From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032
Received for publication, January 31, 2001, and in revised form, April 19, 2001
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ABSTRACT |
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The human cholesteryl ester transfer protein
(CETP) transfers cholesteryl esters from high density lipoproteins to
triglyceride-rich lipoproteins, indirectly facilitating cholesteryl
esters uptake by the liver. Hepatic CETP gene expression is
increased in response to dietary hypercholesterolemia, an effect that
is mediated by the activity of liver X receptor/retinoid X receptor
(LXR/RXR) on a direct repeat 4 element in the CETP
promoter. In this study we show that the orphan nuclear receptor LRH-1
also transactivates the CETP promoter by binding to a
proximal promoter element distinct from the DR4 site. LRH-1 potentiates
the sterol-dependent regulation of the wild type
CETP promoter by LXR/RXR. Small heterodimer partner, a
repressor of LRH-1, abolishes the potentiation effect of LRH-1 but not
its basal transactivation of the CETP promoter. Since this
mode of regulation of CETP is very similar to that recently reported for the bile salt-mediated repression of Cyp7a
(encoding the rate-limiting enzyme for conversion of cholesterol into
bile acid in the liver), we examined the effects of bile salt feeding on CETP mRNA expression in human CETP
transgenic mice. Hepatic CETP mRNA expression was
repressed by a diet containing 1% cholic acid in male mice but was
induced by the same diet in female mice. Microarray analysis of hepatic
mRNA showed that about 1.5% of genes were repressed, and 2.5%
were induced by the bile acid diet. However, the sexually dimorphic
regulatory pattern of the CETP gene was an unusual
response. Our data provide further evidence for the regulation of
CETP and Cyp7a genes by similar molecular mechanisms, consistent with coordinate transcriptional regulation of
sequential steps of reverse cholesterol transport. However, differential effects of the bile salt diet indicate additional complexity in the response of these two genes.
The cholesteryl ester transfer protein
(CETP)1 catalyzes the
transfer of cholesterol ester from HDL to triglyceride-rich
lipoproteins (1). CETP is expressed in liver, intestine, and a number
of peripheral tissues, such as adipose (1). In humans and animals, plasma CETP and tissue mRNA levels are increased in response to high fat, high cholesterol diets or endogenous hypercholesterolemia. These increases are due to elevated CETP gene transcription
especially in the liver (2, 3). Transgenic mice expressing human CETP, controlled by its natural flanking region, also increase expression of
CETP in response to hypercholesterolemia (4). The mechanism of this
effect was recently shown to involve the transcription factor LXR,
binding as a heterodimer with RXR to a site in the CETP
proximal promoter, a direct repeat of a nuclear receptor binding
sequence separated by 4 nucleotides (DR4 element, LXR Liver receptor homologue-1 (LRH-1) is a mouse homologue of the orphan
nuclear receptor fushi tarazu F1 (Ftz-F1) from Drosophila. CYP7A promoter binding factor (CPF), the human homologue of LRH-1, was
found to transactivate the human CYP7A promoter (19). LRH-1 and CPF bind to an extended nuclear hormone receptor-binding site as
monomers. LRH-1 has also been shown to act as a competence factor,
enhancing the ability of LXR Cell Culture and Transfection--
HEK293 cells were maintained
in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at
37 °C. 70-80% confluent cells were transfected using LipofectAMINE
transfection reagent (Life Technologies, Inc.) as described previously
(10). 0.2 µg of reporter DNA, 25 ng of pCMV-RL (Renilla)
(Promega), and 100 ng of receptors (CMX-hRXR Plasmid Construction--
LRH-1 and SHP-1 were cloned by reverse
transcriptase-polymerase chain reaction from mouse liver RNA and
subcloned into pcDNA3.1 (Invitrogen). The 10-amino acid epitope
from human c-MYC (EQKLISEEDL), which can be recognized by the
monoclonal antibody 9E10, was used for tagging the LRH-1
gene to produce Myc-LRH fusion protein. Myc-LRH was subcloned into the
pcDNA3.1 expression vector.
Dietary and mRNA Studies in Human CETP Transgenic
Mice--
Human CETP transgenic mice (C57BL/6J background, 10-15
weeks old), expressing CETP controlled by its native flanking region (3.4-kb NFR) (4), were fed standard rodent chow diets or chow diets
supplemented with 1% cholic acid (CA) (TD00548, Harlan Teklad) for 5 days. The animals were housed under a 12-h light/dark cycle. Total RNA
was isolated using Trizol Reagent (Life Technologies, Inc.) and pooled
from four female or four male mice for each diet. Poly(A)+
RNA was prepared using Qiagen Oligotex mRNA purification Kit. Northern blotting was carried out as described (25). The blots were
exposed to a PhosphorImager screen and visualized with Molecular Dynamics PhosphorImager system. The intensity of the bands was quantified using an ImageQuant tool IQMac version 1.2.
Microarray Printing and Hybridization--
The Escherichia
coli bacterial colonies harboring 1248 unique cDNA or EST
clones were individually grown in 96-well microplates (Corning Costar
Co.). The cDNA inserts were polymerase chain reaction-amplified with vector-derived primers directly using 5 µl of bacterial lysates and subsequently purified in the microplates. A GMS 417 Arrayer (Genetic MicroSystems) was then used to deposit purified polymerase chain reaction products in 3× SSC onto polylysine-coated glass slides
(Sigma). The printed cDNA microarrays were then processed as
described (26). The fluorescence-labeled cDNA probes for array
hybridization were prepared from purified liver mRNA (4 µg),
pooled from 4 mice fed with chow diet or chow diet supplemented with
1% CA, by using Superscript II reverse transcriptase (Life Technologies, Inc.) and fluorescent Cy3- or Cy5-dUTP (Amersham Pharmacia Biotech). Hybridization of both Cy5- and Cy3-labeled probes
in 4× SSC, 0.3% SDS, 50% formamide to the same microarray was
carried out in a sealed, humid hybridization cassette (TeleChem International) for about 14 h at 42 °C. After washing and
drying, the slides were scanned with a confocal array scanner GMS 418 (Genetic MicroSystems). The fluorescence signals of Cy5- and Cy3-tagged cDNA spots on arrays were quantified using an ArrayVision software package (Imaging Research). Two independent microarray hybridization experiments were carried out to analyze the gene expression profile in
control transgenic mice or cholic acid-fed mice. The ratios of Cy5/Cy3
(+CA/ Electrophoresis Mobility Shift Assays--
Myc epitope-tagged
LRH-1 protein was translated in vitro using the TNT
Quick-Coupled Transcription/Translation System (Promega). Double-stranded oligonucleotides with HindIII overhangs,
corresponding to wild type CETP LRH-binding site (LRHBS)
(5'aggaagaccctgctgc3') and mutated LRHBS
(5'aggaagaGcAtgctgc3') or Cyp7a LRH
element (LRHE) (20), were used in gel shift experiments. 2 µl of
lysates expressing Myc-LRH or luciferase (control) were mixed with
~50 fmol of 32P-end-labeled LRHBS fragment in a volume of
20 µl of binding buffer (75 mM KCl, 20 mM
HEPES, pH 7.9, 2 mM dithiothreitol, 10% glycerol, 2 µg
of poly(dI-dC), 30 pmol of nonspecific single-stranded
oligonucleotides). Reactions were incubated at room temperature for 20 min, and protein-DNA complexes were resolved on 5% polyacrylamide gels
at 140 V for 1 h. For competition experiments, ~50-fold molar
excess of unlabeled competitor DNA relative to labeled DNA were added
to the reaction mixture before the addition of the labeled probe. In
antibody experiments, the protein lysates were first incubated with 0.4 µg of Myc epitope antibody 9E10 (sc-40, Santa Cruz Biotechnology) or
control monoclonal anti-actin antibody (sc-8432, Santa Cruz Biotechnology) for 10 min without the labeled DNA, followed by 20 min
of incubation in the presence of labeled DNA.
Potentiation of LXR Function by LRH-1--
Whereas LXRs induce
robust sterol activation of a deleted or multicopy version of the
CETP promoter (9), activation of the intact, single copy
CETP promoter by sterol was either modest (LXR
Mutation of the LXR-binding site (DR4) in the CETP promoter abolished
both the sterol-dependent increase in promoter activity, and the amplification of this effect by LRH (Fig.
3). However, this mutation did not affect
the increase in basal promoter activity attributable to LRH.
LRH Binding to the CETP Promoter Is Required for Potentiation of
Sterol Induction: Mapping the LRH-binding Site--
The increase in
basal activity by LRH, independent of the DR4 mutation (Fig. 3),
suggests that LRH binds to a site distinct from the LXR-binding region.
Consistent with this idea, mutagenesis of the zinc finger DNA binding
domain of LRH (C107S, C200S, and S128G) abolished both the increase in
basal activity mediated by LRH and also the potentiation of sterol
induction mediated through LXR (data not shown). There are several
potential LRH-binding sites (YCA(A/G)GGYCR) in the proximal CETP
promoter (Fig. 4). Deletional mutagenesis
localized the basal LRH response to a region between
We next showed binding of LRH to this site. Gel shift analysis showed
specific binding of LRH to a DNA fragment containing the SHP Abolishes the Sterol Potentiation Effect of LRH-1 on LXR in the
CETP Promoter--
LRH-1 has been shown to interact with and be
repressed by small heterodimer partner (SHP), an orphan nuclear
receptor (20). Since LRH-1 also regulates CETP gene
expression, we analyzed the effect of SHP on the expression of the
CETP gene by co-transfecting SHP with RXR, LXR, and LRH in
293 cells. As shown in Fig. 8, LRH specifically increased the response to sterols but not the RXR ligand,
9-cis-retinoic acid. SHP abolished the
LXR-dependent induction of CETP promoter
activity by sterols. Increasing the amounts of RXR
To see if SHP also represses the basal transactivation activity of
LRH-1 on the CETP promoter, increasing amounts of SHP
plasmid were co-transfected with LRH. However, the
LRH-1-dependent increase of the reporter activity was not
repressed by expression of SHP (Fig. 9).
Therefore, the inhibitory effect of SHP specifically abolished the
potentiation effect of LRH-1, while not affecting the increase in basal
promoter activity induced by LRH-1 (Fig. 8 and 9). These results
indicate that SHP only represses the LRH-1 competence effect on the
CETP promoter in a sterol-dependent manner.
Regulation of CETP Gene by Bile Salt in CETP Transgenic
Mice--
It has been proposed that the repression of LRH-1 activity
by SHP may mediate the bile acid repression of Cyp7a (20,
27). Since CETP expression seems to be regulated by LRH-1
and SHP in a similar fashion to Cyp7a, we analyzed the
regulation of CETP gene expression by bile acid in CETP
transgenic mice. CETP transgenic mice expressing CETP controlled by its
native promoter ( Microarray Analysis of Hepatic mRNA from Mice Fed a 1% Cholic
Acid Diet--
In order to gain further insights into the effects of
bile acids on gene expression, we carried out microarray analysis using mRNA from male and female mice. The microarrays contained about 1,200 cDNAs and ESTs, enriched for genes expressed in liver. Also, a substantial number of transcription factors expressed in liver were
represented. About 2.5% of genes were induced, and 1.5% genes were
repressed by dietary cholic acid, with parallel effects in both sexes
(Supplemental Table 1). We and others (30) have found that the large
majority of genes with altered expression on the microarrays show
similar or larger changes in expression when assessed by Northern
analysis. As an example, SR-BI, a high density lipoprotein receptor
(31), showed 1.7- or 2.6-fold induction by the diet, in male and female
mice, respectively. Northern blot analysis confirmed the results (Fig.
10).
Among the 1200 genes analyzed, 11 genes (0.9%) displayed sexually
dimorphic changes in mRNA expression, in response to the bile
acid-enriched diet (Table I). The most
common response was a higher induction in male than in female mice. The
opposite pattern of response, i.e. female greater than male,
was only demonstrated by the human CETP gene and by one EST.
Notable among the dimorphically induced genes was a TNF We have shown that the orphan nuclear receptor LRH-1 binds and
transactivates the human CETP promoter. In 293 cells, the
native CETP promoter showed modest sterol induction in the
presence of LXR The effect of LRH-1 was strikingly specific for the
LXR-dependent sterol induction of CETP
expression. The mechanism of the LRH-1 potentiation effect required the
binding of LRH-1 to a site ( Recently, considerable evidence has been obtained to support the idea
that LXR/RXR coordinates the regulation of genes involved in reverse
cholesterol transport (9), such as ABCA1 (10, 38),
CETP (9), Cyp7a (7, 8), and apoE (39).
It appears that ABCA1 does not need LRH-1 as a competence
factor for the regulation by LXR/RXR, since the LXR/RXR-binding site is
sufficient to mediate sterol induction in cultured cells in the context
of the native promoter (10). ApoE and ABC1 are involved in the initial
step of cholesterol efflux from macrophage foam cells, resulting in
enrichment of HDL particles with cholesterol and cholesteryl ester.
CETP transfers cholesteryl esters between lipoproteins in the
bloodstream, and thereby facilitates the transport of HDL cholesterol
ester into the liver. Cyp7a converts cholesterol into bile acids in the
liver, leading to excretion from the body.
The mechanism of regulation of CETP by LXR/RXR and LRH is
strikingly similar to the regulation of Cyp7a by these
factors. The sterol induction of Cyp7a has also been shown
to require LRH-1 as a potentiation or competence factor (20). The
competence effect requires binding of LRH-1 to both CETP and
Cyp7a promoters. Similar to its effects on the human
CETP promoter, LRH-1 (CPF) transactivates the human
CYP7A promoter in 293 cells (19). However, little basal
transactivation of the rat Cyp7a promoter by LRH-1 was seen
in 293 cells (20). In the rat Cyp7a promoter, LRH-1 not only
increased the induction by sterol but also the additive or synergistic
induction by both sterols and 9-cis-retinoic acid. However,
in the CETP promoter, LRH-1 only increased the induction by
sterol. These results imply that the competence mechanism of LRH-1 on
CETP and Cyp7a might be slightly different.
Cyp7a is not only regulated by hydroxysterols, but also is
under feedback control by bile salts in the entero-hepatic circulation. The nuclear receptor FXR was recently identified as a receptor for the
bile acid, chenodeoxycholic acid (40-42). Binding of FXR to the
promoters of several bile salt-induced genes, such as ileal bile
acid-binding protein and phospholipid transfer protein, resulted in
bile acid-dependent promoter activation (40, 43). It has been suggested that FXR/RXR may also mediate the bile acid repression of Cyp7a; however, since FXR/RXR does not bind the bile acid
response element in the Cyp7a promoter, an indirect
mechanism is implied (44). Disruption of FXR has provided
strong evidence for a role of FXR in up-regulating expression of genes
such as the ileal bile acid transporter and the basal bile salt
exporter (29). FXR knock-out mice also failed to repress
hepatic Cyp7a mRNA levels in response to a cholic
acid-containing diet (29), suggesting a role for this factor in bile
salt-mediated gene repression.
Recently, it has been proposed that FXR induces SHP, which then
interacts with LRH-1, preventing its activation of the Cyp7a promoter (20, 27). SHP, a transcriptional repressor, was induced by
bile acid treatment as a result of activity of FXR/RXR on the SHP
promoter; furthermore, SHP directly interacts with LRH-1 and represses
LRH-1 activity in cell culture (20, 27). The abolition of LRH
facilitation of the LXR-mediated sterol response by SHP was strikingly
similar in the CETP and Cyp7a promoters (this
study and (20)). A weak or absent effect of SHP on basal
promoter activity in the presence of LRH also appears to be
similar for CETP and Cyp7a (20). However, in
previous studies (4) we observed that a high fat, high cholesterol diet
containing bile salts induced hepatic CETP mRNA
expression, whereas Cyp7a mRNA is repressed in response
to the same diet. In the present study a somewhat different response of
CETP and Cyp7a genes to a chow diet supplemented
with 1% cholic acid was seen. Whereas Cyp7a mRNA was
repressed by the bile salt-supplemented diet in both sexes,
CETP mRNA was repressed in males but induced in female mice.
In an attempt to understand these different responses, we characterized
the responses of about 1200 different cDNAs and ESTs to the bile
salt diet. The majority of differentially expressed genes were either
induced or repressed in parallel fashion in both sexes. However, 11 genes gave a sexually dimorphic response, with the majority showing an
induction in males but not in females. Thus, the CETP
response is unusual and is unlikely to be mediated by a simple
mechanism, such as induction of a single transcription factor in one
sex but not the other. The array results provide an intriguing hint for
how the differential response could be mediated. Thus, the
TNF In summary, we have shown that the competence factor, LRH, enhances the
LXR-mediated sterol response of the CETP promoter, probably
contributes to the moderate tissue specificity of this response (46),
and provides a way for the CETP promoter to respond to
sterols, independent of RXR agonists. Both of these effects, i.e. tissue-specific responses and responses to LXR/RXR that
are independent of RXR ligands, may be general properties of competence factors in transcriptional responses. The interaction of LXR and LRH in
the sterol-dependent induction of the CETP and
Cyp7a promoters, and the repression of this effect by SHP,
is strikingly similar for CETP and Cyp7a,
supporting the idea of coordinate regulation of these two genes in the
liver (9). However, the divergent responses of these and other genes to
diets containing bile salts highlights the complexity of the in
vivo response to this challenge.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
384 to
399). This
response was seen with both LXR
and LXR
, related nuclear hormone
receptors that bind and are activated by specific hydroxylated sterols
at physiological concentrations (5, 6).
also transactivates the Cyp7a gene, encoding
cholesterol 7
-hydroxylase, the rate-limiting enzyme in the pathway
converting cholesterol to bile acids (7). Furthermore, disruption of
LXR
in mice abolished the induction of Cyp7a
expression by dietary cholesterol (8). Based on these data, we proposed
that LXRs may coordinate the regulation of genes involved in different
steps of reverse cholesterol transport (9). This idea was further supported by the demonstration that ABCA1 is up-regulated by
sterols in an LXR-dependent fashion, due to the interaction
of LXR with a DR4 element in the proximal promoter of the
ABCA1 gene (10). ABCA1, the gene that is mutated
in Tangier disease, mediates phospholipid and cholesterol efflux from
macrophages to apoA-I (11-15). Recently, additional LXR-regulated ABC
transporters were shown to be mutated in sitosterolemia, implying a
role for these molecules in excretion of sitosterol and cholesterol
from intestinal cells (16). In addition to stimulating reverse
cholesterol transport and cholesterol excretion, LXRs activate the
promoter of SREBP-1c, indicating a role in the regulation of fatty acid
synthesis (17, 18).
/RXR
to mediate a sterol response on
the Cyp7a gene (20). This interaction is abolished by small
heterodimer partner 1 (SHP-1), just as SHP-1 negates the interaction of
DAX1 with SF-1 (21) and represses the activity of several nuclear
receptors (22-24). Since CETP and Cyp7a are both
regulated by LXR/RXR, we investigated the role of LRH-1 and SHP-1 in
the regulation of CETP gene expression. Our study shows a
marked similarity between the effects of these factors on the CETP and Cyp7a promoters. This supports the idea
of coordinate regulation of the sequential metabolic steps mediated by
CETP and CYP7A. However, divergent responses of these two genes to a
cholic acid-containing diet indicate additional complexity in the
in vivo response to bile acids.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, CMX-hLXR
,
CMV-LXR
, CMV-LRH, and CMV-SHP) were used in each transfection
experiment. Empty pcDNA3.1 expression vector (Invitrogen) was used
as control and to maintain equal amounts of DNA (0.625 µg per well in
24-well plate) for each transfection. The transfected cells were
cultured in 5% LPDS medium (consists of Dulbecco's modified
Eagle's medium with 5.1 lipoprotein deficient serum) in the presence
of 2 µg/ml 22(R)-hydroxycholesterol (Sigma) or vehicle
alone for 24 h. The luciferase activities were measured using
Promega Dual Luciferase assay system. Reporter constructs were
constructed as described previously (9). Each experiment was carried
out in duplicate.
CA) fluorescence intensity of each cDNA sample were
normalized with actin signal ratios obtained from the same measurements
and were averaged from two hybridization results.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) or
non-existent (LXR
) (Fig. 1). To see if
the competence factor LRH might increase the sterol activation, as
reported recently for the Cyp7a promoter (20), we
co-transfected LXR/RXR and LRH in 293 cells. For both LXR
and
LXR
, this resulted in a significant increase in
sterol-dependent activation of promoter activity (Fig. 1).
LRH alone increased basal promoter activity (about 3-fold) but did not
provide a sterol response (Fig. 1). In contrast to these findings with
the CETP promoter, the ABC1 promoter was well activated by sterols in the presence of LXR/RXR, and the response was
not further increased by co-expression of LRH (Fig.
2), showing that the LRH effect is
promoter-specific.
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Fig. 1.
Potentiation of LXR function by LRH-1.
HEK293 cells were co-transfected with reporter construct NFR-luc in
which the luciferase reporter gene is controlled by the proximal
CETP promoter (from 1 to 570) and equivalent amounts (100 ng) of either control plasmid or LXR
, LXR
, RXR
, and/or LRH-1.
The transfected cells were cultured in LPDS + vehicle or LPDS + 2 µg/ml 22(R)-hydroxycholesterol for 24 h, and
luciferase activities were measured. Relative luciferase activity,
which was obtained by normalizing to NFR-Luc basal activity, is shown.
Results (mean ± S.D.) are shown for three independent
experiments, carried out in duplicate. The fold induction of reporter
gene activity by sterol is shown above the filled
bars.
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Fig. 2.
ABC1 promoter is not
transactivated by LRH-1. The ABC1-Luc reporter, in which the
luciferase reporter gene is controlled by 1-kb ABC1 proximal
promoter, was co-transfected with equivalent amounts (100 ng) of
control plasmid or LXR , RXR
, and/or LRH-1 in HEK293 cells.
Relative luciferase activities are presented. Three independent
experiments were performed in duplicate. The fold induction by sterols
is shown.
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Fig. 3.
Mutation of the LXR DR4 element in the
CETP promoter abolishes the sterol induction and LRH
competence effect but not the basal transactivation by LRH.
M3L-Luc reporter, in which the luciferase reporter gene is controlled
by the intact CETP promoter bearing mutations in DR4, was
co-transfected with equivalent amounts (100 ng) of either control
plasmid or LXR , RXR
, and/or LRH-1 in HEK293 cells. Three
independent experiments were carried out in duplicate, and relative
luciferase activities are presented.
60 to
88 (Fig.
5). Moreover, whereas a large deletion of
the region between
90 to
370 did not affect the LRH potentiation of
sterol induction by LXR (Supplemental Fig. 1), deletion of the
60 to
88 region abrogated this response (Fig.
6a). These findings indicate
that the same LRH-binding region (
75 to
83) is involved in the
induction of basal activity and in the amplification of the LXR
response to sterols. To verify further this conclusion, the LRH site
was mutated by changing nucleotides CCC to
GCA (Fig. 4), which markedly reduced the
impact of LRH on sterol induction of the CETP promoter
activity (Fig. 6b).
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Fig. 4.
Sequence of the CETP
promoter region from 1 to
430. The LXR-binding DR4
element and the functional LRH element (consensus sequence in
non-coding strand) are shown in bold. The mutated bases in
the LRHBS are shown by arrows. Several putative LRH-binding
sites that did not contribute to the LRH effect are
underlined.
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Fig. 5.
Deletion or mutation of the LRH element at
60 to
88 abolishes the basal transactivation of the CETP
promoter by LRH-1. CETP reporter constructs containing
various deletions or mutations were co-transfected with LRH-1 in 293 cells, and luciferase activities were measured after 24 h. The
fold transactivation by LRH-1 was obtained by normalizing the
luciferase activities in the presence of LRH-1 to that in the absence
of LRH-1. Three independent experiments (mean ± S.D.) were
carried out in duplicate, p < 0.0005, for the
comparison of Nfr-Luc with
58-370-Luc,
60-88-Luc, or
mLRHE-Luc.
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Fig. 6.
The LRH element at 75 to
83 is required
for potentiation of sterol induction and transactivation of the
CETP promoter by LRH-1. A, deletion
from
60 to
88 abolishes both the basal transactivation of the
CETP promoter by LRH-1 and the LRH-1 potentiation effect on
sterol induction. Reporter construct
60-88-Luc was co-transfected
with receptors and assayed for luciferase activities as in Fig. 1.
Value is greater than 0.1 for the comparison of luciferase
activity (+ sterols) for LXR
/RXR versus LXR
/RXR/LRH,
or LXR
/RXR versus LXR
/RXR/LRH. B, mutation
of the LRH element (
75 to
83) (see Fig. 4) markedly reduces the
basal transactivation and potentiation activity of LRH-1 on the
CETP promoter. The mLRHE-Luc reporter construct was
co-transfected with receptor plasmids and assayed for luciferase
activities as in A. Value is greater than 0.1 for the
comparison of luciferase activity (+ sterols) for LXR
/RXR
versus LXR
/RXR/LRH. Value is greater than 0.1 for
LXR
/RXR versus LXR
/RXR/LRH. Three independent
experiments were carried out in duplicate for all the reporter
constructs. 22HC, 22(R)-hydroxycholesterol.
75 to
83
region (LRHBS) of the CETP promoter (Fig.
7, arrow). In vitro
translated, Myc epitope-tagged LRHs were used in the gel shift
experiments. The Myc-tagged protein showed full potentiation of the
sterol induction of the CETP promoter (not shown) and bound specifically to the CETP LRHBS (Fig. 7, a and b,
lane 2, arrow). This specific band was reduced in
competition assays, using either the CETP LRHBS or the Cyp7a LRHRE (20)
(Fig. 7a, lanes 3 and 5), whereas the mutant CETP
LRHBS was unable to compete for binding (Fig. 7a, lane 4).
This same mutation abolished the functional effects of LRH (Fig.
6b). A factor from reticulocyte lysates also bound
specifically to the CETP LRHBS (Fig. 7a, lane 1, indicated by asterisk). However, this gel shift band was not competed
by the Cyp7a LRHRE. An antibody to the Myc epitope, but not a control antibody (anti-actin), specifically reduced the intensity of the shifted band resulting from the binding of Myc-LRH to the CETP LRHBS
(Fig. 7b, lanes 3 and 4, arrow).
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Fig. 7.
LRH-1 binds to the CETP
LRHBS in vitro. Gel mobility shift analysis
was carried out to analyze the interaction of LRH-1 with a functional
LRH element ( 75 to
83) in CETP promoter. a, a
16-base pair fragment (
87 to
72) containing the CETP
LRHBS was labeled and incubated with in vitro translated
Myc-LRH lysates (lanes 2-5 and 3) or control
lysate (lane 1). The band resulting from the binding of
Myc-LRH to LRHBS is indicated by the arrow and was
specifically competed away by unlabeled CETP LRHBS and
Cyp7a LRERE (lanes 3 and 5). Mutated
LRHBS was unable to compete for the binding (lane 4). A
specific shifted band resulting from the binding of a factor from
reticulocyte lysates was also obtained (lane 1, indicated by
the asterisk). b, Myc epitope antibody 9E10
specifically blocks the binding of Myc-LRH to CETP LRHBS
(lanes 3 and 4).
, while other
factors were held constant, did not eliminate the repression by SHP
(data not shown), suggesting that the abolition of sterol induction by
SHP is not mediated through RXR. These results are consistent with
previous studies showing a direct interaction between SHP and LRH (20)
and suggest that SHP might bind to a region of LRH involved in
interactions with LXR. The 3.4-kb full-length CETP promoter
was regulated similarly by LRH and SHP (Supplemental Fig. 2). The only
difference was that expression of LXR
/RXR
and LXR
/RXR
led
to about 2.5- and 3-fold sterol induction, respectively, in the 3.4-kb
promoter, a more robust response than seen with the shorter promoter
fragment (Fig. 1). LRH further increased the induction fold to about
4-fold for LXR
/RXR
and 6-fold for LXR
/RXR
.
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Fig. 8.
SHP abolished the sterol potentiation effect
of LRH-1 on LXR in the CETP promoter. The NFR-Luc
reporter was co-transfected with equivalent amount of either control
plasmid or plasmid expressing LXR , LXR
, RXR
and/or LRH, SHP
(100 ng). Cells were then cultured in LPDS in the presence or absence
of 5 µM 22(R)-hydroxycholesterol
(22RHC), 5 µM 9-cis-retinoic acid
(9cRA), or both (22RHC/9cRA) for 24 h. Three
independent experiments were carried out in duplicate.
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Fig. 9.
SHP does not abolish the basal
transactivation of the CETP promoter by LRH-1.
NFR-Luc reporter was co-transfected with various amounts of plasmid
expressing LRH and/or SHP. Cells were then cultured in LPDS medium for
24 h and assayed for luciferase activities.
3.4 kb) (25) were fed a chow diet with or without
1% cholic acid for 5 days. Northern blot analysis was carried out to
analyze the expression of several genes that are regulated by bile
acids (Fig. 10). As expected,
Cyp7a expression was completely repressed by dietary cholic
acid. Cyp8b (encoding 12
-Hydroxylase), also regulated by
LRH-1 (28), was repressed by cholic acid feeding as reported (29). The
cholic acid-containing diet also led to an increase in SHP mRNA and
a decrease in Cyp27 expression (20, 29). For all of these
genes a similar response was seen in male and female mice.
Intriguingly, however, the response of CETP mRNA was sexually dimorphic. In male mice, CETP mRNA was decreased upon cholic acid feeding, whereas in female mice the bile salt diet led to a 2-fold induction of CETP mRNA.
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[in a new window]
Fig. 10.
Northern blot analysis of mRNA from male
(M) and female (F) mice fed a chow
diet or a chow diet supplemented with 1% cholic acid. Mice
expressing the human CETP transgene, controlled by its
native promoter, were fed with either rodent chow diet or chow diet
containing 1% cholic acid for 5 days. Liver poly(A)+
mRNA was prepared from total RNA pooled from four mice per diet per
sex. 2 µg of poly(A)+ RNA was loaded, and gene expression
was analyzed by Northern blot, using the cDNA probes shown.
G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
-induced
protein, suggesting a possible differential effect of TNF
on target
genes. Hepatic TNF
is induced by the bile acid-enriched diet, and
both CETP and Cyp7a are repressed by TNF
(32-34).
Genes dimorphically regulated by dietary CA, revealed by microarray
analysis, in human CETP transgenic mice
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/RXR
or LXR
/RXR
(Fig. 1 and Supplemental
Fig. 2). The expression of LRH-1 enhanced the response of LXR/RXR to
sterols. The principal tissue expressing LRH-1 is the liver, which also
is a major site where CETP is expressed (4). Even though CETP is
expressed in peripheral tissues, the sterol regulation in peripheral
tissue is less pronounced than in liver (4). A requirement for LRH-1 in
the sterol induction mediated by LXR might confer tissue specificity for the induction of genes by sterols, since the ubiquitously expressed
LXR
could otherwise mediate sterol induction of CETP gene. Additional physiological significance might be that LRH-1 abolishes the requirement for RXR ligands, such as
9-cis-retinoic acid or docosahexanoic acid (35), in order to
obtain significant LXR-mediated sterol induction of gene expression.
Thus, in response to endogenous hypercholesterolemia,
24(S),25-epoxycholesterol, which is synthesized from a shunt
pathway (36) and has been shown to act as an LXR ligand (6), may bind
and activate LXR/RXR without the requirement for additional RXR
ligands. This effect is clearly shown in Fig. 8 and Supplemental Fig.
2. The presence of LRH increased the induction of CETP by
sterol but had no additional incremental effect as a result of the
addition of 9-cis-retinoic acid.
75 to
83) (LRHBS) on the
CETP promoter, and the DNA binding ability is required for
the potentiation function of LRH-1, since mutation of the LRH-1
DNA-binding zinc finger motif abolished this effect (not shown). The
activity of LRH-1 on the CETP promoter might enhance ligand
binding to LXR or cooperatively work with LXR/RXR to recruit
ligand-dependent co-activators. The co-activator SRC-1
(nuclear receptor co-activator) has been shown to interact with
LXR
/RXR upon binding of 22(R)-hydroxycholesterol and/or 9-cis-retinoic acid (37). It is possible that
sterol-dependent binding of co-activator is enhanced either
by the LRH/LXR activation or by retinoid binding to RXR. This would
explain why in the presence of sterols the expression of LRH or
activation by 9-cis-retinoic acid achieved similar promoter
activity (Fig. 8).
-induced protein 2 was increased in males and decreased in
females, suggesting that there could be a dimorphic effect of TNF
signaling in male versus female mice. Hepatic TNF
expression is induced by the cholic acid diet (it is probably made in
Kupffer cells) (33), and TNF
has been shown to repress both
CETP (34) and Cyp7a (32). If the effect of TNF
is larger in male mice, this could explain why CETP is
repressed in this sex. One has to then hypothesize an additional factor
that up-regulates CETP in response to bile acids and only
becomes apparent in females because of a lessened TNF
effect. The
related gene PLTP is up-regulated by the bile salt diet,
through an FXR mechanism (43), and it is conceivable that
CETP could be similarly regulated. Induction of TNF
and
interleukin-1 by the bile salt diet has also been suggested as a
possible mechanism of repression of Cyp7a (33, 45), but this
response was not dimorphic. There could be additional mechanisms of
repression of Cyp7a, such as that proposed involving FXR,
SHP, and LRH, affecting both sexes.
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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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Table 1 and Figs.
1 and 2.
To whom correspondence should be addressed: College of Physicians
& Surgeons, 8-401, Division of Molecular Medicine, Dept. of Medicine,
Columbia University, New York, NY 10032. Tel.: 212-305-5789; Fax:
212-305-5052; E-mail: yl220@columbia.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M100912200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CETP, cholesteryl
ester transfer protein;
HDL, high density lipoproteins;
NFR, native
flanking region;
CA, cholic acid;
LRH, liver receptor homologue;
LRHBS, LRH-binding site;
kb, kilobase pair;
FXR, farnesoid X receptor;
LXR, liver X receptor;
RXR, retinoid X receptor;
CPF, CYP7A promoter binding
factor;
SHP-1, small heterodimer partner 1;
kb, kilobase pair;
LRHE, LRH element;
TNF, tumor necrosis factor
;
EST, expressed sequence
tag;
SHP, small heterodimer partner.
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