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Interaction in 17ß-Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression1
Veterans Affairs Palo Alto Health Care System (C.L., T.E.A., F.B.K., J.L.), Palo Alto, California 94304; Pharmacia Corp. (M.R.B.), St. Louis, Missouri 63017
Address all correspondence and requests for reprints to: Jingwen Liu, Ph.D. (154P), Vetarans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and LDLR promoter constructs, we show that the specific
interaction of ER with the transcription factor Sp1 bound to the
LDLR promoter is responsible for the activation of LDLR transcription
by estrogen. We demonstrate that 1) mutations to abrogate the binding
of Sp1 to its recognition sequences present in repeat 1 and repeat 3
elements of the LDLR promoter completely abolish the ER-mediated
activation of the LDLR promoter activity; 2) mutations that abolish the
selective DNA-binding activity or inactivate the C-terminal
transcription activation function (AF2) of ER had no effect on the
ability of ER to activate LDLR transcription; however,
transcriptional activation was completely lost by deletion of the
N-terminal transcription activation region (AF1); 3) a subregion of AF1
(amino acids 67–139) was further identified to be important for ER
to activate the LDLR promoter; and 4) ER enhanced the formation of
Sp1-repeat 3 DNA complexes. We also show that mutation at the
sterol-responsive element-1 site diminishes the activity of ER on
LDLR transcription, thereby suggesting that the sterol-responsive
element-1-binding protein may interact with the Sp1-ER complex to
trans-activate LDLR gene transcription. This study for
the first time provides a molecular basis for an understanding of the
regulation of LDLR transcription by estrogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ethinyl estradiol increased hepatic LDLR messenger
RNA and protein levels (8, 9, 10, 11). LDLR expression is mainly controlled at the transcriptional level under the influence of the intracellular cholesterol content (12, 13). When cellular cholesterol levels rise, LDLR transcription is reduced, and the amount of LDLR on the cell surface decreases. When cellular cholesterol storage is depleted, LDLR transcription is activated. This leads to increased expression of LDLR on the cell surface of hepatocytes, thereby reducing plasma LDL cholesterol levels through increased binding of LDL particles to the receptor and subsequent internalization of the ligand-receptor complex.
The promoter region and the cis-acting elements that control basal and cholesterol-mediated transcription of LDLR have been localized to three GC-rich imperfect 16 bp direct repeats (14, 15, 16, 17). These three repeats lie within 100 bp upstream of the transcriptional start site. Repeat 1 (R1) and repeat 3 (R3) contain Sp1-binding sites that support the basal transcriptional activity of the LDLR. Interactions of Sp1 to the proximal binding site in R3 and to the distal binding site in R1 are required for the normal maximal transcriptional activity of the LDLR gene. Interruption of Sp1 binding to either repeat severely decreases basal transcription. For example, a 3-bp deletion in the R1 region of the LDLR promoter was identified in a patient with a clinical diagnosis of familial hypercholesterolemia (18). Sp1 binding to the mutated R1 sequence was abolished, and LDLR gene expression in fibroblasts from the patient was severely decreased.
Cholesterol regulation of LDLR transcription is mediated through a 10-bp sequence (5'-ATCACCCCAC-3') within repeat 2 (R2), designated sterol-responsive element-1 (SRE-1) (19, 20). Under low intracellular cholesterol conditions, SRE-1-binding proteins, SREBP1 and SREBP2, can bind to the SRE-1 sequence and activate transcription (21, 22). Recent studies showed that the interaction of SREBPs with SRE-1 increases the binding of Sp1 to R3, thereby resulting in a synergistic activation (23, 24).
To understand the beneficial effect of estrogen on the plasma LDL
cholesterol level, a number of studies, using a liver hepatoma cell
line HepG2 as an in vitro system, were carried out to
directly examine the effects of estrogen on LDLR expression.
Semenkovich et al. reported that treatment of HepG2 cells
with 10 µg/ml (38 µM) 17ß-estradiol
(E2) increased the number of LDLR on the cell
surface by measuring [125I]LDL binding and
ligand blotting (25), whereas a subsequent study reported
by Wade et al. showed that E2 at
concentrations up to 500 ng/ml (1.9 µM) did not
increase LDLR expression in HepG2 cells (26). However,
another study using 10 µg/ml E2 showed that
LDLR promoter activity was increased in HepG2 cells (27).
Apparently, very high and unphysiological concentrations of
E2 (38 µM) were required
to produce an effect on LDLR expression in HepG2 cells, which was
probably due to a lack of adequate expression of estrogen receptors
(ERs). The actions of estrogen are mediated primarily by ER
(28) and ERß (29, 30). Liver cells express
only the ER (29, 30). Although HepG2 cells retain many
liver-specific functions, the expression of ER is below the level of
detection (31). With transfection of an ER expression
vector into HepG2 cells, LDLR promoter activity was increased by
E2 at a much lower physiological concentration (1
nM) (32). Collectively, these
studies suggest that E2 can increase LDLR
expression through activation of gene transcription; however, the
cis-acting regulatory elements and the
trans-acting factors that mediate the effect of estrogen on
LDLR transcription have not been clearly characterized.
The current report shows that ER- and
E2-mediated activation of LDLR promoter activity
in HepG2 cells requires functional Sp1-binding sites. Mutations
disrupting Sp1 binding to R1 and R3 completely abolished
E2 activity on the LDLR promoter. By using
vectors containing mutations or deletions of different functional
domains of ER, we identified a region within activation function
domain 1 (AF1) of ER that was important for activation of LDLR
transcription by ER. This functional region overlaps a domain of AF1
that was recently shown to be required for ER to activate a
synthetic promoter that contains an Sp1-binding site (33).
Electrophoresis mobility shift assays (EMSA) demonstrated that the
binding of Sp1 to R3 was enhanced by ER. Taken together, these
studies for the first time document that the effect of estrogen on LDLR
transcription is mediated through specific interactions between ER
and the transcription factor Sp1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SC-543) were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), for
use in EMSA. Human recombinant Sp1 was purchased from Promega Corp. (Madison, WI). Human recombinant ER was obtained from
PanVera (Madison, WI). E2 and anti-ß-actin
monoclonal antibody were purchased from Sigma (St. Louis,
MO).
Plasmid vectors
LDLR promoter luciferase reporters pLDLR234,
pLDLR234-R1, pLDLR234-R2, pLDLR234-R3,
and pLDLR234-R1/R3 have been previously described
(34). Briefly, vector pLDLR234-R2 contains a
two-base mutation (underlined) within the core SRE-1
region (AAAATCACCCCACTGC to AAAATCACggCACTGC). The
promoter activity of this vector was decreased to 12% of the wild-type
promoter and was no longer regulated by cholesterol. The vector
pLDLR234-R3 contains a two-base substitution
(underlined) within the core Sp1 site
(AAACTCCTCCCCCTGC to AAACTCtTtCCCCTGC). This
mutation abolished Sp1 binding to R3 and decreased promoter activity to
7% that in the wild-type vector. The vector pLDLR234-R1
contains the same two-base substitution (underlined)
within the core Sp1 site as the R3 mutant (AAACTCCTCCTCTTGC to
AAACTCtTtCTCTTGC). The mutation of R1
eliminated Sp1 binding and decreased LDLR promoter activity to 10% of
the wild-type activity. The vector pLDLR234-R1/R3
contains mutations within both R1 and R3.
The wild-type human ER expression vector (pRST7hER) and a luciferase
reporter vector (pMTVERE4-LUC) containing four copies of the estrogen
response element (ERE) were provided by Dr. Kathy Fosnaugh
(Ligand Pharmaceuticals, Inc., San Diego, CA). The ER
mutant expression vectors AF1-DBD-X, X-DBD-X, AF1-X-AF2, and X-DBD-AF2
have been previously described (31) and were provided by
Dr. Douglas C. Harnish (Wyeth-Ayerst Laboratories, Inc.,
Radnor, PA). The ER mutant expression vectors HE1, HE2, HE3, HE10,
HE18, and HE19 have been previously described (35) and
were provided by Dr. Pierre Chambon (IGBMC, Strasbourg, France).
Preparation of nuclear extracts and EMSAs
HepG2 cells were seeded at 4.5 x 106
cells/100-mm dish 3 days before harvesting. Nuclear extracts were
prepared by the method of Dignam et al. (36),
except buffer A was supplemented with 1 mM
Na3VO4 and 1 µg/ml each
of pepstatin and leupeptin. Nuclear extracts were quick-frozen by
liquid nitrogen and stored in aliquots. Protein concentrations were
determined using a modified Bradford assay using BSA as a standard
(Pierce Chemical Co., Rockford, IL). Protein
concentrations of nuclear extracts from different preparations were
typically 2–3 mg/ml. Oligonucleotide probes were annealed and
end-labeled with T4 polynucleotide kinase in the presence of
[
-32P]ATP.
Each binding reaction was composed of 10 mM HEPES (pH 7.8),
2 mM MgCl2, 2 mM
dithiothreitol, 80 mM NaCl, 10% glycerol, 1 µg
poly(dI-dC), 1 µg BSA, and 0.1–5 µg nuclear extract in a final
volume of 20 µl. Nuclear extracts were incubated with 0.4–0.5 ng
32P-labeled, double stranded, synthetic
oligonucleotide probe (40–80 x 103 cpm)
for 10 min at room temperature. For EMSA with recombinant human Sp1,
the reaction mixture contained 10 mM Tris-HCl (pH 8.0), 1
mM MgCl2, 0.5 mM EDTA,
0.5 mM dithiothreitol, 50 mM NaCl, 6%
glycerol, 1 µg poly(dI-dC), 2 µg BSA, and 1–10 ng
affinity-purified Sp1 in the presence or absence of recombinant human
ER (0.5–2 pmol) in a final volume of 20 µl. The reaction mixtures
were loaded onto a 6% polyacrylamide gel and run in TBE buffer (90
mM Tris, 90 mM borate, and 2 mM
EDTA, pH 8.3) at 20 mA for 2 h at 4 C. The gels were dried and
visualized on a PhosphorImager. For supershift assays, antibody was
incubated with recombinant Sp1 for 30 min at room temperature before
addition of the probe.
The sequences of EMSA probes are as follows, and the consensus binding sites of Sp1 are underlined: R23, 5'-TTTGAAAATCACCCCACTGCAAACTCCTCCCCCTGCT-3'; R23D, 5'-TTTGAAAATCACCCCACTGCAAACTCtTtCCCCTGCT-3'; and R1, 5'-TTCGAAACTCCTCCTCTTGCAGTGAGGTGAAGACATTTG-3'. The oligonucleotide R23D contains the same mutation as the vector pLDLR234-R3. The mutated nucleotides in R23D are in lowercase italic.
Transient transfection assays
HepG2 cells cultured in a 24-well plate at a density of
0.12 x 106 cells/well were transiently
transfected with plasmid DNA by the method of calcium phosphate
coprecipitation with 20 µg total DNA in each precipitation in the
ratio of 10 µg LDLR-luciferase reporter, 5 µg ER expression
vector, and 5 µg pRSV-ß-galactosidase. The ratio of pLDLR reporter
and ER (10:5) was shown to give a maximal response to
E2 at both 1-µM and
100-nM concentrations in pilot experiments that examined
the ER dose-dependent effect on pLDLR promoter activity. After
40-min precipitation, 50 µl precipitate containing 880 ng DNA were
added to each well. The cells were incubated with the DNA precipitate
for 4 h, washed with PBS, and refed with fresh medium containing
10% charcoal-absorbed FBS without or with various concentrations of
E2. After 40-h treatment, cells were lysed, and
luciferase and ß-galactosidase activities were assayed. Absolute
luciferase activity was normalized against ß-galactosidase activity
to correct for transfection efficiency.
Statistical analysis
Comparisons of experimental data were analyzed by a two-tailed
Student’s t test. P < 0.05 was considered
to indicate a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in HepG2 cells and
the exogenous expression of ER after transient transfection, total
cell lysates were prepared from HepG2 cells, mock-transfected or
transfected with pRST7hER. Cell lysate from MCF-7 cells that express
normal ER was also prepared to provide a positive control. Fifty
micrograms of soluble protein from each sample were loaded on a 10%
SDS gel and separated by electrophoresis, transferred to polyvinylidene
membrane, and blotted with anti-ER polyclonal antibody. Figure 1
shows that HepG2 cells transfected with
pRST7hER express ER at a level similar to MCF-7 cells, whereas no
ER was detected from mock-transfected HepG2 cells. Immunoblotting
with anti-ß-actin antibody showed comparable amounts of soluble
protein in each sample. To determine the effect of
E2-activated ER on LDLR transcription, HepG2
cells were transfected with LDLR promoter luciferase reporter construct
pLDLR234 with control DNA or with pRST7hER and then treated with
different concentrations of E2 for 40 h.
Analysis of luciferase activities in transfected cells shows that
E2 produced a dose-dependent activation of LDLR
promoter activity in ER-transfected cells. The promoter activity was
increased 3-fold by E2 at 1 nM and
was increased 5-fold at 1 µM. In contrast, the LDLR
promoter activity in HepG2 cells transfected with a negative control
vector was not changed by E2 in the same
concentration range. To determine the kinetics of the
E2 action, E2 (100
nM) was added at different times after transfection, and
all cells were harvested after 48 h posttransfection. The results
show that the LDLR promoter activity was increased 3-fold at 24 h
and reached a plateau (3.7- to 4-fold) after 32-h treatment with
E2. These experiments established that
E2 activates LDLR transcription of HepG2 cells in
a time- and a dose-dependent manner and is mediated through ER.
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in activation of LDLR
transcription-mediated activation of LDLR promoter
activity, we compared the activity of E2 on the
wild-type vector pLDLR234 with the activities of
E2 on mutant vectors that either abolish the
SREBP binding or the Sp1 binding. Figure 2 shows that the stimulatory effect of
E2 on LDLR promoter activity was partially
reduced from 4.7- to 1.9-fold by mutation in the SRE-1 site
(pLDLR234-R2). The E2 activity was
also significantly diminished in the construct carrying mutations in
either R1 (pLDLR234-R1, 2.4-fold) or R3
(pLDLR234-R3, 2.0-fold). In contrast,
E2 activity in cells transfected with
pLDLR-R1/R3 was completely lost (1.3 ± 0.2-fold),
thereby demonstrating that interference of Sp1 binding to both R1 and
R3 eliminates the activity of E2 on LDLR
transcription. These results suggest that although a functional SRE-1
is required for maximal E2 activation,
interaction of Sp1 with the LDLR promoter may be critically
involved in the ER-mediated activation of LDLR transcription.
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transcription activation function 1 (AF1) is required for
activation of LDLR transcription and
E2 activate LDLR transcription, vectors
expressing wild-type ER or ER with a deletion in the N-terminal
transcription activation function (AF1), point mutations in the DNA
binding domain, or point mutations in the C-terminal transcription
activation function (AF2), were individually cotransfected with
pLDLR234 into HepG2 cells. Figure 3A
shows that inactivation of the AF2 functional domain (AF1-DBD-X) did
not affect the ability of E2 to stimulate LDLR
promoter activity. Similarly, E2 activity was
retained in the vector that contains mutations within the DNA-binding
domain (AF1-X-AF2) that convert ER binding selectivity from an ERE
to a glucocorticoid response element (37). In contrast,
deletion of the AF1 functional domain (X-DBD-AF2) completely abolished
E2 activity. Likewise, mutant vector (X-DBD-X)
containing the AF1 deletion and AF2 mutations did not respond to
E2. The lack of E2-ER
response on LDLR promoter activity by deletion of AF1 was not due to
the absence or poor expression of the mutant receptor, as Western blot
analysis showed comparable levels of expressions for wild-type ER
and mutant ER in cells transfected with the various vectors (Fig. 3B). To demonstrate the promoter-specific effect of the AF1 deletion,
vectors pRST7hER and X-DBD-AF2 were cotransfected into HepG2 cells with
pMTVERE4-LUC, which contains four copies of ERE. Stimulation of cells
with E2 produced 7.5- and 2.8-fold increases in
luciferase activities in cells transfected with the wild-type ER and
the X-DBD-AF2, respectively. This demonstrates that the AF1 deletion
impaired the trans-activating activity of ER, but did not
completely eliminate the ability of ER to bind to ERE and to
trans-activate. These results suggest that the N-terminal
region of ER (AF1) is critically involved in the interaction of
ER and LDLR promoter, possibly through the Sp1-binding sites.
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responsible for
activation of LDLR transcription were cotransfected into HepG2 cells
with pLDLR234, and the transfected cells were treated with
E2 for 40 h. Figure 4 shows that deletion of amino acids
9–62 (HE1) lowered the E2 activation of pLDLR234
from 4.4- to 3-fold, retaining 60% activity of the wild-type ER.
Deletion of amino acids 67–131 (HE2) decreased
the E2 activity to 1.7-fold, and deletion of
amino acids 132–198 (HE3) nearly eliminated the ER activity.
Similar to HE3, constructs HE18 and HE19 that contain deletions of
amino acids 1–139 and 1–178 did not activate LDLR promoter activity.
In contrast, deletion of amino acids 141–170 (HE10) had no effect on
the induction of LDLR promoter activity by ER and
E2. These data suggest that the region
encompassing amino acids 67–139 is important for ER to activate
LDLR transcription through Sp1-binding sites. A recent study has
localized the ER and Sp1 interaction to a similar region of AF1
(amino acids 79–117) (33).
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enhances Sp1 binding to repeat 3 element of the LDLR
promoter, EMSA using a labeled oligonucleotide probe R23
containing the R2 and R3 elements was performed with purified
recombinant Sp1. Figure 5A shows a
dose-dependent binding of Sp1 to the probe (lanes 2, 4, and 6). Figure 5B shows the specific interaction of Sp1 with the R23 probe, as binding
of Sp1 to the probe was inhibited by competition with a 100-fold molar
excess of the unlabeled oligonucleotide R23 (lane 2), but was not
inhibited by oligonucleotide R23d that contains a CTCCTCCCCC to
CTCtTtCCCC double nucleotide substitution
(underlined) within the core Sp1 binding site (lane
3). Furthermore, the complex was completely supershifted by anti-Sp1
antibody (lane 4). Addition of purified recombinant ER (1 pmol) to
the reaction mixture increased the intensity of the Sp1-DNA complex
approximately 2-fold within the concentration range of Sp1 tested (Fig. 5A, lanes 3, 5, and 7), but formation of additional complexes of
different mobility were not detected. The enhancement of Sp1 binding
was also ER dose dependent (Fig. 5C, lanes 1–4). The enhancement by
ER of Sp1-binding activity within the context of HepG2 cells was
also detected by EMSA using HepG2 nuclear extracts (Fig. 5D). The
increased DNA-binding activity of Sp1 in the presence of ER was also
observed using oligonucleotide probe containing the R1 sequence of the
LDLR promoter (data not shown). These results are consistent with other
studies using different GC-rich regulatory elements showing that ER
enhanced the DNA-binding activity of Sp1 without formation of the
Sp1/ER-DNA ternary complex (38, 39, 40, 41).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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complex uncouples from the heat shock
proteins and binds, as homodimers or heterodimers, to a specific ERE
present in the promoter region of target genes, thereby activating gene
transcription. It is also well known that ER can regulate the
transcription of genes whose promoters do not contain consensus ERE
sites. In such cases, direct interactions of ER with other
transcription factors bound to their cognate sites in the promoters
have been demonstrated (42, 43, 44, 45). The promoter region of
the LDLR does not contain a functional ERE site; thus, the effect of
E2-ER on LDLR transcription is most likely
mediated through specific interaction of ER with other transcription
factors that regulate LDLR transcription through their recognition
sequences present in the promoter region. In this study we demonstrate
that the ER/Sp1 interaction through Sp1-binding sites present in the R3
and R1 sequences are responsible for activation of LDLR promoter
activity in HepG2 cells when functional ER is present.
We show that the activity of E2 on the LDLR
promoter was significantly impaired by mutations that interrupt Sp1
binding to R1 (pLDLR234-R1) or R3 (pLDLR234-R3)
sequences, whereas inhibition of Sp1 binding to both repeats completely
abolished the effect of ER on LDLR transcription (vector
R1/R3). These data suggest that Sp1-binding sites in the
promoter are the critical regulatory cis-acting elements
that mediate the activation of LDLR transcription by ER. We also
showed that mutation at the SRE-1 site (pLDLR234-R2), which
abolished SREBP binding, partially inhibited the activity of ER on
LDLR promoter activity. Although E2 activity was
decreased, the promoter activity in cells stimulated with
E2 was still statistically significantly higher
than that in controls (P = 0.004). Previously, using a
synthetic promoter reporter construct that contains multiple copies of
R2 and R3 in tandem, Croston et al. showed that mutation
within the SRE-1 site drastically decreased basal and
E2-stimulated promoter activities in HepG2 cells
cotransfected with ER. Based on those results, it was concluded that
the effect of E2 was mediated through the SRE-1
site despite the fact that the activity of the promoter with SRE-1
mutation in E2-stimulated cells was still higher
than the control value (
2-fold) (32). The synthetic
promoters used by Croston et al. were created to be much
more sensitive to signaling through the SRE-1 site, and therefore,
differences are likely to be magnified in their system. As the native
LDLR promoter system is used in the current study, it is highly likely
that results from the present study more accurately reflect the
physiological regulation of the LDLR. It has been shown that SREBP
increases the binding of Sp1 to R3 in an in vitro study
(23). This observation was further confirmed in intact
cells, showing that activation of SREBP by sterol depletion results in
an increased binding of Sp1 to a site adjacent to SRE-1 in the LDLR
promoter (24). Therefore, it is possible that mutation of
SRE-1 indirectly affected the action of ER on the LDLR promoter by
affecting the binding of a Sp1-ER complex to the R3 element.
Alternatively, ER may form a trans-activation complex
with Sp1 and SREBP. Requirement of another transcription factor to
interact with the ER/Sp1 complex to trans-activate has been
described in the regulation of the E2F1 gene
(39). Due to the extremely low amounts of SREBP present in
the HepG2 nuclear extracts, we were not able to detect the binding of
SREBP to the R23 probe in the presence or the absence of ER.
Therefore, the effect of SREBP on formation of the Sp1/ER-DNA
complex could not be directly examined in this study.
The critical involvement of Sp1 binding sites of the LDLR promoter in
ER-mediated LDLR transcription suggests a direct interaction between
ER and Sp1. It has been shown that different functional domains of
ER are involved in interactions of ER with different
transcription factors or cofactors in a cell type- and
promoter-specific manner (46). The ER mutant vector,
AF1-X-AF2, expresses ER with point mutations in the DNA-binding
domain, which had been shown to convert the selectivity of DNA binding
of ER from the ERE to the glucocorticoid response element
(37). Cotransfection of AF1-X-AF2 with pLDLR234 produced
an E2 response similar to that of wild-type
ER. In addition, our gel shift assays could not detect direct
binding of ER to the labeled R23 probe, but showed strong binding of
ER to the ERE probe (data not shown). Moreover, it has been
previously shown that the ER/Sp1 interaction does not require the
DNA-binding domain of ER (38). Together, these results
demonstrate that regulation of LDLR transcription by ER does not occur
through direct binding to the promoter element. Similar to the DBD
mutation, inactivation of AF2 (mutant AF1-DBD-X) had no effect on
ER-mediated activation of LDLR transcription. In contrast, the
activity of ER on the LDLR promoter was completely lost when the AF1
domain was deleted (X-DBD-AF2). Interestingly, a recent report using
the same mutant vectors showed that the ER-mediated suppression of
apoA1 promoter activity in HepG2 cells was not affected by AF1
deletion, but was affected by mutations that inactivate DBD or AF2
(31). Thus, the loss of ER activity by AF1 deletion is
LDLR promoter specific. The different effects of AF1 deletion on LDLR
vs. apoA1 further illustrate the promoter-specific
regulation by ER.
Interactions of ER with Sp1 have been demonstrated in several
promoters that contain GC-rich elements, such as
E2F (39) and Bcl-2
(41). A recent study has localized a region of AF1 (amino
acids 79–117) that is important for activation at an Sp1 element
(33). By using deletion constructs, we showed that
deletions of amino acids 67–139 drastically reduced the activity of
E2-activated ER on the LDLR promoter, whereas
deletion of amino acids 141–170 had no effect. Thus, it appears that
the functional region of ER (aa 67–139) for activating the LDLR
promoter overlaps with a functional region (aa 79–117) that is
required for activation of a synthetic promoter containing an Sp1 site.
These data strongly support our conclusion that the Sp1 binding sites
of the LDLR promoter constitute the primary ER-responsive
element.
Finally, gel shift assays were performed to demonstrate an enhancement
of Sp1 binding to the LDLR promoter by ER. The binding of Sp1 to the
R23 probe was increased by ER in a dose-dependent manner. The
intensities of the Sp1-DNA complex varied between different
experiments. Typically, a 2- to 3-fold increase in Sp1 binding in the
presence of ER was detected. This is presumably caused by enhancing
the on rate of Sp1-[32P]R3 formation by ER
as described in other studies (38, 39, 40, 41). Our findings that
ER enhances Sp1 DNA-binding activity without formation of a
Sp1/ER-R23 ternary structure are consistent with a number of studies
in which Sp1/ER-DNA ternary complex was not detected by EMSA
(33, 38, 39, 40, 41).
In this study we provide strong evidence demonstrating that regulation
of LDLR transcription by ligand-activated ER is primarily mediated
through the Sp1-binding sites present in the R1 and R3 sequences of the
LDLR promoter. This regulation requires the AF1 functional domain of
ER. We hypothesize that upon E2 binding ER
undergoes a conformational change that exposes the AF1 domain to Sp1
and perhaps also to SREBP, thereby resulting in
trans-activation of the LDLR gene transcription. However,
our studies could not exclude the possibility that other cofactors
might also be recruited in the Sp1/ER complex during the activation
process. Previously, it has been shown that in MCF-7 cells,
E2 and the estrogen antagonists
4'-hydroxytamoxifen and ICI 182,780 induced a synthetic promoter (pSp1)
activity that contains a consensus Sp1-binding site presumably through
Sp1/ER interaction. However, we found that 4'-hydroxytamoxifen and
two other ER-selective modulators, namely idoxifen and raloxifen,
inhibited E2-stimulated LDLR promoter activity in
HepG2 cells cotransfected with the wild-type ER. The different
activities of ER-selective modulators on pSp1 in MCF-7 cells and on
pLDLR in HepG2 cells suggest the possible involvement of different
cofactors in these two assay systems.
Previously, our laboratory has identified a sterol-independent
regulatory element, located downstream of the SRE-1- and
Sp1-binding sites, that is responsible for cytokine oncostatin M- and
cAMP-mediated activation of LDLR transcription (47). In
addition, several studies have linked mitogen-activated protein kinase
activation with LDLR transcription (31, 48, 49). The
current studies demonstrate another route of activation of LDLR
transcription by ER. Together, these studies illustrate that LDLR
transcription is regulated by multiple mechanisms, including
sterol-dependent and sterol-independent pathways.
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Acknowledgments |
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mutant
vectors and giving technical advise concerning our transfection
experiments. |
Footnotes |
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Received October 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ethinyl estradiol. J Biol Chem 254:11367–11373-ethinyl estradiol. Proc Natl Acad Sci USA 83:792–796/ß)-dependent activation at GC-rich (Sp1) promoter
elements. J Biol Chem 275:5379–53871 gene expression: role of estrogen
receptor-Sp1 complex. Mol Endocrinol 12:882–890This article has been cited by other articles:
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 F. Zou, R. K. Gopalraj, J. Lok, H. Zhu, I-F. Ling, J. F. Simpson, H. M. Tucker, J. F. Kelly, S. G. Younkin, D. W. Dickson, et al. Sex-dependent association of a common low-density lipoprotein receptor polymorphism with RNA splicing efficiency in the brain and Alzheimer's disease Hum. Mol. Genet., April 1, 2008; 17(7): 929 - 935. [Abstract] [Full Text] [PDF]
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 S. Khan, F. Wu, S. Liu, Q. Wu, and S. Safe Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells J. Mol. Endocrinol., October 1, 2007; 39(4): 289 - 304. [Abstract] [Full Text] [PDF]
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H. Zhu, H. M. Tucker, K. E. Grear, J. F. Simpson, A. K. Manning, L. A. Cupples, and S. Estus A common polymorphism decreases low-density lipoprotein receptor exon 12 splicing efficiency and associates with increased cholesterol Hum. Mol. Genet., July 15, 2007; 16(14): 1765 - 1772. [Abstract] [Full Text] [PDF]
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 F. Stossi, V. S. Likhite, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Estrogen-occupied Estrogen Receptor Represses Cyclin G2 Gene Expression and Recruits a Repressor Complex at the Cyclin G2 Promoter J. Biol. Chem., June 16, 2006; 281(24): 16272 - 16278. [Abstract] [Full Text] [PDF]
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M L Panno, L Mauro, S Marsico, D Bellizzi, P Rizza, C Morelli, M Salerno, F Giordano, and S Ando' Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor {alpha} through its interaction with Sp1 J. Mol. Endocrinol., February 1, 2006; 36(1): 91 - 105. [Abstract] [Full Text] [PDF]
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 K. Kim, R. Barhoumi, R. Burghardt, and S. Safe Analysis of Estrogen Receptor {alpha}-Sp1 Interactions in Breast Cancer Cells by Fluorescence Resonance Energy Transfer Mol. Endocrinol., April 1, 2005; 19(4): 843 - 854. [Abstract] [Full Text] [PDF]
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L. Bjornstrom and M. Sjoberg Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes Mol. Endocrinol., April 1, 2005; 19(4): 833 - 842. [Abstract] [Full Text] [PDF]
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Y-L Zhao, W-D Han, Q Li, Y-M Mu, X-C Lu, L Yu, H-J Song, X Li, J-M Lu, and C-Y Pan Mechanism of transcriptional regulation of LRP16 gene expression by 17-{beta} estradiol in MCF-7 human breast cancer cells J. Mol. Endocrinol., February 1, 2005; 34(1): 77 - 89. [Abstract] [Full Text] [PDF]
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R. O'Lone, M. C. Frith, E. K. Karlsson, and U. Hansen Genomic Targets of Nuclear Estrogen Receptors Mol. Endocrinol., August 1, 2004; 18(8): 1859 - 1875. [Abstract] [Full Text] [PDF]
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P. M. Smith, A. Cowan, and B. A. White The Low-Density Lipoprotein Receptor Is Regulated by Estrogen and Forms a Functional Complex with the Estrogen-Regulated Protein Ezrin in Pituitary GH3 Somatolactotropes Endocrinology, July 1, 2004; 145(7): 3075 - 3083. [Abstract] [Full Text] [PDF]
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X.-H. Li and D. E. Ong Cellular Retinoic Acid-binding Protein II Gene Expression Is Directly Induced by Estrogen, but Not Retinoic Acid, in Rat Uterus J. Biol. Chem., September 12, 2003; 278(37): 35819 - 35825. [Abstract] [Full Text] [PDF]
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 Y. Jiang, Y. E. Liu, A. Lu, A. Gupta, I. D. Goldberg, J. Liu, and Y. E. Shi Stimulation of Estrogen Receptor Signaling by {gamma} Synuclein Cancer Res., July 15, 2003; 63(14): 3899 - 3903. [Abstract] [Full Text] [PDF]
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S. Khan, M. Abdelrahim, I. Samudio, and S. Safe Estrogen Receptor/Sp1 Complexes Are Required for Induction of cad Gene Expression by 17{beta}-Estradiol in Breast Cancer Cells Endocrinology, June 1, 2003; 144(6): 2325 - 2335. [Abstract] [Full Text] [PDF]
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X. Hu, L. Cherbas, and P. Cherbas Transcription Activation by the Ecdysone Receptor (EcR/USP): Identification of Activation Functions Mol. Endocrinol., April 1, 2003; 17(4): 716 - 731. [Abstract] [Full Text] [PDF]
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 J. Liu, F. Zhang, C. Li, M. Lin, and M. R. Briggs Synergistic Activation of Human LDL Receptor Expression by SCAP Ligand and Cytokine Oncostatin M Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 90 - 96. [Abstract] [Full Text] [PDF]
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M. Jakacka, M. Ito, F. Martinson, T. Ishikawa, E. J. Lee, and J. L. Jameson An Estrogen Receptor (ER){alpha} Deoxyribonucleic Acid-Binding Domain Knock-In Mutation Provides Evidence for Nonclassical ER Pathway Signaling in Vivo Mol. Endocrinol., October 1, 2002; 16(10): 2188 - 2201. [Abstract] [Full Text] [PDF]
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F. Zhang, T. E. Ahlborn, C. Li, F. B. Kraemer, and J. Liu Identification of Egr1 as the oncostatin M-induced transcription activator that binds to sterol-independent regulatory element of human LDL receptor promoter J. Lipid Res., September 1, 2002; 43(9): 1477 - 1485. [Abstract] [Full Text] [PDF]
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D. Lopez, M. D. Sanchez, W. Shea-Eaton, and M. P. McLean Estrogen Activates the High-Density Lipoprotein Receptor Gene via Binding to Estrogen Response Elements and Interaction with Sterol Regulatory Element Binding Protein-1A Endocrinology, June 1, 2002; 143(6): 2155 - 2168. [Abstract] [Full Text] [PDF]
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 C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]
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