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Originally published In Press as doi:10.1074/jbc.M107000200 on October 17, 2001

J. Biol. Chem., Vol. 276, Issue 50, 47154-47162, December 14, 2001
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Antagonism of the Actions of Peroxisome Proliferator-activated Receptor-alpha by Bile Acids*

Christopher J. Sinal, Michung YoonDagger, and Frank J. Gonzalez§

From the Laboratory of Metabolism, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 24, 2001, and in revised form, October 15, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The peroxisome proliferator-activated receptor-alpha (PPARalpha ) is a ligand-activated transcription factor that regulates the expression of a number of genes critical for fatty acid beta -oxidation. Because a number of substrates and intermediates of this metabolic pathway serve as ligand activators of this receptor, homeostatic control of fatty acid metabolism is achieved. Evidence also exists for PPARalpha -dependent regulation of genes encoding critical enzymes of bile acid biosynthesis. To determine whether the primary products of bile acid biosynthesis, cholic acid and chenodeoxycholic acid, were capable of modulating PPARalpha function, a variety of in vivo and in vitro approaches were utilized. Feeding a bile acid-enriched diet significantly reduced the degree of hepatomegaly and induction of target genes encoding enzymes of fatty acid beta -oxidation caused by treatment with the potent PPARalpha ligand Wyeth-14,643. Convergent data from mechanistic studies indicate that bile acids interfere with transactivation by PPARalpha at least in part by impairing the recruitment of transcriptional coactivators. The results of this study provide the first evidence in favor of the existence of compounds, normally found within the body, that are capable of antagonizing the physiological actions of PPARalpha . The impact of PPARalpha antagonism by endogenous bile acids is likely to be limited under normal conditions and to have only minimal effects on bile acid homeostasis. However, during certain pathophysiological states where intracellular bile acid concentrations are elevated, meaningful effects on PPARalpha -dependent target gene regulation are possible.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptors (PPARs)1 are members of a large family of ligand-activated transcription factors with essential roles in mammalian development, physiology, and homeostasis. Three genetically and functionally distinct PPAR isoforms occur in mammals, PPARalpha (NR1C1), PPARbeta (NR1C2), and PPARgamma (NR1C3) (1-4). Ligand-bound PPARs modulate target gene expression by binding to DNA response elements composed of a direct repeat of the hexameric core motif AGGTCA separated by a single base pair (DR-1). High affinity binding of PPARs to these PPAR-response elements (PPREs) occurs after heterodimerization with the retinoid X receptor (RXR). PPAR·RXR heterodimers interact with PPREs located in the promoter region of genes implicated in a number of physiological processes including fatty acid metabolism, inflammation, adipogenesis, carcinogenesis, and development. PPARalpha , in particular, exerts regulatory control over the expression of numerous genes encoding proteins involved in lipid oxidation and transport (1, 5, 7).

By using transient transfection assays, a number of structurally diverse compounds have been shown to activate PPARalpha in a selective manner. Among these are anti-hyperlipidemic drugs used for the treatment of hypertriglyceridemia, including clofibrate, bezafibrate, ciprofibrate, and the potent experimental PPARalpha agonist Wyeth-14,643 (Wy-14,643) (8-10). Treatment of rodents with these compounds, collectively termed peroxisome proliferators, causes an increase in the size and number of peroxisomes, hepatomegaly, and after chronic administration, tumor formation (2, 11, 12). PPARalpha is also activated by a variety of natural long chain fatty acids, particularly polyunsaturated fatty acids such as docosahexaenoic, linoleic, linolenic, and arachidonic acids (13, 14). No single high affinity natural ligand has been identified for PPARalpha , raising the possibility that PPARalpha may serve as a generalized cellular sensor of fatty acid flux. Consistent with this hypothesis, enzymes such as acyl-CoA oxidase (ACOX), bifunctional enzyme (BE), and thiolase, which are involved in peroxisomal beta -oxidation of long chain fatty acids, are known targets for PPARalpha regulation (15-17). PPARalpha also affects the processes of fatty acid transport and uptake through positive regulation of genes encoding the fatty acid transport protein, fatty-acid translocase (CD36), and the liver cytosolic fatty acid-binding protein (18). Other PPARalpha target genes involved in lipid homeostasis are CYP4A microsomal omega -hydroxylases (19, 20), lipoprotein lipase (21), and apolipoproteins AI, AII, and CIII (22, 23). The expression of PPARalpha and many of its target genes is up-regulated in response to pathophysiological stresses, such as starvation, that are associated with the release of fatty acids into the circulation from adipose tissue as part of the process for mobilizing energy stores (24). Generation of a PPARalpha -null mouse definitively established the role of PPARalpha as the mediator of the pleitropic effects of peroxisome proliferators (17). This model has also confirmed the importance of PPARalpha in lipid homeostasis. In the absence of PPARalpha , cellular fatty acid flux is impaired due to reduced mitochondrial fatty acid import and beta -oxidation, leading to hepatic lipid accumulation in animals fed a high fat diet (25).

In addition to lowering serum triglyceride levels, peroxisome proliferator anti-hyperlipidemia drugs such as bezafibrate also lower plasma cholesterol levels in humans (26). Hepatic bile acid biosynthesis from cholesterol represents a quantitatively important route of cholesterol elimination from the body. The rate-limiting step in this process is catalyzed by the hepatic microsomal enzyme cholesterol 7alpha -hydroxylase (CYP7A1) (27). Sterol 12alpha -hydroxylase (CYP8B1), another hepatic microsomal enzyme, is the major determinant of the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA; Fig. 1), the major products of bile acid biosynthesis, in the bile (28). Thus, changes in the expression of CYP7A1 and CYP8B1 affect both the quantity and quality of bile acid biosynthesis, factors that can influence both the elimination of cholesterol from the liver and the absorption of dietary lipids from the intestine. Recent studies indicate that PPARalpha may exert regulatory control over CYP7A1 (29-31) and CYP8B1 (32), thereby potentially extending the physiological roles of this receptor. Feedback and feed-forward regulation is a recurrent means by which homeostatic control over essential biochemical pathways is achieved. Thus, we hypothesized that the end products of bile acid biosynthesis, CA and CDCA, were capable of affecting PPARalpha function. By using in vivo and in vitro approaches, we show that bile acids inhibit transcriptional activation by PPARalpha , demonstrating the existence of endogenous antagonists of PPARalpha -dependent signaling.


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  		Fig. 1.   Structures of Wy-14,643, cholic acid, and chenodeoxycholic acid.


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

Animal Treatments-- PPARalpha -null and FXR-null mice were obtained as described previously (17, 33). All other mice used in this study were obtained from our existing colony of wild-type SV129 mice. Mice were housed in a pathogen-free animal facility under a standard 12-h light/12-h dark cycle. Prior to the administration of special diets, mice were fed standard rodent chow and water ad libitum. All diets were prepared by Bioserv (Frenchtown, NJ) and were based upon a standard AIN-93G rodent diet containing 58.6% carbohydrate, 18.1% protein, 7.2% fat, 5.1% fiber, 3.4% ash, and 10% moisture (control diet). Experimental diets consisted of the control diet supplemented with 1% (w/w) CA, 0.1% (w/w) Wy-14,643, or 1% (w/w) CA plus 0.1% (w/w) Wy-14,643. Age- and sex-matched groups of 8-12-week-old animals were used for all experiments and were allowed water ad libitum. At the end of the study, animals were euthanized by carbon dioxide asphyxiation, and tissues were harvested, weighed, and snap-frozen in liquid nitrogen and stored at -80 °C until use. All protocols and procedures were approved by the NCI Division of Basic Sciences Animal Care and Use Committee and are in accordance with National Institutes of Health Guidelines.

Analysis of Target Gene Expression-- Total RNA was prepared using Trizol reagent (Life Technologies, Inc.) and analyzed by electrophoresis on 0.22 M formaldehyde-containing 1% agarose gels. The separated RNA was transferred to GeneScreen Plus membranes (PerkinElmer Life Sciences) by downward capillary transfer in the presence of 20× SSC buffer (3 M NaCl, 0.3 M sodium citrate (pH 7.0)), UV cross-linked, and baked for 2 h at 80 °C. Probe hybridization and washing were performed using standard techniques. Blots were exposed to Phosphor-Imager screen cassettes and were visualized using a Molecular Dynamics Storm 860 PhosphorImager system (Sunnyvale, CA). The cDNA probes used in this study were 32P-labeled by the random-primer method using Ready-to-Go DNA labeling beads (Amersham Biosciences) as described previously (17). Densitometric analysis of the mRNA signals was performed using ImageQuant image analysis software (Molecular Dynamics, Sunnyvale, CA).

Transfection Assays-- The expression vectors, pSG5-mPPARbeta and pSG5-PPARgamma , were generously provided by Walter Wahli (Universite de Luasanne, Luasanne, Switzerland). The expression vectors for murine PPARalpha , pSG5-mPPARalpha , and the PPRE3-tk-luc reporter gene constructs (34) have been described previously. Expression vectors for VP16-mPPARalpha , GAL-CBP, and GAL-NCoR were generously provided by Steve Kliewer (Glaxo Wellcome) and have been described previously (35). The GAL4-UAS luciferase reporter plasmid (pFR-Luc) was obtained from Stratagene (La Jolla, CA). The pSV-beta -Gal control vector (Promega, Madison, WI) was used to normalize transfection efficiency. Hepa 1c1c7 and CV-1 cells were routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 100 units/ml penicillin/streptomycin, and 0.5 mg/ml gentamicin (Life Technologies, Inc.). Cells were seeded in 6-well tissue culture plates (2 × 104 cells/well) 24 h prior to transfection. For all transfections, 200 ng/well of each of the appropriate plasmids were used with the exception of pFR-Luc where 50 ng/well was used. Transfections were performed using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. After 18 h, the culture medium was changed and the test compounds, Me2SO (Sigma), CDCA (Sigma), Wy-14,643 (Chemsyn Science Laboratories, Lenexa, KS), bezafibrate (Sigma), or troglitazone (Sankyo, Tokyo, Japan) were added at 0.1% (v/v). After incubation for 24 h with the test compounds, the cells were washed twice with phosphate-buffered saline and assayed for luciferase and beta -galactosidase activity using commercial kits according to the manufacturer's (Promega, Madison, WI) instructions.

EMSA Analysis-- Murine PPARalpha , PPARbeta , and PPARgamma and human RXRalpha were synthesized in vitro by programming the TNT-coupled transcription/translation system (Promega, Madison, WI) with 1 µg of pSG5-PPARalpha , pSG5-PPARbeta , pSG5-PPARgamma , and pSG5-RXRalpha . An oligonucleotide (obtained from Life Technologies, Inc.) consensus DR-1 element was synthesized with the following sequence: 5'-AGGTCAAAGGTCA-3' along with an oligonucleotide of complementary sequence. The oligonucleotides were mixed (50 ng/µl final concentration) and denatured by heating to 95 °C for 10 min in 0.1 M Tris-Cl, 50 mM MgCl2 (pH 7.9) and allowed to anneal by slowly cooling to room temperature. The annealed oligonucleotides were end-labeled with [gamma -32P]ATP using T4 polynucleotide kinase according to the supplier's (Promega, Madison, WI) instructions. In a total volume of 20 µl of binding buffer (25 mM Tris-Cl (pH 7.5), 40 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol), the following components were combined: 1 µg of poly(DI-dC), 2 µl of each in vitro translation reaction and the indicated concentrations of Wy-14,643, bezafibrate, troglitazone, CA, or CDCA dissolved in Me2SO. After a 20-min incubation at room temperature, 20,000 cpm of the labeled oligonucleotide was added, and the incubation was continued for a further 20 min. Samples were analyzed on 5% non-denaturing polyacrylamide gel, containing 2.5% glycerol, in 0.4× TBE (1× = 89 mM Tris-Cl, 89 mM boric acid, 2 mM EDTA). After drying, the gels were exposed to PhosphorImager screen cassettes and were visualized using a Molecular Dynamics Storm 860 PhosphorImager system (Sunnyvale, CA).

Statistics-- Unless otherwise noted, all values are expressed as the mean ± S.D. All data were analyzed by the unpaired Student's t test for significant differences between the mean values of each group using Statview version 4.5 (Abacus Concepts, Berkeley, CA) for Macintosh.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice fed a diet containing 1% CA for 5 days exhibited no significant increases in liver size as indicated by the liver/body weight ratio in these animals compared with chow-fed controls (Fig. 2A). As expected, feeding mice a chow diet for 3 days followed by a diet containing 0.1% Wy-14,643 for 2 days caused significant hepatomegaly compared with chow-fed mice. In contrast, mice fed the 1% CA diet for 3 days followed by a 1% CA, 0.1% Wy-14,643 diet for 2 days exhibited a significant reduction in the magnitude of hepatomegaly compared with mice fed the 0.1% Wy-14,643 diet alone. None of the dietary regimens altered the hepatic expression of PPARalpha mRNA or the mRNA levels for its obligate heterodimerization partner RXRalpha (Fig. 2B). However, administration of the Wy-14,643 diet caused substantial increases, compared with chow-fed mice, of the mRNA levels for CYP4A1, CYP4A3, ACOX, BE, and thiolase, all known to be targets for PPARalpha regulation (15-17). When mice received the combined CA/Wy-14,643 dietary regimen, hepatic target gene induction was reduced substantially compared with Wy-14,643 alone. Mice fed the CA-containing diet also exhibited reduced constitutive levels of the mRNA for these target genes compared with chow-fed mice.


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  		Fig. 2.   Inhibition of Wy-14,643-induced hepatomegaly and target gene expression by cholic acid. Mice were fed normal chow for 5 days, a 1% CA diet for 5 days, chow for 3 days followed by a 0.1% Wy-14,643 diet for 2 days, or a 1% CA diet for 3 days followed by a 1% CA, 0.1% Wy-14,643 diet for 2 days. A, liver/body weight ratios of animals maintained on the indicated diets. B, Northern blot analysis of hepatic mRNA levels for PPARalpha target genes. Numbers are the average (n = 4 for each group) fold change of each mRNA, after correction for the beta -actin signal, relative to that determined for mice fed the chow diet. a, significantly different from Wy-14,643 diet alone, p < 0.05; b, significantly different from chow diet, p < 0.05.

To determine whether the attenuation of Wy-14,643-induced target gene expression was a direct effect on PPARalpha or an indirect effect not mediated by this receptor, target gene responses were also examined in the livers of PPARalpha -null mice. As expected, administration of the Wy-14,643 diet failed to cause induction of CYP4A1, CYP4A3, ACOX, BE, and thiolase mRNA levels in the livers of PPARalpha -null mice (Fig. 3A). Interestingly, reduced levels of CYP4A1, CYP4A3, and thiolase mRNAs were caused by CA or CA/Wy-14,643 feeding to PPARalpha -null mice. However, the magnitude of this effect was not as great as that observed for CA feeding to wild-type mice. These data suggest the existence of PPARalpha -independent mechanism(s) by which bile acids can affect the constitutive expression of some PPARalpha target genes. Bile acids serve as physiological ligands for the farnesoid X-receptor (FXR), a nuclear receptor critical for the regulation of bile acid biosynthesis and transport (33, 36-38). Feeding a CA-enriched diet to mice induces the expression of the small heterodimer partner (SHP) gene, a process dependent upon the presence of a functional FXR (39, 40). SHP is an orphan nuclear receptor that has been shown to inhibit transactivation by a number of nuclear receptors, including PPARalpha (39-43). To determine whether the reduced PPARalpha target gene induction caused by CA feeding was FXR-dependent, either through direct actions on the target gene promoters or indirectly via SHP induction, FXR-null mice were examined. These mice have reduced constitutive SHP expression and lack inducible SHP expression in response to dietary bile acids (33). As shown in Fig. 3B, FXR-null mice exhibited normal PPARalpha hepatic target gene induction in response to the Wy-14,643 diet and experienced a similar attenuation, compared with wild-type mice, of this response when the combined Wy-14,643/CA dietary regimen was applied.


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  		Fig. 3.   Modulation of PPARalpha target gene expression by cholic acid in PPARalpha - and FXR-null mice. PPARalpha -null or FXR-null mice were fed normal chow for 5 days, a 1% CA diet for 5 days, chow for 3 days followed by a 0.1% Wy-14,643 diet for 2 days, or a 1% CA diet for 3 days followed by a 1% CA, 0.1% Wy-14,643 diet for 2 days. Total RNA was prepared from the livers of PPARalpha -null (A) and FXR-null (B) mice and analyzed by Northern blot analysis using the indicated cDNA probes. Numbers are the average (n = 4 for each group) fold change of each mRNA, after correction for the beta -actin signal, relative to that determined for mice fed the chow diet.

To examine the mechanism by which bile acids inhibited the induction of PPARalpha target genes by Wy-14,643, Hepa 1c1c7 cells were transfected with PPARalpha and RXRalpha expression constructs as well as a luciferase reporter construct (PPRE3-tk-luc) containing three copies of the PPRE from the rat ACOX gene. CDCA was used in these assays in place of CA, as the former is more readily taken up into cells (38). Treatment of the transfected cells with CDCA caused a dose-dependent decrease of luciferase activity in the absence of Wy-14,643 treatment (Fig. 4A). Similarly, CDCA treatment also caused a dose-dependent decrease in the magnitude of reporter gene activation caused by 10 µM Wy-14,643 treatment. To determine whether bile acids also interfered with transactivation mediated by other PPARalpha isoforms, experiments were performed using cells transfected with PPARbeta or PPARgamma in place of PPARalpha . As shown in Fig. 4B, treatment of PPARalpha -transfected cells with Wy-14,643 caused a 5-fold increase of reporter gene expression compared with Me2SO-treated cells. However, co-treatment with 50 µM CDCA almost entirely abolished reporter gene activation caused by 10 µM Wy-14,643. In contrast, reporter gene activation of PPARbeta - and PPARgamma -transfected cells treated with the selective agonists bezafibrate (10 µM) and troglitazone (10 µM) was unaffected by co-treatment with 50 µM CDCA. CA was without effect on PPARalpha -dependent transactivation, likely due to an inability of this compound to be taken up effectively by the cells.


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  		Fig. 4.   Inhibition of PPARalpha reporter gene expression by bile acids. A, Hepa 1c1c7 cells were transiently transfected with expression plasmids for PPARalpha and RXRalpha and a luciferase reporter construct containing three copies of the PPRE from the rat ACOX gene. After treatment (24 h) with increasing concentrations of CDCA in the presence or absence of 10 µM Wy-14,643, cells were harvested, lysed, and assayed for luciferase activity. Values shown are the fold activation versus cells treated with Me2SO (DMSO) alone (mean ± S.D., n = 4). *, significantly different versus 0.1 µM CDCA + 10 µM Wy-14,643, p < 0.05. #, significantly different versus 0.1 µM CDCA + Me2SO, p < 0.05. B, Hepa 1c1c7 cells were transiently transfected with expression plasmids for PPARalpha , PPARbeta or PPARgamma , and RXRalpha and a luciferase reporter construct containing three copies of the PPRE from the rat ACOX gene. After treatment (24 h) with the Me2SO (DMSO), 10 µM agonist (Wy-14,643, PPARalpha ; bezafibrate, PPARbeta ; troglitazone, PPARgamma ) or 10 µM agonist plus 50 µM CA or CDCA, cells were harvested, lysed, and assayed for luciferase activity. Values shown are the fold activation versus cells treated with Me2SO alone (mean ± S.D., n = 4). *, significantly different versus Me2SO, p < 0.05.

EMSAs were used to determine whether CA or CDCA interfered with the binding of in vitro translated PPARs to a consensus DR-1 sequence (AGGTCAAAGGTCA). As shown in Fig. 5A, PPAR·RXRalpha , but not PPAR or RXRalpha homodimers, exhibited a constitutive DNA binding activity in the absence of treatment with any of the test compounds. Consistent with published reports (19, 44, 45), treatment of the PPARalpha ·RXRalpha heterodimers with increasing doses of Wy-14,643 had no discernible effect on DNA binding (Fig. 5B). The bile acids CA and CDCA, either alone, or in combination with Wy-14,643, had no effect on DNA binding by PPARalpha ·RXRalpha . Similarly, DNA binding by PPARbeta -RXRalpha or PPARgamma ·RXRalpha heterodimers was unaffected by treatment with the appropriate ligands (bezafibrate and troglitazone, respectively) or with the bile acids CA or CDCA (Fig. 5C). These data indicate that the inhibition of PPARalpha -dependent transactivation by bile acids does not occur at the level of DNA binding.


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  		Fig. 5.   EMSA analysis of PPAR·RXRalpha protein-DNA complexes. EMSAs were performed using in vitro translated PPARalpha , PPARbeta , PPARgamma , and RXRalpha as described under "Materials and Methods." A, PPAR·RXR heterodimers, but not homodimers, bind to a consensus DR-1 sequence (AGGTCAAAGGTCA). B, bile acids do not affect PPARalpha binding to the consensus DR-1 sequence. In vitro translated PPARalpha and RXRalpha protein were added to all lanes. Numbers indicate the concentration (in µM) of Wy-14,643, CA, or CDCA used in each experiment. C, bile acids do not affect PPARbeta or PPARgamma binding to the consensus DR-1 sequence. Either in vitro translated PPARbeta and RXRalpha or PPARgamma and RXRalpha protein were added to each of the indicated lanes. Numbers indicate the concentration (in µM) of ligand (bezafibrate for PPARbeta ; troglitazone for PPARgamma ), CA, or CDCA used in each experiment.

A mammalian two-hybrid assay was used to determine the ability of Wy-14,643 and CDCA to modulate the interaction of PPARalpha . Ligand-induced conformational changes that allow recruitment of coactivators, such as steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP) and dissociation of corepressors such as the nuclear receptor corepressor (NCoR), are obligatory for transactivation by PPARalpha (35, 47-51). The key components of this assay included reporter constructs for full-length murine PPARalpha fused to the transactivation domain of VP16 and the nuclear receptor interaction domains of CBP or NCoR fused to the DNA binding domain of GAL4. Treatment of transfected CV-1 cells with Wy-14,643 caused efficient CBP recruitment as evidenced by an increase in luciferase reporter gene activity (Fig. 6). In contrast, treatment with increasing concentrations of CDCA reduced both basal CBP association and Wy-14,643-induced CBP recruitment. Wy-14,643 treatment also reduced the constitutive level of PPARalpha ·NCoR association as evidenced by a decrease in reporter gene activation. Similarly, 100 µM CDCA treatment caused a significant decrease in NCoR association. CV-1 cells transfected with the PPARalpha construct exhibited no changes in reporter gene activity when treated with 100 µM Wy-14,643 alone or in combination with up to 100 µM CDCA in the absence of cotransfection with CBP or NCoR expression constructs.


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  		Fig. 6.   Inhibition of PPARalpha coactivator recruitment by chenodeoxycholic acid. A mammalian two-hybrid assay was used to detect the ligand-dependent interaction of PPARalpha with CBP and NCoR. Values shown are the fold activation relative to cells treated with Me2SO (DMSO) alone (mean ± S.D., n = 4). *, significantly different versus Me2SO alone, p < 0.05. #, significantly different versus 100 µM Wy-14,643 + Me2SO, p < 0.05.

To determine whether the interaction of bile acids with PPARalpha had the potential to exert meaningful effects on bile acid homeostasis, the expression of genes encoding important enzymes of hepatic bile acid biosynthesis was examined by Northern blotting. These included CYP7A1, a cholesterol 7alpha -hydroxylase that catalyzes the initial and rate-limiting step in the neutral pathway of bile acid biosynthesis (27, 52); CYP27, a sterol 27-hydroxylase that catalyzes the initial step of the acidic pathway of bile acid biosynthesis (52, 53); and CYP8B1, a sterol 12alpha -hydroxylase that participates in both pathways of bile acid biosynthesis (52). Feedback repression of multiple genes encoding enzymes of bile acid biosynthesis, including CYP7A1 and CYP8B1, occurs in an FXR-dependent manner (33). To dissociate the regulatory effects of bile acids mediated by FXR from those that may have been mediated by PPARalpha , both wild-type and FXR-null mice were utilized. As shown in Fig. 7, CA, either alone or in combination with Wy-14,643, caused a substantial reduction of hepatic CYP7A1 mRNA levels in wild-type mice. Essentially identical effects were observed for CYP8B1 mRNA levels in the livers of wild-type mice. As expected, and consistent with earlier work (33), repression of CYP7A1 and CYP8B1 mRNA levels by CA feeding was completely absent in FXR-null mice. Feeding Wy-14,643 alone caused a moderate increase of CYP7A1 mRNA levels in the livers of both wild-type and FXR-null mice. Concomitant feeding of CA completely abolished the inducing effects of Wy-14,643 in wild-type but not FXR-null mice. In contrast to CYP7A1, CYP8B1 mRNA levels were reduced by feeding Wy-14,643 alone, to both wild-type and FXR-null mice. Also in contrast to CYP7A1, concomitant feeding of CA reversed the repressive effects of Wy-14,643 feeding on hepatic CYP8B1 mRNA levels in FXR-null but not wild-type mice. Hepatic CYP27 mRNA levels were not substantially affected by CA feeding alone but were more markedly decreased by feeding the Wy-14,643 diet. These effects were similar for both wild-type and FXR-null mice.


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  		Fig. 7.   Expression genes encoding enzymes of bile acid biosynthesis in wild-type and FXR-null mice. Mice were fed normal chow for 5 days, a 1% CA diet for 5 days, chow for 3 days followed by a 0.1% Wy-14,643 diet for 2 days, or a 1% CA diet for 3 days followed by a 1% CA, 0.1% Wy-14,643 diet for 2 days. Total RNA was prepared from the livers of wild-type and FXR-null mice and analyzed by Northern blot analysis using the indicated cDNA probes. Numbers are the average (n = 4 for each group) fold change of each mRNA, after correction for the beta -actin signal, relative to that determined for mice fed the chow diet.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that dietary bile acid administration can inhibit, in vivo, both the physiological and gene expression outcomes of exposure to the well characterized PPARalpha ligand Wy-14,643. Furthermore, the basal expression of PPARalpha target genes, presumably activated by endogenous ligands, was also inhibited by dietary bile acid. The experiments with PPARalpha -null mice indicated that bile acids can affect the basal expression of some of these genes by a mechanism(s) independent of PPARalpha . For example, whereas CYP4A1, CYP4A3, and thiolase mRNA levels were decreased by CA feeding in PPARalpha -null mice, ACOX and BE mRNA levels were unaffected. In contrast, the constitutive expression of all of these genes was suppressed when wild-type mice were used. Thus, the effects of CA on the constitutive expression of PPARalpha target genes is promoter-specific and is most likely related to the quantitative importance of PPARalpha in the normal expression of these genes. Various nuclear receptors, in addition to PPARalpha , are capable of binding to the DR-1 elements located in the regulatory regions of PPARalpha target genes. For example, HNF-4alpha has been shown to interact with DR-1 elements in the CYP4A1 gene promoter (54). Evidence exists for the inhibition of HNF-4alpha transactivation by bile acids involving either activation of the mitogen-activated protein kinase pathway (55), repression of HNF-4alpha expression (56), or activation of SHP gene expression (42, 56). It is unknown if any of these mechanisms contributed to the reduction of CYP4A1, CYP4A3, and thiolase mRNA levels caused by CA feeding to PPARalpha -null mice. However, given that SHP induction is lacking in FXR-null mice, but CA repression of PPARalpha target genes is maintained, a mechanism dependent upon SHP induction is not likely.

In contrast to the observed effects of CA feeding on the basal expression of PPARalpha target genes in PPARalpha -null mice, the inducible expression of these genes was uniformly decreased by bile acid feeding to wild-type mice. The data presented in this study provide convergent evidence for a PPARalpha -dependent mechanism underlying this effect. First, bile acid inhibition of PPARalpha -dependent target gene induction was maintained in FXR-null mice. FXR-dependent induction of SHP by bile acids has been shown to be an important mechanism by which CYP7A1 repression is achieved in the liver. Studies have shown that SHP dimerizes with liver receptor homologue-1 (LRH-1) and inhibits its ability to constitutively transactivate the CYP7A1 gene (39, 40). Although SHP has been shown to inhibit the activity of various nuclear receptors in cell culture transactivation assays (41, 42, 57, 58), it is unlikely that a similar mechanism could account for our in vivo data because the effects of bile acid feeding on PPARalpha target gene activation are preserved in FXR-null mice. Second, consistent with the in vivo data, CDCA effectively inhibited the expression of a PPRE-luciferase reporter construct in transactivation assays. This effect was selective for PPARalpha as indicated by the inability of CDCA to inhibit PPARbeta - or PPARgamma -dependent transactivation in transient transfection assays. CA was without effect in these assays, most likely due to a lack of efficient uptake of this tri-hydroxylated bile acid by the cultured cells (36-38, 56). Finally, EMSA analysis revealed that neither CA nor CDCA inhibited DNA binding by PPARalpha . Although a physicochemical effect, independent of direct CA or CDCA binding with PPARalpha , cannot be ruled out, it is likely that these bile acids are capable of binding to PPARalpha . However, it is unlikely that this receptor-ligand complex is competent for transactivation as indicated by the inability of CDCA to stimulate PPARalpha recruitment of coactivators such as CBP.

PPARalpha is activated by a diverse group of chemical compounds including endogenous fatty acids such as linoleic acid and exogenous synthetic peroxisome proliferators such as Wy-14,643 and clofibrate. A common feature exhibited by most of these compounds is the presence of a free carboxylic acid function that may be present in the parent structure or may be unmasked by metabolic conversion (59). Homology model building (60) and x-ray crystallography of the PPARalpha ligand binding domain (61) indicate that ionization of this carboxylate anion at physiological pH allows electrostatic interactions with positively charged amino acids (e.g. lysine) and thereby stabilizes ligand binding. Alternatively, a biostere of a carboxylic acid group, such as a tetrazole or sulfonamide moiety, is present within the structures of other PPARalpha activators such as Fornesafen and LY171883. Most PPARalpha activators also have at least one aromatic ring or alkene bond that may engage in hydrophobic interactions with non-polar amino acids contained within the PPARalpha ligand binding pocket. As CA and CDCA possess a free carboxylic acid, it is clear that both of these compounds exhibit at least one feature common to well characterized PPARalpha activators. In contrast, whereas CA and CDCA have extensive hydrophobic regions, the absence of a conjugated alkene bond system within their structures delineates these bile acids from established PPARalpha activators. Although binding of CA or CDCA to PPARalpha has not been demonstrated in this study, the data presented are in support of this type of interaction. It is likely that the carboxylic acid group and hydrophobic nature of the bile acids promotes binding to PPARalpha . However, as indicated by the mammalian two-hybrid assay, the conformational change in PPARalpha elicited by this interaction is not compatible with the recruitment of coactivator proteins. Thus, CA and CDCA appear to act as antagonists of PPARalpha function.

The critical role of PPARalpha for the regulation of lipid metabolism is well established. This function is achieved, in large part, as a result of direct transcriptional regulation of many genes involved in this process. Experimental evidence also exists in support of a role for PPARalpha in the modulation of bile acid metabolism. The major route for hepatic cholesterol elimination is the bile, either through direct secretion or by metabolic conversion to bile acids. The initial and rate-limiting step in the conversion of cholesterol to bile acids via the neutral pathway is catalyzed by CYP7A1 (27). Regulation of the expression of the CYP7A1 gene occurs primarily at the transcriptional level and is affected by a number of hormonal and dietary factors including cholesterol and bile acids. Recent studies (29, 31) have suggested a potential role for PPARalpha in regulating CYP7A1 expression. However, these studies are in apparent conflict as evidence has been presented in support of both PPARalpha -dependent activation (31) and repression (29) of human and murine CYP7A1 reporter gene constructs in transient transfection assays. Our data indicate that hepatic CYP7A1 mRNA levels are modestly increased (2-3-fold) by feeding a diet containing Wy-14,643. However, this activation was unaffected by concomitant CA feeding to FXR-null mice suggesting that the effects of Wy-14,643 on CYP7A1 expression are PPARalpha -independent or that bile acids cannot effectively modulate PPARalpha activation of this promoter. Regardless of explanation, the similar levels of hepatic CYP7A1 mRNA in wild-type compared with PPARalpha -null mice (29) indicate that PPARalpha is unlikely to directly exert a significant regulatory influence under normal physiological conditions. Fibrate treatment lowers CYP7A1 catalytic activity in patients with hyperlipoproteinemia (62); however, no evidence exists for a direct effect of PPARalpha on the CYP7A1 gene promoter. CYP7A1 gene expression is positively affected by cholesterol and cholesterol derivatives both in vivo in rodent models (27, 63-65) and in transient transfection assays (6, 46, 66). Thus, it is possible that the effect of fibrates on this gene may be due in large part to the cholesterol-lowering actions of these drugs.

CYP8B1 is another enzyme of the bile acid biosynthesis pathway whose level of activity determines the ratio of cholic to chenodeoxycholic acid in the bile. Changes in biliary bile composition can have important effects on the intestinal absorption and biliary excretion secretion (28). Dietary administration of Wy-14,643 has been shown to result in increased hepatic levels of CYP8B1 mRNA and an increased proportion of biliary cholic acid in wild-type but not PPARalpha -null mice (32). Fasting also caused increased levels of CYP8B1 mRNA in wild-type but not PPARalpha -null mice. Metabolic processes that operate under conditions such as fasting and diabetes result in the production of free fatty acids that are natural ligands for PPARalpha . Thus, these data suggest that under these conditions bile with a higher proportion of the more hydrophobic (versus CDCA) and more effective solubilization agent, CA, would be produced resulting in enhanced absorption of dietary lipids from the intestine. In contrast, our data indicate that hepatic CYP8B1 mRNA levels are decreased by Wy-14,643 administration. The reason for this discrepancy is unknown; however, it is worth noting that the induction of CYP8B1 caused by fasting in mice is relatively modest (2-fold), and the PPRE within the promoter region of the gene does not exhibit a strong interaction with PPARalpha in EMSAs (32). CYP8B1 mRNA levels are also repressed by dietary bile acid administration, a mechanism that requires a functional FXR (33). In FXR-null mice, repression of CYP8B1 mRNA levels by Wy-14,643 is essentially reversed by concomitant dietary CA administration, consistent with the effects of bile acids on the expression of well characterized PPARalpha target genes observed in this study. Hepatic CYP27 mRNA levels were decreased slightly by Wy-14,643 administration to both wild-type and FXR-null mice. As CA feeding alone also caused a decrease of CYP27 mRNA levels in both wild-type and FXR-null mice, it is unclear if the repression of CYP27 mRNA levels caused by Wy-14,643 was affected by dietary bile acid.

Overall, our data do not provide strong support for a major role for PPARalpha in regulating bile acid homeostasis. This is due, in part, to the relatively modest effects of Wy-14,643 administration, particularly when compared with the effects of dietary CA administration, on the expression of genes encoding critical enzymes of bile acid biosynthesis. Furthermore, interactions between Wy-14,643 and CA, similar to those observed for other PPARalpha target genes, were generally only revealed when FXR-null mice were used, i.e. when the dominant effects of FXR-mediated bile acid repression of genes involved in bile acid biosynthesis are absent. Taken together, these data indicate that bile acid antagonism of PPARalpha function does not represent a significant mechanism by which bile acid homeostasis is achieved. It is also unlikely that bile acids exert a substantial effect on PPARalpha -dependent regulation of more responsive target genes, such as those involved in the beta -oxidation of fatty acids, under normal conditions. However, under certain pathophysiological conditions, such as cholestatic syndromes, where bile acids accumulate to extreme levels, bile acids may cause significant impairment of PPARalpha -dependent activation of these genes. This would be expected to exacerbate the severity of the disease state by virtue of disregulation of critical metabolic pathways affecting fatty acid utilization. Further investigation of this phenomenon will have important implications regarding the function of PPARalpha under physiological and pathophysiological states. Furthermore, the identification of bile acids as a structural class of molecules capable of antagonizing PPARalpha function represents an important finding relevant to the design and use of therapeutic agents that utilize PPARalpha as a molecular target.

    FOOTNOTES

* 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 Biology, Mokwon University, 800 Doan-dong, Seo-gu, Taejon, 302-729, Korea.

§ To whom correspondence should be addressed: Laboratory of Metabolism, Bldg. 37, Rm. 3E24, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9067; Fax: 301-496-8419; E-mail: fjgonz@ helix.nih.gov.

Published, JBC Papers in Press, October 17, 2001, DOI 10.1074/jbc.M107000200

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXR, retinoid X receptor; Wy-14, 643, Wyeth-14,643; ACOX, acyl-CoA oxidase; BE, bifunctional enzyme; CA, cholic acid; CDCA, chenodeoxycholic acid; FXR, farnesoid X-receptor; EMSA, electrophoretic mobility shift assay; CBP, CREB-binding protein; NCoR, nuclear receptor corepressor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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