Received for publication, February 1, 2001, and in revised form, February 21, 2001
Estrogens used in hormone replacement therapy
regimens may increase the risk of developing breast cancer.
Paradoxically, high consumption of plant-derived phytoestrogens,
particularly soybean isoflavones, is associated with a low incidence of
breast cancer. To explore the molecular basis for these potential
different clinical outcomes, we investigated whether soybean
isoflavones elicit distinct transcriptional actions from estrogens. Our
results demonstrate that the estrogen 17
-estradiol effectively
triggers the transcriptional activation and repression pathways with
both estrogen receptors (ERs) ER
and ER
. In contrast, soybean
isoflavones (genistein, daidzein, and biochanin A) are ER
-selective
agonists of transcriptional repression and activation at physiological
levels. The molecular mechanism for ER
selectivity by isoflavones
involves their capacity to create an activation function-2 surface of
ER
that has a greater affinity for coregulators than ER
.
Phytoestrogens may act as natural selective estrogen receptor
modulators that elicit distinct clinical effects from estrogens used
for hormone replacement by selectively recruiting coregulatory proteins
to ER
that trigger transcriptional pathways.
 |
INTRODUCTION |
Estrogens are used in hormone replacement therapy
(HRT)1 to prevent hot
flashes, urogenital atrophy, and osteoporosis in postmenopausal women
(1, 2). HRT also may prevent heart disease (3), Alzheimer's disease
(4), and colon cancer (5). Unfortunately, HRT has not lived up to its
potential to improve the health of women, because estrogens have
been associated with an increased incidence of breast (6, 7) and
endometrial cancer (8). This relationship has hampered compliance with
HRT severely and has sparked an intense pursuit for selective estrogen
receptor modulators (SERMs) that have a safer profile (9, 10).
Recently, raloxifene has been approved for the prevention and treatment of osteoporosis (11). Raloxifene is classified as a SERM because it
exhibits agonist activity in some tissues such as the bone (12, 13) and
acts as an antagonist in other tissues including the breast (14).
Although these effects are extremely desirable, raloxifene also
increases hot flashes (15), is weaker than estrogens at increasing bone
mineral density (16), and does not improve cognitive function (17) or
prevent hip fracture (13). Thus, the quest for superior SERMs for HRT
continues to be intense.
There also is a growing interest in using dietary natural plant
estrogens (phytoestrogens), particularly those found in soy products,
as a potential alternative to the estrogens in HRT (18). Interest in
phytoestrogens has been fueled by observational studies showing a lower
incidence of menopausal symptoms, osteoporosis, cardiovascular disease,
and breast and endometrial cancers in Asian women who have a diet rich
in soy products (19-24). Consistent with epidemiological studies are
the findings that soy phytoestrogens prevent mammary tumors (25, 26)
and bone loss (27, 28) in rodents and atherosclerosis of coronary
arteries in monkeys (29). Soy protein relieves hot flashes in
postmenopausal women (30) and attenuates bone loss in the lumbar spine
of perimenopausal women (31). Furthermore, a high intake of dietary
phytoestrogens is associated with a lower incidence of breast cancer in
women (19). Many postmenopausal women are taking phytoestrogens in an
effort to alleviate menopausal symptoms without increasing their risk
of developing breast cancer. Moreover, many women with a history of
breast cancer take phytoestrogens to control menopausal symptoms
(32, 33) because estrogens are contraindicated.
The isoflavones, genistein, daidzein, and biochanin A, which are
abundant in soybeans (34) and available widely as herbal tablets, are
especially popular among postmenopausal women. Despite their popularity
and putative health benefits it is clear that we need to know much more
about the molecular mechanisms, safety, and efficacy of isoflavones
before they can be recommended to postmenopausal women as an
alternative to estrogens for HRT. However, it is clearly important to
elucidate the molecular mechanisms whereby isoflavones may elicit
distinct clinical actions from estrogens used in HRT. Isoflavones have
a structure similar to that of 17
-estradiol (E2) and are
capable of binding to the two known estrogen receptors, ER
and ER
(35-37). Compared with ER
, ER
exhibits a 7-30-fold greater
binding affinity for genistein, whereas E2 binds to ER
and ER
with equal affinity (38, 39). The relatively selective
binding of genistein to ER
indicates that isoflavones may produce
distinct clinical effects from estrogens by selectively triggering
ER
-mediated transcriptional pathways or differentially triggering
transcriptional activation or repression pathways by ER
.
To test this hypothesis, we compared the effects of isoflavones and
E2 on transcriptional repression and activation in the presence of ER
or ER
. Our data demonstrate that isoflavones selectively trigger the transcriptional pathways of ER
, particularly transcriptional repression. In addition to selectively binding to
ER
, our results suggest that the ER
selectivity of isoflavones involves their capacity to induce an activation function-2 (AF-2) surface of ER
that has greater affinity for coregulators such as
glucocorticoid interacting receptor protein 1 (GRIP1) (40) compared
with ER
. Phytoestrogens may act as natural SERMs by selectively recruiting coregulators that trigger ER
-mediated transcriptional pathways.
 |
MATERIALS AND METHODS |
Plasmids--
Human ER
and ER
were provided by P. Chambon
and J.-A. Gustafsson, respectively (41). Gal-GRIP1 and GST-GRIP1 were
provided by M. Stallcup (42). Three copies of the
125 to
82 human
TNF-
promoter fragment (43) or one copy of the ERE from the frog vitellogenin A2 gene (5'-TCAGGTCACAGTGACCTGA-3'; vitA2-ERE) were ligated into the polylinker upstream of
32 to +45 herpes simplex thymidine kinase (tk) promoter linked to luciferase (TNF-RE tkLuc and
ERE tkLuc, respectively). A synthetic oligonucleotide containing the
17-nucleotide Gal-responsive element (5'-CGGAGTACTGTCCTCCG-3') was
inserted in between the G and C of the AP-1-like site (5'-TGAGCTCA-3') at the
105 to
95 region of the TNF-RE and cloned upstream of the
32 to + 45 tk promoter (Gal-TNF-RE tkLuc).
Cell Culture, Transfection, and Luciferase Assays--
U937,
U2OS, MDA-MB-435, and MCF-7 cells were obtained from the cell culture
facility at the University of California, San Francisco. U937 cells
were maintained as described previously (44), whereas U2OS, MDA-MB-435,
and MCF-7 cells were maintained and subcultured in phenol red-free
Dulbecco's modified Eagle's medium/F-12 media containing 5% fetal
bovine serum, 2 mM glutamine, 50 units/ml penicillin, and
50 µg/ml streptomycin. For experiments, cells were collected,
transferred to a cuvette, and then electroporated with a Bio-Rad gene
pulser as described previously (41) using 3 µg of reporter plasmid
and 1 µg of ER
or ER
expression vectors. After electroporation,
the cells were resuspended in media and plated at 1 ml/dish in 12-well
multiplates. The cells were treated with E2, genistein,
daidzein, or biochanin A (Sigma-Aldrich) 3 h prior to exposure to
5 ng/ml TNF-
(R & D Systems) for 24 h at 37 °C. Cells were
solubilized with 200 µl of 1× lysis buffer, and luciferase activity
was determined using a commercially available kit (Promega). The
concentration of hormone required to produce a half-maximal induction
(EC50) or inhibition (IC50) of luciferase activity was calculated with the Prism curve-fitting program (Graph Pad
Software, version 2.0b). For proliferation studies, parental MCF-7
cells were subcloned at 1 cell/well in the presence of 0.1 nM E2, and the fastest growing clone was
selected for experiments. These cells expressed exclusively ER
as
determined by reverse transcription polymerase chain reaction (RT-PCR).
The cells were plated in duplicate at a density of 25,000 cells/35-mm
plate in tissue culture medium containing 3% stripped fetal bovine
serum. One day after plating they were treated with increasing
concentrations of E2 or genistein. The medium was changed
every other day, and E2 or genistein was added to the
medium. After 8 days the cells were counted with a Coulter counter. All
experiments presented in the figures were performed at least three
times, and the data were similar between experiments.
RT-PCR--
U20S cells were grown in Dulbecco's modified
Eagle's medium/F-12 supplemented with 3% stripped fetal bovine serum.
Cells were treated for 24 h with 10 nM E2
or 1 µM genistein and then exposed to 5 ng/ml TNF-
for
1 h at 37 °C and 5% CO2. Total RNA was prepared using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. First-strand cDNA synthesis was performed using oligo(dT) primers (Life Technologies, Inc.) and Maloney murine
leukemia virus(H
) reverse transcriptase (Promega) as
recommended by the manufacturer. PCR amplification using primers for
the human TNF-
(sense 5'-GAGTGACAAGCCTGTAGCCCATGTTGTAGCA-3' and
antisense 5'-GCAATGATCCCAAAGTAGACCTGCCCAGACT-3') or
glyceraldehyde-3-phosphate dehydrogenase gene (sense
5'-TGATGACATCAAGAAGGTGGTGAAG-3' and antisense 5'-TCCTTGGAGG
CCATGTGGGCCAT-3') was performed using the HotStarTaq PCR kit (Qiagen)
or Ready-To-Go PCR beads (Amersham Pharmacia Biotech). The PCR products
were visualized on a 1.5% agarose gel stained with ethidium bromide.
MDA-MB-453 Stable Cell Line--
The ER-negative human breast
cancer cell line, MDA-MB-453 (45), was transfected by electroporation
with pcDNA 6/V5-His (Invitrogen) vector containing human
ER
. The cells were maintained in 10 µg/ml blastocidin (Invitrogen)
until resistant colonies formed. Individual clones were obtained after
the cells were plated into 96-well dishes at 1 cell/well in the
presence of blastocidin. The expression of ER
and blastocidin-S
deaminase, which confers resistance, was confirmed by RT-PCR in the
clonal stable cell line.
Glutathione S-Transferase Pull-down Assays--
GST pull-down
assays were performed as described previously (46). Briefly, human
ER
and ER
were transcribed and translated in vitro
using the TNT T7 Quick Coupled Transcription/Translation system
(Promega) and [35S]methionine. For each binding reaction,
a 2-µl aliquot of translation product was incubated with
Escherichia coli-expressed GST-GRIP1 immobilized to
glutathione-Sepharose beads (Amersham Pharmacia Biotech) in the
presence of vehicle control (0.1% ethanol), E2, or
genistein. The samples were rocked gently at 4 °C for 2 h. After extensive washing of the beads, the labeled proteins were eluted
with SDS-polyacrylamide gel electrophoresis loading buffer and
separated on a 12% SDS-polyacrylamide gel. The radiolabeled bound ERs
were detected by autoradiography and analyzed using the Storm
phosphorimaging system and ImageQuant software (Molecular Dynamics).
 |
RESULTS |
Estrogens Selectively Repress the TNF-
Promoter through
ER
--
To investigate the effects of isoflavones on
transcriptional repression, we used the
125 to
82 region (43) of
the TNF-
promoter (TNF-
-responsive element, (TNF-RE)) because
this region mediates TNF-
activation and E2 repression
(41). E2 produced a profound dose-dependent
repression of TNF-
activation of the TNF-RE upstream of a minimal tk
promoter (TNF-RE tkLuc) with either transfected ER
(Fig.
1A) or ER
(Fig.
1B) in U937 cells. Daidzein and biochanin A had no effect on
TNF-
activation of the TNF-RE with ER
, whereas genistein produced
a minor repression at 1 µM (Fig. 1A). In
contrast, all three isoflavones produced a large repression (30-60%)
of TNF-
activation of TNF-RE in the presence of ER
(Fig.
1B). Genistein is the most potent isoflavone and is about
65-fold weaker than E2 at repression (IC50 = 8.5 versus 0.13 nM). The isoflavones are more
effective also at triggering transcriptional activation of a classical
estrogen response element (ERE) in U937 cells with ER
(Fig.
2B) compared with ER
(Fig. 2A). However, isoflavones are about 10-300-fold more potent
at triggering transcriptional repression compared with transcriptional activation with ER
(genistein, IC50 = 8.5 nM, EC50 = 55 nM; daidzein, IC50 = 0.072 µM, EC50 = 1.2 µM; biochanin A, IC50 = 0.17 µM, EC50 = 50 µM).

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Fig. 1.
Isoflavones selectively repress transcription
of the TNF-RE through ER . Three copies of
the 125 to 82 region of the TNF- promoter were cloned upstream
of the minimal tk promoter (TNF-RE tkLuc). U937 cells were transfected
with 3 µg of TNF-RE tkLuc and 1 µg of expression vector for human
ER (A) or ER (B). After transfection, the
cells were treated for 24 h with TNF- (5 ng/ml) in the presence
of increasing concentrations of E2, genistein, daidzein, or
biochanin A, and luciferase activity was measured. TNF- activated
the TNF-RE tkLuc by 5-10-fold in the absence of drugs. Each data point
represents the mean of triplicate samples ± S.E. RLU,
relative luciferase units.
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Fig. 2.
Isoflavones selectively activate
transcription of an ERE through ER . A
single copy of the vitellogenin A2 ERE upstream of the minimal tk
promoter (ERE tkLuc) was transfected into U937 cells with either 1 µg
of expression vector for human ER (A) or ER
(B). After transfection, the cells were treated for 24 h with increasing concentrations of E2, genistein,
daidzein, or biochanin A, and luciferase activity was determined. Each
data point represents the mean of triplicate samples ± S.E.
RLU, relative luciferase units.
|
|
Genistein Decreases TNF-
mRNA in Bone Cells--
The effect
of genistein on endogenous TNF-
gene expression was investigated in
a human osteosarcoma cell line (U20S) because these cells express ER
and ER
, as demonstrated by RT-PCR (data not shown), and TNF-
is
involved in the pathogenesis of osteoporosis (47). U20S cells were
treated with E2 or genistein for 24 h and then exposed
to TNF-
for 1 h. TNF-
produced a profound induction of
TNF-
mRNA as determined by RT-PCR that was repressed markedly by
E2 or genistein (Fig.
3A). The observation that
genistein inhibits endogenous TNF-
mRNA in untransfected cells
demonstrates that repression of TNF-
transcription by genistein is
physiological and not caused by nonspecific squelching of
transcriptional factors by transfected ERs. To determine which ER
isoform is responsible for repressing the endogenous TNF-
gene, we
transfected U2OS cells with ER
or ER
. Although the endogenous ERs
are capable of repressing the native TNF-
gene, they are not present
in high enough levels to repress the large number of transfected
plasmids containing the TNF-RE. Fig. 3B shows that genistein
is very effective at repressing the TNF-RE in cells transfected with
ER
but not ER
. These results indicate that genistein represses
the endogenous TNF-
gene through ER
even though U2OS cells also
express ER
.

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Fig. 3.
A, genistein inhibits endogenous TNF-
gene expression in human osteosarcoma cells. U20S cells were treated
with 10 nM E2, 1 µM genistein
(Gen) or ethanol (Cont) for 24 h and then
exposed to TNF- (5 ng/ml) for 1 h. Total RNA was isolated, and
the expression of TNF- mRNA was determined by RT-PCR. Ethidium
bromide staining shows a 444-base pair PCR product of the TNF- gene
and a 217-base pair product for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), which was used as internal control
for the quality of RNA prepared. B, genistein selectively
represses transcription of the TNF-RE in U2OS cells through ER . U2OS
cells were transfected with TNF-RE tkLuc (3 µg) and 1 µg of
expression vector for either ER ( ) or ER ( ). The cells were
treated for 24 h with increasing concentrations of genistein and
then assayed for luciferase activity. Each data point represents the
mean of triplicate samples. The S.E. was < 10%.
|
|
Isoflavones Are Weak ER
Agonists in Breast Cancer
Cells--
Our results indicate that isoflavones selectively promote
ER
-mediated transcription. To explore the activity of genistein on
ER
in breast cancer cell lines, we compared the effects of E2 and genistein on ER
activation of ERE tkLuc in an
ER-negative breast cancer cell line (MDA-MB-453) stably transfected
with ER
and on the proliferation of MCF-7 cells, which express
endogenous ER
but not ER
as determined by RT-PCR (data not
shown). Similar to transiently transfected U937 and U20S cells,
genistein is much weaker than E2 at activating an ERE in
the ER
MDA-MB-453 stable cells (Fig.
4A) and stimulating the
proliferation of MCF-7 cells (Fig. 4B). Thus, genistein is a
weak ER
agonist in cells transiently (U937 and U20S) or stably
(MDA-MB-453) transfected with ER
and in cells that express
endogenous ER
(MCF-7).

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Fig. 4.
A, genistein is weak at activating an
ERE in ER -stable MDA-MB-453 cells. ERE tkLuc (3 µg) was
transfected into MDA-MB-453 stably transfected with ER . Cells were
treated for 24 h with increasing concentrations of E2
or genistein, and luciferase activity was measured. B,
genistein is weak at stimulating the growth of MCF-7 breast cancer
cells. MCF-7 cells were plated at a density of 25,000 cells/35-mm plate
in tissue culture medium containing 3% stripped fetal bovine serum.
One day after plating they were treated with increasing concentrations
of E2 or genistein. After 8 days the cells were counted
with a Coulter counter. Each data point represents the mean of
duplicate samples. The S.E. was < 10%. RLU, relative
luciferase units.
|
|
Isoflavones Selectively Recruit GRIP1 to ER
--
A potential
explanation for ER
-selective activity is that isoflavones induce a
functional AF-2 surface in ER
but not ER
because we showed
previously that the AF-2 surface is required for repression (41).
Consistent with this hypothesis is the observation that an ER
with a
mutation in helix 3 (K314A) of the AF-2 surface failed to promote
repression in response to genistein (Fig.
5). Because binding of coregulatory
proteins (48, 49) to the AF-2 surface is required for repression by
E2 (41), we compared the effects of E2 and
genistein on functional interactions that occur at the TNF-RE between
the coregulator, GRIP1 (40), and ER
or ER
. For these studies, a
Gal response element was inserted in the center of an AP-1-like site in
the TNF-RE (Gal-TNF-RE), which is essential for TNF-
activation and
E2 repression (41). Gal-GRIP1 was used for these studies
instead of Gal-ER because ERs do not bind directly to the TNF-RE (41)
but may be tethered to the TNF-RE through coregulators such as GRIP1.
Gal-GRIP1 activated Gal-TNF-RE tkLuc ~20-fold (data not shown).
E2 is extremely potent at inhibiting Gal-GRIP1 activation
of Gal-TNF-RE tkLuc in the presence of either ER
(Fig.
6A) or ER
(Fig.
6B) (IC50 = 28.5 pM for ER
, and
IC50 = 1.5 pM for ER
). In contrast,
genistein is much more potent at repressing Gal-GRIP1 activation with
ER
(IC50 = 49 pM) compared with ER
(IC50 = 1.8 µM). Furthermore, at saturating
levels (10 µM), genistein produced a 33% repression with
ER
compared with a maximal 72% repression with ER
at only 10 nM.

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Fig. 5.
Genistein requires a functional AF-2 surface
for repression by ER . U937 cells were
transfected with 3 µg of TNF-RE tkLuc and 1 µg of human wild-type
ER or a helix 3 activation function-2 mutant (K314A in human
ER 530). Cells were treated for 24 h with TNF- (5 ng/ml) in the absence or presence of 100 nM genistein, and
luciferase activity was measured. Each data point represents the mean
of triplicate samples ± S.E. RLU, relative luciferase
units.
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Fig. 6.
Genistein selectively represses Gal-GRIP1
activation of Gal-TNF-RE through ER . A
synthetic oligonucleotide that contained the 17-nucleotide
Gal-responsive element was inserted in between the G and C of the
AP-1-like site (5'-TGAGCTCA-3') at the 105 to 95 region of the
TNF-RE. Because the AP-1-like site is destroyed, this construct is
inactive in the absence of Gal-GRIP1. Cells were transfected with
Gal-GRIP1, Gal-TNF-RE tkLuc, and ER (A) or ER
(B) and then treated with increasing concentrations of
E2 or genistein for 24 h, and luciferase activity was
determined. Gal-GRIP1 activated Gal-TNF-RE tkLuc by about 20-fold in
the absence of E2 or genistein. Each data point represents
the mean of triplicate samples ± S.E. RLU, relative
luciferase units.
|
|
These results suggest that genistein creates an AF-2 surface in ER
that permits the binding of GRIP1 more efficiently compared with ER
.
To investigate this hypothesis directly, glutathione S-transferase-GRIP1 pull-down assays were performed with
either 35S-labeled ER
or ER
in the presence of
E2 or genistein. A similar dose-dependent
increase in binding of ER
or ER
to GRIP1 was observed with
E2 (Fig. 7A). In
contrast, genistein is more effective at enhancing the interaction
between GRIP1 and ER
(Fig. 7B). At 10 µM,
binding of ER
to GRIP1 is 2-fold greater than with ER
. These
findings demonstrate that genistein creates an AF-2 surface in ER
that has a higher affinity for GRIP1 than that in ER
.

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Fig. 7.
Genistein is more effective at recruiting
GRIP1 to ER .
[35S]Methionine-labeled ER or ER was synthesized in
an in vitro transcription/translation system and then
incubated with E. coli-expressed glutathione
S-transferase-GRIP1 in the presence of increasing
concentrations of E2 (A) or genistein
(B). [35S]methionine-labeled ER or ER
bound to glutathione S-transferase-GRIP1 was separated by
SDS-polyacrylamide gel electrophoresis. The dried gels were exposed to
x-ray film (top) and a phosphorscreen and then scanned with
a PhosphorImager (bottom). The first lane on the
autoradiograms represents the input of
[35S]methionine-labeled ER or ER in the binding
reaction. This is a single experiment that is representative of six
experiments that produced similar results.
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|
 |
DISCUSSION |
Estrogens in HRT improve menopausal symptoms but are associated
with an increased risk of breast (6, 7) and endometrial cancer (8). To
overcome the uterotropic effects of estrogens, women with a uterus are
treated also with progesterone in HRT regimens. Unfortunately, the
addition of progesterone may increase the risk of breast cancer further
(50, 51) and attenuate potential benefits of estrogens on the
cardiovascular system (52). The current challenge is to discover
estrogens that retain their ability to prevent menopausal symptoms
without promoting breast cancer or requiring progesterone for
endometrium protection. The development of more ideal estrogens for HRT
requires a greater understanding of how different estrogenic compounds
differentially regulate gene activation and repression by ER
and
ER
.
We have shown that isoflavones elicit distinct transcriptional actions
from estrogens. E2 effectively triggers both ER
- and ER
-mediated transcriptional activation or repression pathways. In
contrast, our results demonstrate that isoflavones are weak ER
agonists and potent ER
agonists because they are effective only at
triggering transcriptional activation or repression with ER
. The key
question is how do isoflavones elicit distinct transcriptional actions
from estrogens despite the fact they both bind to the same binding
pocket of ER
and ER
(53-55)? One possibility is that isoflavones
bind to ER
more effectively than to ER
. In fact, ER
has a
30-fold greater affinity for genistein compared with ER
(39).
However, this difference in binding affinity is unlikely to account
entirely for the distinct transcriptional actions of isoflavones
because we observed that isoflavones were over a 1,000-fold more potent
at triggering transcriptional activity with ER
compared with ER
.
Furthermore, at saturating levels (10 µM), genistein was
less effective at repressing GRIP1 activation of Gal-TNF-RE tkLuc with
ER
and recruiting GRIP1 to ER
, compared with ER
. These studies
indicate that the divergent transcriptional actions of estrogens and
isoflavones probably also result from differences in their ability to
recruit coregulators and trigger transcriptional functions of ER
or
ER
. These data are consistent with the discoveries that coregulator
proteins (48, 49) are required for both transcriptional activation and
repression by ERs (41, 56, 57).
E2 nonselectively recruits coregulators to ER
and ER
,
whereas isoflavones selectively recruit coregulators to ER
. By
recruiting coregulators such as GRIP1 to both ERs, E2
effectively triggers transcriptional activation and repression pathways
for both ER
and ER
. Undoubtedly, E2 elicits its full
spectrum of beneficial and adverse effects by triggering all
transcriptional pathways of ERs. In contrast, at physiological levels
(0.55-0.86 µM) (58) genistein is very weak at recruiting
GRIP1 to ER
, but it is potent at recruiting GRIP1 to ER
. By
selectively recruiting coregulators to ER
, isoflavones would only
trigger ER
-mediated transcriptional pathways. These results suggest
that isoflavones should be effective at eliciting the clinical effects
that are mediated by ER
but not ER
. Moreover, isoflavones are
10-300-fold more potent at triggering transcriptional repression
compared with activation. These results indicate that it may be
possible to develop transcriptional activation or repression-selective
estrogens for HRT. It is unclear why genistein recruits GRIP1 more
effectively to ER
than to ER
. However, the binding of GRIP1 may
stabilize the genistein-ER
complex more effectively than the
genistein-ER
complex because the binding of coregulators has been
shown to slow the rate of dissociation of an agonist from the
ER-coregulator complex (59).
The lack of regulation of ER
-mediated genes and the potent
repression of ER
-mediated genes by isoflavones may account for the
low incidence of menopausal symptoms, osteoporosis, cardiovascular disease, and breast and endometrial cancer in Asian countries (19,
21-24). For example, our studies suggest that ER
-mediated repression of the TNF-
gene may be an important mechanism whereby isoflavones may prevent osteoporosis because excessive production of
TNF-
after menopause is thought to lead to osteoporosis (47). Genistein also protects against vascular injury in ovariectomized female rats through ER
(60). We have shown also that E2
produces a robust stimulation of proliferation of breast cancer (MCF-7) cells. ER
undoubtedly mediates this effect because these cells only
express ER
. Furthermore, it is likely that ER
mediates the
proliferative effects on endometrial cells because these cells do not
express ER
(61). Based on these findings, we hypothesize that
ER
-selective estrogens such as isoflavones may prevent some menopausal symptoms and conditions but will be less likely to elicit
stimulatory effects on breast and endometrial cells compared with
estrogens present in current HRT regimens that also trigger ER
-transcriptional pathways. Consistent with this hypothesis are the
observations that isoflavone-rich soy protein relieves menopausal
symptoms (30, 62) but does not exert estrogenic effects on the
endometrium in postmenopausal women (62, 63). Furthermore,
isoflavone-rich soy protein does not induce proliferation in
endometrial and mammary tissue in postmenopausal female macaques (64).
Understanding how natural estrogens and synthetic SERMs elicit
selective clinical effects is a key to the development of safer estrogens for HRT. We have shown that isoflavones elicit distinct transcriptional actions from estrogens by selectively recruiting coregulators to ER
. These data are consistent with the observation that helix 12 of the AF-2 surface exists in a different position when
genistein is bound to ER
(55) compared with E2-bound
ER
(53) or ER
(55). Our results suggest that isoflavones may act
as natural SERMs, which may be safer than estrogens in current HRT
regimens because they selectively trigger the transcriptional pathways
of ER
. Estrogens in HRT also trigger ER
transcriptional pathways,
which may promote the proliferation of breast and endometrial cells.
We thank P. Chambon, J.-A. Gustafsson, and M. Stallcup for providing plasmids and Keith Yamamoto and Paul Webb for
critical review of the manuscript.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100953200
The abbreviations used are:
HRT, hormone
replacement therapy;
SERM, selective estrogen receptor modulator;
E2, 17
-estradiol;
ER, estrogen receptor;
AF-2, activation function-2;
GST, glutathione S-transferase;
TNF, tumor necrosis factor;
tk, thymidine kinase;
RT, reverse transcription;
PCR, polymerase chain reaction;
TNF-RE, TNF responsive element;
ERE, estrogen response element.
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