The Promoter of the Rat 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene Contains a Tissue-Specific Estrogen-Responsive Region
Luciano Di Croce,
Guillermo P. Vicent,
Adali Pecci,
Giovannella Bruscalupi,
Anna Trentalance and
Miguel Beato
Institute for Molecular Biology and Tumor Research (IMT)
D-35037 Marburg, Germany
Department of Cellular and
Developmental Biology Università "La Sapienza" 00185
Rome, Italy
Department of Biology Università di
Roma 3 00146 Rome, Italy
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ABSTRACT
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The isoprenoid metabolic pathway is mainly
regulated at the level of conversion of 3-hydroxy-3-methylglutaryl
coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA
reductase. As estrogens are known to influence cholesterol metabolism,
we have explored the potential regulation of the HMG CoA reductase gene
promoter by estrogens. The promoter contains an estrogen-responsive
element-like sequence at position -93 (termed Red-ERE), which differs
from the ERE consensus by one mismatch in each half of the palindrome.
A Red-ERE oligonucleotide specifically bound estrogen receptor in
vitro and conferred receptor-dependent estrogen responsiveness to
a heterologous promoter in all cell lines tested. However, expression
of a reporter driven by the rat HMG CoA reductase promoter was induced
by estrogen treatment after transient transfection into the breast
cancer cell line MCF-7 cells but not in hepatic cell lines expressing
estrogen receptor. Estrogen induction in MCF-7 cells was dependent on
the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384.
A functional cAMP-responsive element is located immediately
upstream of the Red-ERE, but cAMP and estrogens inhibit each other in
terms of transactivation of the promoter. Similarly, induction by
estrogens was inhibited by micromolar concentrations of cholesterol,
likely acting via changes in occupancy of the sterol-responsive element
located 70 bp upstream of the Red-ERE. Thus, within its natural
context, Red-ERE is able to mediate hormonal regulation of the HMG CoA
reductase gene in tissues that respond to estrogens with enhanced cell
proliferation, while it is not operative in liver cells. We postulate
that this tissue-specific regulation of HMG CoA reductase by estrogens
could partially explain the protective effect of estrogens against
heart disease.
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INTRODUCTION
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Isoprenoid biosynthesis provides the cell with essential compounds
such as cholesterol, dolichols, ubiquinon, and prenylated proteins. The
common precursor of these compounds, mevalonate, is produced by the
activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase
(1), a 97-kDa glycoprotein that resides in the endoplasmatic reticulum,
where the enzyme is also degraded (2). Cholesterol represents the
predominant product of the biosynthetic pathway and plays a primary
role in membrane biogenesis and in steroid hormone biosynthesis. The
other isoprenoid compounds produced in branched paths of the main
biosynthetic pathway are involved in protein glycosylation (dolichol),
energetic processes (ubiquinon), cell membrane traffic (Rab), and
nuclear structure (lamins) (3). The level of HMG CoA reductase can be
modulated at the level of gene transcription (4, 5), mRNA stability (6, 7), translation (8, 9), and/or enzyme degradation (3, 10). Furthermore,
the specific activity of HMG CoA reductase is reversibly regulated by
phosphorylation/dephosphorylation mechanisms and by oxidation events
(11).
To maintain an adequate cholesterol concentration, the cell changes the
level of the endogenous synthesis and exogenous supply through a
feedback regulation of HMG CoA synthase, HMG CoA reductase, and
low-density lipoprotein receptor (LDLr). Regulation of the HMG CoA
reductase controls the endogenous cholesterol production, while LDLr
controls the exogenous uptake as well as the supply to the extrahepatic
tissues (3). The sterol-regulatory element (SRE) present in the
promoters of these genes mediates the transcriptional regulation by
sterols (12, 13). The transcription factors that bind this element at
low cholesterol concentrations (SRE-binding proteins, SREBPs) have been
characterized (14, 15, 16). However, metabolic changes detectable in
different physiological states suggest the occurrence of additional
regulatory mechanisms involving several hormones. For instance, TSH is
reported to induce the transcription of the HMG CoA reductase gene via
cAMP (17).
Estrogens have been strongly connected to the very low incidence of
heart disease in women and with the hypolipidemic effect caused by an
enhanced LDLr function. Furthermore, estrogens have been reported to
affect the metabolism of isoprenoid compounds in various species
displaying species-specific differences (18, 19, 20, 21). The variable effects
on the HMG CoA reductase observed in the rat liver after estrogen
treatment could be explained by differences in the circadian rhythm and
feeding status of the experimental animals (22, 23, 24). In our hands,
estrogen administration to rats caused an early increase of LDLr and a
late increase of HMG CoA reductase, detectable only after 5 days (24).
In vitro experiments showed that estrogens induce HMG CoA
reductase activity as well as cholesterol synthesis (25, 26). Direct
transcriptional and posttranscriptional mechanisms have been proposed
to explain the estrogen effects (27), even though indirect mechanisms
involving changes in cholesterol content acting via SRE or by changes
in second messenger levels (cAMP, Ca++, or inositol
triphosphate) cannot be excluded (28).
Little is known about the molecular mechanism(s) of estrogen effects on
isoprenoid metabolism. It is generally accepted that estrogens act by
interacting with their intracellular receptor, the estrogen receptor
(ER), which is a ligand-dependent member of the nuclear receptor family
(29, 30). The ER binds to specific DNA sequences, called
estrogen-responsive elements (EREs), that share the palindromic
consensus sequence GGTCAnnnTGACC (31) and are located in the promoter
or enhancer regions of many estrogen-regulated genes. To explore
the possibility of a direct effect of estrogenic hormones on the HMG
CoA reductase gene, we screened the sequence of the rat gene for the
presence of potential EREs. Starting at position -93, we found an
ERE-like sequence, CGTCAGGCTGAGC (hereafter denoted Red-ERE for HMG CoA
reductase-ERE). Here we show that ER binds specifically to the Red-ERE
and that the isolated Red-ERE confers estrogen responsiveness to a
heterologous promoter in all cell lines tested. Thus, the Red-ERE
behaves as a bona fide ERE. However, a reporter plasmid
carrying the native HMG CoA reductase promoter sequences up to -323,
including the Red-ERE, is transactivated by estrogens in the breast
cancer cell line MCF-7, but not in two hepatic cell lines transfected
with ER expression vectors. The activity of the Red-ERE is modulated by
adjacent cis-acting elements, in particular by a
sterol-responsive element and a cAMP-responsive element. Our results
are compatible with a function of the Red-ERE in mediating hormonal
induction of the rat HMG CoA reductase gene in peripheral tissues that
respond to estrogens with cell proliferation. Estrogen regulation is
tissue-specific as it does not operate in hepatic cell lines expressing
a functional ER.
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RESULTS
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The Red-ERE Binds ER
in Vitro
A computer comparison of the promoter sequences of human, hamster,
mouse, and rat HMG CoA reductase genes showed a high degree of homology
(Fig. 1
), in particular in the region of
the SRE, and revealed the presence of a potential estrogen responsive
element (ERE) close to a cAMP-responsive element (CRE) (Fig. 1
). The ERE-like sequence in the rat HMG
CoA reductase gene promoter (Red-ERE) between -81 and -93
(CGTCAGGCTGAGC, see Fig. 1
) is well conserved
and presents two mismatches (bases underlined) when compared
with the palindromic consensus ERE identified within the Xenopus
laevis vitellogenin A2 gene (cons-ERE; GGTCAnnnTGACC) (31).

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Figure 1. Alignment of HMG CoA Reductase Promoter Sequences
from Human, Hamster, Mouse, and Rat
The rat HMG CoA reductase promoter from -277 to +63 is shown aligned
with the corresponding regions of the human, hamster, and mouse
promoters. The SRE, the CRE, and the potential ERE are
boxed. The asterisks denote conserved
bases, while the diamond denotes the major transcription
start site.
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To test whether the putative Red-ERE is able to bind ER in
vitro, we performed band shift experiments using nuclear extracts
from the estrogen-responsive MCF-7 cell line and an oligonucleotide
including HMG CoA promoter sequences from -98 to -77 (Red-ERE). The
consensus ERE from the Xenopus vitellogenin A2 gene (32, 33)
was used as positive control. A complex with the same retarded mobility
as with the consensus ERE (cons-ERE) was detected with the Red-ERE
oligonucleotide (Fig. 2A
, lanes 1 and 3).
Both complexes were competed by an excess of the specific, but not
unspecific, unlabeled oligonucleotides (Fig. 2A
, lanes 2 and 4, and
data not shown). Densitometric analysis showed that ER binds with
higher affinity to the cons-ERE than to the Red-ERE (see below).

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Figure 2. Specific Binding of ER to the Red-ERE and Its
Relative Affinity
A, Nuclear extracts from MCF-7 cells were incubated with a
32P-radiolabeled oligonucleotide containing the cons-ERE
(lanes 1 and 2) or the Red-ERE (lanes 3 and 4), and specific binding
was determined by EMSA. Competition with a 200-fold molar excess of
cons-ERE (lane 2) or Red-ERE (lane 4) was performed as specificity
controls. The arrow indicates the ER/DNA complexes. B,
EMSA was performed using either recombinant ER (lanes 17) or MCF-7
nuclear extract (MCF-7 NE; lanes 9 and 10). Equal amounts of
32P-labeled Red-ERE (lanes 1, 4, and 5), cons-ERE (lanes 2
and 610) or PRE (lane 3) were added to each reaction with a 200-fold
molar excess of competitor Red-ERE (lanes 4 and 6) or competitor
cons-ERE (lanes 5, 7, and 10). The arrow indicates the
ER/DNA complexes. C, The ability of the Red-ERE to compete with the
32P-labeled cons-ERE for binding to the ER was analyzed by
adding increasing amounts (0, 10, 20, 100, 200, and 500-fold molar
excess) of competitor Red-ERE. The arrow indicates the
ER/cons-ERE complexes. D, 32P-labeled cons-ERE was
incubated with increasing amounts (0, 0.2, 0.4, 2, 4, and 10 pmol) of
either cons-ERE or Red-ERE to measure their relative affinities to ER
in nuclear extracts of MCF-7 cells. The amount of the complexes
ER/cons-ERE in the absence of competitor was set at 100%. The
SE was less than 3%.
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The retarded complexes observed with nuclear extracts were likely due
to binding of ER as a similar complex could be formed using recombinant
ER
expressed in baculovirus-infected insect cells (34) (Fig. 2B
, compare lanes 1 and 2 with 9), and a supershift was observed in the
presence of an antibody specific for ER
(data not shown). Moreover,
formation of these complexes was also effectively inhibited by addition
of an excess of unlabeled cons-ERE or Red-ERE oligonucleotides (Fig. 2B
, lanes 47). Importantly, the recombinant ER
did not bind an
oligonucleotide containing the related sequence of a
progesterone-responsive element (PRE) (Fig. 2B
, lane 3),
demonstrating its specific binding to the ERE oligonucleotides.
These experiments confirm the involvement of ER in the retarded
complexes and indicate the presence of an heretofore undetected
ER-binding site in the promoter of the rat HMG CoA reductase gene.
Relative Affinity of ER
for Red-ERE and cons-ERE
Since the Red-ERE exhibits two mismatches when compared with the
cons-ERE, it was important to determine whether the Red-ERE was able to
compete for the binding of the ER to the cons-ERE. We performed binding
experiments with MCF-7 nuclear extracts using the labeled cons-ERE and
increasing amounts (10- to 500-fold molar excess) of unlabeled Red-ERE
(Fig. 2C
). The specifically retarded band was significantly reduced in
the presence of a 20-fold excess (Fig. 2C
, lane 3) and was almost
abolished by a 500-fold excess of unlabeled Red-ERE (Fig. 2C
, lane
6).
To determine the relative affinities, we compared the ability of
unlabeled cons-ERE and Red-ERE oligonucleotides to compete for the
binding of ER to the labeled cons-ERE sequence (Fig. 2D
). The cons-ERE
oligonucleotide competed very effectively for ER binding: a 50%
reduction in ER-DNA complex formation was observed with 0.4 pmol of the
unlabeled cons-ERE oligonucleotide. In contrast, about 0.9 pmol of the
Red-ERE oligonucleotide was required to reduce by 50% binding of ER to
the labeled cons-ERE. These results indicate that ER binds to the
Red-ERE with approximately half the affinity as to the consensus
ERE.
Red-ERE Confers Estrogen Responsiveness to a tk-Minimal
Promoter in All Tested Cell Lines
We next investigated whether, as reported for a cons-ERE, the
Red-ERE is able to confer estrogen responsiveness to a heterologous
promoter. To this aim, a single copy of either the cons-ERE or the
Red-ERE was inserted in front of a minimal herpes simplex virus
thymidine kinase (tk) promoter linked to the firefly luciferase gene
and transiently transfected in Cos-1 cells along with an expression
vector for ER
(Fig. 3A
). The cells
were incubated with 100 nM of either 17ß-estradiol or the
pure steroidal antiestrogen ICI 164,384. The level of promoter activity
in cells treated with the solvent alone (ethanol, EtOH) was 1015%
higher than the value observed in cells treated with ICI. As a negative
control, we used the wild-type tk promoter, which did not respond to
hormone treatment. In contrast, estradiol induced the expression of the
transfected construct containing the cons-ERE (pTK-cons-ERE) by about
12-fold, whereas the Red-ERE containing reporter (pTK-Red-ERE) was
induced about 5-fold. This difference in induction efficiency
correlates with the observed lower affinity of ER for the Red-ERE
compared with the cons-ERE (see above).

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Figure 3. Influence of Estrogen on HMG CoA Reductase Promoter
Activity in Mammary and Liver Cells
A, Vectors containing a single copy of Red-ERE or cons-ERE in front of
a tk minimal promoter driving the luciferase gene
(pTK-cons-ERE or pTK-Red-ERE, respectively) were transfected into
either Cos-1 cells or HepG2 cells, along with expression vector for the
human ER (pHEGO). The cells were incubated for 48 h with 100
nM ICI 164,384 (ICI) or 100 nM 17ß-estradiol
(Estrogen) or with solvent alone (ethanol, EtOH). The luciferase
activity (expressed in arbitrary units) is shown relative to the
activity in cells treated with EtOH; the values represent the mean of
four independent determinations calculated after normalization for
cotransfected pRSV-lacZ. The SE was less than 3%. The
empty expression was used as a negative control (pTK). B, CAT assays
were performed using MCF-7 cells transfected with the plasmid
pHMGRed-CAT containing sequences of the HMG CoA reductase promoter
(-323 to +442) linked to a CAT reporter gene. Cells were treated with
solvent alone (ethanol, EtOH) or with 100 nM ICI 164,384 or
with 17ß-estradiol (indicated by E) at concentrations of 100
nM or 10 nM, as indicated. CAT activity is
shown relative to the activity in cells treated with ICI. The values
represent the mean ± SE of three independent
determinations calculated after normalization for the
cotransfected reference plasmid pRSV-luc. C, Plasmid containing either
the wild-type construct (pHMGRed-luc) or the Red-ERE-deleted promoter
(pHMGRed ERE) was transfected into MCF-7 cells or HepG2 cells. The
cells were incubated with solvent alone (ethanol, EtOH) or with 100
nM ICI 164,384 (ICI) or with 100 nM
17ß-estradiol (Estrogen). The values shown represent the mean ±
SE of three separate determinations calculated after
normalization for the cotransfected reference plasmid pRSV-lacZ.
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The Red-ERE-tk reporter was also tested in liver cells (HepG2)
cotransfected with an expression vector for ER
. As in Cos-1 cells,
we reproducibly observed a 4-fold response to estrogens that was lower
than the response found with the cons-ERE-tk reporter (Fig. 3A
). The
Red-ERE-tk reporter was also induced by estrogens when transfected in
MCF-7 cells (data not shown). Our results confirm the ability of the
isolated Red-ERE to confer estrogen responsiveness to a heterologous
promoter in various cell types and, therefore, identify the
corresponding sequence as a bona fide ERE.
The rat HMG CoA Reductase Gene Promoter Is Induced by Estrogens in
MCF-7 Cells but Not in Hepatic Cell Lines
To investigate whether the Red-ERE is functional within the
context of the HMG CoA reductase gene promoter, we used the reporter
plasmid pHMGRed-CAT containing the chloramphenicol acetyl transferase
(CAT) gene of Escherichia coli driven by a rat promoter
fragment spanning from 323 bp upstream to 442 bp downstream of the
major transcription start site. This reporter was transiently
transfected in the estrogen-responsive MCF-7 cell line. Figure 3B
shows
that the HMG CoA reductase gene promoter responded to 17ß-estradiol
stimulation in a dose-dependent manner. The average induction was
2-fold at 10 nM and 3-fold at 100 nM of
17ß-estradiol when compared with cells treated with the antiestrogen
ICI. A similar estrogen response was observed in a rat endometrial cell
line that express ER (35) (data not shown). Deletion of the potential
ERE element (pHMGRed
ERE-luc) completely eliminated the
hormone-dependent transactivation of the promoter but had no influence
on the activity of the reporter in the presence of ethanol or
antiestrogen (Fig. 3C
). These results demonstrate the estrogen
responsiveness of the rat HMG CoA reductase gene promoter and are
compatible with the notion that the hormonal effect is mediated by
the Red-ERE sequence.
Since the liver is the main tissue involved in the body cholesterol
homeostasis, we next investigated the effects of estrogen
administration on the HMG CoA reductase promoter transiently
transfected in two different hepatic cell lines: the human HepG2 cell
line and the rat Fto cell line. As these cells do not contain
sufficient levels of endogenous ER, an expression vector for ER
was
cotransfected. In neither case did we observe an effect of estrogens on
expression of the HMG CoA reductase promoter (Fig. 3C
and data not
shown) although the transfected ER
was shown to be functionally
active on either a canonical ERE reporter or on the Red-ERE in front of
a heterologous promoter (Fig. 3A
) and activated the HMG CoA reductase
gene promoter in Cos-1 cells (Fig. 3C
). Note that the basal activity of
the HMG CoA reductase promoter in hepatic cells was consistently 8- to
10-fold higher than in other cell lines, as previously observed (36).
This could be explained by the presence in different cells of different
ratios of SREBP isoforms (14, 37) or by the presence of specific
hepatic transcription factor(s). Thus, the HMG CoA reductase promoter
is selectively responsive to estrogens in estrogen target cells, such
as mammary and endometrial cell lines, but not in hepatic cell
lines.
Modulation of ERE Function by Cholesterol Levels
The HMG CoA reductase promoter encompasses an SRE that is known to
mediate regulation of the promoter in response to changes in
cholesterol levels (12) and to be the target of SREBP (38). To explore
a possible cross-talk between these signaling pathways and estrogen
regulation, we tested the effect of cholesterol on hormone-independent
and estrogen-induced expression of the promoter in transfection
assays. In MCF-7 cells, addition of 25 µM
25-OH-cholesterol to the medium had a weak inhibitory effect on
promoter activity in the presence of antiestrogen but caused an almost
complete inhibition of estrogen transactivation (Fig. 4A
). In hepatic cells the same
concentration of cholesterol caused a 50% reduction in HMG CoA
reductase promoter activity, and the promoter remained unresponsive to
estrogen-activated ER
(data not shown). Interestingly, when a
down-mutation of the SRE site was introduced in the HMG CoA promoter
(
SRE), cholesterol addition failed to prevent estrogen
transactivation in MCF-7 cells (Fig. 4A
). These results confirm the
strong inhibitory influence of signals acting via the SRE on
transcriptional regulation of the rat HMG CoA reductase promoter and
furthermore demonstrated that these signals can modulate the inductive
action of estrogens (3, 38).

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Figure 4. Influence of Cholesterol and cAMP on the Estrogen
Induction of HMG CoA Reductase Promoter
A, MCF-7 cells were transfected with a reporter plasmid containing the
CAT gene under the control of the rat HMG CoA reductase promoter,
either wild-type (pHMGRed-luc) or carrying the SRE deletion ( SRE).
Cells were treated with solvent alone (ethanol, EtOH) or 100
mM of ICI 164,384 (ICI) or 17ß-estradiol (E), in the
presence or absence of 25 µM 25-OH-cholesterol as
indicated below each bar. Values represent CAT activity
in arbitrary units. The mean ± SE values from three
independent determinations were calculated after normalization for
cotransfected pRSV-lacZ. B, MCF-7 cells were transfected with a
reporter plasmid containing the CAT gene under the control of the rat
HMG CoA reductase promoter and treated with solvent alone (ethanol,
EtOH), 100 nM ICI 164,384 (ICI), 100 nM
17ß-estradiol (E), 1 mM (Bu)2cAMP (cAMP), or
various combinations as indicated below each bar. Values
represent CAT activity expressed in arbitrary units and represent the
mean ± SE calculated from four separate
determinations after normalization for cotransfected pRSV-lacZ. C,
MCF-7 cells were cotransfected with a plasmid containing the wild-type
HMG CoA reductase promoter, and with a plasmid carrying four tandem
copies of a canonical CRE (p4xCRE), and treated with 100 nM
17ß-estradiol (E) as indicated below each bar. The
values represent CAT activity expressed in arbitrary units and
represent the mean ± SE from four separate
determinations calculated after normalization for cotransfected
pRSV-lacZ. D, EMSA with 32P-labeled oligonucleotide
encompassing both the Red-ERE and CRE sequences and nuclear extracts
from MCF-7 cells. A 200-fold excess of Red-ERE (lane 2), cons-ERE (lane
3), or CRE (lane 4) was added as competitor. The top
arrow indicates the ER/DNA complex while the lower
arrow indicates the CRE-containing retarded complex.
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Modulation of ERE Function by cAMP
The sequence TGACGTAG (17), which is very similar (with only two
mismatches) to the consensus CRE (39), is found immediately upstream of
the Red-ERE in the HMG CoA reductase promoter. Since cAMP is known to
influence the expression of the HMG CoA reductase gene (17), we tested
its influence on the activity of the pHMGRed-CAT reporter in the
presence or absence of estrogens and antiestrogens (Fig. 4B
).
(Bu)2cAMP (1 mM) alone induced the activity of
the reporter to about the same level as 100 nM
17ß-estradiol. However, simultaneous addition of cAMP and
17ß-estradiol did not lead to a further increase in activity,
suggesting that the two inducers cannot act additively or
synergistically. In fact, the two inducers appear to compete, as shown
by the results obtained in the presence of the pure antiestrogen ICI,
which leads to a further increase in cAMP induction of the reporter
(Fig. 4B
). This suggests that the effect of cAMP may be inhibited in
part by remaining estrogenic activities in the culture medium, or by a
ligand-independent activation of ER.
To test whether estrogen induction is inhibited by the basic activity
of the cAMP-signaling pathway, we performed oligonucleotide competition
studies. MCF-7 cells were cotransfected with the pHMGRed-CAT reporter
plasmid along with an excess of a plasmid carrying four tandem copies
of a canonical CRE (p4xCRE), which should bind endogenous CRE-binding
protein (CREB) and thus prevent its interaction with the CRE on the
transfected HMG CoA promoter (Fig. 4C
). Whereas this plasmid had little
effect on the activity of the HMG CoA reductase reporter in the absence
of estrogens, it doubled the estrogen response, increasing it from
3-fold in the absence of the CRE plasmid to 6-fold in its presence
(Fig. 4C
). These findings are compatible with an inhibitory effect of
endogenous cAMP levels on estrogen induction of the HMG CoA reductase
promoter.
To explore the mechanism of the mutual inhibition, we performed
electrophoretic mobility shift assays (EMSAs) with a DNA fragment
encompassing both the Red-ERE and CRE sequences. In the absence of
competitor DNA, two complexes with different mobilities were observed
using MCF-7 nuclear extract (Fig. 4D
, lane 1). The slower complex
likely corresponded to ER binding, as it was effectively reduced by the
addition of unlabeled Red-ERE or cons-ERE (Fig. 4D
, lanes 2 and 3,
respectively). The faster complex probably involved proteins
interacting with the CRE, since it was markedly reduced by an excess of
unlabeled CRE (Fig. 4D
, lane 4). Under no conditions did we find
indications for a ternary complex containing both ER- and
CRE-associated factors. Thus, both sites are occupied independently and
no indication for simultaneous or cooperative binding is found. Our
data are compatible with a mutually exclusive binding of ER and CREB,
or related factors, to the HMG CoA reductase promoter region.
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DISCUSSION
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The nucleotide sequence of rat HMG CoA reductase promoter is
highly conserved between species (see Fig. 1
). This conservation is
84% over the regulatory region that includes SRE, CRE, and ERE,
suggesting an important role for all of these sites in regulating
expression of the HMG CoA reductase gene. The involvement of the CRE
site (17) as well as the SRE site (38) in regulation of the gene is
well documented. Here, we have investigated the molecular basis for
estrogen regulation of HMG CoA reductase gene expression. The Red-ERE
is able to bind specifically ER from MCF-7 nuclear extracts as well as
recombinant ER
. The observation that the retarded complexes exhibit
the same mobility on the Red-ERE as on the cons-ERE and that it is
specifically supershifted in the presence of monoclonal antibody
confirm that a homodimer of ER
binds to the HMG CoA reductase
promoter. Quantitative competition experiments reveal that ER
binds
the Red-ERE with 50% of the affinity observed for the consensus ERE,
likely reflecting the two mismatches present in the Red-ERE.
The Red-ERE not only binds ER but also acts as a functional ERE in
front of a heterologous promoter when transfected in a variety of
cells, including two hepatic cell lines. The Red-ERE is functional in
the context of the HMG CoA reductase promoter in mammary and
endometrial cells. Estrogens (10100 nM) induce the
expression of the promoter in MCF-7 cells, and this effect is abolished
by mutations of the Red-ERE. However, whereas the estrogen induction of
the isolated Red-ERE was 6-fold, the response of the native HMG CoA
reductase promoter was only 3-fold, suggesting that other sequences
within the promoter interfere with ERE function.
Since the original discovery of an ERE in the Xenopus
vitellogenin A2 gene (31), only one other gene has been found
containing a 100% identical ERE element (40). In contrast, several
estrogen-responsive genes have been shown to utilize degenerate EREs
(41, 42, 43, 44, 45). Thus, it seems that imperfect EREs are more frequently used
than consensus ERE, although canonical EREs confer stronger activation
by estrogens. One possible reason for the higher frequency of imperfect
EREs is that they may facilitate fine tuning of the hormone induction
by other signal transduction pathways impinging on nearby regulatory
elements of the promoter.
In contrast with the results obtained with classical estrogen target
cells, like mammary and endometrial cells, the HMG CoA reductase
promoter does not respond to estrogen treatment in two hepatic cell
lines. This is not due to a defect of the transfected ER, since in
these cells, the isolated Red-ERE is able to confer estrogen
responsiveness to the tk promoter. Thus, the Red-ERE is potentially
functional in liver cells but is inactive in the context of the HMG CoA
reductase promoter. These data suggest a molecular explanation for the
lack of estrogen induction of cholesterol biosynthesis in liver cells
in contrast to MCF-7 cells (46).
Among other potential regulatory elements, the HMG CoA reductase
promoter contains a SRE and a CRE element. The SRE seems to be
functional in mammary cell lines since addition of cholesterol
inhibited the estrogen response of the promoter, and this effect was
dependent on the integrity of the SRE site. Thus, feedback inhibition
by cholesterol appears to play a dominant role in the regulation of the
HMG CoA reductase promoter. Transient transfection experiments
demonstrated that estrogens and the signals acting via the CRE do not
synergize but, on the contrary, appear to inhibit each other. Using a
fragment of the HMG CoA reductase promoter encompassing the ERE and CRE
sequences, no simultaneous binding of CREB and ER
was detected. As
the two elements are separated by only 2 bp, steric hindrance could
preclude simultaneous binding of ER and CREB to the promoter (47). A
similar scenario has been postulated to explain the lack of
simultaneous binding of progesterone receptor and NF1 to the mouse
mammary tumor virus (MMTV) promoter DNA (48). Alternatively, the
cross-talk can be mediated by complex interaction with the integrator
CBP/p300, which is known to interact directly with ER (49), CREB (50),
and SREBP (51) and exhibits histone acetyltransferase activity
(52).
Our results with liver cell lines are in good agreement with our
previous observations that while estrogen treatment in vivo
affects the protein level of HMG CoA reductase in rat liver, it does
not modify the level of mRNA (24). The lack of induction either
in vivo (rat liver) or in vitro (hepatic cell
lines) could result from interplay between the estrogen and other
signal transduction pathways targeting the HMG CoA reductase promoter
(53). Here both the SRE and/or the CRE could play a role. The
inhibition by cholesterol of the estrogen induction of the HMG CoA
reductase promoter observed in transfection assays offers a possible
explanation for the lack of hormonal induction in hepatocytes. The
increased intracellular cholesterol level observed in liver after
estrogen treatment (24, 54), and the estrogen induction of the LDL
receptor expression, could exert a feedback-inhibitory effect on
transcription of the HMG CoA reductase gene by modifying the ratio of
different SREBP isoforms present in liver cells (14, 37). On the other
hand, Osborne and co-workers (4) have shown that the CRE site in the
HMG CoA reductase promoter is strongly protected from deoxyribonuclease
I (DNaseI) digestion by nuclear liver proteins. Deletion of this region
almost completely abolished transcription. In contrast, no major effect
was observed when the region covering the ERE site was deleted. These
results are in agreement with our finding that the ERE is not active in
liver and suggest that cAMP may be a main regulator in this tissue.
Alternatively, in hepatocytes, the high basal expression level of the
HMG CoA reductase promoter (10-fold) might preclude any further
physiological induction.
Heart disease has been related to an increase in blood cholesterol
levels and subsequent deposit in the vascular walls. If estrogens were
able to induce the HMG CoA reductase promoter in the liver, the
cholesterol flux from the liver would increase in response to
estrogens. However, we demonstrated here that HMG CoA reductase is not
estrogen inducible in liver cells, thus allowing estrogens to exert
other protective effects in peripheral tissues (e.g.
vasodilatation, heart activity moderation) without affecting
cholesterol production. Additionally, in estrogen target cells, such as
mammary cells and endometrial cells, the induction of the HMG CoA
reductase expression would facilitate cholesterol synthesis that may be
required for the proliferative response of these cells to estrogens.
Thus, the difference in estrogen responsiveness of the HMG CoA
reductase gene in liver and classical estrogen target cells could
explain in part of the low incidence of coronary heart disease in
females (55). However, as the ERE in the human HMG CoA reductase
promoter exhibits an additional mismatch, experiments with the human
promoter as well as studies on the estrogen regulation of the
endogenous HMG CoA reductase gene in mammary gland would be required to
support the physiological significance of our findings.
 |
MATERIALS AND METHODS
|
---|
Chemicals and Materials
Cell culture media and sera were purchased from Life Technologies, Inc. (Gaithersburg, MD).
[14C]-Chloramphenicol and [32P]-
-ATP
were from Amersham Pharmacia Biotech (Arlington Heights,
IL). 17ß-Estradiol, 25-OH-cholesterol, and (Bu)2cAMP were
purchased from Sigma Chemical Co. (St. Louis, MO). ICI
164,384 was kindly provided by A. E. Wakeling, Zeneca Pharmaceuticals. Restriction enzymes were purchased
from Roche Molecular Biochemicals (Foster City,
CA).
Reporter Plasmids
The plasmid pHMGRed-CAT, containing a 765 bp PstI
fragment (from -323 to +442) of the rat HMG CoA reductase promoter
cloned into the pEMBL-8-CAT vector, was kindly provided by M. Bifulco.
For construction of the luciferase reporter, pHMGRed-luc, the
PstI fragment of pHMGRed-CAT, was cloned into the
SmaI site of the pXP2 vector (56). p
HMGRed-luc was
generated by deleting the Red-ERE. Briefly, pHMGRed-luc was linearized
with EspI, which cleaves at the center of the Red-ERE, and
subsequently treated with S1 nuclease (1 U/µg DNA for 30 min at 37 C)
before religation. The resulting plasmid, p
HMGRed-luc, lacked the
ERE as confirmed by DNA sequence analysis. pERETK-luc and pRedTK-luc
were generated by cloning oligonucleotides containing the ERE-consensus
(5'-GATCCGTCAGGTCACAGTGACCTGATG-3') or the Red-ERE
(5'-TAGGCCGTCAGGCTGAGCAGCC-3'), respectively, into the SmaI
site of the pTK81-luc vector (56).
SRE plasmid contains the HMG CoA reductase promoter with a mutated
SRE (5'-TGGCGGTG-3'). The
oligonucleotide-directed mutagenesis was performed according to
the method described by Osborne (5).
EMSAs
EMSAs were performed with synthetic oligonucleotides. In
addition to the ERE-consensus and the Red-ERE oligonucleotides, the
following oligonucleotides were used as competitors for EMSA:
5'-TCGAGTGCCTAGAGAACAAACTGTTCTGACTCAAC-3', encompassing a progesterone-
responsive element (47), and
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' containing a
cAMP-responsive element (17). An oligonucleotides containing the HMG
CoA reductase promoter sequence from -111 to -72 was used for EMSA to
detect complexes with ER and CREB. The complementary strands were
annealed in equimolar amounts (10 nmol each) in 100 µl of annealing
buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 30
mM KCl), by denaturation (5 min at 95 C) and cooling down
to room temperature. Double-stranded oligonucleotides were radiolabeled
using T4 polynucleotide kinase and [32P]-
-ATP. Nuclear
extracts containing ER were prepared from MCF-7 cells treated
for 2 h with 10 nM 17ß-estradiol, as previously
described (57). Binding reactions were carried out in 30 µl reaction
buffer containing 10 mM Tris-HCl, pH 7.5, 0.1
mM EDTA, 0.1 mM dithiothreitol, 10% glycerol,
5 µg BSA, 1 mM MgCl2, 90 mM NaCl,
0.10.5 ng radiolabeled DNA probe, and 2 µg calf thymus DNA.
Specific competition assays were performed by adding 10- to 500-fold
molar excess of unlabeled oligonucleotides. Five-microliter aliquots of
the nuclear extract were added to the binding reaction and incubated
for 30 min at room temperature. For the detection of retarded
complexes, the reaction mixture was subjected to electrophoresis for
3 h on 4% polyacrylamide gels (acrylamide-bisacrylamide ratio,
30:1) at 11 V/cm in 0.5 x TBE buffer (Tris-borate-EDTA).
Results were visualized by autoradiography of the dried gel and
analyzed using a PhosphorImager and ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA). The monoclonal
ER
antibody (C-314, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) was included in the incubation mixture for supershift
experiments.
Transfection Assays
MCF-7, Cos-1, Fto, and HepG2 cells were cultured in DMEM
supplemented with 10% FCS. For transfection, 5 x 105
cells were plated on 60-mm plates. After 24 h, the medium was
replaced by DMEM without phenol red supplemented with 10%
charcoal-stripped FCS. Transfections were performed 48 h later
using the diethylaminoethyl-dextran method (48) for MCF-7 cells
and the calcium phosphate precipitation method (35) for Cos-1, Fto, and
HepG2 cells. The amount of reporter plasmid used for each assay was 3
µg. For the experiments performed in Cos-1, Fto, and HepG2 cells, 0.2
µg of the expression vector for the human
-ER [pHEGO, (58)] was
cotransfected. As an internal reference for transfection efficiency, 3
µg of the pRSV-lacZ plasmid were also cotransfected. After 24 h
the medium was replaced by fresh medium containing the appropriate
hormones or the corresponding solvent, and cells were cultured for
additional 48 h. Cell extracts were prepared by three cycles of
freezing and thawing. Protein concentration was measured by Bradford
assay. CAT, luciferase, and ß-galactosidase activities were performed
as described previously (31, 59).
Computer Analysis
Comparison and alignment of the different promoters were
performed using the Wisconsin Package (Genetics Computer Group, Inc., Madison, WI) installed on a OpenVMS AXP station.
Screening for the potential EREs was performed using MatInspector
software (60).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank P. Venditti, V. A. Raker, and J. Klug for
the helpful discussions. We are grateful to A. Scholz for providing the
recombinant ER and the p4xCRE vector and to A. E. Wakeling for the
generous gift of antiestrogen ICI 164,384.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Miguel Beato, Institute for Molecular Biology, Phillips University, D-35033 Marburg, Germany.
L.D.C. was partially supported by a European Molecular Biology
Organization short-term fellowship. This research was supported
by a grants from the Deutsche Forschungsgemeinschaft, the Fonds der
chemischen Industrie, and the European Union to M.B., and from
MURST (40%60%) to A.T.
Received for publication December 15, 1998.
Revision received April 29, 1999.
Accepted for publication May 18, 1999.
 |
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