Distinct mechanisms of action of selective estrogen receptor
modulators in breast and osteoblastic cells
Mark E.
Nuttall,
George B.
Stroup,
Paul W.
Fisher,
Daniel P.
Nadeau,
Maxine
Gowen, and
Larry J.
Suva
Department of Bone and Cartilage Biology, SmithKline Beecham
Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
Raloxifene and idoxifene are
selective estrogen receptor modulators (SERMs) that exhibit
tissue-specific agonist or antagonist properties via interactions with
the estrogen receptor (ER). Both compounds are similarly
osteoprotective in the ovariectomized rat in vivo as assessed by
measurement of bone mineral density, urinary pyridinium cross-links,
and serum osteocalcin, suggesting a similar mechanism of action.
However, we have identified a fundamental difference in this mechanism
via the estrogen response element (ERE) in osteoblast-like cells. With
the use of ERE-luciferase reporter constructs, raloxifene, like the
complete ER-antagonist ICI-182780, acts as an antagonist via
the ERE in osteoblastic cells. In contrast, idoxifene, like
17
-estrogen itself and 4-OH-tamoxifen, acts as an agonist in
osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780
and raloxifene inhibited the ERE-dependent agonist activity of
17
-estradiol and idoxifene in osteoblastic cells. In contrast, in
breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had
no agonist activity and, indeed, raloxifene and idoxifene were potent
antagonists of ERE-mediated 17
-estradiol action, indicating an
ERE-dependent mode of action in these cells. Although these SERMs
exhibit a similar antagonist activity profile in breast cells, they can
be distinguished mechanistically in osteoblastic cells.
raloxifene; selective estrogen receptor modulator; estrogen; bone; breast
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INTRODUCTION |
THE PROTECTIVE
EFFECTS of 17
-estradiol on the skeleton, and thus its utility
in postmenopausal osteoporosis, have attracted considerable clinical
attention (13, 18). Estrogen can effectively prevent the
rapid bone loss that occurs in postmenopausal women and in clinically
relevant animal models, such as the ovariectomized (Ovx) rat (2,
26). Although clinically useful in preventing bone loss,
estrogen therapy has been reported to be linked to an increased risk of
tissue-specific side effects including endometrial hyperplasia, which
may result in uterine cancer, and proliferative effects in mammary
tissue, which may result in an increased risk of breast cancer
(1, 4, 7, 8). Because of the known and suspected risks of
estrogen therapy, it has been estimated that in the United States
<40% of women on estrogen replacement therapy will continue treatment
beyond 1 yr (18, 31).
The ideal postmenopausal "estrogen" would reproduce the beneficial
effects of estrogen on vasomotor symptoms, skeletal tissue, and the
cardiovascular system without producing the adverse effects of estrogen
on reproductive tissues (15). This concept has led to the
development of selective estrogen receptor modulators (SERMs) (16, 41). A SERM is defined as a compound that exhibits
estrogen agonism in one or more target tissues such as bone or liver
and exhibits antagonism and/or minimal agonism (i.e., clinically
insignificant) in reproductive tissue (15, 16, 31, 39,
41). Although a number of SERMs have reached later stages of
clinical development, only raloxifene is approved for the treatment and
prevention of osteoporosis. Since the failure of idoxifene and
levormeloxifene in the clinic was associated with unwanted reproductive
tissue side effects, we decided to investigate the mechanism for the tissue-selective activity of raloxifene with idoxifene and other estrogen receptor (ER) ligands.
We reported previously (26) that idoxifene, like
raloxifene, lowers cholesterol levels in Ovx rats, decreases uterine
weight in intact rats, and is osteoprotective in vivo. We have now
extended these studies to directly compare the SERMs idoxifene and
raloxifene in the Ovx rat in vivo. The effects of idoxifene
and raloxifene in vivo were examined by dual-energy X-ray
absorptiometry (DXA) measurement of bone mineral density, serum
osteocalcin, and urinary pyridinium cross-link excretion as markers of
bone turnover. In addition, we also have evaluated the molecular
mechanism of action of raloxifene and idoxifene as well as that of
17
-estradiol, 4-OH-tamoxifen, and the pure antagonist ICI-182780
(37) in osteoblast-like and breast cells in vitro.
We utilized reporter constructs containing estrogen response elements
(ERE X5, ERE X3) upstream of the luciferase reporter gene as an
indicator of the estrogenic agonist activity of the ER ligands. This
approach measures directly the effects of estrogen agonists or
antagonists on gene expression and extends classic ER binding studies,
since receptor binding does not necessarily correlate with the
modulation of gene expression in a given tissue (14). We
report that both raloxifene and idoxifene are similarly osteoprotective
in vivo. Interestingly, the results in vitro support data previously
reported for a lack of raloxifene activity via the ERE (15,
40). Our data provide important information that distinguishes
the molecular mechanism of action of idoxifene as an ERE-mediated
agonist from that of raloxifene and point to differences suggesting
that the clinically acceptable profile of raloxifene in bone may be due
to non-ERE-mediated effects.
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MATERIALS AND METHODS |
In Vivo Experiments
All procedures were reviewed and approved by the Animal Care and
Use Committee at SmithKline Beecham Pharmaceuticals. Virgin female
Sprague-Dawley rats (Charles River) were used at the age of 8-9 mo
following an acclimation period of at least 1 mo. Before either
sham operation or ovariectomy was performed, proximal tibial bone
mineral density (BMD) was determined by DXA with the use of a Hologic
QDR-1000 (Hologic, Waltham, MA) equipped with high-resolution scanning
software. Animals were maintained anesthetized with isoflurane while
placed prone on an acrylic block. The hind legs were maintained in
exterior rotation with adhesive tape, and hock joints were arranged at
a 90° angle to aid reproducibility. Surgery was done after the
collection of 24-h urine samples for the determination of baseline
pyridinium cross-link excretion by ELISA (product no. 8001; Metra
Biosystems, Palo Alto, CA).
Groups of Ovx rats (n = 8/group) were given either
idoxifene (0.5 mg/kg), raloxifene (1.0 mg/kg), or vehicle [1% aqueous
solution (wt/vol) of carboxymethyl cellulose] by oral gavage once
daily for 28 days. The doses chosen were based on the previously
reported maximum effective dose (3, 26). A group of
sham-operated rats was dosed with vehicle.
During the course of treatment the following measurements were made:
proximal tibial BMD (expressed as a percentage of the baseline BMD for
each animal), serum osteocalcin (Biomedical Technologies, Stoughton,
MA), and pyridinium cross-link excretion (measurements normalized to creatinine).
In Vitro Experiments
Cell culture and materials.
All chemicals were purchased from Sigma (St. Louis, MO) unless
otherwise stated. The cell lines used in this study were human breast
cells MCF-7 (36), human osteosarcoma cells MG-63
(6), and rat osteosarcoma cells Ros 17/2.8
(20). All cell lines were grown routinely in Dulbecco's
modified Eagle's medium (DMEM; GIBCO-BRL, Grand Island, NY)
supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL)
supplemented with penicillin (10 U/ml) and streptomycin (100 mg/ml)
(GIBCO-BRL) plus 1.1 mM calcium chloride and 25 mM HEPES. All
experiments were performed in phenol red-free Eagle's modified minimal
essential medium (MEM) containing 10% heat-inactivated,
charcoal-dextran-stripped fetal bovine serum (Hyclone, Logan, UT) and
supplemented as described above. All cell lines were cultured at
37°C in a humidified atmosphere of 95% air-5%
CO2. At confluence, cells were subcultured after
exposure to trypsin-EDTA (GIBCO-BRL). The MMTV-ERE(X5)-Luc
and C3-ERE(X3)-Luc constructs were a kind gift
from D. McDonnell (Duke University, Durham, NC). The
MMTV-ERE(X5)-Luc construct comprises the mouse mammary tumor
virus promoter in which the glucocorticoid response elements have been
replaced with five copies of a 33-base pair vitellogenin estrogen
response element [positive strand of oligonucleotide: 5'-AATTCAAAGTCAGGTCACAGTGACCTGATCAAA, upstream of the luciferase reporter gene MMTV-ERE(X5)-Luc] (38). The
C3-ERE(X3)-Luc construct is the natural complement 3 containing three nonconsensus EREs cloned upstream of the luciferase
reporter gene (25). These constructs were used for
transfection in both osteoblast-like and breast cells. The
Renilla luciferase vector was used to correct for
transfection efficiency using the dual-luciferase detection method
(Promega, Madison, WI). The osteocalcin promoter (pOCZCAT) was a kind
gift from J. Lian (University of Massachusetts, Worcester, MA).
This construct was transfected into Ros 17/2.8 cells as described in
Transient transfections, and chloramphenicol
acetyltransferase (CAT) activity was determined with the use of a CAT
ELISA (Boehringer Mannheim, Indianapolis, IN) as described in the
manufacturer's protocol. Results are expressed as absorbance units
(405 nm). ICI-182780 and raloxifene were synthesized at SmithKline Beecham.
Transient transfections.
Cells were seeded in either six-well plates at 1.5 × 105 cells/well or in 24-well plates at 1.5 × 104 cells/well in phenol red-free medium. DNA was
introduced into the cell lines by the Lipofectin method (Life
Technologies, Gaithersburg, MD). Briefly, cells were cotransfected with
1 µg of either MMTV-ERE(X5)-Luc or
C3-ERE(X3)-Luc per well in 24-well plates and 25 ng of the control Renilla luciferase vector (pRL-CMV) (Promega) to
monitor transfection efficiency and were then incubated overnight.
Transfection medium was removed, and the cells were incubated for
48 h with or without ligands. Cell lysates were prepared as
described in the manufacturer's protocol for dual-luciferase reporter
assay (Promega). Briefly, cells were washed in PBS and then lysed with 100 µl/well passive lysis buffer for 15 min on a rocking platform. Lysates were centrifuged for 30 s at 14,000 g, and the
clear lysate was transferred to a tube before reporter enzyme analysis.
Samples (20 µl) were transferred to a 96-well luminescence detection
plate and reacted with 100 µl of each assay reagent (Promega). Each assay reagent was injected by using a microlumat LB96P luminometer (Wallac, Gaithersburg, MD). Results are expressed as a percentage of
maximum agonism (100%) from triplicate wells for each ligand concentration.
Cell proliferation.
MCF-7 cells were seeded at 1.5 × 105 cells/well in
six-well plates and treated with ligands (100 nM) for 48 h. Cell
counts were performed in triplicate under light microscopy by using a hemacytometer.
Statistical Analysis
Biochemical marker data from in vivo studies as well as all in
vitro data were analyzed using one-way ANOVA followed by Dunnett's multiple comparisons test. BMD data were analyzed by repeated-measures ANOVA. All statistical analyses were carried out using Statistica for
Windows (version 5.1; Statsoft, Tulsa, OK).
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RESULTS |
In Vivo Studies With Idoxifene and Raloxifene
Four weeks after surgery, the Ovx group had ~7% lower BMD in
the proximal tibia than the sham controls. This loss was significantly prevented by treatment with either raloxifene or idoxifene (Fig. 1). The difference between the two
treated groups was not significant, and values for either group were
not significantly different from those for shams.

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Fig. 1.
Idoxifene and raloxifene prevent bone loss in the
proximal tibia of ovariectomized (Ovx) rats. Data are bone mineral
densities expressed as percent baseline values (means ± SE). Mean
baseline densities were 0.260, 0.260, 0.259, and 0.261 g/cm2 for the sham, Ovx, Ovx + idoxifene
(I), and Ovx + raloxifene (R) groups, respectively. BMD, bone
mineral density. * P < 0.01 vs. Ovx as determined
by repeated-measures ANOVA.
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Ovariectomy also resulted in an increase in biochemical markers of bone
turnover, including urinary pyridinoline (Fig.
2A) and serum osteocalcin
(Fig. 2B). Both raloxifene and idoxifene caused a decrease
in urinary pyridinoline from the Ovx level. There was no statistical
difference in excreted cross-link levels between idoxifene- and
raloxifene-treated animals. In addition, both compounds reduced
osteocalcin to the same extent relative to the Ovx group.

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Fig. 2.
Idoxifene and raloxifene decrease bone turnover in Ovx
rats. Rats were treated as described in MATERIALS AND
METHODS. Pyridinyl cross-links were measured by ELISA in a 24-h
urine sample collected on day 12 (A). Osteocalcin
was measured in serum collected on day 14 (B).
Data are presented as means ± SE. * P < 0.05 vs. Ovx as determined by Dunnett's multiple comparisons test.
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In Vitro Studies With ER Ligands
Raloxifene and the pure antagonist ICI-182780 had no demonstrable
direct agonist activity on the MMTV-ERE(X5)-Luc construct transfected into Ros 17/2.8 cells (Fig.
3). In contrast, idoxifene and
17
-estradiol were both potent agonists via the ERE in Ros 17/2.8
cells (Fig. 3). We reported previously (26) that
idoxifene, like 17
-estradiol, was an agonist through this response
element in both rat and human osteoblast-like cells (26).
4-OH-tamoxifen appeared to be a mixed agonist/antagonist, showing a
biphasic effect with a decrease in agonist activity at higher
concentrations (Fig. 3). We confirmed that these observations were not
dependent on the number of EREs in the construct by using the
endogenous complement 3 promoter that contains three nonconsensus EREs
(Fig. 4).

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Fig. 3.
Effects of 17 -estradiol, idoxifene, raloxifene,
tamoxifen, and ICI-182780 on estrogen response element (ERE)-dependent
transcriptional activity in rat osteoblast-like cells (Ros 17/2.8). Ros
17/2.8 cells were transfected with the MMTV-ERE(X5)-Luc
construct as described in MATERIALS AND METHODS. Cells were
treated with either vehicle [dimethylformamide (DMF)],
17 -estradiol (E), idoxifene (I), raloxifene (R), 4-OH-tamoxifen (T),
or ICI-182,780 (ICI) (10 nM-10 µM) for 48 h.
17 -Estradiol, idoxifene, and 4-OH-tamoxifen showed significant
increases in reporter gene activation as ERE agonists, while raloxifene
and ICI-182780 exhibited no activity. These observations are averages
of duplicate experiments, and the maximum range was ±11%. Results are
typical of at least 6 independent experiments.
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Fig. 4.
Effects of 17 -estradiol, idoxifene, raloxifene,
tamoxifen, and ICI-182780 on complement 3-ERE-dependent transcriptional
activity in Ros 17/2.8 cells. Ros 17/2.8 cells were transfected with
the C3-ERE(X3)-Luc construct as described in MATERIALS
AND METHODS. Cells were treated with either vehicle (DMF),
17 -estradiol, idoxifene, raloxifene, 4-OH-tamoxifen, or ICI-182780
(100 nM) for 48 h. 17 -Estradiol, idoxifene, and 4-OH-tamoxifen
showed significant increases in reporter gene activation as ERE
agonists, while raloxifene and ICI-182780 exhibited no activity. Data
are presented as means ± SD; n = 3. * P < 0.05 compared with vehicle alone. Results are
typical of at least 3 independent experiments.
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With the C3-ERE(X3)-Luc construct, idoxifene,
17
-estradiol, and 4-OH-tamoxifen were all agonists, whereas
raloxifene and ICI-182780 had no demonstrable agonist activity (Fig.
4). These observations are similar to reports by us and others using
the endogenous ERE from the complement 3 gene linked to luciferase (25, 26). To confirm the ERE-dependent agonist activity of idoxifene, we transfected Ros 17/2.8 cells with a control luciferase reporter and the osteocalcin promoter construct (pOCZCAT) lacking an
ERE. 1,25-Dihydroxyvitamin D3 was used as a positive control because it
has been shown to directly upregulate this promoter via the osteocalcin
vitamin D responsive element (42). No increase in reporter
gene (CAT) activity was observed in the presence of 100 nM
17
-estradiol, idoxifene, raloxifene, 4-OH-tamoxifen, or ICI-182780,
suggesting that an intact ERE is required for transcriptional activity
in these cells (Fig. 5A). This
concentration of ligand was effective at inducing ERE-dependent
transcription (Fig. 3). No transcriptional activation was observed with
idoxifene when the same reporter vector lacking the ERE was used (Fig.
5B). We next examined the effects of these ER ligands in
human osteoblast-like (MG-63) cells transfected with the
MMTV-ERE(X5)-Luc construct, where the profile was similar to
that observed in rat osteoblast-like cells, suggesting that these
effects are not species specific (Fig.
6).

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Fig. 5.
Effects of 17 -estradiol, idoxifene, raloxifene,
4-OH-tamoxifen, and ICI-182780 on transcriptional activity of
non-ERE-containing reporter/promoter constructs in Ros 17/2.8 cells.
A: Ros 17/2.8 cells were transfected with pOCZCAT as
described in MATERIALS AND METHODS. Cells were treated with
either vehicle (DMF), 17 -estradiol, idoxifene, raloxifene,
ICI-182780, or 1,25-dihydroxyvitamin D3 (vitamin D3) (100 nM) for
48 h. Only 1,25-dihydroxyvitamin D3 stimulated transcriptional
activity of the osteocalcin promoter. Data are expressed as means ± SD; n = 3. * P < 0.05 compared
with vehicle control. Results are typical of at least 4 independent
experiments. B: cells were transfected with the
ERE-containing plasmid (ERE) and the host plasmid lacking the ERE
(non-ERE) and treated with idoxifene (100 nM) as described in
A. Results are the average of duplicate experiments. The
maximum range was ±9%.
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Fig. 6.
Effects of 17 -estradiol, idoxifene, raloxifene,
4-OH-tamoxifen, and ICI-182780 on ERE-dependent transcriptional
activity in human osteoblast-like cells (MG-63). MG-63 cells were
transfected with the MMTV-ERE(X5)-Luc construct as described
in MATERIALS AND METHODS. Cells were treated with either
vehicle (DMF), 17 -estradiol, idoxifene, raloxifene, 4-OH-tamoxifen,
or ICI-182780 (100 nM) for 48 h. 17 -Estradiol, idoxifene, and
4-OH-tamoxifen showed significant increases in reporter gene activation
as ERE agonists, while raloxifene and ICI-182780 exhibited no activity.
Data are expressed as means ± SD; n = 3. * P < 0.05 compared with vehicle control. Results
are typical of at least 3 independent experiments.
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In contrast to the effects seen in osteoblast-like cells,
17
-estradiol was the only ligand exhibiting measurable agonist activity in human breast cancer cells (MCF-7) transfected with the ERE
reporter (Fig. 7). 17
-Estradiol was a
potent agonist through the ERE in human breast cells.

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Fig. 7.
Effects of 17 -estradiol, idoxifene, raloxifene,
4-OH-tamoxifen, and ICI-182780 on ERE-dependent transcriptional
activity in human breast cells (MCF-7). MCF-7 cells were transfected
with the MMTV-ERE(X5)-Luc construct as described in
MATERIALS AND METHODS. Cells were treated with either
vehicle (DMF), 17 -estradiol, idoxifene, raloxifene, or ICI-182780 (1 nM-5 µM) for 48 h. Only estrogen showed significant
increases in reporter gene activation. Data are expressed as means ± SD; n = 3. * P < 0.05 compared
with vehicle control. Results are typical of at least 3 independent
experiments.
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To confirm the estrogen selectivity in breast cancer cells, we also
examined MCF-7 proliferation. 17
-Estradiol, but not the other
ligands, stimulated breast cancer cell proliferation (Fig. 8). These data may suggest that
ERE-dependent activity is directly responsible for the increase in
MCF-7 cell proliferation (Fig. 8). Although there is a correlation
between ERE-dependent agonist activity and cell proliferation, these
effects could also be through non-ERE-containing genes. None of the
ligands, including 17
-estradiol, had any effect on the proliferation
of either rat or human osteoblast-like cells in vitro (data not shown).

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Fig. 8.
Effects of 17 -estradiol, idoxifene, raloxifene,
4-OH-tamoxifen, and ICI-182780 on proliferation of MCF-7 cells. MCF-7
cells were grown as described in MATERIALS AND METHODS and
were treated with ligands (100 nM) for 48 h. Cell counts were
measured by hemacytometer. Data are expressed as means ± SD;
n = 3. * P < 0.05 compared with
vehicle alone. Results are typical of at least 3 independent
experiments.
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Because of the lack of agonism of idoxifene and raloxifene in MCF-7
cells in vitro, we next tested the ability of idoxifene and
raloxifene to antagonize 17
-estradiol-induced ERE-dependent transcriptional activity (Fig. 9). Both
raloxifene and idoxifene were able to completely suppress 100 nM
17
-estradiol-induced transcriptional activation of the
MMTV-ERE(X5)-Luc construct in MCF-7 cells (Fig. 9). In
addition, both raloxifene and idoxifene suppressed
17
-estradiol-stimulated proliferation of MCF-7 cells (data not
shown). This is similar to the antagonism of 17
-estradiol shown
previously for both 4-OH-tamoxifen and ICI-182780 (10, 27). Our data indicate that idoxifene functions as a full
agonist via the ERE in osteoblast-like cells (Fig. 3) and is a
functional antagonist in breast (Fig. 9).

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Fig. 9.
The effect of cotreatment with either idoxifene or
raloxifene on 17 -estradiol-induced transcriptional activation
through the MMTV-ERE(X5)-Luc construct in MCF-7 cells. Cells
were transfected as described in Fig. 3 and cotreated with
17 -estradiol (100 nM) plus either idoxifene or raloxifene. The value
100% represents agonism of 17 -estradiol alone at 100 nM. Both
compounds dose-dependently inhibited the ERE-dependent agonist activity
induced by estrogen. Data are expressed as means ± SD;
n = 3. * P < 0.05 compared with
estrogen agonism alone. Results are typical of at least 3 independent
experiments.
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Given the clear cell-specific differences between raloxifene and
idoxifene, we next examined the ability of idoxifene and raloxifene to
antagonize 17
-estradiol-stimulated transcriptional activity in
osteoblast-like Ros 17/2.8 cells. Idoxifene had no effect on the
agonist activity of 17
-estradiol (100 nM), whereas raloxifene
dose-dependently inhibited the ERE-dependent transcriptional activity
of 17
-estradiol in osteoblast-like cells (Fig.
10).

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Fig. 10.
The effect of cotreatment with either idoxifene or
raloxifene on 17 -estradiol-induced transcriptional activation via
the MMTV-ERE(X5)-Luc construct in Ros 17/2.8 cells. Cells
were transfected as described in Fig. 3 and cotreated with
17 -estradiol (100 nM) plus either idoxifene or raloxifene. The value
100% represents agonism of estrogen alone at 100 nM. Raloxifene
dose-dependently inhibited the ERE-dependent agonist activity induced
by 17 -estradiol. In contrast, idoxifene had no effect. Data are
expressed as means ± SD; n = 3. * P < 0.05 compared with estrogen alone. Results
are typical of at least 3 independent experiments.
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To investigate this difference in antagonism/agonism further, we
compared the ability of raloxifene and idoxifene to antagonize 17
-estradiol-stimulated transcription compared with that of the pure
antagonist ICI-182780. Raloxifene and ICI-182780 (500 nM) both
inhibited the agonist activity of 100 nM 17
-estradiol in osteoblast-like cells (Fig. 11)
similarly to their antagonist activity in MCF-7 cells (Fig. 9). In
contrast, idoxifene (500 nM) had no effect on the agonist activity of
17
-estradiol in osteoblast-like cells (Fig. 11). With the use of
submaximal agonist concentrations of 17
-estradiol (<0.1 nM),
idoxifene was able to enhance the observed 17
-estradiol-stimulated
transcription (data not shown). It is likely that idoxifene is
stimulating target gene activation in the presence of low
concentrations of estradiol by simply increasing the amount of
available ligand of the receptor and thereby enhancing the occupancy of
the receptor. In addition, both raloxifene and ICI-182780 (500 nM)
completely inhibited 100 nM idoxifene-stimulated transcriptional
activity in osteoblast-like cells (Fig. 11), indicating that both
raloxifene and ICI-182780 are acting as antagonists of idoxifene- and
17
-estradiol-stimulated transcription. These data highlight the
mechanistic differences between raloxifene and idoxifene and suggest
that the osteoprotective effects of both may be mediated by both ERE-
and non-ERE-dependent mechanisms.

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Fig. 11.
The effect of cotreatment with selective estrogen
receptor modulators (SERMs) on transcriptional activation via the
MMTV-ERE(X5)-Luc construct in Ros 17/2.8 cells. Cells were
transfected as described in Fig. 3 and cotreated with 17 -estradiol
(100 nM) plus idoxifene, raloxifene, or ICI-182780 (500 nM). Unlike
raloxifene and ICI-182780, which inhibited agonist activity, idoxifene
cotreated with 17 -estradiol maintained ER-mediated ERE luciferase
reporter activation. The agonism observed with idoxifene (100 nM) alone
was completely inhibited in the presence of either raloxifene (500 nM)
or ICI-182780 (500 nM). Data are expressed as means ± SD;
n = 3. * P < 0.05 compared with
vehicle alone. Results are typical of at least 3 independent
experiments.
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DISCUSSION |
It was shown previously that raloxifene, idoxifene,
4-OH-tamoxifen, and ICI-182780 bind to the ER (17, 21) and
that binding of ligands to nuclear receptors produces either agonist or
antagonist effects, depending on the specific cellular context
(14, 21). The fact that raloxifene and idoxifene are able
to function as dose-dependent antagonists of 17
-estradiol-stimulated
transcription in human breast cancer cells and appear to have different
activities in osteoblast-like cells suggests that this in vitro system
is a viable model in which to investigate cell-specific differences between these ER ligands. The differences in potency of estrogen between breast and osteoblast cells suggest that there could be other
important differences between the mechanism of action of this ER ligand
in these different tissues. Together, these data support the notion
that both raloxifene and idoxifene, like 4-OH-tamoxifen and the pure
antagonist ICI-182780, may have potential utility in
estrogen-responsive breast cancer treatment (7).
Interestingly, although both raloxifene and idoxifene are
osteoprotective in vivo, their mechanism of action in bone cells appears quite distinct. The fact that both compounds are ER ligands suggests a similar mechanism of action in bone. However, raloxifene is
an antagonist and idoxifene is a potent agonist via the ERE in
osteoblast-like cells. At all doses examined here, raloxifene antagonized 17
-estradiol-stimulated transcriptional activity in
osteoblast-like cells (Figs. 9 and 10). We confirmed the data generated
through the MMTV-ERE(5)-Luc construct using the
complement 3 gene promoter, which contains three non-consensus EREs and
has been shown to be regulated by ER ligands in vivo (25).
It is therefore possible and, given the clinical data, highly likely that there are distinct mechanisms for the bone-sparing effects of both
raloxifene and idoxifene. These observations suggest that, although
raloxifene and idoxifene are osteoprotective, there may be fundamental
mechanistic differences in the reproductive tissue. The lack of agonism
by raloxifene via the ERE in Ros 17/2.8 and MG-63 cells suggests that
either raloxifene is substantially less potent than idoxifene
(concentrations of raloxifene higher than those reported here could not
be used due to inherent cell toxicity) or that the molecular mechanism
of action of raloxifene is different from that of either idoxifene or
17
-estradiol (15, 40). The data presented here provide
mechanistic support for the clinical efficacy of raloxifene. These data
indirectly support the existence of the raloxifene response element
(RRE) or related elements through which raloxifene has been shown to
exert its effects in osteoblast-like cells (40, 41).
Raloxifene has been shown to act as an agonist via a non-ERE sequence
located in the 5'-untranslated region of the human transforming growth
factor TGF
3 promoter (41) and, potentially, other
target genes. In human and rat osteoblast-like cells, raloxifene, in
contrast to idoxifene, inhibited ERE-containing promoter activity and
exhibited pure estrogen antagonism. This distinguishes the mechanism of
action of raloxifene from that of idoxifene. These data demonstrate
that idoxifene acts as an agonist in osteoblast-like cells via the ERE,
similarly to the agonist activity of 17
-estradiol in these cells
(Figs. 3 and 6).
The activity of 4-OH-tamoxifen in osteoblast-like cells is in agreement
with the clinical data of ER-mediated partial agonist activity in bone
and the cardiovascular system (19, 34). 4-OH-tamoxifen also has been shown to be both an agonist and antagonist of
17
-estradiol action in osteoblast-like cells (11, 30).
It would be interesting to examine whether bone-specific transcription
factors modulate the actions of these ER ligands in a tissue-specific
manner, as has been previously reported for the effects of Cbfa 1 on
estrogen effects in osteoblast-like and non-osteoblast-like cells
(33).
We propose a model (Fig. 12) to
illustrate the mechanistic differences between raloxifene and idoxifene
in osteoblast-like cells. Our data suggest that idoxifene acts as an
agonist via the ERE, whereas raloxifene is an antagonist via this
element (Fig. 12). The osteoprotective action of raloxifene (agonism)
is presumably mediated by the RRE or other non-ERE elements. The model
highlights the different mechanistic pathways that ultimately contribute to the osteoprotective effects of raloxifene and idoxifene and provides insight into the features that distinguish the two SERMS
clinically.

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|
Fig. 12.
A
model for the action of 17 -estradiol, idoxifene, and raloxifene via
17 -estradiol response element-dependent and -independent mechanisms
in osteoblast-like cells. This schematic illustrates the similar
mechanism of action of idoxifene and 17 -estradiol via either
ERE(X5)- or ERE(X3)-dependent mechanisms in osteoblastic cells. In
osteoblastic cells, raloxifene can act as either an agonist via the RRE
(or RRE-like sequence) or an antagonist via the ERE(X5) or ERE(X3). The
pathway for idoxifene and 17 -estradiol is the same, whereas
raloxifene has a fundamentally different mechanism of action.
|
|
This mechanistic explanation for the activity of ER ligands has been
further complicated by the discovery of another ER isoform (ER
),
which has different ligand specificities and tissue distribution (9, 23). Recent studies on conformation of the isoforms of the ER have revealed that ligand binding induces distinct
conformational changes to the receptors (14, 22, 29). The
design of ligands and the elucidation of the reasons for
tissue-specific expression of functional activity are areas of
considerable research interest (22). In addition, the
identification of nuclear hormone coactivators and corepressors greatly
impacts the tissue selectivity of the SERMs (35). There is
also increasing evidence of estrogen(s) having a non-genomic mechanism
of action (5) as well as activity through nonclassic EREs
such as AP-1 (28). Evidence is also accumulating to
support the existence of a plasma membrane ER (32) and
involvement of estrogen in proteosome-dependent degradation of the ER
(24). Added to this are data suggesting that estrogens and
the SERMs can induce osteoclast apoptosis and thereby inhibit bone
resorption (12, 26). It is possible that SERMs could exert
their effects through a variety of mechanisms and have multiple target
cells, which may contribute to tissue selectivity, and therefore the in
vivo effects on bone could be through a variety of transcriptional
and nontranscriptional mechanisms in multiple cell types that cannot be
accurately represented as in vitro models. Nonetheless, idoxifene
utilizes a classic ERE-dependent pathway in osteoblast-like cells and
breast cells, whereas raloxifene, which has a similar mechanism of
action in breast cells, has at least in part a distinct
non-ERE-dependent mechanism of action in osteoblast-like cells. There
are clearly mechanistic cell-specific differences between ER ligands
that result ultimately in the same physiological end point of bone mass preservation.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. E. Nuttall, Dept. of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, UW2109, 709 Swedeland Road, PO Box 1539, King of
Prussia, PA 19406 (E-mail: Mark_E_Nuttall{at}sbphrd.com).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 April 2000; accepted in final form 13 June 2000.
 |
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