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The Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Paul J. Shughrue, Department of Functional Morphology, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: shughrp{at}war.wyeth.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol all
significantly increased the level of PR mRNA, although the degree of
induction varied with each compound. The injection of tamoxifen,
raloxifene, toremifene, droloxifene, clomiphene, GW 5638, or ICI
182,780 had no significant estrogenic effect on PR gene expression at
the dose evaluated. In contrast, when tamoxifen or raloxifene, but not
ICI 182,780, was administered in the antagonist mode, a significant
dose-related decrease in the estradiol-induced level of PR mRNA was
seen in the preoptic area. The results of these studies clearly
demonstrate that PR mRNA expression in the rat preoptic area is rapidly
stimulated by a small dose of 17ß-estradiol. Moreover, the present
report has also shown that the estrogenic nature of compounds such as
tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, and GW 5638
cannot be predicted by their activity in peripheral tissues. Together,
the results of these studies provide important information about the
central activity of estrogens and provide evidence for their
tissue-specifc actions in the rat. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A variety of natural and synthetic compounds have been shown to have estrogenic properties in the rat. Interestingly, a group of compounds called "antiestrogens" have been shown to exhibit mixed estrogen agonist and antagonist properties (18). The selective estrogen agonist or antagonist activity appears to be determined by the tissue, cellular background, and promoter context (19, 20, 21, 22). For example, tamoxifen is a partial agonist in the uterus (23, 24, 25) and bone (26, 27, 28) but an antagonist in breast cancer cells (29, 30, 31, 32, 33). To date, little is known about the activity of many estrogenic and antiestrogenic compounds in the brain. Since the action of estrogens in the brain is important for the treatment of postmenopausal symptoms, including hot flashes (34, 35) and perhaps for the prevention of Alzheimer’s disease (36, 37), a clear understanding of the central action of the antiestrogens could provide insight about the therapeutic benefits or liabilities of these compounds in women. The major goal of this study was to characterize the exquisite sensitivity of PR gene expression by estrogen and use this activity to assess the estrogenic nature of a variety of partial agonists using in situ hybridization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization
A fragment (bases 2177–2992) of the rat PR complementary DNA
(cDNA) was amplified using PCR and the rPR-2 plasmid (38) as a
template. The fragment was subcloned into a pCRII plasmid (Invitrogen,
San Diego, CA), excised with EcoRI, and then subcloned into
the EcoRI site of a pBluescript plasmid (Stratagene, La
Jolla, CA). The resulting plasmid (PR-815) contained an 815-bp fragment
of the rat PR cDNA. The PR-815 plasmid was linearized with
HindIII (sense; control) or BamHI (antisense) and
used to generate[35S]UTP-labeled complementary RNA (cRNA)
probes for in situ hybridization.
The in situ hybridization methodology used for these studies has been described previously (39). Briefly, 20-µm cryostat sections were collected on Silane-coated slides, dried, and then stored at -80 C. At the time of processing, the desiccated slide boxes were warmed to room temperature, postfixed in paraformaldehyde, treated with acetic anhydride, and then delipidated and dehydrated. Processed section-mounted slides were hybridized with 100–200 µl of an antisense or sense (control) riboprobe (6 x 106 dpm/slide)-50% formamide hybridization mix and incubated overnight at 55 C in an open air-humidified slide chamber. In the morning, the slides were immersed in 2xSSC (0.3 M NaCl, 0.03 M sodium citrate; pH 7.0)/10 mM dithiothreitol, treated with ribonuclease A (RNase A; 20 µg/ml) and washed (2x 30 min) at 65 C in 0.1xSSC to remove nonspecific label. After dehydration, the slides were apposed to BioMax (BMR-1; Eastman Kodak, Rochester, NY) x-ray film for 3 days and then dipped in NTB2 nuclear emulsion (Eastman Kodak; diluted 1:1 with 600 mM ammonium acetate). The slides were exposed for 4–6 weeks in light-tight black desiccated boxes, photographically processed, stained in cresyl violet, and coverslipped. The slides from all animals were hybridized, washed, exposed, and photographically processed together to eliminate differences due to interassay variation in conditions.
Evaluation
The medial preoptic nucleus (Fig. 1
) was selected for statistical
evaluation because the level of PR mRNA in this brain region appears to
be highly sensitive to estrogenic action. Film autoradiographic images
were used to evaluate the intensity of hybridization signal. Relative
optical density measurements of PR hybridization signal were obtained
from film autoradiograms with a computer-based image analysis system
(C-Imaging Inc., Pittsburgh, PA). The results from two sequential
sections per animal were averaged and statistically evaluated.
Numerical values are reported as the mean ± SE.
Two-way ANOVA was used to test for differences in the level of PR mRNA,
and all statements of nondifference in Results imply that
P > 0.05. The computer program StatView (Abacus
Concepts Inc., Berkeley, CA) was used for statistical analysis of
data.
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Exp 2. The results from the first experiment clearly indicated that PR mRNA was rapidly modulated by estradiol after a single injection. To ascertain how fast PR mRNA was induced by estradiol, a second study examined a broad range of survival times. A new vehicle was also used [50% dimethylsulfoxide (DMSO), 40% PBS, and 10% ethanol] to ensure that the estradiol was rapidly available to the brain. Ovariectomized rats were injected sc with 2 µg/kg of 17ß-estradiol (Sigma) or 400 µl of vehicle on postovariectomy day 12. The animals (n = 4 per group) were then exposed to a lethal dose of CO2 0.5, 1, 2, 4, 6, 8, 10, or 12 h after injection and their brains collected, frozen on dry ice, and processed for in situ hybridization.
Exp 3. The results of the second time course study revealed that a 6-h survival after a single injection of estradiol was the best point to study the effects of estrogen on PR mRNA. A dose-response curve was now needed to determine how much estradiol was required to modulate PR gene expression. Preliminary studies (data not shown) revealed that a dose of 1 µg/kg or greater was capable of eliciting a maximal stimulation of PR mRNA in the rat preoptic area. Therefore, a dose of 1 µg/kg was selected as the highest dose. Rats (n = 4 per group), ovariectomized for 12 days, were injected sc with 25, 50, 100, 300, 500, or 1000 ng/kg of 17ß-estradiol or 400 µl of vehicle (50% DMSO, 40% PBS, and 10% ethanol) at 0900 h. Six hours after injection, the animals were exposed to a lethal dose of CO2, and their brains were collected, frozen on dry ice, and processed for in situ hybridization.
Evaluation of estrogens
Exp 4: agonist activity. The purpose of the second series of
studies was to evaluate the agonistic activity of a selected group of
compounds that are estrogenic, antiestrogenic, or have mixed activity
in peripheral tissues. A pharmacological dose of compounds was used to
ensure maximal induction of PR mRNA. Rats (n = 4 per group) were
ovariectomized for 12 days and injected sc with 2 mg/kg of
17ß-estradiol (Sigma), 17-estradiol (Sigma), diethylstilbestrol
(DES; Sigma), 4-OH-tamoxifen (Research Biochemicals International,
Natick, MA), raloxifene (keoxifene or LY156758; synthesized at
Wyeth-Ayerst), toremifene (NK622; synthesized at Wyeth-Ayerst),
droloxifene (3-OH-tamoxifen; Research Biochemicals International), GW
5638 (3-[4-(1,2-diphenylbut-1-enyl) phenyl] acrylic acid; Ref. 40;
synthesized at Wyeth-Ayerst), clomiphene (Sigma), ICI 182,780 (Zeneca
Pharmaceuticals, Wilmington, DE), or 400 µl of vehicle (50% DMSO,
40% PBS, and 10% ethanol) at 0900 h. Six hours after injection,
the animals were exposed to a lethal dose of CO2, and
brains were collected, frozen, and processed for in situ
hybridization.
Exp 5: antagonist activity. The results of Exp 4 revealed that tamoxifen, raloxifene, and ICI 182,780 are not capable of modulating PR mRNA in the rat hypothalamus. Therefore, a final study evaluated the antagonistic properties of these compounds. Adult ovariectomized rats (n = 4 per group) were sc injected with 30, 300, or 3000 µg/kg of 4-OH-tamoxifen or raloxifene, or with 2 mg/kg of ICI 182,780 or vehicle alone (50% DMSO, 40% PBS, and 10% ethanol) at 0800 h. At 0900 h, rats were injected sc with 300 ng/kg of 17ß-estradiol or vehicle (-control). Six hours after the injection of 17ß-estradiol (1500 h), the animals were exposed to a lethal dose of CO2, and their brains were collected, frozen on dry ice, and processed for in situ hybridization.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). When ovariectomized rats were treated
with 5, 50, or 250 µg/kg of estradiol for 6 h, a dramatic
increase (P < 0.0001 vs. control) in the
level of PR mRNA was detected in the preoptic area of all treatment
groups. Interestingly, when the level of PR mRNA was assessed 24 h
after a single injection, a dose-related difference was observed.
Treatment of animals with 5 µg estradiol resulted in a weak, but not
significant, induction of PR mRNA, while 50 µg estradiol moderately
(P < 0.001) enhanced gene expression. Only animals
treated with 250 µg estradiol had a level of hybridization signal
comparable to that seen in animals treated with estradiol for 6 h.
When animals were administered a second dose of estradiol, 24 h
after the initial treatment, the time course of PR mRNA regulation was
similar to that seen after a single injection. That is, the level of
hybridization signal was elevated (P < 0.0001
vs. control) in all treatment groups at 6 h after the
second injection, while only the 250-µg treatment group was different
from control (P < 0.0001) 24 h after the second
injection.
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). When ovariectomized
rats were injected with estradiol, a significant (P <
0.01) increase in the level of PR expression was seen within 2 h
after injection. The level of PR mRNA peaked 4 h after injection
(P < 0.001), remained elevated until 8 h, was
attenuated at 10 h (P < 0.01), and then decreased
to control levels at 12 h postinjection.
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) demonstrated that increasing doses of
17ß-estradiol increased the level of PR mRNA, with a maximal response
at 1000 ng/kg and an EC50 of 93.5 ng/kg.
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). When animals were
injected with 17ß-estradiol or DES, a marked (P <
0.001) increase in the level of PR mRNA was observed in the preoptic
area when compared with controls (Figs. 6 and 7). Interestingly,
17-estradiol also significantly increased the level of hybridization
signal, although the degree of induction was attenuated. Injection of
tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, GW 5638, or
ICI 182,780 had no significant effect on PR gene expression at the dose
evaluated (Figs. 6 and 7). One explanation for this
observation was that the 6-h survival time was not optimal for some of
the antiestrogens, even though a high percentage DMSO vehicle was used.
To address this issue, the efficacy of tamoxifen and raloxifene in the
hypothalamus was compared with 17ß-estradiol 6, 12, 18, and 24 h
after a single injection (data not shown). The results of this study
clearly demonstrated that tamoxifen and raloxifene did not exhibit
estrogenic agonist activity (i.e. PR mRNA levels were not
statistically different from controls) at any of the time points
investigated, while the changes in the level of PR mRNA after treatment
with 17ß-estradiol were as expected based on the previous time course
experiments.
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). In contrast, when animals were administered
tamoxifen or raloxifene 1 h before estradiol treatment, the level
of PR mRNA was significantly attenuated in a dose-dependent maner (Fig. 8). Evaluation of the antagonistic properties of tamoxifen and
raloxifene at three different concentrations (30 µg to 3 mg) has also
provided information about the relative potencies of these compounds.
When animals were treated with 30 or 300 µg of tamoxifen or
raloxifene before estradiol, only a slight reduction in the level of PR
mRNA was observed. Treatment with the highest dose tested (3 mg) was
most effective at antagonizing the action of estradiol in the
hypothalamus, but resulted only in a 55–72% reduction in PR mRNA
expression. Together, these observations suggest that tamoxifen and
raloxifene are weak estrogen antagonists in the rat hypothalamus. In
contrast, when animals were treated with 2 mg/kg of ICI 182,780 1
h before injection of estradiol, the level of PR mRNA was attenuated,
but not significantly different, when compared with estradiol alone
(data not shown). However, it is known that ICI 182,780 does not cross
the blood-brain barrier (41).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of PR mRNA detected in the present study, compared with that found in previous in situ hybridization (10), autoradiographic (3, 4), and immunocytochemical (6, 7) studies, demonstrates that the present observations are in good agreement with the known topography of PR mRNA and protein in the preoptic area of female rodents. While the level of PR mRNA detected in the present study and previously (10) was more abundant than that seen in earlier rodent studies (8, 9, 17), this discrepancy in hybridization signal is probably caused by the use of human PR cRNA riboprobes for rodent studies, short probe length, and/or differences in methodological approach.
Although several studies have examined the regulation of PR mRNA by estrogen in the rodent brain, the use of a variety of estrogen treatment regimens has made it difficult to ascertain the time course and duration of estrogen action. The results of the present studies clearly demonstrated that the level of PR mRNA was elevated in the preoptic area within 2 h after a single injection of estradiol, remained elevated until 10 h postinjection, and then declined to a level similar to that of control ovariectomized animals. If animals received a second injection of estradiol 24 h after the first, the level of PR mRNA was again augmented as seen the previous day. However, if animals were administered a pharmacological dose of estradiol, the level of PR mRNA remained elevated for at least 24 h after a single injection. A dose-response study revealed that as little as 25 ng/kg of estradiol was sufficient to significantly increase the level of PR expression in ovariectomized rats, with a maximal response seen with 1 µg/kg and a calculated EC50 of 93.5 ng/kg. The rapid modulation of PR mRNA reported herein is in good agreement with earlier work that studied changes in PR mRNA over the rat estrous cycle (10). Simerly et al. (10) found that the level of PR mRNA in the anteroventral periventricular nucleus (AVPV) was low on the morning of proestrus and then increased significantly by the afternoon of proestrus. These changes in the level of PR mRNA seen between the morning and afternoon of proestrus clearly demonstrate that under normal physiological conditions the expression of PR mRNA is significantly augmented in the AVPV within a short period of time. Additional studies revealed that the treatment of ovariectomized females with estradiol pellets for 7 days significantly increased the level of PR mRNA in the AVPV, indicating that changes in circulating estradiol were responsible for modulating PR expression in the rat hypothalamus (10). A previous study also evaluated changes in the level of PR expression in the arcuate and ventromedial nuclei of the rat hypothalamus after treatment with estrogen (8). The results of a time course study indicated that a single injection of estradiol benzoate enhanced the level of PR mRNA within 4 h of treatment, although a significant increase in expression was not observed until 24 h postinjection (8). Unfortunately, no data were available for the time points between 4 and 24 h. In contrast, we first detected a significant increase in PR expression 2 h after a single injection of 17ß-estradiol. The discrepancy between these studies is most likely due to differences in hormone treatment. The present studies used 17ß-estradiol dissolved in 50% DMSO, a vehicle intended for rapid systemic delivery of substances, while Romano et al. (8) used estradiol benzoate dissolved in sesame oil. Therefore, the slow systemic uptake of estrogen from oil could account for the delay in the induction of PR mRNA seen in the central hypothalamus. Alternatively, the time course for the regulation of PR mRNA by estrogen in the preoptic area, as compared with the arcuate and ventromedial nuclei, may represent a region-specific difference in the induction of gene transcription in the rat brain.
The fact that estradiol regulates PR binding in vivo and in vitro has been known for many years (for review, see Refs. 42 and 43). Sar and Stumpf (1) were the first to clearly show that the pretreatment of ovariectomized rats with estradiol markedly enhanced the nuclear uptake and retention of radiolabeled progesterone in the preoptic area and central hypothalamus. Subsequent receptor-binding assays showed that estrogen implants or injections increased the level of progestin binding in the rodent hypothalamus in a dose- and time-dependent manner (44, 45, 46, 47, 48). The present results are in good agreement with these biochemical studies, although differences in the method of estradiol treatment make it difficult to compare changes in the expression of PR mRNA with changes in protein.
With an understanding of how estradiol modulates the level of PR mRNA
in the rat preoptic area, a number of compounds with putative
estrogenic properties were evaluated in this assay. When ovariectomized
animals were treated with tamoxifen, raloxifene, toremifene, the GW
5638, clomiphene, or ICI 182,780, no significant increase in the level
of PR mRNA was observed in the rat preoptic area. In contrast,
17ß-estradiol, 17-estradiol, and DES all markedly increased PR
gene expression in ovariectomized animals. To determine whether the
failure of tamoxifen and raloxifene to stimulate PR mRNA was due to the
short survival time after treatment, a time course study was conducted.
The efficacy of tamoxifen, raloxifene, and 17ß-estradiol on the
expression of PR mRNA in the rat preoptic area was assessed 6, 12, 18,
and 24 h after a single injection. This study clearly demonstrated
that, regardless of survival time, tamoxifen and raloxifene were not
capable of stimulating PR mRNA, while treatment with 17ß-estradiol
modulated PR expression as expected. When tamoxifen and raloxifene were
evaluated in the antagonist mode, i.e. administered 1 h
before estradiol, a dose-related decrease in the estradiol-induced
level of PR expression was observed. Together, these data demonstrate
that both tamoxifen and raloxifene act as weak antagonists in our PR
mRNA expression assay. The present observations are in good agreement
with previous studies that also showed that tamoxifen and raloxifene
act as antagonists in the brain. When tamoxifen was injected into rats
or implanted into certain brain regions, it antagonized
estrogen-induced reproductive behavior (25, 49, 50, 51), maternal behavior
(52), and the induction of PR (25, 51). Similarly, implantation of
raloxifene into specific regions of the rat hypothalamus has been shown
to block lordosis (53) and the surge of LH on the afternoon of
proestrus (54). In contrast, ICI 182,780 appears to be unable to
penetrate the blood-brain barrier in the rat (41). It is interesting to
note that in vitro, the affinity (55) and potency (22) of
tamoxifen and raloxifene on the ER is similar to that of
17ß-estradiol. These observations contradict the present finding that
a high dose of tamoxifen and raloxifene (100- to 1000-fold higher than
17ß-estradiol) was required to produce significant antagonist effects
in the brain and suggest that tamoxifen and raloxifene are rapidly
metabolized in vivo or have difficulty crossing the
blood-brain barrier.
Molecular analysis of different compounds has shown that estradiol,
tamoxifen, raloxifene, and ICI 164,384 all differentially modulate the
conformation of the ER and its transcriptional activity (22). In
addition, the cellular background and promoter context may also
determine the nature and magnitude of a compound’s estrogenic
properties (19, 20, 21, 22). In vivo, these differences and others
appear to impart the tissue-specific estrogenic activity of compounds.
That is, a compound such as tamoxifen is an estrogen antagonist in
breast cancer cells (29, 30, 31, 32, 33), weakly agonistic in the uterus (23, 24, 25, 56), and estrogenic in bone (26, 27, 28, 56). However, additional factors
may also be involved in determining the tissue selectivity of
estrogens. For example, a second ER (ERß), recently cloned by Kuiper
and colleagues (57), appears to have a different distribution in some
rat tissues, when compared with ER mRNA (Ref. 55 and P. J.
Shughrue and I. Merchenthaler, unpublished observations). Analysis of
the distribution of ERß mRNA in the rat brain with in situ
hybridization revealed that ERß mRNA was present in brain regions
that expressed ER mRNA, including the preoptic area, as well as
regions where ER mRNA is sparse or absent (58, 59). In addition,
ER mRNA has been detected in brain regions that do not express ERß
mRNA (58, 59). These observations and the finding that 17ß-estradiol
modulates the expression of PR mRNA in the preoptic area of the ER
knockout mouse (60) suggest that estradiol is capable of regulating
genes by interacting with ER and ERß. Therefore, the specificity
of compounds for ER vs. ERß or the ability of compounds
to differentially modulate the activity of these ERs (agonist
vs. antagonist activity) (61) could also impart
tissue-specific estrogenic activity.
To date, little is known about the activity of tamoxifen and raloxifene in the human brain. Interestingly, data from several clinical trials have shown that both compounds significantly increase the incidence of hot flashes (62, 63, 64), consistent with the belief that tamoxifen and raloxifene also act as estrogen antagonists in the human brain. The results of these studies further suggest that tamoxifen and raloxifene may have additional central nervous system liabilities in postmenopausal women if they are estrogen antagonists in the brain. Since estrogen replacement therapy may be capable of abating or delaying the onset of Alzheimer’s disease in women (see Refs. 36 and 37), the long-term use of an antiestrogenic compound might hasten the progression of this disease. While there are currently no data to support this hypothesis, these issues clearly need to be critically examined in human trials.
Received June 27, 1997.
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S. A. Haywood, S. X. Simonian, E. M. van der Beek, R. J. Bicknell, and A. E. Herbison Fluctuating Estrogen and Progesterone Receptor Expression in Brainstem Norepinephrine Neurons through the Rat Estrous Cycle Endocrinology, July 1, 1999; 140(7): 3255 - 3263. [Abstract] [Full Text]
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 B. J. Orr-Walker, A. M. Horne, M. C. Evans, A. B. Grey, M. A. F. Murray, A. R. McNeil, and I. R. Reid Hormone Replacement Therapy Causes a Respiratory Alkalosis in Normal Postmenopausal Women J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 1997 - 2001. [Abstract] [Full Text]
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 Z. Nawaz, G. M. Stancel, and S. M. Hyder The Pure Antiestrogen ICI 182,780 Inhibits Progestin-induced Transcription Cancer Res., January 1, 1999; 59(2): 372 - 376. [Abstract] [Full Text] [PDF]
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D. C. Skinner, N. P. Evans, B. Delaleu, R. L. Goodman, P. Bouchard, and A. Caraty The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor PNAS, September 1, 1998; 95(18): 10978 - 10983. [Abstract] [Full Text] [PDF]
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