PPARalpha /RXRalpha -binding site in the murine Cyp7a1 gene promoter.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptors (PPARs)1 belong to the superfamily of ligand-activated nuclear hormone receptors and regulate a wide spectrum of genes involved in lipid homeostasis (1-3). The regulation of gene transcription by PPARs involves their heterodimerization with 9-cis-retinoic acid receptor alpha  (RXRalpha ) and binding to specific sequences known as peroxisome proliferator response elements (PPREs) in the proximal regions of target genes (2, 4, 5). PPREs consist of a direct repeat of the nuclear receptor hexameric DNA recognition motif (5'-AGGTCA-3') separated by one nucleotide, a configuration termed as DR1. Fatty acids and fibrates, which are known ligands for PPARalpha (6-11), have been shown to regulate genes involved in cholesterol metabolism such as those encoding apoA-I (12), apoA-II (13), apoC-III (14) and 3-hydroxy-3-methylglutaryl-CoA synthase (15).

The liver plays an important role in maintaining whole body cholesterol homeostasis by regulating the biosynthesis of cholesterol, its uptake from plasma, storage, and catabolism to bile acids (16). The conversion of cholesterol to bile acids in the liver represents an important route by which cholesterol is eliminated from the body. Cholesterol 7alpha -hydroxylase (cyp7a) is the rate-controlling enzyme of the major pathway involved in this process (17). The gene encoding cyp7a has been cloned from the rat (18, 19), mouse (20, 21), human (22, 23), and hamster (24). Several studies have shown that cyp7a gene expression is regulated by a variety of hormonal (25-28) and nutritional (29-36) factors. Although phosphorylation has been suggested to regulate cyp7a (30, 37, 38), the abundance of cyp7a mRNA remains the best predictor of cyp7a enzyme activity.

The proximal promoter region of the cyp7a gene contains sequences that resemble nuclear hormone response elements. It was shown recently that induction of the rat Cyp7a1 gene in response to oxysterols involves the nuclear receptor LXRalpha (39), whereas repression of cyp7a gene transcription by bile acids appears to be mediated via the farnesoid X receptor (40). Other nuclear hormone receptors have also been shown to interact with the cyp7a gene promoter (41-44). Given the number of nuclear hormone receptor-binding sites in the cyp7a gene promoter, it would seem that this class of transcription factors plays a major role in regulating cyp7a gene expression.

We previously demonstrated that dietary fats influence the response of the murine Cyp7a1 gene to dietary cholesterol (35, 36). It is well known that fatty acids regulate gene transcription via PPARalpha /RXRalpha (2, 7). Here we evaluated the role of fatty acids in regulating the cyp7a gene expression and whether PPARalpha /RXRalpha is involved in the process.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning and Construction of Recombinant Plasmids-- Genomic clones containing the human CYP7A1 gene were isolated from a human chromosome 8 library (American Type Culture Collection, Manassas, VA). A genomic clone containing the entire murine Cyp7a1 gene was described previously (21). Gene chimeras containing the proximal promoter region of the human CYP7A1 gene (nt -372 to +61) or the murine Cyp7a1 gene (nt -412 to +77) were generated from the cloned cyp7a genomic genes and linked to the chloramphenicol acetyltransferase (CAT) structural gene sequence in pCAT-Basic (Promega Corp., Madison, WI). The primary structures of the in vitro amplified human CYP7A1 and murine Cyp7a1 gene fragments were confirmed by sequencing. The rabbit antiserum to PPARalpha and plasmids encoding murine PPARalpha and RXRalpha were described previously (45).

Mutant derivatives of the human CYP7A1 gene promoter were constructed using mutagenic primers by in vitro DNA amplification and the plasmid containing the wild-type gene chimera as template. The 3'-half-site of the human CYP7A1 Site II regulatory element was converted into an XhoI site,2 and a point mutation was introduced into the 3'-half-site of the human CYP7A1 Site I element. The primary structures of the mutagenized promoters were verified by sequencing.

Cell Culture and Transfections-- McArdle RH-7777 rat hepatoma cells (46) were maintained in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were plated (60-mm culture dishes) at equal densities 16-18 h prior to transfection and grown in medium containing 20% delipidated fetal calf serum. The cells were transfected when cultures were at 50-60% confluency using the calcium phosphate coprecipitation procedure (47) with a mixture of plasmids that contained 2.5 µg of plasmid pCMV-beta -gal (encoding beta -galactosidase) as a control for transfection efficiency, cyp7a-CAT gene chimeras, and expression vectors encoding PPARalpha and RXRalpha . All samples were complemented with sonicated salmon sperm DNA to equalize the mass of total DNA (20 µg) added to each dish. One hour after the introduction of DNA, the transfected cells were treated with either fatty acids (stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2); Sigma-Aldrich, Oakville, Ontario, Canada) complexed to lipid-free bovine serum albumin (BSA) or WY 14,643 (BIOMOL Research Labs Inc., Plymouth Meeting, PA) dissolved in dimethyl sulfoxide at a final concentration of 100 or 50 µM, respectively. The control cells received either lipid-free BSA or Me2SO alone. In initial experiments, we found that fatty acids dissolved in ethanol were toxic when added to the culture medium of transfected McArdle RH-7777 cells at concentrations >= 100 µM. However, the equivalent concentrations of fatty acids were well tolerated when added to cell culture medium as a complex with BSA and were therefore used in all subsequent experiments. Extracts of transfected cells were prepared 36-48 h after the introduction of DNA and assayed for both CAT and beta -galactosidase activities by standard methods. The CAT reaction products were separated by TLC, and radioactivity of acetylated [14C]chloramphenicol was quantitated using a Fuji BAS1000 phosphoimager. The CAT activity values were normalized to the beta -galactosidase activity, which was not affected by the treatments.

Electrophoretic Mobility Shift Assays-- The murine PPARalpha and RXRalpha proteins were synthesized in vitro using a coupled transcription/translation system (Promega Corp.) with a standard scale of synthesis consisting of 25 µl of reaction mixture and 0.5 µg of DNA template. Fragments produced by restriction enzyme digestion of the human CYP7A1 gene promoter were first tested for binding to PPARalpha /RXRalpha by electrophoretic mobility shift assays. The sequences of the promoter fragments that showed binding to PPARalpha /RXRalpha were analyzed, and regions that bore similarity to the consensus PPRE sequence were further characterized. The following synthetic double-stranded oligonucleotides bear natural sequences from the cyp7a gene promoters and were used in electrophoretic mobility shift assays: nt -150 to -123 of the human CYP7A1 gene (human Site II, 5'-TACCTGTGGACTTAGTTCAAGGCCAGTT-3'), nt -73 to -54 of the human CYP7A1 gene (human Site I, 5'-CTTTGTCAACCAAGCTCAAG-3'), and nt -73 to -55 of the murine Cyp7a1 gene (murine Site I, 5'-CTGGTCACCCAAGTTCAAG-3'). The altered residues in the mutagenized derivatives of these sequences are described in the figure legends. The oligonucleotides were synthesized with a 4-nt 5'-extension (5'-AATT-3') to allow for radiolabeling of annealed oligonucleotides by fill-in reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. Radiolabeled double-stranded oligonucleotides (0.1 pmol, approx 15,000 cpm) were incubated in a total volume of 40 µl for 15 min at 25 °C with 4 µg of poly(dI·dC), 4 µg of BSA, and 1 µl of recombinant PPARalpha and RXRalpha in binding buffer (10 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 7% glycerol, 150 µM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Double-stranded oligonucleotides containing the rat acyl-CoA oxidase PPRE (45) and the rat tyrosine aminotransferase glucocorticoid response element (48) were used as competitors for PPARalpha /RXRalpha binding. In competition experiments, increasing amounts (5-50-fold molar excess) of unlabeled acyl-CoA oxidase PPRE, murine Cyp7a1 Site I, or human CYP7A1 Site I were added to reaction mixtures and incubated at room temperature for 10 min prior to the addition of radiolabeled double-stranded oligonucleotides. After an additional 15 min of incubation at room temperature, DNA-protein complexes were separated by electrophoresis on a 4% polyacrylamide gel in 0.5× Tris borate electrophoresis buffer at 4 °C and visualized by autoradiography or phosphoimaging. In some experiments, 1-2 µl of rabbit anti-PPARalpha antiserum (45) was added after incubation of the radiolabeled probes with the reticulocyte lysates. The reaction was left on ice for an additional 30-60 min prior to electrophoresis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fatty Acids Stimulate the Murine Cyp7a1 and Human CYP7A1 Gene Promoters via PPARalpha /RXRalpha -- We observed that mice consuming a semipurified high fat (20%) diet in the absence of exogenous bile acids had a significantly higher (2-3-fold, p < 0.01) abundance of Cyp7a1 mRNA compared with mice consuming a chow diet (5% fat content). To investigate whether fatty acids directly regulate cyp7a gene transcription through PPARalpha /RXRalpha , gene chimeras containing the murine Cyp7a1 or human CYP7A1 proximal promoter regions linked to the CAT structural gene were transfected into rat hepatoma cells in the absence or presence of plasmids encoding PPARalpha and RXRalpha . In the presence of PPARalpha /RXRalpha , the basal activity of the murine Cyp7a1 gene promoter was significantly lower (p < 0.001) than that of the human CYP7A1 gene promoter (Fig. 1A). Addition of stearic acid (18:0), oleic acid (18:1), or linoleic acid (18:2) complexed to lipid-free BSA to the culture medium at a concentration of 100 µM increased the activities of both the murine Cyp7a1 and human CYP7A1 gene promoters as reflected by the rise in CAT activity in lysates of treated cells (Fig. 1B). These results indicate that fatty acids stimulate the cyp7a gene promoter of both species.


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  		Fig. 1.   Human CYP7A1 and murine Cyp7a1 gene promoters are stimulated by PPARalpha /RXRalpha activators. A, CAT activity in hepatoma cells transfected with equal molar amounts of the human or murine cyp7a-CAT gene chimera (white and black bars, respectively). The CAT activity was normalized to beta -galactosidase activity encoded by a cotransfected beta -galactosidase expression vector. The results shown represent the means ± S.E. of three independent experiments performed in triplicate. B, response of the human CYP7A1 and murine Cyp7a1 gene promoters (white and black bars, respectively) to fatty acids in the presence of PPARalpha /RXRalpha . The cyp7a-CAT gene chimeras and plasmids encoding PPARalpha , RXRalpha , and beta -galactosidase (internal reference standard) were cotransfected into hepatoma cells. Fatty acids (stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2)) complexed to lipid-free BSA were added to the cell culture medium to a final concentration of 100 µM. Control cells received lipid-free BSA (no FA). C, response of the human CYP7A1 and murine Cyp7a1 gene promoters to WY 14,643 in the presence of PPARalpha /RXRalpha . WY 14,643 was added to culture medium to a final concentration of 50 µM (black bars). Control cells received Me2SO (carrier) alone (white bars). For both B and C, the average normalized CAT activity value in the absence of ligands was assigned a value of 1. The results are the means ± S.E. of two independent experiments performed in triplicate.

Interestingly, the response of the murine Cyp7a1 gene promoter to fatty acids was consistently greater than that of the human CYP7A1 gene promoter. To verify the observed difference in the response of the murine Cyp7a1 and human CYP7A1 gene promoters to fatty acids, cells transfected with the chimeric plasmids were treated with WY 14,643, a potent and well characterized synthetic activator of PPARalpha (6, 8, 10). As shown in Fig. 1C, the response of the murine Cyp7a1 gene promoter to WY 14,643 was significantly greater (11-fold activation) compared with the human CYP7A1 gene promoter (1.5-fold activation). These results show that although the human CYP7A1 gene promoter has a higher basal activity compared with the murine Cyp7a1 gene promoter, the human CYP7A1 gene is less responsive to regulation by activated PPARalpha /RXRalpha .

The cyp7a Gene Promoter Contains a PPARalpha /RXRalpha -binding Site-- Analysis of the human CYP7A1 and murine Cyp7a1 gene promoter fragments produced by restriction enzyme digestion revealed that PPARalpha /RXRalpha can bind to the cyp7a promoter somewhere in the region between nt -255 and -47 (data not shown). Inspection of the sequence revealed that this region of the cyp7a gene promoter contains a DR1 motif that resembles a PPARalpha /RXRalpha -binding site. This DR1 motif spans nt -144 to -132 and nt -158 to -146 of the human CYP7A1 and murine Cyp7a1 gene promoters, respectively. The sequence of this site, which we have designated as Site II, is invariant among the promoter regions of the human, murine, rat, and hamster cyp7a genes (Fig. 2A). To determine if Site II could function as a PPRE, we tested its ability to interact with PPARalpha /RXRalpha by electrophoretic mobility shift assay. Double-stranded oligonucleotides containing the Site II sequence displayed strong binding to PPARalpha /RXRalpha heterodimers (Fig. 2B, left arrow). Interaction of the probe with the recombinant transcription factors was not observed when either PPARalpha or RXRalpha was omitted from the binding reactions (data not shown). Addition of anti-PPARalpha antibodies to the binding reaction containing PPARalpha /RXRalpha resulted in the formation of a complex with greatly reduced mobility (Fig. 2B, right arrow), demonstrating that the complex contains PPARalpha . The binding of Site II to PPARalpha /RXRalpha was competed efficiently by the well characterized acyl-CoA oxidase PPRE (45), but not by a glucocorticoid response element (48) (Fig. 2C, left panel). Mutagenesis of the 3'-half-site of Site II drastically reduced binding to PPARalpha /RXRalpha (Fig. 2C, right panel). These data demonstrate that PPARalpha /RXRalpha can interact with the cyp7a gene promoter.


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  		Fig. 2.   The cyp7a gene promoter contains a conserved PPARalpha /RXRalpha -binding site. A, shown is a schematic (top) illustrating the approximate location of the Site II element of the cyp7a gene promoter. The sequences of the Site II elements of the human (GenBankTM/EBI Data Bank accession number L20569), murine (this work and L20569), rat (J02926), and hamster (L04690) cyp7a gene promoters and of the mutant human CYP7A1 Site II element (mut Site II) are shown below. The coordinates shown are based on a numbering system in which the most distal transcription start site is assigned +1. The rat acyl-CoA oxidase (AOx) PPRE and the consensus PPRE sequences are also shown for comparison. The dots represent identical residues, and the lowercase letters represent residues in the DR1 spacer region. bp, base pairs. B, PPARalpha /RXRalpha binding to the cyp7a Site II element (left arrow) was demonstrated by electrophoretic mobility shift assay. Addition of anti-PPARalpha antiserum (last lane) resulted in the formation of a complex with greatly reduced mobility (right arrow). C, the specificity of the binding was tested by adding a 5- or 50-fold molar excess of competitor (glucocorticoid response element (GRE) or acyl-CoA oxidase PPRE) relative to the radiolabeled probe in the binding reactions. The binding of PPARalpha /RXRalpha heterodimers to wild-type human CYP7A1 Site II (arrows) relative to mutant Site II is shown in the right panel. D, mutagenesis of the 3'-half-site of the human CYP7A1 Site II element abolished responsiveness to WY 14,643 (black bars). The wild-type and mutant cyp7a-CAT gene chimeras were introduced into hepatoma cells along with plasmids encoding PPARalpha , RXRalpha , and beta -galactosidase. The normalized CAT activity values in the absence of PPARalpha /RXRalpha ligands (white bars) were assigned a value of 1. Results shown represent the means ± S.E. of three independent experiments performed in triplicate.

Next, we evaluated the role of Site II in the regulation of the cyp7a gene promoter by ligand-activated PPARalpha /RXRalpha . The 3'-half-site of Site II (a DR1 element) was mutagenized, and then CAT gene chimeras containing the human CYP7A1 gene promoter with either the wild-type or mutagenized Site II element were transfected into hepatoma cells along with expression vectors encoding PPARalpha and RXRalpha . The activities of the wild-type and mutant Site II elements were compared in the presence and absence of 50 µM WY 14,643. As shown in Fig. 2D, the cells transfected with the gene chimera containing the wild-type human CYP7A1 Site II showed a small but reproducible increase (1.5-fold, p < 0.05) in CAT activity, whereas cells transfected with the CYP7A1 gene chimera containing mutagenized Site II showed no response to WY 14,643. These results demonstrate that PPARalpha /RXRalpha can regulate the cyp7a gene promoter through Site II. As the sequence of Site II is identical in both human CYP7A1 and murine Cyp7a1 gene promoters, the basis for the more robust response of the murine Cyp7a1 gene promoter to fatty acids was unclear.

The Murine Cyp7a1 Gene Promoter Contains an Additional PPARalpha /RXRalpha -binding Site-- The LXRalpha /RXRalpha heterodimers bind to a tandem repeat of a nuclear hormone-binding sequence motif separated by four unconserved residues (i.e. a DR4 element termed the LXR response element) (49). Region -72 to -57 of the murine Cyp7a1 gene promoter contains a DR4 element that is similar to the LXR response element found in the rat Cyp7a1 gene promoter (39). Alignment of murine and rat DR4 elements shows a single nucleotide difference in the spacer region, but the 5'- and 3'-half-sites are identical (Fig. 3A). Inspection of the murine DR4 sequence (designated as Site I) suggests the existence of a possible DR1 element embedded within DR4. The DR1 element is configured in such a way that it shares a common 3'-half-site with the DR4.


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  		Fig. 3.   The murine Cyp7a1 gene promoter contains an additional PPRE. A, shown is a schematic (top) illustrating the approximate location of the Site I element of the cyp7a gene promoter. The sequences of the Site I elements of the cyp7a gene promoters from different species are shown below. The dots represent identical residues, and the lowercase letters represent residues in the spacer regions. B, the PPARalpha /RXRalpha heterodimers bound to murine Cyp7a1 Site I (left arrow) were demonstrated by electrophoretic mobility shift assay. Addition of anti-PPARalpha antiserum (last lane) resulted in the formation of a complex with greatly reduced mobility (right arrow). C, the specificity of PPARalpha /RXRalpha binding (left arrow) was tested by adding a 5-50-fold molar excess of the rat acyl-CoA oxidase (AOx) PPRE or the human Site I element relative to the radiolabeled probe in the binding reactions.

Comparison of the murine, human, rat, and hamster cyp7a gene promoters revealed that the sequence of Site I is not conserved (Fig. 3A). Electrophoretic mobility shift assays using oligonucleotides corresponding to the murine Site I element demonstrated a strong interaction with PPARalpha /RXRalpha , whereas the corresponding Site I element of the human CYP7A1 gene promoter was unable to bind PPARalpha /RXRalpha (Fig. 3B, left arrow). Binding of PPARalpha was confirmed by the formation of a complex with greatly reduced mobility in the presence of anti-PPARalpha antibodies (Fig. 3B, right arrow). Consistent with these results, the binding of PPARalpha /RXRalpha to murine Cyp7a1 Site I could be competed with the acyl-CoA oxidase PPRE, but not with the human CYP7A1 Site I element (Fig. 3C). These results indicate that the Site I element of the murine Cyp7a1 gene promoter is configured to enable binding of PPARalpha /RXRalpha .

Alteration of a Single Nucleotide in the Human CYP7A1 Site I Regulatory Element Allows PPARalpha /RXRalpha Binding-- The human CYP7A1 Site I differs from the murine Cyp7a1 Site I by three nucleotide residues (Fig. 4A). We designed three different double-stranded oligonucleotides containing the human CYP7A1 Site I sequence in which each of the variant residues was replaced with the corresponding residue in the murine Cyp7a1 Site I sequence. Changes from T-to-G and from A-to-C in the 5'-half-site of the DR4 element and in the spacer region, respectively, did not promote binding of human CYP7A1 Site I to PPARalpha /RXRalpha (Fig. 4B, left panel). In contrast, the C-to-T substitution in the 3'-half-site of human CYP7A1 Site I enabled binding to PPARalpha /RXRalpha (Fig. 4B, left panel), and the binding was competed by both the acyl-CoA oxidase PPRE and murine Cyp7a1 Site I (Fig. 4B, right panel).


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  		Fig. 4.   Functional analysis of the human CYP7A1 Site I regulatory element. A, the murine Cyp7a1 and wild-type and mutant human CYP7A1 Site I sequences are shown. The dots represent residues identical to the human sequence, and the lowercase letters represent substituted residues. B, PPARalpha /RXRalpha binding to wild-type and mutant CYP7A1 Site I elements was evaluated by electrophoretic mobility shift assay (left panel, arrow). The specificity of PPARalpha /RXRalpha binding to human CYP7A1 Site I (C to T) (right panel, arrow) was tested by adding a 5- or 50-fold molar excess of either the rat acyl-CoA oxidase (AOx) PPRE or the murine Cyp7a1 Site I element relative to the radiolabeled probe in the binding reaction. C, the cyp7a-CAT gene chimeras with a wild-type or mutant Site I element were introduced into hepatoma cells along with plasmids encoding PPARalpha , RXRalpha , and beta -galactosidase. The left panel shows the relative CAT activity normalized to beta -galactosidase activity encoded by a cotransfected beta -galactosidase expression vector in the absence of PPARalpha activators (white bars). The right panel shows the response of the cyp7a promoters to WY 14,643 (black bars). The normalized CAT activity values in the absence of PPARalpha /RXRalpha ligands (white bars) were assigned a value of 1. Results shown represent the means ± S.E. of three independent experiments performed in triplicate.

To demonstrate the functional significance of the human CYP7A1 Site I C-to-T mutation, a cyp7a-CAT gene chimera containing mutagenized human CYP7A1 Site I was created and analyzed for its response to WY 14,643-activated PPARalpha /RXRalpha in hepatoma cells. Analysis of CAT activity in extracts of cells transfected with the gene chimera carrying this mutation revealed a marked reduction of the basal promoter activity as compared with cells transfected with the wild-type version of the human CYP7A1 promoter (Fig. 4C, left panel). This result, taken together with the results shown in Fig. 1A, suggests that the low basal activity of the murine Cyp7a1 gene promoter compared with the wild-type human CYP7A1 gene promoter is attributable to the sequence of the Site I element. As observed before, the wild-type human CYP7A1 gene promoter showed a small (1.5-fold) but consistent stimulation in the presence of WY 14,643. In contrast, the human CYP7A1 gene promoter containing the Site I C-to-T mutation responded significantly better (3.5-fold, p < 0.01) (Fig. 4C, right panel). These results illustrate that a single nucleotide difference in the 3'-half-site of Site I can alter the basal activity of the cyp7a promoter as well as determine the ability of Site I to interact with PPARalpha /RXRalpha .

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Both the murine Cyp7a1 and human CYP7A1 gene promoters were induced when the culture medium of cells transfected with gene chimeras containing these promoters was supplemented with fatty acids. The stimulatory effect was observed even with stearic acid, a fatty acid that interacts weakly with PPARalpha (9, 11). This could be explained by the fact that metabolism of fatty acids in the transfected cells can produce metabolites that are more potent activators of PPARalpha than the parental fatty acids. Inspection of the proximal region of the cyp7a gene promoters from various mammalian species revealed the existence of an invariant DR1 motif that is a possible binding site for PPARalpha /RXRalpha . Electrophoretic mobility shift assays using recombinant PPARalpha and RXRalpha revealed that this sequence (termed Site II) was capable of binding PPARalpha /RXRalpha in vitro. Since mutagenesis of the human Site II element abolished the stimulatory effect of fatty acids on the human CYP7A1 gene promoter, we surmised that this site is capable of acting as a target for PPARalpha -mediated signaling.

Although both fatty acids and WY 14,643 reproducibly stimulated the human CYP7A1 gene promoter in our experimental system, the magnitude of the stimulation was modest. Fibrates have generally been shown to increase the concentration of cholesterol in human and rat bile, and this effect appears to be correlated with the reduction of cholesterol 7alpha -hydroxylation activity in both species (50-53). However, it should be noted that some fibrates have only minimal effects on the lithogenic index of bile or cyp7a activity (50, 53). Significant changes in the activities of other cholesterol-metabolizing enzymes have also been demonstrated (50, 51), and these effects likely also contribute to changes in the lithogenic index of bile.

Recently, it was discovered that human liver contains significantly lower levels of PPARalpha mRNA compared with murine liver (54). Measurement of PPARalpha /RXRalpha DNA-binding activity in liver extracts suggested that human liver contains an order of magnitude less activity compared with murine liver (54). These observations raise the possibility that PPARalpha may not play a significant role in regulating human CYP7A1 gene expression. The conserved Site II element of the cyp7a gene promoter is also capable of binding other transcription factors (42-44) and thus may not be available for PPARalpha /RXRalpha interaction in vivo. The reduction of cholesterol 7alpha -hydroxylation activity observed in humans and rats after fibrate therapy may therefore be due to an indirect effect of PPARalpha -mediated signaling. A transgenic mouse strain bearing the human CYP7A1 gene should aid in clarifying the significance of PPARalpha function in regulating human CYP7A1 gene expression.

In contrast, the murine Cyp7a1 gene promoter was highly responsive to fatty acids and WY 14,643 in transfected cells. Since the murine Cyp7a1 gene promoter did not contain other sequences that resemble the typical PPARalpha /RXRalpha -binding site (a DR1 motif) (2, 5, 45), the basis for the enhanced response of the murine Cyp7a1 gene promoter in transfected cells was not immediately obvious. Mapping of the murine proximal gene promoter using restriction fragments as probes in gel mobility shift assays surprisingly revealed that the fragment containing the LXRalpha /RXRalpha -binding site (LXR response element, a DR4 motif) (49) could also bind PPARalpha /RXRalpha in vitro. Unlike Site II, the sequence that makes up the murine DR4 element (termed Site I) of the Cyp7a1 gene promoter is not conserved among the different mammalian species. The human and murine Site I sequences are non-identical; and unlike murine Site I, the human variant is not capable of binding PPARalpha /RXRalpha . However, the substitution of a single residue at the 3'-half-site of the human Site I sequence with that found in the same position of the murine Site I element enabled PPARalpha /RXRalpha binding. In addition, this substitution substantially improved the response of the human CYP7A1 gene promoter to WY 14,643 in the presence of PPARalpha /RXRalpha in transfected cells. The magnitude of the response of the modified human CYP7A1 gene promoter did not reach that observed for the murine Cyp7a1 gene promoter, but this may be due to other sequence differences between the two promoters. Thus, we attribute the robust response of the murine Cyp7a1 gene promoter to fatty acids and WY 14,643 to the existence of a second PPARalpha /RXRalpha -binding site. Studies done on mice that are homozygous for a disrupted Scp2 gene (encodes sterol carrier protein 2 and sterol carrier protein X) (55) provide additional support for the involvement of PPARalpha in the regulation of murine Cyp7a1 gene expression. The expression of the Cyp7a1 gene in these mice is increased, coincident with the enhanced expression of hepatic genes known to be regulated by PPARalpha (55). This effect has been ascribed to the accumulation of phytanic acid, a metabolite that can act as a ligand for and activator of PPARalpha (56).

Previously, we demonstrated that the fat background of the diet had a significant impact on the response of the murine Cyp7a1 gene promoter to dietary cholesterol (35, 36). The stimulatory effect of dietary cholesterol on the murine Cyp7a1 gene is likely mediated via LXRalpha , a nuclear hormone receptor that is activated by oxysterols (39, 57), since inactivation of the LXRalpha gene in mice abolishes the response (58). Based on the present study, it appears that PPARalpha /RXRalpha and LXRalpha /RXRalpha bind to overlapping sequences in the murine Cyp7a1 gene promoter. The exclusive interaction of these transcription factors with the murine Site I element may explain why fats have an apparent influence on the stimulatory effect of dietary cholesterol. The exact requirements dictating the preferential binding of either of these transcription factors to the murine Site I element remain to be elucidated. In any case, the data suggest that fats are important physiological regulators of murine Cyp7a1 gene expression.

The inhibitory effect of bile acids on cyp7a gene expression is a well documented phenomenon (17). The discovery that bile acids serve as ligands for the farnesoid X receptor provides an explanation for the repressive effect of bile acids on the transcription of bile acid-responsive genes (40, 59, 60). A recent study demonstrated that ligand-bound farnesoid X receptor is capable of antagonizing transactivation by LXRalpha (60), although the precise mechanism by which this effect is accomplished remains to be described. It will be of interest to determine also if the stimulatory effect of ligand-activated PPARalpha on murine Cyp7a1 gene expression is influenced by the farnesoid X receptor.

In summary, we demonstrated that the murine Cyp7a1 and human CYP7A1 gene promoters are differentially responsive to regulation by fatty acids via PPARalpha /RXRalpha in transfected cells. The basis for the differential sensitivity is apparently due to a variation in a critical residue in the Site I element of the cyp7a gene promoter. The murine Site I variant enables PPARalpha /RXRalpha binding and allows the murine Cyp7a1 gene promoter to be more responsive to regulation by fats. Mice may have either maintained or acquired more sophisticated control of cyp7a gene expression through evolution to allow greater flexibility in regulating bile acid synthesis.

    ACKNOWLEDGEMENTS

We thank Dr. R. A. Rachubinski (University of Alberta) for providing antiserum to PPARalpha and plasmids encoding recombinant PPARalpha and RXRalpha . We also thank V. Drover for assistance in some of the experiments.

    FOOTNOTES

* This work was supported by Grant MT-14812 (to L. B. A.) from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Biochemistry, BT 3012, Memorial University of Newfoundland, St. John's, NF A1B 3X9, Canada.

§ Senior Medical Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: 303 Heritage Medical Research Centre, Dept. of Biochemistry, University of Alberta, Edmonton, AB T6G 2S2, Canada. Tel.: 780-492-5251; Fax: 780-492-3383; E-mail: luis.agellon@ualberta.ca.

2 V. Drover and L. B. Agellon, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PPARs, peroxisome proliferator-activated receptors; RXRalpha , 9-cis-retinoic acid receptor alpha ; PPRE, peroxisome proliferator response element; cyp7a, cholesterol 7alpha -hydroxylase; LXR, nuclear oxysterol receptor; CAT, chloramphenicol acetyltransferase; nt, nucleotides; BSA, bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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