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Endocrinology Vol. 140, No. 9 3928-3937
Copyright © 1999 by The Endocrine Society

ARTICLES

Effect of Estrogen Agonists and Antagonists on Induction of Progesterone Receptor in a Rat Hypothalamic Cell Line

Susan L. Fitzpatrick, Thomas J. Berrodin, Susan F. Jenkins, Deborah M. Sindoni, Darlene C. Deecher and Donald E. Frail

Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087

Address all correspondence and requests for reprints to: Dr. Susan L. Fitzpatrick, Women’s Health Research Institute, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: fitzpas2{at}war.wyeth.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen is essential in the hypothalamus for the central regulation of reproduction. To understand the molecular mechanism(s) of estrogen action in the hypothalamus, immortalized rat embryonic hypothalamic cell lines were characterized for steroid receptors and subcloned. Scatchard analysis of the D12 subclone demonstrated one high affinity estrogen receptor-binding site (Kd = 31.3 ± 1.9 pM) with a Bmax of 30.8 ± 0.8 fmol/mg. Estrogen receptor-{alpha} protein was identified by Western blot and gel shift analyses. Treatment with estradiol (48 h) stimulated progesterone receptor (PR) messenger RNA expression and binding to [3H]R5020, a synthetic progestin. Because the agonist or antagonist activity of estrogen mimetics can be cell type dependent, the activities of various estrogen mimetics were determined in D12 cells. ICI 182,780 (IC50 = 0.63 nM), raloxifene (IC50 = 1 nM), enclomiphene (IC50 = 77 nM), and tamoxifen (IC50 = 174 nM) inhibited the induction of PR by estradiol, and none of these compounds significantly stimulated PR when given alone. In contrast, 17{alpha}-ethynyl estradiol (EC50 = 0.014 nM), zuclomiphene (EC50 = 100 nM), and genistein (EC50 = 17.5 nM) functioned as estrogen agonists in these cells. In addition, the estrogen-induced progesterone receptor activated a progesterone response element reporter construct in response to progestins. Thus, the D12 rat hypothalamic cell line provides a useful model for characterizing tissue-selective estrogenic compounds, identifying estrogen- and progesterone-regulated hypothalamic genes, and understanding the molecular mechanisms of steroid action in various physiological processes mediated by the hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN (E2) plays an important role in the hypothalamus by stimulating the synthesis and release of LHRH (1), which triggers the LH surge and ovulation. Sexual behavior (e.g. lordosis) in female rodents is regulated by the stimulation of the progesterone receptor (PR) by estrogen in the hypothalamus (2). Furthermore, in women, estrogen may regulate the set-point of the putative thermal regulatory center in the hypothalamus, which could explain the onset of hot flushes often seen at menopause (3, 4). Finally, estrogen can stimulate the expression of a number of genes in the hypothalamus. These include the receptors for progesterone (5, 6, 7) and oxytocin (8, 9), the transcription factor c-fos (10, 11), and the neuropeptides enkephalin (12, 13), oxytocin (14, 15, 16), and LHRH (17, 18, 19).

The majority of the actions of estrogen are believed to be mediated by nuclear estrogen receptors (ER), as the effects can be modulated by agonists and antagonists of these receptors. Classically, estrogen has been shown to bind to a nuclear ER, now referred to as ER-{alpha}, which then dimerizes, binds DNA, and activates gene transcription. Recently, a second ER gene, ER-ß, has been identified that shares homology to ER-{alpha} in the DNA- and ligand-binding domains, but is dissimilar in the N-terminal AF-1 trans-activation region (20, 21). The tissue distribution of ER-ß messenger RNA (mRNA) differs from that of ER-{alpha} (22, 23), with the former primarily expressed in prostate, ovary, and brain. ER-ß protein binds 17ß-estradiol with high affinity, like ER-{alpha}, but there are some differences in relative binding affinities for other ligands (22) as well as differences in transcriptional activity (24). In addition, ER-ß can form homodimers as well as heterodimerize with ER-{alpha} (25, 26). A unique physiological ligand or role for ER-ß has not been identified. Thus, there may be both overlapping and distinct physiological roles for ER-{alpha} and ER-ß.

ER-{alpha} and ER-ß mRNAs are present in the hypothalamus but with overlapping and distinct cellular distributions (23). Both ER-{alpha} mRNA and ER-ß mRNA are localized in the medial preoptic area and bed nucleus of the stria terminalis (27), whereas ER-ß mRNA alone is expressed in the paraventricular and supraoptic nuclei and ER-{alpha} mRNA predominates in the arcuate and ventromedial nuclei (23). 17ß-Estradiol (E2) induces PR mRNA expression in the medial preoptic nucleus of ovariectomized rats (5, 6, 7, 28), and this effect appears to be directly through ER, as functional estrogen response elements (ERE) are present in the rat PR promoter (29, 30), and the induction is blocked by ER antagonists (28). In ovariectomized ER-{alpha} knockout animals, PR mRNA is still induced in the preoptic area by estradiol (31), although to a lesser extent than in wild-type ovariectomized mice. This suggests that estradiol induction of PR in the hypothalamus can be mediated by ER-ß or by both receptors or that the receptors can compensate for each other.

The existence of two ER subtypes cannot fully explain the differential effects of various estrogen mimetics in a tissue. For example, tamoxifen is an ER antagonist in breast but an ER agonist in bone and uterus, leading to the concept of tissue-selective estrogens (reviewed in Ref. 32). As tamoxifen binds to ER-{alpha} and ER-ß with similar affinities (22), but results in differences in transcriptional activity (24), additional mechanisms must be involved. The mode of action (agonist vs. antagonist) in a cell can be modulated by tissue-specific metabolism, ligand-induced receptor conformation (33) and DNA binding kinetics (34), promoter elements (35, 36), or coactivator and corepressor proteins (37). The effects of tissue-selective estrogens have been primarily studied in bone, heart, uterus, and breast, with little investigation of their consequences on the hypothalamus.

Studies to elucidate the molecular mechanisms of estrogen action in the hypothalamus have been hindered by the limited number of cell lines available and the lack of cell lines representing different types of hypothalamic nuclei. The best characterized ER-positive hypothalamic cell line is the mouse GT1, an immortalized LHRH-expressing hypothalamic neuronal cell line (38). GT1 cells have been shown to have a high affinity ER (Kd = 0.11 nM) (39) that is ER-{alpha} based on sequencing of RT-PCR products (40) and respond to estrogen by increasing the number of androgen-binding sites (39) and galanin mRNA expression (40). Additional cell lines that represent other estrogen-responsive hypothalamic nuclei have been derived from immortalized embryonic rat hypothalami (41). Paradoxically, estrogen was reported to modulate the proliferation of two of these cell lines in opposing manners, suggesting the potential for distinct estrogen responses in different hypothalamic nuclei.

Because of the limited number of appropriate cell lines to study the biochemical mechanisms of estrogen action in the hypothalamus, we characterized a new rat hypothalamic cell line, referred to as D12, that expresses high levels of ER-{alpha} mRNA and protein and responds to estrogen by inducing the expression of PR mRNA. The D12 cell line represents a new cell-based model for elucidating the actions of estrogen and antiestrogens as well as progestins and antiprogestins in the hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of D12 cells
A collection of cell lines derived from embryonic rat hypothalami immortalized with the entire simian virus 40 (41) was obtained from Dr. Richard Robbins (Yale University, New Haven, CT). Morphological variations in rat hypothalamic cell cultures were noted as was inconsistent ER binding. One cell line was further subcloned by limiting dilution to separate the morphological and biochemical variations, resulting in a subclone with significant ER binding. This D12 subclone had a uniform morphology and reproducible binding in early passages. Periodically, new cells were thawed when the response to estrogen (low inducible R5020-binding activity) diminished.

Cell culture
D12 cells were routinely grown at 36 C in a humidified chamber with 5% CO2 in DMEM-Ham’s F-12 (1:1; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS (Life Technologies, Inc.), 100 U/ml penicillin (Life Technologies, Inc.), 100 U/ml streptomycin (Life Technologies, Inc.), and 1 mM GlutaMAX-1 (Life Technologies, Inc.). For experiments, the cells were plated in DMEM-Ham’s F-12 medium lacking phenol red and supplemented with 2% or 10% charcoal-stripped FCS (HyClone Laboratories, Inc., Logan, UT) and antibiotics. One or 2 days after plating, cells were treated with 17ß-estradiol (Sigma Chemcial Co., St. Louis, MO), raloxifene (synthesized in-house), enclomiphene (Hoechst Marion Roussel, Inc., Kansas City, KS), zuclomiphene (Hoechst Marion Roussel, Inc.), tamoxifen (Sigma Chemcial Co.), 17{alpha}-ethynyl estradiol (Sigma Chemical Co.), genistein (Research Biochemical, Inc., Natick, MA), and/or ICI 182,780 (Zeneca Pharmaceuticals, Mereside Alderley Park, UK) for 48 h. For competition assays, a range of hormone treatments (0.1 nM to 1 µM) was tested in the absence (agonist mode) or presence (antagonist mode) of 10 nM estradiol. For other experiments, cells were treated in the absence or presence of 10 nM estradiol or as otherwise indicated. Dimethylsulfoxide (DMSO; 0.01–0.1%), the solvent used for test compounds, served as the control treatment.

MCF-7 (human breast carcinoma) and T-47D (human breast carcinoma) cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM-Ham’s F-12 (1:1) with 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM GlutaMax-1 (all cell culture products from Life Technologies, Inc.).

Isolation of animals tissues
Ovarian, uterine, and hypothalamic tissues were obtained from adult Sprague Dawley female rats (Taconic, Germantown, NY). Lung tissue from adult rats was purchased from Harlan (Harlan Sprague Dawley, Inc., Indianapolis, IN). Animals were housed in light-controlled rooms with 12-h light, 12-h dark cycles and given food and water ad libitum. All protocols had the approval of the Radnor animal care and use committee, Wyeth-Ayerst Research (Radnor, PA).

RNA isolation and Northern blots
Total RNA was isolated using Trizol (Life Technologies, Inc.; tissue samples required homogenization in Trizol as directed by the manufacturer), treated with deoxyribonuclease (Roche Molecular Biochemicals), extracted with phenol chloroform, and precipitated with ethanol. Polyadenylated [poly(A)+] RNA was isolated from total RNA using the FastTrack 2.0 Kit (Invitrogen, San Diego, CA). Rat liver poly(A)+ RNA was purchased from CLONTECH Laboratories, Inc. RNA (5 µg, except 2 µg for ovary) was size separated on formaldehyde-containing agarose (1%) gels with 0.2 M 3-[N-morpholino]propanesulfonic acid, 0.05 M NaOAc, and 0.1 M EDTA buffer (pH 7). Molecular size markers (0.24–9.5 kb) (Life Technologies, Inc.) were included for size determination. The RNA was transferred to Hybond N+ (Amersham Pharmacia Biotech, Arlington Heights, IL) nylon membrane. Probes were prepared by random priming with [32P]deoxy-CTP (NEN Life Science Products, Boston, MA) and the free [32P]deoxy-CTP was removed. The denatured probe was diluted in Rapid-hyb buffer (Amersham Pharmacia Biotech; 2 x 106 cpm/ml) before adding to the blot. Hybridization and washes were carried out at 65 C (Hybaid oven). The final wash (1–2 x, 15 min, 65 C) was performed with 0.1 x SSC (saline sodium citrate; 1 x = 30 mM sodium citrate, pH 7.5, and 0.15 M NaCl) and 0.1% SDS buffer.

For ER-{alpha}, full-length rat ER-{alpha} complementary DNA (cDNA) was used as probe, and for ER-ß, rat ER-ß cDNA nucleotides (nt) 417-1874 (accession no. RNU57439) was used as probe. The signal from the human PR-B probe (3174-bp cDNA beginning 176 bp 5' to ATG for PR-B; Dr. Nancy Wiegel, Baylor College of Medicine, Houston, TX) was quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The blot was stripped and reprobed with rat A1, a nonestrogen regulated gene (42).

Transfection
D12 cells were seeded in 24-well plates at a density of 40,000 cells/well in phenol red-free DMEM-Ham’s F-12 containing 10% charcoal-stripped FBS (Life Technologies, Inc.) and antibiotics. The next day the cells were treated with DMSO (0.1%) or 10 nM E2 for 48 h. T47D cells were plated in RPMI 1640 medium containing 10% FBS and bovine insulin (0.2 IU/ml; Life Technologies, Inc.) at a density of 100,000 cells/well. Each cell line was transfected in OptiMEM medium (Life Technologies, Inc.) with LipofectAmine (3 µl/well; Life Technologies, Inc.) and 0.8 µg/well E1bPRE (PRE, progesterone response element) luciferase (43) or mouse mammary tumor virus (MMTV) luciferase plasmid DNA (provided by Dr. Keith Marske, Ligand Pharmaceuticals, Inc., San Diego, CA). After 6 h, the medium was replaced with fresh growth medium containing 2% charcoal-stripped FBS with or without 1.0 µM 6{alpha}-methyl-17{alpha}-hydroxyprogesterone acetate (MPA; Sigma). Twenty hours later, the cells were washed twice in PBS, overlayed with 60 µl Reporter Lysis Buffer (Promega Corp., Madison, WI) and lysed by freeze/thaw. The luciferase assay was performed using 20 µl extract according to the manufacture’s instructions and quantified in a EG&G Berthold MicroLumat LB 96 P luminometer (Wallac, Inc., Gaithersburg, MD). Background values were subtracted from each data point. Data are expressed as the mean counts ± SD of triplicate values.

Protein isolation
D12 cells were scraped in Dulbecco’s PBS (BioWhittaker, Inc., Walkersville, MD; Life Technologies, Inc.). The cells were pelleted by centrifugation and lysed by homogenization (BioSpec Products, Inc. Tissue-Tearor, Dremel, Racine, WI) in ice-cold 20 mM HEPES (pH 7.8), 1 mM EDTA, and protease inhibitors (Roche Molecular Biochemicals). The whole cell soluble fraction was obtained by ultracentrifugation, and the protein concentration was determined by the Bradford assay (Pierce Chemical Co., Rockford, IL; Bio-Rad Laboratories, Inc., Hercules, CA). Whole cell extracts from human T47D cells and rat (ovariectomized) uterine tissue were prepared the same way. MCF-7 cell nuclear extract was prepared as described by Dignam et al. (44). Rat protein from hypothalamus and uterus were prepared by mincing the tissues and homogenizing in ice-cold HEPES-EDTA in the presence of protease inhibitors as done for the D12 cells.

Electrophoretic mobility shift assays (EMSAs)
Soluble whole cell extracts, prepared in HEPES-EDTA buffer, from D12 cells (25 µg), or nuclear extracts from MCF-7 cells (10 µg) were incubated with an {alpha}-32P-labeled ERE from the vitellogenin gene (vERE; CCAAAGTCAGGTCACAGTGACCTGATCAAAGTTAATGTAACCT-CA; 30,000 cpm) in the presence of 20 mM HEPES (pH 7.8), 80 mM KCl, 10% glycerol, 2 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.2 µg/µl polyd(I-C) (Pharmacia Biotech, Piscataway, NJ), and 0.025 µg/µl denatured herring sperm DNA (Sigma Chemical Co.) for 1 h at room temperature. For ER supershift experiments, SRA 1000 monoclonal antiserum [targeted to the hinge region of human ER-{alpha} amino acids (aa) 287–300 (StressGen, Victoria, Canada) or AB1–3 polyclonal antiserum (Wyeth-Ayerst Research) against the AB domain of human ER-{alpha} aa 1–177] were incubated with extracts for 1 h at room temperature, then probe was added for an additional hour at room temperature. Bound and free vERE were separated by electrophoresis on nondenaturing 6% polyacrylamide gels (0.5 x Tris, boric acid, EDTA) at 4 C. Gels were dried and exposed to XAR film (Eastman Kodak Co., New Haven, CT) with an intensifying screen at -80 C.

Western blots
Proteins were fractionated by electrophoresis on 8% SDS-PAGE. The gel was transferred to an Immobilon-P (Millipore Corp., Bedford, MA) polyvinylidene difluoride membrane overnight, blocked with 5% Carnation dry milk (Nestlé USA, Solon Ohio) and incubated 2 h with a 1:1000 dilution of mouse monoclonal antiserum raised against human ER-{alpha} (aa 578–595; this epitope is not found in ER-ß; Chemicon 464, Chemicon International, Temecula, CA) or human PR (aa 922–933; Chemicon 462). Colored protein mol wt markers (Amersham Pharmacia Biotech) were included for size determination. The blot was then incubated 1 h with a 1:10,000 dilution of goat antimouse IgG conjugated to horseradish peroxidase (Roche Molecular Biochemicals), washed, and detected with chemiluminescence (Amersham Pharmacia Biotech).

Binding assays
D12 protein extracts (100–200 µg) were incubated in triplicate with 1–500 pM [125I]16{alpha}-iodo-3,17ß-estradiol (NEN Life Science Products) with or without 1 µM diethylstilbesterol (DES; Sigma Chemical Co.) for 2 h at room temperature. Free ligand was removed with a 5% Norit SX-4 charcoal (EM Science, Gibbstown, NJ)-0.05% dextran T70 (Pharmacia, Uppsala, Sweden) suspension, and the bound radioactivity was determined with an ICN (Costa Mesa, CA) 10/600 {gamma}-counter.

Fresh soluble protein extracts (100–200 µg) were incubated in triplicate with (10–6000 pM) [3H]R5020 (NEN Life Science Products) and 100 mM dexamethasone with or without 1 µM unlabeled R5020 (NEN Life Science Products; for Scatchard plots) or 10 nM [3H]R5020 with or without 1 µM (100-fold excess) unlabeled R5020. Binding was performed for 1–2 h at room temperature under equilibrium conditions. Unbound ligand was removed by charcoal as described above, and the bound radioactivity was quantified in a Beckman Coulter, Inc. (Fullerton, CA), LS 6500 scintillation counter.

Statistical analysis
A three-parameter logistic model with parameters Kd, binding capacity (Bmax), and slope was fitted to evaluate the two-site saturation models. If the slope estimate indicated a one-site model (slope not significantly different from 1), the slope was locked to 1, and the analysis was rerun to provide a linear Rosenthal plot. In contrast, if the slope differed significantly from 1, a curvilinear plot was generated, and the two-site saturation model was run to determine the binding parameters of each binding site. Using this procedure for the data report herein, we were able to demonstrate that a one-site model was suited for our data. Values reported for Kd and Bmax were based on a one-site analysis where C = 1, using our customized JMP programs. The customized JMP applications were developed by Biometrics Research (Wyeth-Ayerst Laboratories, Inc., Princeton, NJ). Graphical representation for the Rosenthal plots (inset) was generated using SigmaPlot (Jandel Corp., San Rafael, CA) linear regression analysis of the estimated mean data for bound (femtomoles per mg protein) vs. bound/free. Data points shown for the saturation plot are the mean disintegrations per min for the total, nonspecific, and specific binding, and the hyperbola single rectangular, two-parameter equation (SigmaPlot) was used to generate the curve fit.

Statistical analysis was performed on R5020 data using a scripted (Krishnendu Ghosh, Wyeth-Ayerst Research) JMP package (SAS Institute, Inc., Cary, NC). Square root transformation with Huber weighting was used. Values are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of ER in D12 hypothalamic cells
Detection of ER protein by Western blot analysis. To determine whether the D12 cell line contained ER, RNA was isolated from cells treated with DMSO or 10 nM estradiol for 2 days and probed with rat ER-{alpha} or rat ER-ß cDNAs. As shown by Northern blot analysis, both ER-{alpha} (Fig. 1Go, left panel) and ER-ß (Fig. 1Go, right panel) mRNAs were expressed in D12 cells, and the expression was not regulated by estradiol treatment. Note that the ER-{alpha} blot was exposed for only 1 day, whereas the ER-ß blot was exposed for 1 week.



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  		Figure 1. ER-{alpha} and ER-ß mRNA expression in D12 cells. Poly(A)+ RNA (5 µg) isolated from D12 cells treated with DMSO or 10 nM E2 for 2 days. As a positive control, poly(A)+ RNA from rat ovary (2 µg) and rat liver (5 µg) were included on the blot. ER-{alpha} mRNA was detected on a Northern blot using a rat ER-{alpha} cDNA probe (left panel), whereas ER-ß was detected using a rat ER-ß cDNA probe (right panel). The ER-{alpha} blot was exposed to autoradiographic film for 1 day at -80 C, whereas the ER-ß blot was exposed to film for 1 week at -80 C. The mol wt size markers (in kilobases) are indicated on the left.

 
To determine whether ER-{alpha} and/or ER-ß protein was present in these cells, whole cell extracts were isolated from the same cells. The proteins, along with protein extracts from rat hypothalamus, rat pituitary, and human MCF-7 breast cells, were analyzed by Western blot with Ab 464, an ER-{alpha} monoclonal antiserum. As expected, a predominant band corresponding to the known molecular mass of ER-{alpha} (67 kDa) was detected in the hypothalamus and MCF-7 cells (Fig. 2Go). A comigrating band was also detected in D12 cells, indicating the presence of ER-{alpha}. The amount of ER-{alpha} protein was not altered by estradiol treatment. Using a commercially available antiserum to ER-ß 6–629(6–629, Upstate Biotechnology, Inc., Lake Placid, NY) or our in-house ER-ß antibody, RB 2.3, we were not able to detect ER-ß protein by Western blot or EMSA (data not shown).



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  		Figure 2. Expression of ER-{alpha} protein in D12 cells. Soluble protein extracts (50 µg) from human MCF-7 cells, D12 cells treated without and with E2 (10 nM) for 2 days, and rat hypothalamus were size separated and immunoblotted. ER-{alpha} protein was detected with the monoclonal antiserum, Ab 464. The migration of protein size markers (in kilodaltons) is shown on the left.

 
Scatchard analysis of ER in D12 cells. To further substantiate the presence of ER in D12 cells, whole cell extracts were incubated with increasing amounts of [125I]16{alpha}-iodo-3,17ß-estradiol in the absence and presence of excess unlabeled DES. Specific, saturable binding sites were observed (Fig. 3Go). Scatchard analysis (Fig. 3Go, inset) revealed a single high affinity binding site with a slope of 0.97. Therefore, binding parameters were determined using a locked slope of 1 as indicated in Materials and Methods. Linear regression gave a Kd of 31.3 ± 1.9 pM and a Bmax of 30.8 ± 0.8 fmol/mg protein.



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  		Figure 3. Saturation of [125I]16{alpha}-iodo-estradiol binding to D12 cell extracts. Varying concentrations of radioligand (1–500 pM) with or without 1 µM DES were incubated with protein extracts (100 µg) from D12 cells for 2 h at room temperature. Total (•), nonspecific ({circ}), and specific ({blacksquare}) binding were determined as described in Materials and Methods. Data are the mean of triplicate determinations. Line estimations for the saturation isotherms were generated by the SigmaPlot (Jandel Scientific) curve-fitting program using hyberbolic, single rectangular, two-parameter fitting. Inset, Saturation transformation generated by JMP analysis revealed a Kd value of 31.3 ± 1.9 pM, and a Bmax value of 30.8 ± 0.8 fmol/mg protein.

 
Characterization of ER in D12 cells by EMSA. To determine whether ER from D12 cells could bind DNA, an EMSA was performed. A consensus ERE oligonucleotide from the vitellogenin promoter was labeled with [32P]deoxy-CTP and incubated with whole cell extracts from D12 cells. As shown in Fig. 4Go, protein from D12 cells bound the ERE oligonucleotide (lane D12; -), and this complex was supershifted in the presence of monoclonal antiserum (SRA 1000, which recognizes the hinge region; +a) or polyclonal antiserum (AB 1–3, which recognizes the TAF-1 domain; +b) to ER-{alpha}. These results are identical to those obtained using extracts prepared from MCF-7 cells, a cell line known to contain ER (Fig. 4Go).



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  		Figure 4. Binding of D12 extracts to a consensus ERE. EMSAs were performed with a 32P-labeled ERE oligonucleotide probe (30,000 cpm) and D12 whole cell extracts (25 µg) or MCF-7 nuclear extracts (10 µg). Extracts were incubated in the absence (-) or presence (+) of SRA 1000, an ER-{alpha} monoclonal antiserum (a), or AB1–3, an ER-{alpha} polyclonal antiserum (b), for 1 h at room temperature. Labeled probe was then added for an additional 1 h at room temperature. Bound and free complexes were separated by electrophoresis on 5% native polyacrylamide gels performed at 4 C.

 
Induction of PR in D12 cells
Effects of estradiol on PR mRNA levels. Induction of a target gene, PR, known to be regulated by estrogen in the hypothalamus (5, 6, 7), was examined to determine whether the D12 cell ER was functional. Poly(A)+ RNA isolated from control and E2-treated D12 cells, rat lung, and rat uterine tissue was size separated, transferred to membrane, and probed with a rat PR cDNA fragment (Fig. 5AGo). The results were normalized against the expression of A1 (42), a gene not regulated by estrogen (Fig. 5BGo). As expected, PR mRNA was undetectable in the lung (Fig. 5AGo, lane 1), but multiple transcripts, characteristic of PR mRNA (45), were observed in the uterine sample (Fig. 5AGo, lane 2). D12 cells grown in charcoal-stripped serum (- E2) contained low levels of PR mRNA (Fig. 5AGo, lane 3). Addition of E2 (10 nM) for 2 days increased PR mRNA levels 4.5-fold (Fig. 5AGo, lane 4).



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  		Figure 5. Induction of PR mRNA by estradiol in D12 cells. Poly(A)+ RNA (5 µg) was isolated from rat lung, uterus, and D12 cells (treated without and with 10 nM E2 for 48 h) and analyzed by Northern blot for PR (A). Markers in kilobases are shown on the left. The blot was stripped and reprobed with 32P-labeled A1 cDNA (49 ) (B).

 
Effects of estradiol on PR protein levels. Soluble protein was isolated from untreated D12 cells, 10 nM E2-treated D12 cells, rat uterine tissue, and human T47D cells and analyzed by Western blot to determine whether the induction of PR by E2 also occurred at the protein level (Fig. 6Go). In rat uterus and T47D cells, which served as positive controls, both PR-A (46) and PR-B (46) were detected. D12 cells grown in the absence of estrogen have low levels of both PR-A and PR-B. Treating the cells for 2 days with estradiol increased the levels of both PR-A and PR-B approximately 4-fold.



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  		Figure 6. Induction of PR protein by estradiol treatment of D12 cells. Whole cell proteins (25–100 µg) from D12 rat cells treated without and with 10 nM E2, rat uterus, and human T47D cells were size separated and immunoblotted with a mouse monoclonal PR antiserum Chemicon 462. Protein mol wt markers are shown on the left. PR-A and PR-B are indicated by arrows.

 
Estradiol increases PR binding activity. E2 induction of PR was quantified using a ligand binding assay. Whole cell extracts from D12 cells were incubated with [3H]R5020, a synthetic progestin, in the absence (total counts per min) or presence (nonspecific counts per min) of a 100-fold molar excess of unlabeled R5020. D12 cells grown in charcoal-stripped serum without exogenous estrogen had low specific R5020 binding (Fig. 7AGo, control). Treating the cells for 2 days with 10 nM E2 resulted in a 7- to 8-fold increase in specific R5020 binding (Fig. 7AGo, +E2). This induction was reduced to control levels when the cells were cotreated with 10 nM E2 and the estrogen antagonist ICI 182,780 (1 µM; Fig. 6Go, A and E2+ICI). Basal R5020 binding activity was observed in D12 cells grown in the presence of ICI alone (Fig. 7AGo, +ICI).



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  		Figure 7. Effect of estradiol treatment on [3H]R5020 Binding to D12 extracts. A, D12 cells were treated with E2 (10 nM) alone, with ICI 182,780 (1 µM) alone, or with the two in combination for 48 h. Untreated D12 cells served as a control. The whole cell protein extracts (100 µg) were incubated with 10 nM [3H]R5020 in the absence (total) or presence (nonspecific) of 100-fold molar excess of unlabeled R5020 for 2 h at room temperature. Nonspecific binding was subtracted from total binding to calculate specific binding. The data represent the mean and SD of triplicate values from a representative experiment. B, D12 cells were treated with increasing concentrations of E2 for 48 h. Whole cell protein extracts (150 µg) were incubated with 10 nM [3H]R5020 in the absence (total, {blacksquare}) or presence (nonspecific, •) of a 100-fold molar excess of unlabeled R5020 for 2 h at room temperature. Nonspecific binding was subtracted from total binding to calculate specific binding ({blacktriangleup}). Results are the mean and SD of triplicate values.

 
D12 cells were treated with increasing concentrations (0.1 pM to 100 nM) of E2 for 2 days to determine the potency of E2 in inducing the PR. R5020 binding assays were performed as mentioned above. As indicated by the specific counts per min (Fig. 7BGo), maximal induction of R5020 binding occurred at 1 nM E2, with an EC50 of 23 pM. The EC50 is similar to the binding affinity of E2 to its receptor in these cells.

Effects of estrogen antagonists and agonists on R5020 binding. Compounds that bind to the ER and display tissue-selective agonist or antagonism were tested in D12 cells. To test for antagonist activity, D12 cells were treated for 2 days with 0.1 nM E2 (EC80) in the absence or presence of increasing concentrations (0.1–1000 nM) of test compound. To test for agonist activity, D12 cells were treated for 2 days with increasing concentrations (0.1–1000 nM) of test compound. The average IC50 and efficacy (percentage) for each compound tested are shown in Table 1Go. Raloxifene, enclomiphene (geometrical isomer of clomiphene), tamoxifen, and ICI 182,780 reduced R5020 binding induced by E2, and no apparent agonist activity was observed. In contrast, zuclomiphene (geometrical isomer of clomiphene) and 17{alpha}-ethynyl estradiol stimulated R5020 binding, and no antagonist activity was observed. The natural product phytoestrogen, genistein, acted as an agonist with an EC50 of 17.5 nM in D12 cells.


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  		Table 1. Antagonist and agonist activities Antagonist mode

 
Characterization of PR activity in D12 cells
R5020 binding affinity and specificity. To demonstrate the binding activity of PR in D12 cells, increasing concentrations of [3H]R5020 were incubated with protein extracts from estrogen-treated D12 cells in the absence or presence of excess unlabeled R5020 (1 µM). To prevent binding of [3H]R5020 at high concentrations to the glucocorticoid receptor, 100 nM dexamethasone was included in the reaction. As shown in Fig. 8Go, specific, saturable binding sites were observed. Scatchard analysis (Fig. 8Go, inset) revealed a single, high affinity binding site with a slope of 0.93. Therefore, binding parameters were generated using a locked slope value of 1, as indicated in Materials and Methods. Linear regression gave a Kd of 536 ± 30.8 pM and a Bmax of 290 ± 5.2 fmol/mg protein. To validate the specificity of this interaction, compounds that bind to various other steroid receptors were included in the binding reaction. Binding was competed by unlabeled R5020 (1 µM) or progesterone (1 µM), but not by dexamethasone (50 nM), dihydrotestosterone (100 nM), or aldosterone (20 nM; data not shown).



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  		Figure 8. Saturation of [3H]R5020 binding to D12 extracts. Protein extracts (150 µg) from E2-treated D12 cells were incubated with increasing concentrations (10–6000 pM) of [3H]R5020 as described in Fig. 6Go. Dexamethasone (100 nM) was also included in the binding reactions. Total (•), nonspecific ({circ}), and specific ({blacksquare}) binding values are indicated and are the mean and SD of triplicate values. Line estimations for the saturation isotherms were generated by the SigmaPlot (Jandel Scientific) curve-fitting program using hyberbolic, single rectangular, two-parameter fitting. Inset, Saturation transformation generated by JMP analysis revealed a Kd value of 536 ± 30.8 pM and a Bmax value of 290 ± 5.2 fmol/mg protein.

 
Estrogen-induced PR regulates PRE activity. To demonstrate the functionality of the estrogen-induced PR, D12 cells were transfected with PRE/glucocorticoid response element-containing expression vectors. D12 cells were treated for 48 h with 10 nM E2 to induce PR expression, then transfected with E1bPRE- or MMTV-PRE-luciferase plasmids that contain PRE elements within the context of different promoters. The luciferase assay was performed after treatment with MPA (1 µM) for 20 h. Hormone treatment enhanced luciferase activity 3.8- and 2.7-fold in treated cells transfected with E1bPRE-luciferase and MMTV-luciferase, respectively (Fig. 9Go). This is much lower than the 400- to 600-fold induction seen in T47D cells and is attributed at least partially to the higher basal PRE activity in D12 cells. Similar induction of PRE activity by other progestins (R5020 or levonorgestrel) was observed in D12 cells, and the induction was inhibited by the addition of the antiprogestin, RU 486 (data not shown).



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  		Figure 9. Estrogen-regulated PRE activity in transfected D12 cells. D12 cells treated with 10 nM E2 for 48 h or untreated T47D cells were transfected with E1bPRE- or MMTV-luciferase plasmids. Six hours later the DNA was removed, and the cells were treated with or without 0.01 or 1.0 µM MPA for an additional 20 h. Luciferase activity was quantified in a luminometer. Error bars represent the mean ± SD of triplicate values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogen is a key modulator of the hypothalamus, particularly for regulating reproductive function. Several difficulties emerge when trying to elucidate the mechanism of action of estrogen in the hypothalamus. First, the effect of estrogen may be direct or indirect. A number of genes have been identified that are activated by the ER in the hypothalamus, although only a few have been shown to contain consensus EREs [e.g. PR (29, 30)]. In contrast, the lack of ER in estrogen-responsive LHRH neurons in vivo suggests an indirect modulation via nongenomic, trans-synaptic, or glial cell-mediated interaction (reviewed in Ref. 19). A second difficulty is the potential number of complexes involved in estrogen action. For example, ER-{alpha} and ER-ß are located in distinct and overlapping regions of the hypothalamus, they can form homo- or heterodimers (25, 26), and each can be modulated by different accessory proteins, e.g. coactivators and corepressors (37). Thus, estrogen may exert cell-specific actions in the hypothalamus of the rat and mouse. Lastly, studies in vitro have been hampered by the limited availability of hypothalamic cell lines and cell lines representing different hypothalamic cell types. The best characterized hypothalamic cell line, GT1, was derived from a mouse tumor expressing LHRH (38). Unlike their endogenous counterpart (reviewed in Refs. 17, 18, 19), these cells contain measurable ER-{alpha} mRNA and bind [3H]estradiol with high affinity (Kd = 0.11 nM; Bmax = 6.2 fmol/mg protein) (39, 40). Induction of androgen-binding sites (39) and galanin mRNA (40) by estradiol indicates that the ER-{alpha} in these cells are functional.

The data presented herein characterize a new cell line, D12, from a collection of immortalized embryonic hypothalamic cells (41) that are immunopositive for neurofilament, but not glial fibrillary acidic protein (data not shown). D12 cells express ER-{alpha} protein and respond to estradiol by inducing PR mRNA expression. Thus, it is a good model for studying the actions of both estrogen and progesterone in the hypothalamus. The presence of high affinity, functional estrogen receptors was demonstrated by radioligand binding and activity assays. In fact, the lower Kd (35 pM) and EC50 (23 pM; PR induction) in D12 cells compared with those in the uterus (Kd = 820 pM) (47) suggests that D12 cells, and the hypothalamus in general (28), may be more sensitive than the uterus to low concentrations of estradiol. Induction of PR mRNA and protein by estradiol and inhibition by the estrogen antagonist ICI 182,780 indicate that the ER in these cells is functional.

Both ER-{alpha} and ER-ß mRNAs were observed in D12 cells by Northern blot analysis; however, whereas ER-{alpha} protein was readily detectable by Western blot analysis (and EMSA; data not shown), ER-ß protein was not detected by Western blot or EMSA using several commercially available antisera as well as an in-house ER-ß antiserum (data not shown). Indeed, the gel shift band produced from D12 cell extracts was completely supershifted by ER-{alpha} antiserum. No supershift was observed with antiserum against ER-ß (data not shown). Within the hypothalamus, the expressions of ER-{alpha} and ER-ß mRNAs are both distinct and overlapping. Areas such as the arcuate and ventromedial hypothalamic (VMH) nuclei contain predominantly ER-{alpha} mRNA, areas such as the paraventricular and supraoptic nuclei contain predominantly ER-ß mRNA, and areas such as the bed nucleus of the stria terminalis and preoptic areas (POA) contain both ER-{alpha} and ER-ß mRNAs (23, 27). Furthermore, estrogen induction of PR mRNA occurs in hypothalamic areas that contain ER-{alpha}, including the arcuate nuclei, VMH, and the POA (5, 6, 7, 28). However, PR mRNA expression was stimulated by estrogen in the arcuate nuclei, VMH, and POA of ER-{alpha} knockout animals (31, 48), although to a lesser extent than in wild-type animals, suggesting that PR mRNA expression could be regulated by a splice variant of ER-{alpha}, ER-ß, or some other mechanism. Based on the ratio of ER-{alpha}/ER-ß mRNA expression and the induction of PR mRNA by estrogen, the likely source of the D12 cells is the medial basal hypothalamus (arcuate, VMH) or possibly the POA.

Estrogen mimetics can act as agonists or antagonists depending on the cell type in which they are tested. Cellular models to predict the activity of estrogen mimetics in the hypothalamus are lacking. Therefore, the activities of various compounds was assessed in D12 cells by measuring the induction of PR protein and compared with the in vivo activity observed. In D12 cells, tamoxifen, raloxifene, and ICI 182,780 acted as antagonists with no agonist activity. Similarly, tamoxifen and raloxifene demonstrated antagonist activity (inhibition of 17ß-estradiol-induced PR mRNA expression) but no agonist activity in the rat hypothalamic POA in vivo (28). In contrast, zuclomiphene and ethynyl estradiol acted as estrogen agonists in D12 cells by inducing PR ligand binding activity.

The effects of tissue-selective estrogens on estrogen-regulated rodent behavior have been investigated. Maternal behavior, e.g. pup retrieval, can be inhibited by implanting 4-hydroxytamoxifen in the medial POA of the hypothalamus before parturition (49). Sexual behavior can also be modulated by estrogen agonists and antagonists. Lordosis behavior in rats can be induced by treatment with estrogen followed by progesterone. Tamoxifen antagonizes the estrogen effect and inhibits lordosis (50, 51, 52, 53) as well as the induction of PR mRNA (28, 50, 53). Similarly, raloxifene can block lordosis (54) and the LH surge (55). Therefore, both tamoxifen and raloxifene appear, at least in the cases studies, to act as estrogen antagonists in rats as well as humans. As similar antagonistic action was observed in D12 cells, the ability of a compound to modulate PR regulation in D12 cells might be predictive of the estrogenic activity of the compound in vivo.

In humans, tamoxifen has been shown to act as an estrogen agonist in bone but an estrogen antagonist in breast (reviewed in Ref. 32). Clinical studies of breast cancer patients treated with tamoxifen suggest that it acts as an estrogen antagonist in the brain, as some women experience hot flushes during treatment (56). Raloxifene is an estrogen agonist in bone and has little agonist activity in the uterus (reviewed in Ref. 32). Clinical studies with raloxifene suggest that it also acts as an estrogen antagonist in the hypothalamus, as patients experienced hot flashes at certain doses (57). ICI 182,780, a pure antiestrogen, did not induce or inhibit hot flushes in postmenopausal women with breast cancer (58), probably because the compound reportedly does not cross the blood-brain barrier (59).

Furthermore, because of the interest in phytoestrogens and their possible use as a chemopreventive agents or to prevent bone loss after menopause, the ability of genistein, an isoflavone, to induce PR expression was assayed. In D12 cells, genistein acted as an estrogen agonist and at a concentration (EC50 = 17.5 nM) that is 1000-fold lower than the concentration required (10–300 µM) for genistein to act as a tyrosine kinase inhibitor (60, 61). Soy products, which are a rich source of genistein, and ipriflavone, a synthetic isoflavone, have been shown to have estrogenic activity in bone and in the cardiovascular system in animals and humans (reviewed in Refs. 62, 63). In several studies, soy products had weak estrogenic activity in the brain by reducing the incidence of hot flushes in postmenopausal women (62, 63).

D12 cells can also be used to study the activity of progestins. PR protein synthesis was proportional to the exposure of the cells to estrogen, and synthesis was inhibited in the presence of ICI 182,780. Both PR-A and PR-B protein were detected by Western blot. PR protein was also detected in a binding assay using a synthetic progesterone agonist, R5020. Finally, PR protein induced by E2 was shown to regulate expression of two PRE luciferase constructs, suggesting that D12 cells could be used to identify and characterize progesterone-induced genes in the hypothalamus.

In conclusion, estrogen plays a critical role in the hypothalamus by regulating reproductive functions in both rodents and humans and, among others, modulating the incidence of hot flashes in postmenopausal women. D12 immortalized embryonic hypothalamic cells serve as an excellent model to help elucidate the molecular mechanisms of estrogen and progesterone activities in the hypothalamus. The response of D12 cells to estrogen agonists and antagonists mimics that seen in rat hypothalamus in vivo and might predict the mode of action of new compounds in humans.


   Acknowledgments
 
We thank Krishnendu Ghosh, Chen-Shian Suen, Linda Shanno, Paul Shughrue, and Istvan Merchanthaler for their input into this work.

Received January 8, 1999.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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