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a Medical Sciences and
b Program in Neural Science, Indiana University, School of Medicine, Bloomington, Indiana 47405
c Department of Obstetrics and Gynecology, Indiana University, School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ER. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ER expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17ß. Uteri were collected and analyzed for changes in ER mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ER. Immunohistochemical analysis was performed with a polyclonal antibody specific to ER. Expression of ER in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ER mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ER mRNA occurs independent of new protein synthesis. A decrease in ER mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ER expression. The results indicate that down-regulation of ER in the epithelia and up-regulation of stromal ER play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ER mRNA and protein levels in various target tissues can be influenced by several physiological factors, including estrogens. The effect of E2 on ER concentration varies among different tissues and cell types. In liver cells, for example, E2 up-regulates ER [1]. In estrogen-responsive human breast cancer cells and rat fibroblasts [2–5], ER is down-regulated by E2. The rat uterus has served as an excellent model for studying the effect of E2 on ER mRNA and receptor protein expression. Both up- and down-regulation of ER by E2 have been reported in rat uterus, depending upon the physiological state of the animal and experimental system employed [6–13], making direct comparisons of results difficult. Work by a number of investigators [6–10,12] demonstrated in rats that E2 has a biphasic effect on total uterine ER mRNA and protein levels; ER levels were rapidly depleted after hormone administration followed by a subsequent increase. However, those and similar studies [6–13] investigating the effects of E2 on uterine ER expression in rats have utilized whole uterine homogenates, which represent the combined response of the uterine tissue compartments (the myometrium, endometrial stroma, and luminal and glandular epithelium). Such studies therefore have not revealed the response of the specific uterine cell types. In addition, there are striking differences in the response of the uterus to E2 in adult and sexually immature rodents. In the adult rodent, only the luminal epithelium proliferates in response to E2 [14–19]. In sexually immature rodents, E2 stimulates DNA synthesis and cell proliferation in all uterine compartments [20–24]. The cellular pattern of ER expression may play a key role in the response of immature and mature rat uterus to E2, but neither the kinetics nor the cell-type specific effects of E2 treatment on uterine ER expression have been compared in these experimental models.
The objectives of the present study were to 1) compare uterine ER mRNA levels after administration of E2 to sexually immature and mature rats and 2) establish which cell types respond to E2 with autoregulation of ER in immature and mature rat uterus. RNase protection assay, in situ hybridization, and immunohistochemistry procedures were utilized to meet these experimental objectives.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal studies were performed under protocols and procedures approved by local Institutional Animal Care and Use Committee and in accord with NIH standards established by the Guidelines for the Care and Use of Experimental Animals and by the American Veterinary Medical Association. Mature (150–200 g; n = 20) and immature (~23 days old and 40–45 g; n = 32) female Sprague-Dawley rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Mature animals or immature animals were ovariectomized (OVX) 3 wk or 5 days, respectively, before an experiment. Rats were given animal chow and water ad libitum and maintained on a 12 h light-dark cycle, with lights-on at 0700 h. Animals were injected s.c. in the periscapular area with 4 µg/kg BW of E2 (Sigma Chemical Company, St. Louis, MO) or sesame oil vehicle (Sigma) and sacrificed 0, 3, 6, 24, or 72 h later. Uteri were collected quickly and trimmed of extraneous connective tissues. One uterine horn from each animal was placed immediately in liquid nitrogen and stored at -80°C for RNase protection assays (see below). The other uterine horn from each rat was cryopreserved according to procedures we have described previously [25–27] and used for in situ hybridization and immunohistochemical analyses (see below). The dose of E2 used in this study is based on earlier work demonstrating increases in uterine tissue DNA synthesis as a function of hormone dose in immature and mature rats [24]. Immature animals treated with cycloheximide (5 mg, i.p.; Sigma) or saline vehicle 30 min prior to hormone treatment were killed at 0 or 3 h after administration of E2. This level and route of administration of cycloheximide effectively blocks protein synthesis in the uterus by 95% [7,8].
RNase Protection Assay for ER
Total RNA was isolated using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's protocol. Concentrations of the final preparations were calculated from A260 reading using a Shimadzu UV-1201 spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). An aliquot of all RNA preparations was then analyzed on a 1% agarose gel to ensure integrity before further analysis. A 485-base pair (bp) cDNA fragment of the rat ER gene was amplified by PCR using full length cDNA (pFLER, supplied by Dr. M. Shupnik, University of Virginia Medical Center, Charlottesville) as the template. Primers specific for ER were as follows: forward (bases 74–94): 5'cggaattcATGACCATGACCCTTCACACC3' and reverse (bases 539–559 5'tcccccgggggaTCGATTGTCAGAATTGGACCT3' (base numbers refer to the rat ER sequence, Genbank accession No. X 61098). The forward primer contains an EcoRI site and the reverse primer contains a SmaI site (listed in small letters). The amplified ER cDNA fragment was then cloned into the EcoRI-SmaI sites of pBD-gal4 Cam vector (Stratagene Cloning Systems, La Jolla, CA). The plasmid was linearized by NotI. Antisense riboprobes were generated from linearized templates using the MAXIscript kit (Ambion, Austin, TX), T7 RNA polymerase and the incorporation of [-32P]UTP (New England Nuclear, Boston, MA; 800 Ci/mmol) according to the manufacturer's protocol. The rat ER antisense probe was 240 nt in full length and produced a 210 nt specific protected fragment. The probe detects only ER. The sequence of the probe corresponds to the N-terminal A/B domain, which is highly variable between ERs, and shows only 33% homology with the ERß sequence. An antisense riboprobe specific for rat cyclophilin, used to normalize lanes for differences in loading among lanes, was generated from the template pTRI-cyclophilin (Ambion; a probe length of 165 nt) and produced a protected fragment of 103 nt. Cyclophilin mRNA expression does not appear to be up-regulated by E2 in the rat uterus [28,29]. In addition, RNA probes were derived from c-fos cDNA in a pBluescript plasmid, as we have described [26]; the plasmid was linearized with NcoI. Protection assays were performed using the RPA II kit (Ambion). Samples were hybridized according to the manufacturer's protocol. Free riboprobe was removed by digestion with a 1:100 dilution of a mixture of RNase A (250 U/ml) and RNase T1 (10 000 U/ml) for 30 min at 37°C. Samples were precipitated and resuspended in 80% formamide (v:v), 0.1% (w:v) xylene cyanol, 0.1% (w:v) bromophenol blue, and 2 mM EDTA, and separated on 6% (w:v) polyacrylamide, 8 M urea gels in single-strength TBE buffer. The gels were exposed to x-ray film (Biomax AR; Eastman Kodak Company, Rochester, NY) to -70°C for appropriate time using intensifying screens. The optical densities of autoradiograms from the protected bands of the RNase protection assay for both ER and cyclophilin of the individual animals were measured and quantified using an imaging densitometer and Molecular Analyst Software (G5670; Bio-Rad, Hercules, CA) software. The levels of ER were normalized to those of cyclophilin mRNA, and the results were expressed as arbitrary units. Effect of E2 treatment on ER mRNA levels was assessed by one-way ANOVA, and Fisher's least significant difference test was used to determine the significance of differences between control levels and individual treatment means. The effect of E2 in the experiment with cycloheximide was tested using Student's t-test.
In Situ Hybridization for ER
In situ hybridization was performed as we described previously [25–27]. Briefly, frozen uterine sections (10 µm) were mounted on slides, fixed in 4% paraformaldehyde, dehydrated, and stored at -70°C. Before use, slides were refixed in 4% paraformaldehyde, treated with proteinase K (40 µg/slide; 20 µg/ml, 2 ml per slide) for 3 min, fixed again, treated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, dipped in water, dehydrated, and air dried. Full length rat ER (nucleotides 1–2000), containing the entire coding sequence and some 5' and 3' untranslated regions as originally described by Koike and coworkers [30], in pGEM3Z (pFLER) was digested with HindIII and SacII for sense and antisense strands, respectively. 35S-Labeled riboprobes (Amersham; [35S]UTPS, > 1000 Ci/mmol) were synthesized using the Promega Systems kit (Promega, Madison, WI). The riboprobes were subjected to alkaline hydrolysis (40 mM NaHCO3, 60 mM Na2CO3, 5 mM DTT; 60°C, 41 min) to yield 200-bp fragments, purified on a SELECT-D(RF) column (5Prime
3Prime, Inc., Boulder, CO), ammonium acetate (7.5 M) and ethanol precipitated, and resuspended in 0.2 M dithiothreitol (DTT). Probes (3 x 105 cpm/coverslip) in hybridization buffer (50% deionized formamide; 0.3 M NaCl; 20 mM Tris, pH 8.0; 50 mM EDTA, pH 8.0; 10 mM Na3PO4; single-strength Denhardt's; 10% dextran sulfate; 0.5 mg/ml yeast tRNA) containing 200 mM DTT at a concentration of 1 x 106 cpm/µl were added to tissue sections as described [26–28], coverslipped, and incubated at 55°C for 16 h. Coverslips were removed by incubating the slides in 5-strength SSC (single-strength SSC is 0.15 M NaCl and 0.015 M sodium citrate), 10 mM DTT at 55°C for 30 min. The sections were washed in 50% formamide, double-strength SSC, 10 mM DTT at 65°C for 30 min, followed by three washes in 10 mM Tris-HCl, 0.5 M NaCl, 5 mM EDTA (TNE buffer) for 10 min at 37°C. The slides were treated with RNase A (20 µg/ml) and RNase T1 (10 U/ml) for 30 min at 37°C and then washed in TNE buffer for 15 min at 37°C. Slides were washed in 50% formamide, double-strength SSC, 10 mM DTT for 30 min, followed by two 15-min washes in double-strength SSC, 1 mM DTT, and 0.10-strength SSC, 1 mM DTT at 65°C. Slides were dehydrated, dried, then dipped in Ilford Nuclear Emulsion K5 (Polysciences, Warrington, PA), stored with Drierite (Drierite Co., Xenia, OH) at 4°C and developed in 4–5 days.
Immunohistochemistry
Tissues were cryosectioned at 8 µm, mounted on silane-coated slides (Histology Control Systems, Inc., Glen Head, NY) and warmed to room temperature for 20 min. To enhance antigenicity, sections were incubated in 0.05 M glycine-HCl/0.01% EDTA buffer, pH 3.1, at 95°C for 15 min. Sections were allowed to cool to room temperature for 20 min, then washed for 10 min 3 times in PBS 0.5% Tween 20 (PBS-Tween), pH 7.4. After washing, tissues were blocked with undiluted normal goat serum (Sigma) for 20 min, washed 3 times for 5 min in PBS-Tween, then incubated overnight at 4°C in ER-21, a polyclonal antiserum raised against the N-terminus of ER (1:500; gift of Dr. Geoffrey Greene, University of Chicago). ER21 is specific for the first 21 amino acids of ER and recognizes only ER [31]. Control sections were incubated without antibody, or in antibody preabsorbed with a 10-fold excess of peptide immunogen (also provided by Dr. Greene). The next day, sections were washed 3 times in PBS-Tween for 10 min each, then subjected to reaction with secondary antibody (1:400 goat anti-rabbit; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After three additional 5-min washes in PBS-Tween, sections were incubated in ABC Elite reagent (Vector Laboratories) for 1 h, washed 3 times for 5 min each with PBS-Tween, then subjected to reaction with nickel-intensified diaminobenzidine reagent (DAB kit; Vector Laboratories) for 10–15 min. Sections were then rinsed twice in water, allowed to dry, and then dehydrated in graded alcohols and coverslipped.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA in Rat Uterus
To examine the effects of E2 on the expression of uterine ER, adult and immature rats were ovariectomized to reduce endogenous hormones, given a single, physiological dose of E2, and sacrificed at various hours after hormone injection. RNase protection assay was used to quantitate ER mRNA levels in whole uterus. In adult rats, uterine levels of ER mRNA decreased (P < 0.05) to about 40% of control levels by 6 h after E2 treatment (Fig. 1). At 24 h, ER mRNA levels tended to be higher compared to 0 h (P = 0.056), and by 72 h after hormone treatment, levels of ER mRNA in the uterus of mature animals were not different from control levels. E2 treatment of immature rats tended to decrease ER mRNA levels by 3 h compared to controls (P = 0.088) (Fig. 2). ER mRNA levels returned to control levels by 6 h; however, by 24 h, ER transcripts in the uterus were increased (P < 0.01) compared to controls. ER mRNA levels returned to control levels by 72 h after E2 treatment in immature rat uteri. Expression of the c-fos immediate early gene was used as a positive control for the uterine response to E2 in this study, and, as has been well documented [26,32–34], c-fos expression increased at 3 h after E2 treatment and then declined (data not shown). In order to investigate whether the E2-induced decrease in ER mRNA levels requires the synthesis of new proteins, the protein synthesis inhibitor cycloheximide was used. The effect of cycloheximide alone, or in combination with E2, on the steady-state level of ER mRNA was examined using RNase protection assay (Fig. 3). In this study, E2 treatment of immature rats resulted in suppression (P < 0.05) of receptor message by 3 h, and cycloheximide alone had no significant effect on ER mRNA. When combined with E2 treatment, cycloheximide did not abolish the E2-induced decrease (P < 0.05) in ER mRNA levels, suggesting that suppression of ER mRNA by E2 occurs independent of protein synthesis in the rat uterus. In addition, studies by Copland and coworkers [8] showed that treatment of castrated rats with cycloheximide had no effect on the E2-induced decrease in ER protein.
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Uterine Cell-Type Specific Expression of ER mRNA After E2 Treatment
In situ hybridization studies were performed to determine which uterine cell types displayed down-regulation of ER mRNA after E2 treatment. Uteri from the same rats analyzed by RNase protection analysis were used. Using the ER antisense probe on uterine sections from control mature rats, ER mRNA was detected in all uterine tissue compartments, but ER transcripts appeared to be most abundant in the epithelia (Fig. 4B). Uterine sections hybridized with ER sense RNA probe showed only background levels of silver grains (Figs. 4A and 5A). The pattern and intensity of ER mRNA silver grains in uterine sections at 3 h after E2 treatment appeared similar to the 0-h control sections (Fig. 4C). However, at 6 h after E2 treatment, a decrease in silver grains corresponding to ER mRNA was seen in all compartments, most strikingly in the luminal and glandular epithelia (Fig. 4D). By 24 h after E2 administration to mature rats, an increase in silver grains in all the uterine compartments was seen (Fig. 4E). By 72 h after hormone treatment, the constitutive cell type-specific expression pattern of ER transcripts had returned (Fig. 4F).
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In uterine sections from untreated immature rats, the ER antisense probe revealed the presence of silver grains corresponding to ER mRNA in all the uterine compartments, with an abundance of ER silver grains in the luminal and glandular epithelia (Fig. 5). At 3 h after E2 treatment of immature animals, silver grains in the endometrial epithelium appeared to decrease; however, the epithelial signal was more distinct than the surrounding stromal and myometrium (Fig. 5C). The pattern of ER mRNA silver grains at 6 h was similar but more intense than that seen at 3 h after E2 treatment, particularly in the epithelia (Fig. 5D). By 24 h after E2 administration to immature rats (Fig. 5E), silver grains were seen in all uterine compartments, and the difference between epithelial signal and the surrounding stromal signal was not as distinct compared to other time points (Fig. 5). By 72 h, however, the cell type-pattern of ER mRNA expression had returned and was essentially similar to that seen in control animals (Fig. 5, B and E).
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Cell Type-Expression of ER Protein in the Uterus After E2 Administration
Immunostaining for ER in the vehicle-treated (0 h) adult rat uterus was intense and localized to the nucleus but confined almost exclusively to the luminal and glandular epithelial cells (Fig. 6B). Negative controls, sections treated with peptide-neutralized antibody or without primary antibody, were devoid of stain (Figs. 6A and 7A). ER immunostaining at 3 h after E2 treatment appeared to decrease slightly in the epithelia compared to 0 h (Fig. 6, C vs. B); however, ER-positive cells in the periluminal stromal area were detected. By 6 h post-E2 administration (Fig. 6D), ER staining was reduced in nearly all the luminal and glandular epithelial cells and few ER positive cells were detected in the stroma. At 24 and 72 h after E2 administration to adult animals (Fig. 6, E and F), ER immunoreactivity in the uterine epithelia was increased and similar to that seen in the uterine sections at 0 h.
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In uterine sections from untreated immature animals, all the major uterine cell types (myometrium and endometrial stroma and epithelia) showed ER immunostaining (Fig. 7B). At 3 h after treatment with E2 (Fig. 7C), immunostaining for ER in the epithelial cells was faint, but ER immunoreactivity was strong in cells of the myometrium and stroma. The staining at 6 h after E2 treatment (Fig. 7D) was similar to that seen at 3 h, although ER immunostaining was slightly stronger in luminal and glandular epithelial cells. By 24 and 72 h (Fig. 7, E and F), ER immunostaining was seen in all uterine compartments; at 72 h, the pattern of staining was similar to that seen at 0 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were studied in both animal models. Our studies demonstrate that a physiological dose of E2 rapidly and transiently down-regulates ER mRNA and receptor protein in a cell type-specific manner in the rat uterus. Overall, the cellular pattern of homologous ER down-regulation was similar in sexually immature and mature rat uterus; the slight differences in the timing of the effect in the two types of animal may be due to differences in the metabolism of estrogens [35].
To better understand the actions of E2, we used in situ hybridization and immunohistochemical analyses to characterize the uterine cell type-specific pattern of ER mRNA and receptor protein before and after hormone treatment of mature and immature rats. Although several groups have reported that supraphysiological doses of E2 suppress ER mRNA and protein in homogenates of whole rodent uterus [6–13,36], we are not aware of any studies on the response of the specific uterine cell types to physiological doses of E2 in adult or immature rats. After hormone treatment, receptor mRNA and protein suppression occurred primarily in the uterine epithelia of both adult and immature rats. The time course of the decrease in ER immunostaining and ER silver grains in the epithelia coincided with the decrease in whole uterine content of ER mRNA detected by RNase protection assays. It is likely that changes in myometrial and stromal levels of ER also contribute to the RPA data but are below background of the in situ data. In addition however, ER transcripts were still detectable, which agrees with results of others using Northern analysis and RNase protection assays to show that E2 treatment suppresses ER mRNA levels to about 40–50% of control values in rodent uterus [7,10,12,13] and estrogen-responsive cell lines [2–5]. The lack of a significant decrease in one experiment, in which RNase protection assay was used to measure receptor mRNA levels 3 h after E2 treatment of juvenile rats (Fig. 2), is most likely due to the fact that the epithelium accounts for only about 5% of the total uterine RNA [23], and the decrease in ER mRNA occurs mainly in the luminal epithelium in the immature rat uterus. In the adult uterus, the decreased expression in ER mRNA at 6 h occurred throughout the three uterine compartments, making the change apparent even on the total uterine RNA level. The decrease in ER protein after E2 treatment of adult and immature rats occurred primarily in the luminal and glandular epithelium. The periluminal stromal cells in the adult uterus and the majority of stromal cells of the juvenile uterus continued to express ER protein, which would be expected based on the fact that the proliferative response of the uterine epithelium is mediated by stromal ER [37–40]. The ER immunostaining seen throughout the stroma and in the myometrium of the immature rat uterus might be predicted based on the observation that all uterine compartments of immature rodents respond to E2 with increased DNA synthesis and cell division [21–24].
The functional implications of the decrease in ER mRNA and protein in uterine epithelial cells may be linked to DNA synthesis and cell proliferation. In immature rats, a second injection of E2 18 h after an initial hormone treatment caused a delay in epithelial DNA synthesis; the authors concluded that the level of ER must decrease before the onset of DNA synthesis [41]. Our data on epithelial cell expression of ER are compatible with such a hypothesis; furthermore, the initial cell-type specific decrease in ER is very similar to what has been observed for c-jun, an immediate early gene, at both the level of mRNA [25,34] and protein [42]. Perhaps a transient decrease in expression of the transcription factors, ER and c-Jun, plays a role in allowing uterine epithelia to undergo cell proliferation. It must be noted however, that epithelial cell proliferation results from a stromally derived signal following estrogen stimulation [40], and results of both in situ hybridization and immunohistochemistry indicate that there was no change, or even a slight increase in the stromal cell expression of ER. Additional studies will be required to address how the changes in the epithelial ER are related to cell cycle regulation.
While our results on ER immunostaining in the untreated juvenile rat uterus agrees with the constitutive pattern of ER protein expression reported in the immature mouse uterus [43], constitutive ER staining in mature rat uterus appeared slightly different than that reported in adult mice. In the present study, we observed equally strong ER staining in the uterine glandular and luminal epithelium and weak staining in the stroma of adult rat uteri. In ovariectomized adult mice, the glandular epithelium showed stronger ER immunostaining than the luminal epithelium, and the stroma showed strong ER staining [43,44], suggesting that the constitutive patterns of ER expression in adult rats and mice differ subtly. The use of different ER antibodies in those studies [43,44], however, could contribute to differences in ER staining in adult rodent uterus. The time course of E2 down-regulation of ER observed in the present study is similar to that described by others [8,10,12]; however, decreases in ER mRNA have been observed as early as 1 h after a supraphysiological dose of E2 [8,10], and Shupnik et al. [11] observed a decrease in uterine ER mRNA at 1 day, but not at 4 h, after a high dose of E2. Differences in animal models and hormone dose and delivery do not permit direct comparisons of our data with those earlier studies. Studies using estrogen responsive cell lines have also shown rapid down-regulation of ER by E2, occurring 1–4 h after hormone treatment [2–5]. Although continued suppression of ER levels were noted in those in vitro systems [2–5], our results on up-regulation of uterine ER by 24 h after hormone treatment and similar in vivo observations of others [9,12,16,17,45] indicate that E2 can cause a biphasic effect on ER expression in the uterus. Up-regulation of uterine ER by circulating levels of E2 has been reported in rats [13], mice [46], and sheep [47–49], and high doses of E2 increased ER in the rat liver and pituitary [11] and in Xenopus laevis liver [1]. There are no reports on the presence of an ERE in the promoter region of the ER gene, suggesting that, in addition to E2 levels, ER regulation is influenced by tissue-specific determinants or other factors such as nuclear receptor coactivators and corepressors [50,51]. Recently, however, a functional ERE has been reported in the protein coding region of the Xenopus laevis estrogen receptor gene [52].
In an attempt to investigate the mechanism of E2 down-regulation of ER mRNA in rat uterus in vivo, we studied the effect of inhibition of protein synthesis by cycloheximide. Cycloheximide alone had no effect on the level of ER mRNA in rat uterus, and cycloheximide did not block the effect of E2 on ER mRNA levels, suggesting that E2 suppression of receptor mRNA levels occurs independently of protein biosynthesis. Our results, together with observations that E2 decreases ER gene transcription rate in vitro [2–5] and that ER can bind to a portion of its cDNA [53], suggest a primary effect of the ER and support a possible involvement of negative EREs in estrogen receptor gene regulation. To date, however, there are no reports of a negative element that confers ER down-regulation, suggesting that other factors or mechanisms may be involved in autologous ER down-regulation. It is possible that E2 alters the stability of ER mRNA in the uterus, as has been shown to occur in sheep uterus [47,49] and human breast cancer cells [54].
In summary, our data on the cell-type specific expression of ER support observations in whole uterine homogenates that E2 down-regulates ER. We have further shown that autologous ER suppression occurs compartmentally in rat uterus, primarily in the uterine epithelia of sexually immature and mature animals. These observations may have implications on the role of rapid but transient suppression of ER in the mitogenic effects of E2 in rat uterus. In addition, the observation that E2 directly down-regulates expression of its own receptor in specific uterine cell types clearly shows that additional in vivo studies are required to delineate the regulation of ER gene expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work supported by NIH grant CA74748 to K.P.N., the Bert Elwert Research Award to K.P.N., and Department of Defense grant DAMD 17-98-1-8011 to R.M.B. 
2 Correspondence: Kenneth P. Nephew, Medical Sciences, Indiana University, School of Medicine, Jordan Hall, 1001 E. 3rd St., Bloomington, IN 47405-4401. FAX: 812 855 4436; knephew{at}indiana.edu
Accepted: August 24, 1999.
Received: June 4, 1999.
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