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INTRODUCTION |
Cholesterol is a multifunctional molecule that is essential for a
broad array of physiologic processes including membrane biogenesis,
caveolae formation, and the distribution of embryonic signaling
molecules. It is also as an essential precursor in the synthesis of
transcriptionally active lipids including the steroid hormones and
oxysterols (1). Although essential, cholesterol is highly insoluble and
can form deposits that contribute to a variety of diseases including
gallstones and heart disease (2, 3). Indeed, excess circulating
cholesterol is a major risk for atherosclerotic heart disease (3, 4).
This disease is responsible for nearly 500,000 deaths each year in the
United States (42) and is the single largest cause of mortality in industrialized nations. It has been estimated that 10% of the U.S.
population would benefit from cholesterol-lowering therapies (5). This
has prompted an intense search for therapeutic agents that specifically
modulate cholesterol homeostasis.
Cholesterol levels are controlled at a variety of levels including
intestinal uptake, endogenous biosynthesis, transport, and elimination.
The major pathway for cholesterol elimination is via hepatic conversion
of cholesterol into water-soluble bile acids (6) and their subsequent
secretion into the gastrointestinal tract. Approximately 95% of the
secreted bile acids are recycled via intestinal uptake and are returned
to the liver through the portal blood. The remaining 5% of bile acids
are eliminated from the gut thereby forcing the liver to replenish
these losses by converting as much as 0.5 g of cholesterol to bile
acids each day (7). The liver therefore has an enormous capacity to
metabolize cholesterol and therapies that target this process have the
potential to eliminate cholesterol derived from a variety of sources
including diet, synthesis, and atherosclerotic lesions (via the reverse cholesterol transport pathway).
Two metabolic pathways have been identified that convert cholesterol to
bile acids (6). In humans, the classic pathway is responsible for at
least 90% of all bile acid synthesis. The first and rate-limiting step
in this pathway is catalyzed by
CYP7A1,1 a liver-specific
cholesterol 7
-hydroxylase. CYP7A1 transcription is
strongly repressed by its bile acid end products (8). An important
advance in understanding this feedback loop came with the
identification of a member of the nuclear receptor superfamily (FXR,
NR1H4, hereafter referred to as BAR) that suppresses CYP7A1 transcription in response to endogenous bile acids (9-12). Two bile
acid response elements (BAREs) have been identified in the CYP7A1 promoter. However, BAR is unable to bind directly to
either element, suggesting an indirect role for BAR in the regulation of CYP7A1 (13). A mechanism has been proposed whereby BAR
induces the negative transcriptional regulator SHP (small
heterodimer partner), which in turn represses transcription factors
that bind to the CYP7A1 BAREs (14, 15). This mechanism for
CYP7A1 repression was suggested based on experiments using
transiently overexpressed SHP. Because SHP can repress (16, 17) and/or
activate (18) numerous nuclear receptors under these conditions, the
SHP-induction model does not account for the specificity by which bile
acids regulate gene transcription.
Although the mechanisms underlying transrepression by BAR is unclear,
it is well known that BAR activates transcription by binding to
specific response elements (19, 20) as a heterodimer with the nuclear
receptor RXR. Several genes have been identified whose transcription is
activated by BAR including SHP, the ileal bile acid-binding
protein (IBABP), and the hepatic bile salt export pump
(BSEP, ABCB11) (21). These genes are critical for
bile acid homeostasis. IBABP is an intracellular protein expressed in
the distal ileum where the majority of bile acids are reabsorbed. It
has been proposed that IBABP plays a role in transcellular shuttling
and/or buffering the high and otherwise toxic levels of bile acids that
pass through this tissue. BSEP is a canalicular ATP binding
cassette transporter that is responsible for biliary secretion of bile
acids. Indeed, inactivating mutations of this gene result in
progressive familial intrahepatic cholestasis (type 2) and hepatic
cirrhosis (22). Thus, in addition to regulating cholesterol
degradation, BAR plays a more general role in coordinately regulating
bile acid physiology.
BAR also controls other aspects of lipid homeostasis. For example, BAR
agonists reduce triglyceride levels (23, 24) and BAR-null mice have
elevated triglycerides (12). This is potentially related to
BAR-mediated regulation of apolipoprotein CII and/or phospholipid
transfer protein (reviewed in Ref. 21). Regardless of the mechanism, it
appears that BAR activation promotes reciprocal effects on cholesterol
and triglyceride levels.
Given its critical role in repressing CYP7A1-mediated
cholesterol degradation, BAR provides an attractive target for the
development of novel cholesterol lowering agents. However, the effects
of BAR on other target genes implies that a generalized antagonist would produce undesirable effects including elevations in
triglycerides, a lowering of biliary bile acid transport, and/or
cholestasis. Therefore, the most desirable therapeutic agents would be
gene-selective modulators that selectively regulate a subset of
BAR-specific genes. In a search for such compounds we have identified
two classes of BAR modulators. The first class include agonists that
are ~25-fold more potent than naturally occurring bile acids. These
compounds activate BAR and produce the expected regulation pattern on
endogenous target genes. Second, we have identified AGN34 as a
gene-selective BAR modulator (BARM): it acts as
an agonist on CYP7A1, an antagonist on IBABP, and
is neutral on SHP. These data demonstrate that
SHP induction is not required to repress CYP7A1.
More significantly, we provide evidence for gene-selective BARMs, a
finding with important implications for the treatment and prevention of
atherosclerotic heart disease.
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EXPERIMENTAL PROCEDURES |
Reagents--
Full-length proteins were expressed using the pCMX
vector (25) and have been previously described (9).
Gal-L-RAR is the human RAR
ligand binding domain
(Glu156-Ser463, accession number X06614) fused
to the C-terminal end of the yeast Gal4 DNA binding domain.
Gal-L-RXR has been described (9). BAR AF2m contains a
single point mutation (E464A) and RXR AF2m contains a double point
mutation (M454L/L455A) in helix 12 of rat BAR and human RXR
,
respectively. The luciferase reporter constructs were as follows. Mouse
IBABP IR-1 (CCTTAAGGTGAATAACCTTGGGGCTCC) x3 was
used as a reporter for BAR, PPREx3 for PPAR
, -
, and -
,
RE2x3 for CAR and RAR, LXREx3 for LXR, MLVx3 for triiodothyronine, the mouse mammary tumor virus LTR was used for AR, UASGx4
(MH100x4) was used as a Gal4 reporter (9), SPPx3 for
1,25-dihydroxyvitamin D3 (25), EREx1 for ER
(26),
CYP3A4x3 was used for mouse and human PXR (27), and apoB-E4 for
HNF4
. The synthesis of AGN29, AGN31, and AGN34 will be described elsewhere.
Transient Transfection Assay--
CV-1 cells were grown and
transiently transfected as described (9). The data presented were
obtained with mouse BAR but human and rat BAR exhibited qualitatively
similar results.
Northern Analysis--
HepG2 cells were maintained in Eagle's
minimal essential medium with 10% fetal bovine serum, 1 mM
sodium pyruvate, 2 mM L-glutamine, and
nonessential amino acids. Caco-2 cells were maintained in Dulbecco's
modified Eagle's medium with 20% fetal bovine serum. Caco-2 cells
were maintained for 20 days post-confluence to allow differentiation.
One day prior to treatment, confluent HepG2 and differentiated Caco-2
cells were switched to phenol red-free media containing
resin-charcoal-stripped fetal bovine serum and treated for an
additional 24 h with the indicated compounds. Northern blots were
prepared from poly(A)+ RNA and analyzed with the
following probes: human CYP7A1 nucleotides 1617-2576
(accession number M93133), human SHP nucleotides 888-1355
(accession number L76571), human IBABP coding sequence (accession number AI311734), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nucleotides 105-331 (accession number
NM_002046). The RNA levels were quantitated using a GS-800 calibrated
densitometer and Quantity One software (Bio-Rad).
DNA-based Coactivator Recruitment Assay--
This assay was
performed as previously described (9). An optimized DR-1 was used for
RXR homodimers and the mouse IBABP IR-1 response element (10) was used
for BAR·RXR heterodimers. The data presented were obtained with rat
BAR.
Competitive Ligand Binding Assay--
RXR
proteins were
produced using a baculovirus expression system and binding assays were
performed as described previously (28).
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RESULTS |
Identification of Synthetic BAR Agonists--
To begin to identify
BAR-specific modulators, we noted that the synthetic retinoid TTNPB
((E)-4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propen-1-yl)benzoic acid) (Fig. 1A) binds to and
activates BAR (11, 29). A major limitation in the use of TTNPB as a BAR
agonist is that TTNPB is a 1000-fold higher affinity ligand for the
retinoic acid receptor (RAR) (30). Thus, it is important to identify
TTNPB derivatives that lack activity on RAR.

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Fig. 1.
Identification of BAR agonists.
A, chemical structures of CDCA, TTNPB, AGN29, and
AGN31. The shaded circles show the trimethylsilyl (AGN29)
and n-butyl (AGN31) groups that corresponds to the methyl
moiety of TTNPB and the cyclohexane of the BAR-specific ligand CDCA.
B, AGN29 and AGN31 activate BAR. CV-1 cells were
transiently transfected with BAR, RXR, the IBABP IR-1x3 TK-Luc reporter
and CMX- gal as an internal control. After transfection, cells were
treated with the following compounds: 100 µM CDCA, 5 µM AGN29, 5 µM AGN31, 5 µM
TTNPB, and 100 nM LG268. The reporter activity was
normalized to the internal -galactosidase control and the data were
plotted as -fold activation relative to untreated cells.
C, AGN29 and AGN31 are poor activators of RAR. AGN29
and AGN31 exhibit 6.7 and 5.3% of the maximal activation observed with
TTNPB. CV-1 cells were transiently transfected with vectors expressing
Gal4 fused to the ligand binding domain of RAR
(Gal-L-RAR) and the UASGx4 TK-Luc Gal4
reporter. After transfection, the cells were treated as in
B. D, AGN29 and AGN31 activate RXR.
Experimental conditions were the same as C, except that
Gal-L-RXR was used in place of Gal-L-RAR.
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Analysis of the crystal structure of retinoid-bound RAR indicates that
the methyl group in the central isoprene unit (Fig. 1A,
TTNPB, shaded area) interacts with critical
residues in helix 5 of the RAR ligand binding domain (31). We
hypothesized that placement of bulky residues in this position would
create a steric hindrance to RAR binding. At the same time, compounds
with a bulky moiety in this location might retain activity on BAR as
this structure would more closely approximate the cyclohexane ring at
the corresponding position of the BAR ligand chenodeoxycholic acid
(CDCA) (Fig. 1A). To test this hypothesis, AGN29 and AGN31
were synthesized with trimethylsilyl and n-butyl groups in
place of the methyl moiety on the central isoprene unit of TTNPB (Fig.
1A, shaded area). At concentrations of 5 µM, AGN29 (91-fold) and AGN31 (85-fold) were potent and
efficacious activators of the BAR·RXR heterodimer (Fig.
1B). By comparison, TTNPB was less efficacious in activating BAR (65-fold) and CDCA, an endogenous BAR ligand, required 20-fold higher concentrations (100 µM) for optimal activity (Fig.
1B). As predicted, AGN29 and AGN31 were both dramatically
less effective than TTNPB in activating RAR (Fig. 1C) and
had no effect on other nuclear receptors including AR, mouse, and human
PXR, ER
, CAR, LXR
, PPAR
, PPAR
, PPAR
, VDR, and
T3R
(data not shown). Thus, unlike the parent ligand
TTNPB, AGN29 and AGN31 exhibit significant selectivity for BAR.
RXR-specific ligands such as LG268 are known to activate the BAR·RXR
heterodimer (Fig. 1B), however, we have previously
established that this occurs via the RXR subunit and not via BAR (9).
Because TTNPB derivatives were originally used to identify RXR-specific ligands (30), we tested the ability of AGN29 and AGN31 to activate RXR.
Both compounds activated RXR at the same concentrations required to
activate BAR·RXR heterodimers (5 µM) and were inactive
at lower doses (data not shown). Although AGN29 and AGN31 activate RXR, their action is distinct from that of LG268. For example, when compared
with LG268, AGN29 and AGN31 preferentially activate BAR·RXR heterodimers whereas LG268 preferentially activates RXR (compare Fig.
1, B and D).
To further explore the molecular properties of AGN29 and AGN31 we
examined their effects on coactivator recruitment in vitro. The primary function of nuclear receptor ligands is to induce a
conformation change that facilitates recruitment of transcriptional coactivator proteins via the AF2 transactivation domain (32). Previous
coactivator recruitment assays have demonstrated that bile acids and
TTNPB bind directly to BAR (9, 11). We used an electrophoretic mobility
shift assay (9) to determine whether AGN29 and AGN31 also serve as BAR
ligands. Thus, BAR·RXR heterodimers were formed on a
32P-labeled BAR response element and incubated with a
peptide containing the receptor interaction domains of the coactivator
GRIP1. As expected, both AGN29 and AGN31 effectively recruited
coactivator (Fig. 2A,
lanes 1-3). LG268 also recruited coactivator (Fig.
2A, lane 4) but was less effective than AGN29 and
AGN31, a finding that parallels the transactivation data (Fig. 1,
B and D). To explore the subunit requirements of
these compounds, we asked whether the BAR or RXR AF2 was required for
these interactions. Mutation of the BAR AF2 resulted in loss of
activity for both AGN29 and AGN31 whereas mutation of RXR AF2 had no
effect (Fig. 2A, lanes 5-7 and
9-11). In contrast, recruitment by LG268 required the AF2
domain of RXR as well as BAR (Fig. 2A, lanes 8 and 12). Thus, whereas AGN29 and AGN31 can activate an
isolated RXR subunit (Fig. 1D), the results of Fig.
2A demonstrate that BAR is essential for the activity of
AGN29 and AGN31 in the context of the BAR·RXR heterodimer.

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Fig. 2.
AGN29 and AGN31 are high affinity BAR
ligands. A, AGN29 and AGN31 recruit coactivator
via the AF2 domain of BAR. DNA-based recruitment assays were performed
by mixing GST-GRIP, a 32P-labeled IBABP IR-1 probe,
wild-type (wt), or AF2-mutated (AF2m) receptors
and the indicated ligands: 10 µM AGN29, 10 µM AGN31, or 500 nM LG268. B,
dose response for activation of BAR by AGN29 and AGN31. Transfections
were performed as in Fig. 1B with increasing ligand
concentrations. C, dose response for in
vitro coactivator recruitment by AGN29 and AGN31. Recruitment
assays were performed as in A using increasing amounts of
AGN29 (top panel) or AGN31 (bottom panel).
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To determine the relative potency of AGN29 and AGN31, transfected cells
were treated with increasing doses of AGN29 and AGN31 (Fig.
2B). The half-maximal effective concentrations
(EC50) for AGN29 and AGN31 are ~2 µM
compared with >50 µM for CDCA. Similarly, using the
coactivator recruitment assay, AGN29 and AGN31 produced a
dose-dependent increase in the GRIP1-containing complex
(Fig. 2C). The concentration of AGN29 and AGN31 that
promoted GRIP recruitment closely paralleled the potency of these
compounds in transfection assays (Fig. 2B). These results
indicate that AGN29 and AGN31 are not only BAR ligands but are
>25-fold more potent than their endogenous counterparts.
Synthetic Agonists Regulate BAR Target Genes--
BAR regulates
transcription of specific target genes in the liver (CYP7A1,
SHP) and intestine (IBABP, SHP)
(reviewed in Ref. 21). This prompted us to test the effect of AGN29 and
AGN31 on gene expression in HepG2 hepatoma cells and in differentiated intestinal Caco-2 cells. Both cell lines were treated for 24 h with optimal concentrations of AGN29, AGN31 (10 µM), or
CDCA (100 µM). Like CDCA, AGN29 and AGN31 strongly
induced SHP and IBABP expression in Caco-2 cells
(Fig. 3A). Similarly, these
compounds acted as agonists in HepG2 cells and produced the expected
induction of SHP and repression of CYP7A1 (Fig.
3B). AGN29 and AGN31 had no effect on expression of
BAR (data not shown) or the GAPDH internal control (Fig. 3). These results demonstrate that AGN29 and AGN31 are
agonists on endogenous target genes.

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Fig. 3.
AGN29 and AGN31 regulate BAR target
genes. A, Northern blot analysis using
poly(A)+ RNA (1 µg) from differentiated Caco-2 cells
treated with CDCA (100 µM), AGN29 (10 µM),
or AGN31 (10 µM) for 24 h. B, same
as A but with HepG2 cells. The RNA levels were quantified
and the relative changes, normalized for GAPDH levels, are indicated.
The levels of RNA in untreated cells were given an arbitrary value of
1. In the case where there is undetectable expression in untreated
cells, the background was also given an arbitrary value of 1 and hence
the -fold activation that is listed represents a minimal or
underestimate of the actual activity. GAPDH,
glyceraldehyde-phosphate dehydrogenase.
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Identification of a BAR Antagonist--
We next explored the
possibility that retinoid derivatives might serve as BAR antagonists.
Indeed, using the transfection assay described above, AGN34 (Fig.
4A, 1 µM) was
identified as a compound that effectively blocked transcriptional
activation by the endogenous CDCA ligand (50 µM) (Fig.
4B). AGN34 had no effect on basal reporter activity (Fig.
4B). AGN34 also failed to antagonize the nuclear receptors
AR, HNF4
CAR, LXR, mouse and human PXR, PPAR
, PPAR
, PPAR
,
and VDR and exhibited only minimal activity on ER
,
T3R
, and RAR (Fig. 4C). These data
demonstrate that AGN34 is a selective antagonist of BAR activity.

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Fig. 4.
Identification of a BAR-specific
antagonist. A, chemical structure of AGN34.
B, AGN34 prevents CDCA-mediated activation of BAR.
Transfections were performed as in Fig. 1B using CDCA (50 µM) with or without AGN34 (1 µM).
C, AGN34 is a BAR-specific antagonist. CV-1 cells were
transfected with the indicated receptors and corresponding reporter
constructs (see "Experimental Procedures"). The cells were then
treated with receptor-specific ligands alone or with 1 µM
AGN34. Specific ligands were as follows: human AR (1 µM
dihydrotestosterone), mouse PXR (10 µM pregnenolone
16 -carbonitrile), human PXR (10 µM rifampicin), ER
(100 nM 17 -estradiol), human LXR (30 µM
hyodeoxycholic acid methyl ester), mouse PPAR (5 µM Wy
14,643), mouse PPAR (1 µM rosiglitazone), mouse
PPAR (1 µM carbaprostacyclin), human
1,25-dihydroxyvitamin D3 (100 nM VDR), human
T3R (100 nM triiodothyronine), and endogenous RAR
(100 nM Am580). No agonist ligand was required for the
constitutively active receptors CAR and HNF4; for these receptors -fold
repression represents the effect of AGN34 on constitutive
activity.
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AGN34 Is a Direct and "Trans-antagonist"--
Because AGN34
possesses a partial retinoid-like structure, we examined the effect of
this compound on the retinoid receptors RAR and RXR. AGN34 was a weak
antagonist of RAR and other receptors that utilize RXR as an obligate
heterodimeric partner (Fig. 4C). However, AGN34 was an
effective antagonist of RXR homodimers (Fig. 4C). This
prompted us to compare the activity of AGN34 on BAR·RXR and RXR
complexes. Dose-response analysis demonstrated that AGN34 antagonized
both receptor complexes with a half-maximal inhibitory concentration
(IC50) of <10 nM (Fig.
5, A and C). Thus,
the ability to antagonize RXR and BAR at similar doses suggest that
AGN34 acts to trans-antagonize bile acid-activated BAR·RXR by
binding to the RXR subunit.

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Fig. 5.
AGN34 is an antagonist
of BAR and RXR. A, dose response analysis for
antagonism of BAR and RXR. Transfections were as described in the
legend to Fig. 1B using Gal-L-RXR and
UASGx4 TK-Luc or BAR, RXR, and IBABP IR-1x3 TK-Luc. After
transfection, cells were treated with 50 nM LG268
(Gal-L-RXR) or 50 µM CDCA (BAR·RXR) and
increasing doses of AGN34. B, AGN34 is an RXR ligand.
RXR was incubated with 10 nM
[20-methyl-3H]9-cis-retinoic acid
(9-cis-RA) and increasing concentrations of AGN34.
Ligand-bound receptors were isolated with hydroxyapatite resin and the
amount of bound radioactive ligand was determined using a Micro-Beta
counter. Binding in the absence of AGN34 was set at 100%.
C, AGN34 reverses coactivator recruitment. For RXR,
coactivator displacement assays were performed by mixing in
vitro translated RXR (0.6 µl), 75 ng of purified GST-GRIP, and a
32P-labeled DR-1 probe with agonist alone (25 nM LG268) or agonist with increasing concentrations of
AGN34 as indicated (top panel). For BAR, assays were
performed by mixing RXR (0.6 µl) and BAR (0.6 µl), 2 µg of
purified GST-GRIP, and a 32P-labeled IBABP IR-1 probe with
agonist alone (2 µM AGN31) or agonist with increasing
concentrations of AGN34 (bottom panel). The receptor dimer
( ) and receptor-coactivator complexes ( ) were separated by
electrophoresis through nondenaturing polyacrylamide gels.
D, AGN34 is a more effective antagonist than LG754.
Transfections were performed as described in the legend to Fig.
1B and cells were treated with 50 µM CDCA
alone or in combination with 1 µM AGN34 or 1 µM LG754. The luciferase reporter activity was normalized
to the -galactosidase internal control. E, reversal
of coactivator recruitment by LG754. Coactivator displacement assay was
performed as in C, except with increasing
concentrations of LG754. The receptor dimer ( ) and
receptor-coactivator complex ( ) are indicated.
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We used an in vitro radioligand displacement assay to
confirm that AGN34 acts directly on the RXR subunit. RXR was incubated with [20-methyl-3H]9-cis-retinoic
acid and binding was measured in the presence of increasing
concentrations of unlabeled AGN34. We found that AGN34 associates with
RXR with a binding constant of ~2 nM (Fig. 5B). The high in vitro affinity of AGN34 for RXR
closely matches its IC50 for both RXR and BAR·RXR
heterodimers. These findings confirm that AGN34 can trans-antagonize
BAR·RXR via the RXR subunit.
While AGN34 can function via the RXR subunit, this does not exclude the
possibility that it could have additional activities via the BAR
subunit. Indeed, examination of the dose-response curve for BAR·RXR
(Fig. 5A) indicates that AGN34-mediated antagonism is
somewhat biphasic. Antagonism is first seen at low doses corresponding to those associated with RXR binding (
10 nM). A plateau
is seen at intermediate doses (100-1000 nM) and then a
second phase of antagonism is observed at high doses (>1
µM). This pattern implies that AGN34 acts via the RXR
subunit at low doses and via the BAR subunit at higher doses.
To further explore the activity of AGN34 on the BAR subunit, we
examined the effect of low and high dose AGN34 on agonist-induced coactivator recruitment. Whereas agonist ligands recruit coactivators to nuclear receptors, antagonists occupy the ligand binding pocket but
fail to recruit coactivators (33). When agonists and antagonists are
mixed, the antagonist competes with the agonist for binding and
promotes an apparent displacement of coactivator. As expected, the
RXR-specific ligand LG268 induced coactivator recruitment to RXR
homodimers (Fig. 5C, top panel, lanes
1 and 2). Addition of AGN34 resulted in a displacement
of coactivator from RXR homodimers with an IC50 of ~30
nM (Fig. 5C, top panel, lanes
3-10). In contrast, low doses of AGN34 (<30 nM)
resulted in only a partial displacement of coactivator from BAR (Fig.
5C, bottom panel, lanes 1-5). Full coactivator displacement and reappearance of the coactivator-free heterodimer required concentrations greater than 300 nM
(Fig. 5C, bottom panel, lanes 6-10).
Similar results were seen using a mutated RXR with decreased ligand
binding affinity (data not shown) suggesting that AGN34
functions via BAR at these high doses. This biphasic pattern of
coactivator displacement in vitro further demonstrates that
AGN34 antagonizes BAR·RXR via both subunits.
The ability to trans-antagonize BAR·RXR with RXR antagonists raises
the possibility that previously characterized RXR antagonists such as
LG754 (34) might generally serve as BAR·RXR antagonists. However,
LG754 did not effectively antagonize BAR in transfection assays (Fig.
5D) and only displaced coactivator from agonist-occupied BAR·RXR heterodimers at very high concentrations (3 µM)
(Fig. 5E). Thus unlike previous RXR antagonists, AGN34 is
distinct in its ability to trans-antagonize BAR·RXR.
A Gene-specific Bile Acid Receptor Modulator--
The above data
demonstrate that AGN34 is a potent and selective antagonist of BAR that
functions via a unique form of trans-antagonism. This prompted us to
explore the effect of this compound on a variety of endogenous BAR
target genes. Differentiated Caco-2 and HepG2 cells were treated with
100 µM CDCA alone or in combination with 1 µM AGN34. In Caco-2 cells AGN34 dramatically inhibited
bile acid-induced expression of IBABP (Fig.
6A), an effect that was observed with doses of AGN34 as low as 30 nM (data not
shown). These results demonstrate that AGN34 is a potent antagonist of endogenous BAR target genes.

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Fig. 6.
AGN34 is a selective BAR modulator.
A and B, Northern blot analyses were
performed as described in the legend to Fig. 3, except that Caco-2
(A) and HepG2 (B) cells were treated with 100 µM CDCA and/or 1 µM AGN34, as indicated.
The relative change in expression of each gene, normalized to GAPDH, is
indicated. RNA levels for each gene in untreated cells were given a
value of one. C, dose response of AGN34 on
CYP7A1 expression in HepG2 cells. Northern blot analyses
were performed with HepG2 cells treated with ligands as indicated. The
relative change in expression of CYP7A1 is also indicated
with the RNA levels in untreated cells given a value of 1.
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To further examine the activity of AGN34, we tested its effect on
SHP expression. Although SHP is induced by BAR
agonists in both cell lines, the antagonist AGN34 failed to inhibit
bile acid-mediated induction of SHP (Fig. 6, A
and B). Because AGN34 is a highly effective antagonist of
IBABP expression, these findings demonstrate that AGN34 is a
gene-specific antagonist. We next tested the effect of AGN34 on
CYP7A1, a gene whose transcription is repressed by BAR
agonists (Fig. 3B). One would expect that a pure BAR
antagonist would relieve bile acid-mediated suppression of
CYP7A1 expression. In contrast, AGN34 unexpectedly repressed CYP7A1 by itself and further repressed activity in concert
with the CDCA agonist (Fig. 6B). Dose-response experiments
with AGN34 further confirmed that AGN34 repressed CYP7A1 by
itself and in combination with CDCA (Fig. 6C). The dose
response in this assay is consistent with the dose response required to
antagonize BAR activity in transient transfection and coactivator
recruitment assays (Fig. 5, A and C). These
results demonstrate that the activity of AGN34 is dramatically
gene-selective: AGN34 acts as an antagonist on IBABP, has no
effect on SHP, and is an agonist on CYP7A1. Thus, AGN34 provides the first example of a novel class of BAR ligands that
we refer to as gene-selective BAR modulators (BARMs). Moreover, the
ability of AGN34 to repress CYP7A1 without inducing
SHP demonstrates that BAR can repress CYP7A1
through mechanisms that do not require the induction of
SHP.
 |
DISCUSSION |
CYP7A1-mediated conversion of cholesterol to bile acids is the
main route for elimination of cholesterol from the body (6). Interventions that enhance CYP7A1 activity are expected to provide a
powerful approach for treating hypercholesterolemia and
atherosclerosis. Indeed, adenoviral mediated overexpression of
CYP7A1 is sufficient to reduce plasma low density
lipoprotein concentrations by ~60-75% (35). The most commonly used
cholesterol-lowering drugs are the statin class of cholesterol
synthesis inhibitors. These agents are nonspecific in that they inhibit
the synthesis of both cholesterol and its biologically active
precursors. In addition, inhibitors of cholesterol synthesis cannot
eliminate pre-existing cholesterol that arises from dietary or other
sources. Therefore, an urgent need exists for additional therapeutic
strategies. Indeed, coronary arterial disease remains the leading cause
of death in industrialized societies and 36-million Americans require
cholesterol-lowering therapies (5).
It has been well established that CYP7A1 transcription is
strongly repressed by its bile acid end products. Although bile acid-mediated repression is conserved in a variety of mammalian species, this pathway is particularly sensitive in humans (36). Drugs
that antagonize bile acid-mediated repression of CYP7A1 would be particularly useful in stimulating cholesterol elimination in
humans. A key advance in elucidating the molecular events underlying CYP7A1 repression was the demonstration that the nuclear
receptor BAR suppresses CYP7A1 transcription in response to
endogenous bile acids (9-12). In principle, a BAR-specific antagonist
would prevent CYP7A1 repression thereby facilitating further
cholesterol catabolism. Whereas BAR has potent effects on
CYP7A1, this receptor plays a broader role in regulating
lipid homeostasis. For example, BAR activation lowers triglycerides
(23, 24) and stimulates expression of genes involved in biliary bile
acid secretion. Thus, a generalized BAR antagonist has the potential to
induce serious side effects including cholestasis and
hypertriglyceridemia. An attractive means to bypass these effects would
be the identification of antagonists whose activities are limited to a
subset of target genes.
We describe two novel BAR agonists: AGN29 and AGN31. These compounds
are 25-fold more potent than naturally occurring ligands and resulted
in the expected activation (IBABP, SHP) or repression (CYP7A1) of BAR-target genes. AGN29 and AGN31 are derived
from TTNPB, a synthetic retinoid that is a ligand for both BAR and RAR
(11, 29, 30). In contrast, AGN29 and AGN31 are BAR-selective ligands in
that they have weak activity on RAR and fail to activate other nuclear
receptors. Structure-activity studies suggest that their weak activity
on RAR results from the placement of bulky functional groups on the
central isoprene unit. Indeed, analysis of the crystal structure of
retinoid-bound RAR indicates that these bulky residues would clash with
critical residues in helix 5 of the RAR ligand binding domain (RAR
Met272) (31).
Interestingly, AGN29 and AGN31 are unique among BAR ligands in that
they also activate RXR, the heterodimeric partner required for the
formation of an active BAR complex. Thus, AGN29 and AGN31 function
through both subunits of the BAR·RXR heterodimer. This prompted us to
examine whether other retinoid-like compounds might antagonize the BAR
complex. Using transient transfection and in vitro
coactivator recruitment assays, we identified AGN34 as an extremely
potent compound that antagonized BAR·RXR at concentrations as low as
10 nM. Antagonism at these low concentrations resulted from
direct binding to the RXR subunit and subsequent reversal of
agonist-induced coactivator recruitment (Fig. 5C). Because coactivator recruitment requires the AF2 transactivation domain of BAR
(Fig. 2A), the ability to displace coactivator while binding to RXR demonstrates that AGN34 is an allosteric inhibitor of BAR. Although RXR functions as an obligate partner for many receptors, the
effect of AGN34 was specific to BAR (Fig. 4C) and other RXR antagonists were not effective at antagonizing BAR (Fig.
5D). This mode of specific "trans-antagonsim" via a
partner receptor has not been previously described among the nuclear
receptor superfamily.
Although AGN34 antagonizes BAR in transient reporter assays, this
compound functions as a gene-selective modulator in vivo: it
acts as an agonist on CYP7A1, an antagonist on
IBABP, and is neutral on SHP. This divergent
pattern of regulation is reminiscent of the activity of selective
estrogen receptor (ER) modulators or SERMs (37). These compounds elicit
an array of biological effects that are either estrogenic or
antiestrogenic depending on the tissue. For example, SERMs such as
tamoxifen and raloxifene are used for the prevention and treatment of
breast cancer by virtue of their ability to antagonize ER in the
breast. In contrast, tamoxifen and raloxifene act as ER agonists in
other tissues. These unexpected activating properties have significant
advantages as ER agonists have beneficial effects on bone density and
plasma lipoprotein levels. By analogy to the SERMs, AGN34 represents a
selective BARM. The ability to regulate BAR in a gene-specific fashion suggests that future compounds may be identified that selectively enhance cholesterol elimination without promoting negative
effects such as hypertriglyceridemia or cholestasis.
Whereas the divergent activities of SERMs have been appreciated for
over 2 decades the molecular mechanisms that underlie their action are
only now being elucidated (reviewed in Ref. 38). ER regulates
transcription either by binding to specific response elements or
indirectly by tethering to other promoter-bound transcription factors.
In both cases ER agonists recruit transcriptional coactivators to its
targeted promoters. However, in breast cancer cells where corepressor
proteins are more highly expressed, tamoxifen act as an antagonist by
recruiting corepressors. In endometrial cells the same drug acts as an
agonist by recruiting more highly expressed coactivators to ER-tethered
promoters. Thus, the direction of SERM activity is determined in a
combinatorial fashion by at least three factors: the conformation of
the ligand-receptor complex, the promoter context, and the relative
levels of expression of specific coactivator and corepressor proteins.
Like their SERM counterparts, the future development of BARMs will
benefit from a more complete description of the mechanisms by which BAR
regulates gene transcription. Direct binding sites for BAR have been
identified in the promoters of certain target genes (e.g.
IBABP and SHP) (21) but it remains unclear which coactivator proteins and/or transcription factors are utilized by BAR
for in vivo regulation of these genes. In the case of
transrepression, several negative BAREs have been identified in the
CYP7A1 gene but BAR does not interact directly with these
elements (13). Instead, it had been proposed that transrepression
occurs by BAR-mediated stimulation of SHP, which in turn represses
transcription at the negative BARE (14, 15). Our findings demonstrate
that alternative mechanisms must be utilized as CYP7A1
repression occurs without an induction of SHP. Indeed, very recent
studies with SHP-null mice have reached a similar conclusion (39). By
dissociating SHP induction from CYP7A1
repression, AGN34 provides a convenient tool for the future elucidation
of this SHP-independent pathway of transrepression.
While this work was in progress, a plant-derivative known as
guggulsterone was also identified as a BAR antagonist (40, 41).
However, this compound was 1000-fold less potent than AGN34 and was not
specific: guggulsterone partially inhibited many nuclear receptors (40,
41) and we find that it fully inhibited CAR and strongly activated PXR
(data not shown). The activity of guggulsterone on the critical
CYP7A1 target gene was not reported and would be difficult
to assess as other receptor targets (PXR) for guggulsterone also
regulate CYP7A1. Thus, guggulsterone is a low-affinity,
low-specificity antagonist and it is unclear whether it possesses the
gene-selective activity that is characteristic of AGN34.