help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nuttall, M. E.
Right arrow Articles by Gowen, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nuttall, M. E.
Right arrow Articles by Gowen, M.
Endocrinology Vol. 139, No. 12 5224-5234
Copyright © 1998 by The Endocrine Society

ARTICLES

Idoxifene: A Novel Selective Estrogen Receptor Modulator Prevents Bone Loss and Lowers Cholesterol Levels in Ovariectomized Rats and Decreases Uterine Weight in Intact Rats

Mark E. Nuttall, Jeremy N. Bradbeer, George B. Stroup, Daniel P. Nadeau, Sandra J. Hoffman, Hugh Zhao, Sabine Rehm and Maxine Gowen

Departments of Bone and Cartilage Biology, and Safety Assessment (SR), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Address all correspondence and requests for reprints to: Mark E. Nuttall, Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, Pennsylvania 19406. E-mail: Mark-E-Nuttall{at}sbphrd.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg·day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg·day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg·day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats.

In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACCELERATED bone loss secondary to loss of ovarian function at the menopause is well recognized as a major risk factor for osteoporotic fractures in postmenopausal women (1). Postmenopausal bone loss can be prevented or arrested by estrogen replacement therapy (ERT) (1, 2) which also protects against cardiovascular disease (3) in part by improving the serum lipid profile. However, unopposed ERT causes an unacceptable increase in the risk of endometrial cancer and proliferative effects in mammary tissue resulting in an increased risk of breast cancer (4). While this can be counteracted by combining ERT with a low dose of a progestin, withdrawal bleeding and the continuing uncertainty about the effect of estrogen on the risk of breast cancer contribute to poor compliance for long-term use (5). Because of the known and suspected risks of estrogen therapy, it has been estimated that in the United States less than 40% of women on estrogen replacement therapy will continue treatment beyond 1 yr (6). An ideal therapy would retain the desirable skeletal and cardiovascular effects of estrogen, would lack estrogenic activity on the endometrium, and would reduce the incidence of breast cancer.

The concept of selective estrogen receptor modulation has been demonstrated for a number of compounds including tamoxifen (7), raloxifene (8), droloxifene (9), GW 5638 (10), and levormeloxifene (11). However, the clinical utility of these agents will depend on the profile of tissue-specific effects, and the extent to which they are translated into in vivo efficacy. A SERM is defined as a compound that has estrogen agonism on one or more of the desired target tissues such as bone or liver and has antagonism and/or minimal agonism (i.e. clinically insignificant) in reproductive tissue such as the breast or uterus (12, 13, 14). Although tamoxifen does act as a SERM, it is also associated with an increased incidence of endometrial cancer (14). Attempts to improve on the pharmacological profile of tamoxifen have resulted in compounds that differ in their estrogen agonist/antagonist characteristics, including the pure estrogen antagonists (12). This suggests that it may be possible to develop a molecule with a desired profile of tissue-specific agonist/antagonist activities.

Idoxifene (pyrrolidino-4-iodotamoxifen, Fig. 1Go) is a novel SERM that has a 2.5- to 5-fold greater affinity for the estrogen receptor {alpha} than tamoxifen, while being significantly less uterotrophic (15, 16). To determine whether or not idoxifene acts as an estrogen agonist on the skeleton following estrogen withdrawal, we have investigated its effect in the Ovx rat model. Here, we report on the effect of idoxifene on bone loss, uterine weight, and plasma cholesterol using experimental designs for both prevention of bone loss and intervention in progressive bone loss.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 1. The chemical structure of idoxifene (pyrrolidino-4-iodotamoxifen).

 
In addition, we have profiled the action of idoxifene on specific cell-types in culture, in direct comparison to the natural hormone estrogen. We have used the classical ERE to measure directly the effects of estrogen agonists/antagonists to explain the effects in vivo with mechanistic based studies on gene expression in a cell-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vivo experiments
Animals and measurements. All procedures were reviewed and approved by the Animal Care and Use Committee at SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Virgin female Sprague-Dawley rats (Charles River) were used at the age of 7–9 months following an acclimation period of at least 1 month. Immediately before either sham operation or ovariectomy, lumbar spine (L3–L6) and proximal tibial bone mineral densities (lsBMD and ptBMD) were determined. BMD was determined by dual energy x-ray absorptiometry (DXA) using a Hologic QDR-1000 (Hologic, Inc., Waltham, MA) equipped with high resolution scanning software. Quality control of the instrument was carried out each day before sample analysis by scanning both a human anthropomorphic spine phantom (low resolution) and the lumbar portion of a rat spine (high resolution), both of which were embedded in methacrylate polymer. All high resolution scans were done with the sample placed on top of an acrylic block 1.5 inches deep. The x-ray beam was collimated to a diameter of 0.9 mm and line spacing and point resolution were 0.25 mm and 0.127 mm, respectively. Animals were maintained anesthetized with isoflurane while placed prone on the acrylic block. The posterior legs were maintained in exterior rotation with adhesive tape. The hip, knee, and ankle joints were each arranged at a 90° angle to aid reproducibility. The lumbar spine was scanned to encompass vertebrae L3-L6. When performing sequential scans, the current scan was compared with the baseline scan using the software’s "compare" feature. The width of the region of interest was kept constant between individual rats. BMD was calculated by dividing the bone mineral content (BMC) by the projected bone area.

The rats were then segregated into groups (n = 10) that did not differ in their mean values for lsBMD and ptBMD. Surgery was done following the collection of 24 h urine samples for the determination of baseline pyridinium cross-link excretion by ELISA (Metra Biosystems, Palo Alto, CA, product no. 8001).

The following measurements were made at one or more timepoints during each experiment: lumbar spine and proximal tibial BMD (expressed as a percentage of the baseline BMD for each animal); serum total cholesterol; plasma osteocalcin (Biomedical Technologies Inc., Stoughton, MA); pyridinium cross-link excretion (measurements normalized to creatinine and expressed as a percentage of the baseline measurement, having established no significant differences between groups at baseline by unpaired t test).

Groups of ovariectomized rats received, by oral gavage, a single daily dose of either dosing vehicle (1% aqueous solution (wt/vol) of carboxymethyl cellulose), or idoxifene suspended in vehicle. A group of sham-operated rats was dosed with vehicle.

For each parameter, differences between groups were assessed by unpaired Student’s t test using Microsoft Corp. Excel.

Dose response study with idoxifene
Before surgery, baseline measurements were made as described above. Ovariectomized rats received idoxifene at 0, 0.1, 0.2, 0.5, and 1.0 mg/kg·day. A sham-operated group received dosing vehicle. The doses of idoxifene were based on information obtained from preliminary dose-ranging studies. Dosing commenced immediately following surgery. Bone mineral density was measured at weeks 8 and 17. After 2 weeks of treatment, blood samples were collected for the determination of serum cholesterol and plasma osteocalcin and 24 h urine samples were collected for the measurement of pyridinium cross-link excretion. Uterine wet weight was determined at necropsy.

Effect of intervention with idoxifene on progressive loss of BMD
In a separate experiment, rats underwent measurement of baseline BMD and were grouped as described above. Following Ovx or sham operation, the rats were maintained untreated for 4 weeks, after which a significant decrease in lumbar spine and proximal tibial BMD was measured in the Ovx rats. Groups of Ovx rats were then dosed with idoxifene at 0.5 and 2.5 mg/kg·day or dosing vehicle. The sham-operated rats were dosed with vehicle alone. Further measurements of lumbar spine and proximal tibial BMD were made at week 8 (week 4 of dosing) and week 12 (week 8 of dosing) post surgery.

Effects of idoxifene and estrogen on uterine histology
A separate study was done in which groups of 9–10 rats (female Sprague-Dawley, 8–9 months of age) were ovariectomized and treated sc with idoxifene (0.2, 1.0, or 10.0 mg/kg·day), 17ß-estradiol (0.1 mg/kg·day) or vehicle (olive oil) for three months. A sham-operated group received injections of vehicle. Uteri were collected at necropsy and fixed in formalin. A cross-section of each uterine horn was processed, embedded in paraffin, and sectioned at 5 µm and stained with hematoxylin and eosin for light microscopic evaluation.

Effect of idoxifene on uterine wet weight in intact animals
Three groups of 10 rats each (female Sprague-Dawley, 9–10 months of age) were dosed with either vehicle or idoxifene (0.5, 1.5 mg/kg·day) for 8 weeks. Uterine wet weight was determined at necropsy.

In vitro experiments
Cell culture and materials. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Cell lines used in this study were rat osteosarcoma cells: Ros 17/2.8 (17) and the same cell-type stably transfected with the estrogen receptor: Ros.smer (18), human osteosarcoma (MG-63) and Hos TE-85 (Hos) cells (19), endometrial Ishikawa cells (kindly given by Dr. V. C. Jordan, Northwestern University, Chicago, IL) (20). Human osteoclastoma derived osteoclast-enriched populations of cells were used for the apoptosis studies as described previously (21). Ros 17/2.8 cells were grown routinely in Ham’s F-12 medium (Gibco BRL Technologies, Grand Island, NY) supplemented with 5% heat inactivated FBS (Gibco BRL Technologies, Grand Island, NY) supplemented with penicillin (10 U/ml), streptomycin (100 mg/ml) (Gibco BRL Technologies) plus 1.1 mM calcium chloride and 25 mM HEPES. All other cell lines were routinely cultured in DMEM supplemented with 10% heat inactivated FBS (Hyclone Laboratories, Inc., Logan, UT), penicillin (10 U/ml) and streptomycin (100 mg/ml) (Gibco BRL Technologies). All experiments were performed in phenol red free Eagle’s MEM (EMEM) containing 10% heat-inactivated charcoal-dextran stripped FBS (Hyclone Laboratories, Inc., Logan, UT) and supplemented as above. All cell lines were cultured at 37 C in a humidified atmosphere 95% air-5% CO2. At confluence cells were subcultured after exposure to trypsin-EDTA (Gibco BRL Technologies).

A DNA construct that comprised of a mouse mammary tumor virus promoter in which the glucocorticoid response elements have been replaced with five copies of a 33-bp vitellogenin estrogen response element was a kind gift from D. McDonnell, (Duke University). This is upstream of the Luciferase reporter gene (MMTV-ERE-Luc) (22). The renilla-Luciferase vector was used to correct for transfection efficiency using the dual-luciferase detection method (Promega Corp., Madison, WI).

Transient transfections
Cells were seeded in either 6-well plates at 1.5 x 105cells/well or in 24-well plates at 1.5 x 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 2 µg per well in 6-well plates and 140 ng per well in 24-well plates of MMTV ERE-Luc and 25 ng of the control renilla-Luciferase vector (pRL-CMV). Transfection efficiency was corrected for by co-transfection with a renilla-Luciferase vector, which uses a different substrate, coelenterazine, for its bioluminescent readout (Promega Corp.). Cells were incubated overnight. Transfection medium was then removed and cells were incubated for 48 or 72 h with or without hormones as indicated by the figure legends. Cell lysates were prepared as described in the manufacturer’s protocol for dual-luciferase reporter assay to assess transfection efficiency (Promega Corp.). Briefly, cells were washed in PBS and then lysed with 500 µl/well 1 x passive lysis buffer (PLB) for 15 min while rocking sample on a rocking platform. Lysates were centrifuged for 30 sec at 14,000 x 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 Corp.). Each assay reagent was injected by a microlumat LB96P luminometer (Wallac, Gaithersburg, MD), which measured luciferase activity. Results are expressed as relative light units (RLU).

Apoptosis
To assess apoptosis, osteoclast-enriched cell populations were prepared as described previously (21) and grown for 72 h in the presence of either estrogen or idoxifene. Camptothecin was used as a known inducer of apoptosis. Cell-lysates were prepared and a cell-death ELISA was performed (Boehringer Mannheim, GmbH, Germany). We have shown that the results from this ELISA correlate with other markers of apoptosis, such as cellular morphological changes and DNA laddering (not shown).

IL-6 production
Human osteosarcoma (MG-63) cells (19) were grown overnight in phenol redfree DMEM supplemented with 10% charcoal/dextran stripped FBS. Before treating the cells, the medium was removed, cells were washed with PBS and medium supplemented with 2% FBS was added. Cells were then treated as follows: vehicle (dimethylformamide), estrogen (100 nM), idoxifene (100 nM) for 20 h. After 20 h, cells were treated with tumor necrosis factor (TNF{alpha}) (500 U/ml) for another 24 h. IL-6 production was determined by BIOTRAK ELISA (Amersham Life Science, Little Chalfont, UK).

Alkaline phosphatase activity
Cell lysates were obtained by solubilization of the monolayer cells with 0.1% (vol/vol) Triton X-100. Alkaline phosphatase activity was determined by colorimetry using p-nitrophenylphosphate as the substrate (22A ). Results are expressed as optical density absorption at 450 nm for 10 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo studies with idoxifene
Bone mineral density at week 8. At doses greater than 0.1 mg/kg·day, 8 weeks of treatment with idoxifene (Fig. 2AGo) significantly inhibited the Ovx-induced loss of lumbar spine BMD (lsBMD). The 0.5 mg/kg·day dose of idoxifene maintained lsBMD at the sham control value. There were no significant differences among the 0.2, 0.5 and 1.0 mg/kg·day doses. Loss of lsBMD following Ovx was dependent on a decline in lsBMC, which was prevented by treatment with idoxifene; there were no differences among these groups with respect to bone area (data not shown). In the proximal tibia, all doses of idoxifene (Fig. 2BGo) significantly inhibited Ovx-induced loss of BMD. The 0.5 mg/kg·day dose of idoxifene maintained ptBMD at sham control levels.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. The effect of 8 weeks of treatment with idoxifene (A and B) on change in BMD in the lumbar spine (A) and the proximal tibia (B) in ovariectomized rats. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. Ovx control.

 
Bone mineral density at week 17
At week 17 the mean bone area value was lower at both skeletal sites in the Ovx control group compared with the sham control group (data not shown). Consequently, the Ovx-induced reductions in lsBMD and proximal tibial BMD (ptBMD) were due to disproportionately greater decreases in BMC than the decreases in bone area. All doses of idoxifene significantly prevented loss of lsBMD compared with Ovx controls (Fig. 3AGo). A dose of 0.5 mg/kg·day was maximally effective, maintaining lsBMD at sham control levels. Normalization of lsBMD by treatment tended to be due more to inhibition of the effect of Ovx on BMC than on bone area. Idoxifene caused a dose-dependent inhibition of the loss of ptBMD (Fig. 3BGo), the values for doses of >= 0.2 mg/kg·day not differing from sham control. The effect on ptBMD was due to an effect on ptBMC, not bone area.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. The effect of 17 weeks of treatment with idoxifene (A and B) on change in BMD in the lumbar spine (A) and the proximal tibia (B) in ovariectomized rats. **, P < 0.01; *, P < 0.05 vs. Ovx control.

 
Effect of treatment on biochemical parameters of bone turnover
Idoxifene caused a dose-dependent suppression of the increases in both pyridinium cross-link excretion (Fig. 4AGo) and plasma osteocalcin levels (Fig. 4BGo) measured 2 weeks post ovariectomy. The maximal effect was at 0.5 mg/kg·day, which appeared to maintain bone turnover at sham control levels. This was maintained at weeks 8 and 17 (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. The effect of 2 weeks of treatment with idoxifene on urinary excretion of pyridinium cross-links (A) and serum osteocalcin (B) in ovariectomized rats. **, P < 0.01; *, P < 0.05 vs. Ovx control.

 
Effect of intervention with idoxifene on progressive loss of BMD
Figure 5Go shows the effect of Ovx on lumbar spine (A) and proximal tibial (B) BMD, and the effect of intervention with idoxifene (0.5 and 2.5 mg/kg·day) on further changes in BMD. One month after Ovx, BMD at both sites was significantly reduced relative to the sham control group. Commencing treatment with idoxifene at this time completely prevented any further loss of BMD. Both doses of idoxifene were equally effective.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. The effect of intervention with idoxifene (0.5 and 2.5 mg/kg·day p.o.) on progressive bone loss in the lumbar spine (A) and proximal tibia (B) in ovariectomized rats. **, P < 0.01; *, P < 0.05 vs. Ovx control.

 
Effect of idoxifene on cholesterol levels
Idoxifene reduced serum cholesterol levels relative to Ovx controls (Fig. 6Go). Idoxifene was maximally effective at 0.5 and 1.0 mg/kg·day, reducing cholesterol levels below sham control values. This was maintained at weeks 8 and 17 (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. In ovariectomized rats, the effect of idoxifene on serum total cholesterol (measured after 2 weeks of treatment: *, P < 0.05 vs. Ovx control).

 
Effect of idoxifene on the uterus
The uterine wet weight of all Ovx rats was greatly reduced relative to sham control (Fig. 7AGo). The uterine wet weight of Ovx rats treated with idoxifene was slightly greater (P < 0.01) than in vehicle-treated Ovx controls, although importantly, this effect was not dose dependent. Significantly, idoxifene inhibited the estrogenic effect seen in the rat uterus in intact animals at a dose of 1.5 mg/kg·day (Fig. 7BGo).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. Uterine wet weight (measured after 17 weeks of treatment: *, P < 0.001, all treatment groups vs. Ovx control) (A). The effect of 8 weeks of treatment with idoxifene on uterine weight in intact rats. *, P < 0.001 vs. normal (untreated) control (B).

 
Uteri taken from Ovx rats treated for 3 months with either idoxifene (0.2, 1.0, 10.0 mg/kg), 17ß-estradiol (0.1 mg/kg) or vehicle were evaluated histologically (Tables 1Go and 2Go; Fig. 8Go). All idoxifene-treated and vehicle-treated Ovx rats showed myometrial atrophy whereas the majority (8/10) of estrogen-treated Ovx rats showed myometrial hypertrophy. Mild stromal fibrosis was seen in 4/9 idoxifene-rats at 10.0 mg/kg and in 3/8 rats at 1.0 mg/kg compared with the moderate to severe fibrosis seen in all estradiol-treated rats. All sham operated rats (9/9) exhibited moderate stromal fibrosis. Idoxifene caused a dose-dependent reduction in the number of uterine glands and caused atrophy of the lining epithelium of the uterus (endometrial atrophy). The lining epithelium was completely absent in some rats treated with idoxifene at 10.0 mg/kg. In contrast, estradiol treatment caused an increase in the number of endometrial glands, endometrial hyperplasia and dilation of the uterine lumina. Extensive squamous metaplasia was also present in 2/10 estradiol-treated rats. Endometrial hyperplasia and focal squamous metaplasia were present in 2/9 sham control rats.


View this table:
[in this window]
[in a new window]
  		Table 1. Myometrial/stromal findings in uterus of sham or ovariectomized (Ovx) control rats, and ovariectomized idoxifene or estradiol treated rats

 

View this table:
[in this window]
[in a new window]
  		Table 2. Endometrial findings in uterus of sham or ovariectomized (Ovx) control rats, and ovariectomized idoxifene or estradiol-treated rats

 


View larger version (132K):
[in this window]
[in a new window]
  		Figure 8. Variable uterine diameter (A, C, E, G) and endometrial morphology (B, D, F, H) depending on treatment, each shown at the same magnifications. Sham control (A, B) with wide lumen (L) and glands (g) located in a collagenous stroma (s). Myometrium not present at these magnifications. Ovx control (C, D) with atrophy of stroma and myometrium (m); note reduced epithelial height and stromal collagen. Idoxifene-treated rat (E, F; 3 months at 1 mg/kg·day) with myometrial and stromal atrophy, narrow lumen, lacking endometrial glands and reduced epithelial height. Estradiol-treated rat (G, H) with thickened stroma and tall columnar, hyperplastic epithelium lining the glands and endometrial lumen (myometrium not present at these magnifications). Hematoxylin and eosin; A, C, E, G at x10 objective magnification, and B, D, F, H at x40 objective magnification.

 
In vitro studies with idoxifene
Effect of idoxifene on osteoblasts and osteoclasts in vitro. Both estrogen and idoxifene induced ERE-dependent gene expression at all concentrations tested in human and rat osteoblasts (Fig. 9AGo). This suggests that idoxifene is interacting with the osteoblast estrogen receptor to activate the estrogen response element and therefore behaving as an estrogen agonist in osteoblasts. Idoxifene at all concentrations tested was an agonist through this response element in osteoblasts (Fig. 9BGo) These observations were confirmed using the natural ERE-linked to luciferase (23), which gave similar responses to idoxifene and estrogen (not shown). Also, a viral vector not containing EREs was not induced above control values in the presence of idoxifene, indicating that the ERE is required for transcriptional activation upstream of this viral promoter (not shown). To further confirm that the ERE was absolutely required for transcriptional activation by idoxifene we transfected the cells with the osteocalcin promoter (pOCZCAT: -1097 to +34) (24), which does not contain an ERE and idoxifene was unable to transcriptionally activate through any of these promoter/reporter constructs that lacked the ERE (not shown). This extends the classical estrogen receptor binding studies because receptor binding does not necessarily correlate with modulation of gene expression in a given tissue (25).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 9. The effects of idoxifene and estrogen on transcriptional activation through the estrogen response element in human (Hos TE85) and rat osteoblasts (Ros 17/2.8) (Fig. 9AGo). Cells were cultured and transfected with the MMTV ERE Luc reporter plasmid as described in Materials and Methods. ERE-dependent luciferase reporter activation was measured after treatment either with estrogen (100 nM) or idoxifene (100 nM). Values are expressed as mean ± SE (n = 4) (*, P < 0.0002). A dose-response curve in Ros 17/2.8 cells is shown in Fig. 9BGo. Results are expressed in RLU and as fold induction as described in Materials and Methods. The observations are averages of duplicates and the maximum range was ± 9%

 
The estrogen response element-containing construct was transfected into rat osteoblasts (Ros 17/2,8) and into the sister cell-line: Ros.smer, that has the estrogen receptor {alpha} stably overexpressed (18). Transcriptional activation was greater in the Ros.smer cells than the wild-type cells in the presence of either idoxifene or estrogen indicating that estrogen receptor-{alpha} levels determine the magnitude of transcriptional activation and therefore support the effects of this compound are through this receptor (Table 3Go). The number of estrogen receptors in the stably transfected cells is approximately 2000 sites/cell compared with the parent cell line that has approximately 200–500 sites/cell (18). The enhanced transcriptional activity correlates (4- to 6-fold) with the increased number of receptors. This is in agreement with our previously reported gel-shift experiments where we showed that idoxifene will bind to the estrogen receptor-{alpha} in rat osteoblasts (26).


View this table:
[in this window]
[in a new window]
  		Table 3. The MMTV-ERE-Luc construct was transfected into rat osteoblasts (ROS 17/2.8) and into the sister cell line: Ros.smer, which has the estrogen receptor stably overexpressed

 
It has been shown previously that treatment with TNF{alpha} stimulated IL-6 production up to 10,000 fold in osteoblast cell lines (27, 28). Addition of 17ß-estradiol to the cultures resulted in an inhibition of TNF{alpha}-induced production of IL-6 (Fig. 10Go). We tested the ability of idoxifene to inhibit TNF{alpha}-induced IL-6 production in human osteosarcoma MG-63 cells in culture. IL-6 was undetectable without TNF{alpha} stimulation in human osteosarcoma cells. Both idoxifene and estrogen inhibited the TNF{alpha}-induced IL-6 production (Fig. 10Go).



View larger version (10K):
[in this window]
[in a new window]
  		Figure 10. The effects of estrogen and idoxifene on TNF-{alpha} induced IL-6 production in human osteoblasts (MG-63). Human osteosarcoma (MG-63) cells were cultured and treated as described in Materials and Methods. Briefly, cells were pretreated with vehicle (dimethylformamide), idoxifene (100 nM), or estrogen (100 nM) for 20 h and then all samples were treated with human recombinant TNF{alpha} (500 U/ml) for an additional 24 h. TNF{alpha} induced IL-6 activity was measured as optical density values (OD 405 nm) by performing the BIOTRAK IL-6 human ELISA (Amersham Life Science). Idoxifene and estrogen both decreased TNF{alpha} induced IL-6 activity. Results are expressed as mean ± SD, n = 3 (*, P < 0.05).

 
We further evaluated the ability of idoxifene to affect an endogenous estrogen-response in osteoblasts by measuring the activity of the enzyme alkaline phosphatase, an activity that is associated with osteoblast activation and bone formation in vivo (29). Enzyme activity was measured in both wild-type rat osteosarcoma cells (Ros 17/2.8) and in the same cells stably transfected with the estrogen receptor {alpha} (Ros.smer). Estrogen has previously been shown to up-regulate alkaline phosphatase activity to a greater extent in the stably transfected cells (18). Idoxifene, like estrogen, stimulated alkaline phosphatase activity in both rat osteoblast cell lines (Fig. 11Go). Significantly, idoxifene and estrogen both stimulated alkaline phosphatase activity to a greater extent in the cells stably transfected with the estrogen receptor (Fig. 11Go).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 11. The effects of estrogen and idoxifene on alkaline phosphatase activity in rat osteoblasts. Rat osteosarcoma (Ros17/2.8) and rat osteosarcoma cells stably transfected with the estrogen receptor (Ros.smer) were cultured and treated with idoxifene (100 nM) and estrogen (100 nM) for 48 h as described in Materials and Methods. After treatment, lysed samples were examined for alkaline phosphatase (AP) activity by photometric enzyme analysis. AP activity is expressed as optical density values (OD 450 nm). Untreated levels of AP activity were higher in the Ros.smer than the Ros 17/2.8 cells. Both estrogen and idoxifene increased AP activity to a greater extent in Ros.smer cells compared with wild-type cells. This is a representative assay in which duplicate measurements were performed with errors below 20%.

 
To investigate further the estrogenic effects of idoxifene at a biological level we evaluated its effects on the apoptosis of osteoclasts. Recently, there have been reports of the presence of estrogen receptors in osteoclasts and studies that estrogen causes direct effects on osteoclast function (30, 31, 32). There is also evidence that estrogen, either directly or indirectly (i.e. stromal cell mediated) induces osteoclast apoptosis (33). It has been proposed that in the estrogen depleted state, the osteoclast life-span and thus function would increase, leading to increased bone resorption. Similar effects of idoxifene would provide evidence of an estrogenic mechanism in the bone resorbing osteoclast, and thus indicate that idoxifene is acting as an estrogen agonist during both the formation and resorption stages of bone remodeling. Idoxifene was shown to stimulate apoptosis in osteoclast-enriched osteoclastoma derived cells in a dose-dependent manner (Fig. 12Go). The observed results with idoxifene were similar to those with estrogen. Both estrogen and idoxifene at the highest concentrations tested induced apoptosis to the same extent as the topoisomerase inhibitor camptothecin, a known inducer of this process. Morphological changes were seen in the osteoclasts consistent with apoptosis in idoxifene, estrogen and camptothecin treated cells (not shown). This indicates an estrogenic-like induction of apoptosis in human osteoclasts by idoxifene which may play a significant role in the bone sparing effects of this compound in vivo.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 12. The effects of idoxifene, estrogen, and camptothecin on apoptosis in human osteoclastoma cells. Human osteoclastoma derived osteoclast-enriched cell populations were treated with estrogen and idoxifene at 0.1,1, 10, and 20 µM idoxifene or estrogen. Camptothecin (0.1, 1, and 4 µg/ml) was used as a positive control for induction of cell death. Cells were treated for 72 h. Lysed cell samples were examined by the Cell Death Detection ELISA (Boehringer Mannheim), and results were expressed as optical density (OD 405 nm). Idoxifene, estrogen, and the positive control camptothecin all stimulated cell death in the osteoclastoma cells. Results are expressed as mean ± SD, n = 3 (*, P < 0.05).

 
Effect of idoxifene on endometrial cells in vitro
In support of the effects observed in vivo, we investigated the actions of idoxifene in Ishikawa endometrial cells. Estrogen was a potent agonist through the ERE (Fig. 13AGo) which may, at least in part, be due to the high levels of estrogen receptors reported in endometrial cells (34). This agonist activity is in agreement with the clinical observations that estrogen treatment promotes endometrial hyperplasia (35). Idoxifene had no agonist activity through the ERE, and indeed prevented the agonist effects of estrogen through the ERE (Fig. 13Go, A and B). This correlates with the in vivo studies in the intact rats (Fig. 7BGo), where idoxifene blocked estrogenic effects on the uterus. We have also observed similar effects of idoxifene in breast cancer MCF-7 cells (26), suggesting that it is acting as an antagonist in breast tissue, an effect which may provide important therapeutic benefits for this compound.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 13. The effects of idoxifene and estrogen on transcription through the estrogen response element in human endometrial cells (Ishikawa) (Fig. 13AGo). Human endometrial adenocarcinoma (Ishikawa) cells were transfected with the MMTV ERE-Luc reporter plasmid as described in Materials and Methods. Cells were transfected and treated with idoxifene or estrogen for 48 h. ERE-dependent luciferase reporter activation was measured in RLU as described in Materials and Methods. Estrogen showed a significant increase in luciferase reporter activation while idoxifene had negligible agonist activity. Figure 13BGo shows the antagonism by idoxifene of estrogen-induced gene transcription through the estrogen response element. Results are expressed as mean ±SD, n = 3 (*, P < 0.005). Significance in Fig. 13BGo is compared with estrogen alone. Veh, Vehicle; I, idoxifene; E, estrogen.

 
This suggests that 1) the effects of idoxifene are mediated by the estrogen receptor and 2) the reporter gene system used in these studies is capable of utilizing tissue specific factors to modulate the effect of the estrogen receptor on the ERE. This indicates that the model system is a valid surrogate for the likely effect of idoxifene on endogenous estrogen responsive genes and indicates that in the context of endometrial cells idoxifene is a complete antagonist.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using both prevention and intervention experimental designs, we have demonstrated that idoxifene can both prevent and arrest the loss of BMD that occurs in the axial and appendicular skeleton in the ovariectomized rat. Idoxifene protected against Ovx-induced loss of BMD in a dose-responsive manner in both the lumbar spine and the proximal tibia. At the optimal dose of 0.5 mg/kg·day, neither lsBMD nor ptBMD differed from sham control values throughout the 4-month treatment period. Ovx-induced increases in serum osteocalcin and urinary pyridinium cross-link levels were completely inhibited by idoxifene at 0.5 mg/kg·day. This suggests that idoxifene prevented bone loss by suppressing the increase in bone turnover that occurs upon estrogen withdrawal. Intervention studies with idoxifene in osteopenic Ovx rats abruptly arrested bone loss and maintained lsBMD and ptBMD at pretreatment levels during a 2-month dosing period. This result is significant in that it demonstrates the ability of idoxifene to halt progressive bone loss in a situation in which measurable loss has already occurred, a common clinical presentation in postmenopausal women. In this experiment 0.5 and 2.5 mg/kg·day were equally effective confirming that 0.5 mg/kg·day is a sufficient and maximally active dose of idoxifene in this model.

We provide direct evidence that idoxifene is working as an estrogen agonist in osteoblasts in culture, and that the profile of induction of transcription through the ERE in osteoblasts by idoxifene is similar to that of estrogen. To our knowledge these are the first reports of this type of transient reporter experiments without cotransfecting an expression plasmid for the estrogen receptor and therefore may represent a more physiologically relevant cell system. It is not yet known whether idoxifene binds to the recently discovered estrogen receptor; ERß (36), the implications of which are discussed below. In contrast to idoxifene, it has been reported previously that raloxifene is an antagonist of the ERE in osteoblasts (8, 37), and it has a distinct mechanism of action through a raloxifene response element (RRE) (8). It has been proposed that raloxifene is able to exert its biological effect through this non-ERE containing sequence present on the 5'-untranslated region of the human TGFß3 promoter (8). We have confirmed these data indicating no agonist activity of raloxifene through the ERE (not shown). This distinguishes the mechanism of action of idoxifene from that of raloxifene, and aligns idoxifene to the more classical estrogenic mechanism, exerting its biological agonist effect in osteoblasts through the ERE. This is supported by the observation of the direct effects of idoxifene, on osteoblasts, in stimulating alkaline phosphatase activity, a marker associated with bone formation and responsiveness to estrogen.

In addition to the similarities between estrogen and idoxifene in osteoblasts on a molecular level, we have shown also that they have similar biological effects in human osteoclasts. The ability to stimulate osteoclast apoptosis may be related directly to the antiresorptive effects of both estrogen and idoxifene, and it may be the combination of effects on both osteoblasts and osteoclasts that ultimately leads to the bone-sparing ability of these compounds. 17ß-estradiol promotes apoptosis of murine osteoclasts in vitro and in vivo by 2- to 3-fold (33). It has been reported that anti-TGF-ß antibody inhibited TGF-ß-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-ß might mediate this effect. These studies were done on mixed populations of stromal cells and osteoclasts and therefore the effects may be direct or indirect. Stromal cells are required for osteoclast formation, but they could also mediate osteoclast death if estrogen induced them to produce an apoptosis promoting agent. Estrogen or idoxifene may also act directly to promote osteoclast apoptosis. Functional estrogen receptor expression has been demonstrated in highly enriched populations of osteoclasts and a number of effects such as inhibition of resorption and induction of the apoptosis associated c-fos gene have been observed at estrogen concentrations that stimulate apoptosis (33). The authors proposed that a major role of estrogen before menopause and of estrogen replacement after the menopause is to limit osteoclast numbers and activity through promotion of apoptosis of mature osteoclasts and their precursors. This may well be one of the pleiotropic estrogenic effects in vivo that allows idoxifene to prevent bone loss after ovariectomy.

17ß-estradiol inhibited both TNF{alpha}-induced IL-6 production and osteoclast development in primary bone cell cultures derived from neonatal murine calvariae (27). In the same system the TNF{alpha}-stimulated osteoclast development was also suppressed by a neutralizing monoclonal anti-IL-6 antibody and the authors postulated that this provided a mechanistic paradigm by which estrogens exert, at least, part of their antiresorptive influence on the skeleton. Bone and bone marrow cells produce cytokines, such as IL-6, which act by a paracrine mechanism in the bone microenvironment to regulate osteoclast formation. Therefore, the bone loss associated with the removal of estrogen might be due, at least in part, to the removal of these inhibitory effects.

Based on these studies we suggest that idoxifene has estrogen agonist effects in bone, in both the osteoblast and osteoclast and that this is the most likely explanation for the prevention of bone loss and inhibition of bone turnover that is seen in in vivo studies. It is tempting to speculate that, because the effects of estrogen in bone are complex and involve multiple target cells (osteoblast and osteoclast) agonism through the classic-ERE is necessary for the complete pleiotropic effects of an estrogenic-compound in bone.

The uterine histology of Ovx rats treated for 3 months with idoxifene (0.2, 1.0, or 10 mg/kg·day) showed myometrial atrophy, decreased numbers of uterine glands and endometrial atrophy, suggesting a lack of estrogenic activity of idoxifene on this tissue. Furthermore, treatment with idoxifene for more than 4 months caused significant prevention of osteopenia while having only a minor, nondose-dependent effect on uterine weight. This observation has been reported for other SERMs such as GW5638 (10) and raloxifene (37). Previously (16) it was shown that idoxifene, at doses similar to those used in the present studies, inhibited the uterotrophic effect of estradiol in immature rats while having only marginal intrinsic uterotrophic activity, and we have confirmed these studies using idoxifene in the intact rat. Taken together, these data suggest that idoxifene is a selective ER agonist in bone while having no ER agonist activity in the uterus, and in fact, is able to block natural estrogen action in the uterus.

These studies support a therapeutic advantage of idoxifene over estrogen for long-term use for the prevention of postmenopausal disease in women. Idoxifene has no estrogenic-agonist properties in endometrial cells in vitro. Agonism by estrogen in these cells is considered to be the molecular basis of the enhanced proliferation observed clinically during endometrial hyperplasia (38, 39). The effects of estrogen on proliferation of endometrial cells are consistent with the clinical observations of promotion of endometrial hyperplasia (34, 40). The in vitro data are consistent with uterotrophic studies based on the measurement of uterine weight in immature rats and mice, which showed that idoxifene antagonized the effects of estrogen (16). These studies are consistent with both the intact and the Ovx studies presented in this report. In breast cells, estrogen is a strong agonist whereas idoxifene is a potent antagonist (16). Taken together, these results in endometrial cells suggest a favorable therapeutic profile for idoxifene over estrogen in reproductive tissues.

It has been shown that tamoxifen (41) and raloxifene (37) induce conformational changes within the ER that are distinct from estrogen. These specific ligand-receptor complexes were differentially recognized by the cellular transcriptional machinery in a cell-specific context (25). The ER contains two transactivation domains: AF-1 and AF-2 and the relative contributions of these domains differ from cell-to-cell. Tamoxifen functions as an AF-2 antagonist, inhibiting ER activity in cells where AF-2 is dominant. In contrast, it functions as an agonist where AF-1 alone is required. These observations provide a mechanistic basis for cell-specific effects of ER modulators. Idoxifene is an agonist in osteoblasts and osteoclasts that could be explained by this being an AF-1 required environment. However, Willson and co-workers (10) have suggested that this does not strictly correlate in bone where different degrees of AF-1 and AF-2 agonist activity all protect against bone loss. The antagonism in endometrial cells would be explained by a complex between idoxifene and the ER that results in inhibiting ER activity in these cells. This would be analogous to the AF-2 dominant environment in which tamoxifen is a complete antagonist. However, reports of tamoxifen being a mixed agonist/antagonist in the uterus suggest this is not an AF-2 dominant environment.

This mechanistic explanation is probably more complex with the discovery of a new estrogen receptor isoform (ERß) that may have different ligand specificities and tissue distribution and also the discovery of nuclear hormone coactivators and corepressors that may contribute to tissue selectivity of the ER modulators (42). Studies are currently underway to define exactly how idoxifene binds to the ER isoforms in a cell specific context.

The most important benefit of HRT is its cardioprotective effect. Long-term adjuvant treatment with tamoxifen for breast cancer in postmenopausal women was associated with an estrogen-like cardioprotective effect (43) and tamoxifen has also been shown to lower cholesterol in ovariectomized and intact rats (44). The present study demonstrated a dose-responsive reduction in serum total cholesterol following treatment with idoxifene, suggesting that it has the potential for beneficial cardiovascular effects similar to those of HRT or tamoxifen. Although the mechanism of this action of idoxifene has not been studied, it may be similar to that of tamoxifen, which is thought to act by inhibiting cholesterol synthesis (45).

In conclusion, idoxifene showed beneficial effects on bone loss in the axial and appendicular skeleton in the Ovx rat model and has an estrogenic mechanism of action in bone. The pharmacological profile of idoxifene suggests that it behaves like an ER agonist on bone and lipid metabolism while having no ER agonist activity on the uterus at doses that prevent bone loss. This was confirmed in in vitro studies in endometrial cells. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data strongly suggest that although idoxifene may not confer any advantage over estrogen in prevention of osteoporosis, it may provide a better overall profile for the treatment of postmenopausal diseases.

Received May 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lindsay R 1988 Sex steroids in the pathogenesis and prevention of osteoporosis. In: Riggs BL (ed) Osteoporosis: Etiology, Diagnosis and Management. Raven Press, New York, pp 333–358
  2. Barzel US 1988 Estrogens in the prevention and treatment of postmenopausal osteoporosis: a review. Am J Med 85:847–850[Medline]
  3. Grodstein F, Stampfer MJ 1995 The epidemiology of coronary heart disease and estrogen replacement in postmenopausal women. Prog Cardiovasc Dis 38:199–210[CrossRef][Medline]
  4. Daley E, Roche M, Barlow D 1992 HRT: an analysis of benefits, risks and costs. Br Med Bull 42:368–400
  5. Hosking DJ 1990 Osteoporosis: problems for the future. In: Smith R (ed) Osteoporosis, Royal College of Physicians, London, pp 199–208
  6. Barrett-Connor E 1992 Hormone replacement and cancer. Br Med Bull 48:345–355[Abstract/Free Full Text]
  7. Grainger DJ, Metcalfe JC 1996 Tamoxifen: teaching an old drug new tricks?. Nat Med 2:381–385[CrossRef][Medline]
  8. Yang NN, Venugopalan M, Hardikar S, Glasebrook A 1996 Identification of an estrogen response element activated by metabolites of 17ß-estradiol and raloxifene. Science 273:1222–1225[Abstract]
  9. Ke HZ, Simmons HA, Piries CM, Crawford DT, Thompson DD 1995 Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats. Endocrinology 136:2435–2441[Abstract]
  10. Willson TM, Norris JD, Wagner BL, Asplin PB, Brown HR, Jones SS, Henke B, Sauls H, Wolfe S, Morris DC, McDonnell DP 1997 Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone. Endocrinology 138:3901–3911[Abstract/Free Full Text]
  11. Bjarnason K, Skrumsager BK, Kiehr B 1997 Levormeloxifene, a new partial estrogen receptor agonist demonstrates antiresorptive and antiatherogenic properties in postmenopausal women. J Bone Miner Res [Suppl] 12:S346
  12. Kauffman RF, Bryant HU 1995 Selective estrogen receptor modulators. DN& P 8:531–538
  13. Yamashita S, Takayanagi A, Shimizu N 1996 Temporal and cell-type specific expression of c-fos and c-jun protooncogenes in the mouse uterus after estrogen stimulation. Endocrinology 137:5468–5475[Abstract]
  14. Love RR, Cameron L, Connell BL, Leventhal H 1991 Symptoms associated with tamoxifen treatment in postmenopausal women. Arch Int Med 151:1842–1847[Abstract]
  15. McCague R, Leclercq G, Legros N, Goodman J, Blackburn GM, Jarman M, Foster AB 1989 Derivatives of tamoxifen. Dependence of antiestrogenicity on the 4-substituent. J Med Chem 32:2527–2533[CrossRef][Medline]
  16. Chander SK, McCague R, Luqmani Y, Newton C, Dowsett M, Jarman M, Coombes RC 1991 Pyrrolidino-4-iodotamoxifen and 4-iodotamoxifen, new analogues of the antiestrogen tamoxifen for the treatment of breast cancer. Cancer Res 51:5851–5858[Abstract/Free Full Text]
  17. Majeska RJ, Rodan SB, Rodan GA 1980 Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology 107:1494–1503[Abstract]
  18. Migliaccio S, Davis VL, Gibson MK, Gray TK, Korach KS 1992 Estrogens modulate the responsiveness of osteoblast-like cells (Ros 17/2.8) stably transfected with estrogen receptor. Endocrinology 130:2617–2624[Abstract]
  19. Clover J, Gowen M 1994 Are MG-63 and Hos TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype?. Bone 15:585–591[Medline]
  20. Holinka CG, Hata H, Kuramoto H, Gurpide E 1986 Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishakawa line). Cancer Res 46:2771–2774[Abstract/Free Full Text]
  21. James IE, Dodds RA, Lee-Rykaczewski E, Eichman CF, Connor JR, Hart KA, Maleef BE, Lackman, RD, Gowen M 1996 Purification and characterization of fully functional human osteoclast precursors. J Bone Miner Res 11:1608–1618[Medline]
  22. Wen DX, Xu Y-F, Goldman MK, McDonnell P 1994 The A and B isoforms of the human progesterone receptor operate through distinct signalling pathways within target cells. Mol Cell Biol 14:8356–8364[Abstract/Free Full Text]
  23. Beresford JN, Gallagher JA, Russell RGG 1986 1,25-dihydroxyvitamin D3 and human bone-derived cells in vitro: effects on alkaline phosphatase, type I collagen and proliferation. Endocrinology 119:1776–1785[Abstract]
  24. Norris JD, Fan D, Wagner BL, McDonnell DP 1996 Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifene mixed agonist activity. Mol Endocrinol 10:1605–1616[Abstract]
  25. Frenkel B, Mijnes J, Aronow MA, Zambetti G, Banerjee C, Stein JL, Lian JB, Stein GS 1993 Position and orientation-selective silencer in protein-coding sequences of the rat osteocalcin gene. Biochemistry 32:13636–13643[CrossRef][Medline]
  26. Katzenellenbogen JA, O’Malley BW, Katzenellenbogen S 1996 Tripartite steroid hormone receptor pharmacology: interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Mol Endocrinol 10:119–131[CrossRef][Medline]
  27. Nuttall ME, Nadeau D, Prichett WP, Gowen M 1997 Idoxifene, a tissue selective estrogen agonist/antagonist, has a mechanism of action in bone similar to estrogen and distinct from raloxifene. J Bone Miner Res [Suppl]12:S170
  28. Girasole G, Jilka RL, Passeri G, Boswell S, Boder G, Williams DC, Manolagos SC 1992 17ß-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest 89:883–891
  29. Littlewood AJ, Russell J, Harvey G, Hughes DE, Russell RGG, Aarden L, Gowen M 1991 The modulation of the expression of IL-6 and its receptor in human osteoblasts in vitro. Endocrinology 129:1513–1520[Abstract]
  30. Weinreb M, Shinar D, Rodan GA 1990 Different patterns of alkaline phosphatase, osteopontin and osteocalcin expression in developing rat bone by in situ hybridization. J Bone Min Res 5:831–842[Medline]
  31. Oursler MJ, Osdoby P, Pyfferoen J, Riggs BL, Spelsberg TC 1991 Avian osteoclasts as estrogen target cells. Pro Natl Acad Sci 88:6613–6617[Abstract/Free Full Text]
  32. Oursler MJ, Pederson L, Fitzpatrick L, Riggs BL, Spelsberg TC 1994 Human giant cell tumors of bone (osteoclastomas) are estrogen target cells. Proc Natl Acad Sci 91:5227–5231[Abstract/Free Full Text]
  33. Hoyland JA, Mee AP, Brown J, Braidman IP, Mawer EB, Freemont AJ 1996 Use of in situ-reverse transcriptase-polymerase chain reaction to localise oestrogen receptor mRNA in bone. J Bone Miner Res 11:S221
  34. Hughes DE, Dai A, Tifee JC, Li HH, Mundy GR, Boyce BF 1996 Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-ß. Nat Med 2:1132–1136[CrossRef][Medline]
  35. Jensen EV, DeSombre ER 1972 Mechanism of action of the female sex hormones. Annu Rev Biochem 41:203–230[CrossRef][Medline]
  36. Cauley JA, Lucas FL, Kuller FH, Vogt MT, Browner WS, Cummings SR 1996 Bone mineral density and risk of breast cancer in older women: the study of osteoporotic fractures. JAMA 276:1404–1408[Abstract]
  37. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  38. Grese TA, Sluka JP, Bryant HU, Cullinan GJ, Glasebrook AL, Jones CD, Matsumoto K, Palkowitz AD, Sato M, Termine JD, Winter MA, Yang NN, Dodge JA 1997 Molecular determinants of tissue selectivity in estrogen receptor modulators. Proc Natl Acad Sci 94:14105–14110[Abstract/Free Full Text]
  39. Furr BJA, Jordan VC 1984 The pharmacology and clinical uses of tamoxifen. Pharmacol Therap 25:127–205[CrossRef][Medline]
  40. Jordan VC 1992 Overview from the international conference on long-term tamoxifen therapy for breast cancer. J Natl Cancer Inst 84:231–234[Free Full Text]
  41. Gottardis MM, Robinson SP, Satyaswaroop PS, Jordan VC 1988 Contrasting actions of tamoxifen on endometrial and breast tumour growth. Cancer Res 48:812–815[Abstract/Free Full Text]
  42. McDonnell DP, Clemm DL, Herman T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 9:659–669[Abstract]
  43. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivators and corepressors regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657–666[Abstract/Free Full Text]
  44. McDonald CC, Stewart HJ 1991 Fatal myocardial infarction in the Scottish adjuvant tamoxifen trial. Br Med J 303:435–437
  45. Gold E, Stapley S, Goulding A 1994 Tamoxifen and norethisterone: effects on plasma cholesterol and total body calcium content in the estrogen-deficient rat. Horm Metab Res 26:100–103[Medline]
  46. Gylling G, Mantyla E, Miettinen TA 1992 Tamoxifen decreases serum cholesterol by inhibiting cholesterol synthesis. Atherosclerosis 96:245–247[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
C. Lemieux, Y. Gelinas, J. Lalonde, F. Labrie, D. Richard, and Y. Deshaies
The Selective Estrogen Receptor Modulator Acolbifene Reduces Cholesterolemia Independently of Its Anorectic Action in Control and Cholesterol-Fed Rats
J. Nutr., September 1, 2005; 135(9): 2225 - 2229.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. J. Perry, S. Gujra, T. Whitworth, and J. H. Tobias
Tamoxifen Stimulates Cancellous Bone Formation in Long Bones of Female Mice
Endocrinology, March 1, 2005; 146(3): 1060 - 1065.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
D. Vanderschueren, L. Vandenput, S. Boonen, M. K. Lindberg, R. Bouillon, and C. Ohlsson
Androgens and Bone
Endocr. Rev., June 1, 2004; 25(3): 389 - 425.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
G. Arpino, M. Nair Krishnan, C. Doval Dinesh, V. J. Bardou, G. M. Clark, and R. M. Elledge
Idoxifene versus tamoxifen: a randomized comparison in postmenopausal patients with metastatic breast cancer
Ann. Onc., February 1, 2003; 14(2): 233 - 241.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
X. L. Ma, F. Gao, J. Chen, T. A. Christopher, B. L. Lopez, E. H. Ohlstein, and T.-L. Yue
Endothelial protective and antishock effects of a selective estrogen receptor modulator in rats
Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H876 - H884.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
X. L. Ma, F. Gao, C.-L. Yao, J. Chen, B. L. Lopez, T. A. Christopher, J. Disa, J.-L. Gu, E. H. Ohlstein, and T.-L. Yue
Nitric Oxide Stimulatory and Endothelial Protective Effects of Idoxifene, a Selective Estrogen Receptor Modulator, in the Splanchnic Artery of the Ovariectomized Rat
J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 786 - 792.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. E. Nuttall, G. B. Stroup, P. W. Fisher, D. P. Nadeau, M. Gowen, and L. J. Suva
Distinct mechanisms of action of sel