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Biology of Reproduction 60, 1204-1210 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Estrogen Responses in Bovine Fetal Uterine Cells Involve Pathways Directed by Both Estrogen Response Element and Activator Protein-11

J.R. Malayer2,a, J. Chenga, and V.M. Woodsa

a Department of Infectious Disease and Physiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078-2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ER{alpha} or ERß were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ER{alpha} or ERß, treatment with estradiol-17ß (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ER{alpha} and ERß. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ER{alpha} and ERß are present prior to expression of the receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens are key molecules in development, differentiation, and growth, whose actions are mediated by specific cellular receptor proteins localized in the nucleus of the cell (for reviews, see [1–5]). We have previously shown that estrogen receptor (ER) expression in the uterus of the bovine fetus is first detected in the sixth month of pregnancy [6]. It is hypothesized that ER expression at that time is essential for uterine growth and initiation of events in mucosal reorganization associated with uterine gland development.

The presence of ER{alpha} [7] and ERß [8] has now been described in various organ systems, including the bovine uterus [9, 10], and ER{alpha} and ERß have been shown to exert a variety of physiologically important activities. The global nature of estrogen action in growth and development has been demonstrated by examples of naturally occurring ER mutations and through use of transgenic animal technology in mice [11, 12]. Evidence suggests that ERß has a unique set of activities apart from ER{alpha} [13, 14], and that ER{alpha} and ERß may interact as heterodimers [15]. Studies in various tissues have shown that ER expression is the only factor lacking in estrogen-nonresponsive cells to bring about estrogen-dependent target gene expression.

It has been established in certain estrogen target cells that ligand-bound ER has the capacity to act through different types of DNA elements. The classical estrogen response element (ERE) consists of a palindromic sequence of GGTCA in an inverted repeat separated by a three-base "spacer" (), although most estrogen-responsive genes contain ERE sequences that diverge to varying degrees from this consensus sequence. In addition, ER mediates transcription from the activator protein-1 (AP-1) enhancer sequence (TGA GTC A) [16–18]. This "AP-1-directed" mechanism involves the ER in protein-protein interactions partially independent of the ER DNA-binding activity, requires the receptor to be ligand-bound, and involves the transcription factors c-fos and c-jun, which bind the DNA as a dimer at the AP-1 site and mediate the ER action [19]. In addition, it has been established that the receptor may be activated in the absence of estrogen through posttranslational phosphorylation of specific serine residues, resulting in so-called ligand-independent activation [20, 21].

Our objective was to test the hypothesis that cells of the bovine fetal uterus contain components necessary for ERE- and AP-1-mediated transactivation, as well as for ligand-independent activation of ER. In addition, we wished to test whether these components were expressed in the cell prior to ER expression, or coincident with ER expression. To explore these questions, cell cultures were prepared from animals early in the second trimester of pregnancy (Days 110–120), prior to ER expression, and at the end of the second trimester (Days 180–200) after ER expression commenced. Cells collected prior to ER expression were transfected with ER{alpha} or ERß expression vectors and reporter gene constructs containing ERE or AP-1 enhancer elements; cells collected after ER expression were transfected with the same reporter constructs to compare activation of endogenous receptor. These data should contribute to understanding of processes related to uterine growth and maturation involving the ER.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection and Sample Preparation

In order to obtain fetal bovine tissues, entire uteri were removed from pregnant, crossbred beef cows at a commercial abattoir. The gestational age of each fetus was estimated by fetal crown-rump measurement [22]. Fetal uteri were dissected free of adipose and connective tissue, placed in sterile tubes containing phenol red-free Dulbecco's Modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD) containing single-strength antibiotic-antimycotic (ABAM; Gibco-BRL), Hepes (5 mM; Sigma Chemical Co., St. Louis, MO), and sodium bicarbonate (Sigma) and transported to the laboratory.

Fetal reproductive tract tissues were used to prepare primary cell cultures as previously described [23]. The cultures were mixed populations of epithelial and stromal cells that equally expressed vimentin and cytokeratin, as determined by immunostaining [23]. Cells used in these experiments represent pooled samples from three individuals in each of the age ranges described. These cells from the "uterus of the bovine fetus" (UBF cells) were plated in 6-well culture plates (Becton-Dickinson Labware, Franklin Lakes, NJ) on collagen matrices (type I collagen, from calf skin, 10 µg/cm2; Sigma). Cells were maintained in phenol red-free DMEM containing single-strength ABAM, 5 mM Hepes, sodium bicarbonate supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), and insulin (0.6 µg/ml; porcine insulin; Gibco-BRL). Cultures were maintained at 37°C in a humidified atmosphere of 95% air:5% CO2 and fed every 48 h. After one to two passages in culture, cells were frozen for use in experiments described below.

A series of control experiments was carried out in a rat embryo fibroblast cell line (Rat-1 cells; [24]) to determine that transient transfection of gene constructs, described below, resulted in functional expression and hormone response. These cells were maintained in phenol red-free DMEM containing single-strength ABAM, 5 mM Hepes, sodium bicarbonate supplemented with 10% FBS, and insulin (0.6 µg/ml). Cultures were maintained at 37°C in a humidified atmosphere of 95% air:5% CO2 and fed every 48 h.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

RT-PCR was used to detect specific transcripts in RNA obtained from individual uterine tissue samples. Total RNA was extracted from cultures by guanidinium thiocyanate extraction [25]. All solutions and glassware used in preparation and analysis of RNA were treated with diethylpyrocarbonate (Sigma). Two micrograms of total RNA was denatured by heating to 95°C and reverse-transcribed in the presence of random hexamers (pdN6; 100 pmol; Pharmacia, Piscataway, NJ), dATP, dTTP, dCTP, and dGTP (dNTPs; 1 mM; Pharmacia); MgCl2, RNase inhibitor (20 U/reaction; Promega, Madison, WI); and reverse transcriptase (Superscript, 200 U/reaction; Gibco-BRL) at 37°C for 75 min. The reaction was stopped by heating to 95°C. Aliquots of reverse-transcribed cDNA (1–5 µl) were denatured by heating to 95°C and subjected to PCR in the presence of 75 pmol specific primers, MgCl2, dNTPs (1 mM), and Amplitaq DNA polymerase (0.5 U/reaction; Perkin-Elmer, Foster City, CA). Conditions used for PCR were 95°C, 1 min; 56°C, 1 min; 72°C, 1 min; 40 cycles. Products of RT-PCR were resolved on 3% agarose-TAE gels (Tris-acetate [40 mM], EDTA [1 mM]) and visualized by staining with ethidium bromide. ERß products (primers 5'-TTC CCG GCA GCA CCA GTA ACC-3'; 5'-TCC CTC TTT GCG TTT GGA CTA-3'; [8]) were expected to be 262 base pairs (bp). Representative RT-PCR products were excised from agarose and tested by dideoxy chain-termination sequencing (Applied Biosystems, Foster City, CA; Model 373A Automated Sequencer, OSU Recombinant DNA/Protein Resource Facility). The identity of the product was verified in a sequence homology analysis using the Basic Local Alignment Search Tool (BLAST; [26]).

Plasmid Constructs

The plasmid pBLCAT2 [27] contains the minimal thymidine kinase promoter from herpes simplex virus (HSV-tk) driving expression of the coding region of the chloramphenicol acetyltransferase (CAT) reporter gene. To confer estradiol responsiveness, the 15-bp consensus ERE from the Xenopus vitellogenin A2 gene (GGT CAC AGT GAC C) was inserted into the XbaI restriction site, immediately upstream of the HSV-tk, to produce the pERE15 plasmid [27]. Both pBLCAT2 and pERE15 are pUC-derived plasmids and thus contain an AP-1 enhancer in the region 5' of the HSV-tk [28]. To remove this confounding regulatory element, we digested these plasmids with NdeI and EcoO109 as described by Kushner et al. [28], followed by religation, to generate pBLCAT2{Delta}NdeI-EcoO109 and pERE15{Delta}NdeI-EcoO109, hereafter called pBLCAT and pERE-CAT, respectively. The unmodified pBLCAT2 plasmid was used directly to compare effects of the presence of an AP-1 element; for clarity, this plasmid was renamed pAP-1-CAT. This resulted in a series of three CAT reporter plasmids (Fig. 1) containing 1) no enhancer element (pBLCAT, control), 2) an ERE alone (pERE-CAT), or 3) an AP-1 element alone (pAP-1-CAT). The plasmid, pBLCAT3 [27], was also digested with NdeI and EcoO109 to remove the AP-1 element, then religated to produce a negative control plasmid with no promoter or enhancer elements. In order to control for variation in transfection efficiency, the plasmid pSV-ß-gal (Promega) was used to cotransfect the cells. This plasmid served as marker for relative transfection efficiency through basal expression of the ß-galactosidase reporter that was assayed in the same cell extracts used to measure the CAT reporter activity. CAT reporter data were normalized to the ß-galactosidase activity to account for the variation in response due to variation in efficiency of reporter transfection and cell numbers.



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  		FIG. 1. Structure of the expression vectors and CAT reporter constructs used in the study. In the pBKCMV vector, expression of ER{alpha} or ERß was driven by the CMV immediate-early promoter. Reporter constructs were developed from the pBLCAT2 plasmid and contained ERE or AP-1 enhancer elements in the presence of the HSV-tk promoter.

To confer ER expression on Rat-1 cells and cells from Day 110 to 120 of gestation (UBF120 cells), the cDNAs for human ER{alpha} (HEG0; [29]) and rat ERß [8] were inserted into the expression plasmid pBKCMV (Stratagene, La Jolla, CA) to produce pBKCMV-ER{alpha} and pBKCMV-ERß, respectively (Fig. 1). These plasmids contain the human cytomegalovirus (CMV) immediate-early promoter driving expression of the inserted ER cDNA. The 3' end of the cDNA is adjacent to the SV40 3' splice site and polyadenylation signal. Both plasmids were examined by restriction fragment analysis and DNA sequence analysis to verify insertion of full-length cDNA.

Transient Transfection and Hormone Treatments

To examine hormone response, cells were placed in phenol red-free DMEM supplemented with 10% dextran-charcoal-stripped FBS (CS-FBS) for 48 h. Dextran-charcoal stripping was performed as described by Horwitz et al. [30]. Plasmid DNAs were cotransfected by cationic liposome-mediated transfection (Transfectam; Promega) into Rat-1, UBF120 (0.5 µg of each plasmid DNA; 1.5 µg total DNA per culture well), and UBF180 cells (0.5 µg of each plasmid DNA; 1.0 µg total DNA per culture well). The transfection reagent and DNAs were prepared according to manufacturer's recommendations and incubated with the cells for 16 h. After a 24-h recovery period in DMEM supplemented with 10% CS-FBS, cells were re-fed using medium supplemented with 10% CS-FBS and incubated for 48 h. Culture wells of transfected Rat-1 cells were then treated in duplicate for 12 h in the presence of estradiol-17ß (E2; 1.0 nM; Sigma), E2 plus the antiestrogen ICI 182,780 (10 nM; Zeneca Pharmaceuticals, Macclesfield, UK), or ethanol vehicle. Each culture well was then assayed in duplicate for a response characterized by increased CAT expression. The entire experiment was then repeated; reported results are based on three to six replicates of each experiment as described in the figure legends. Similarly, cultures of transfected fetal bovine cells collected at 110–120 days gestational age (UBF120 cells) or at 180–200 days gestational age (UBF180 cells) were treated in duplicate wells for 12 h in the presence of E2 (1.0 nM), E2 plus ICI 182,780 (10 nM), or ethanol vehicle, and each well was assayed in duplicate for a response characterized by increased CAT expression. Reported results are based on three replicates of each experiment. To test for ligand-independent ER activation in fetal uterine cells, cultures of transfected UBF180 cells were treated in duplicate wells for 2 h in the presence of epidermal growth factor (EGF; Promega; 1.0 µM), EGF plus ICI 182,780 (10 nM), or ethanol vehicle. Each well was then assayed in duplicate for a response characterized by increased CAT expression. Reported results are based on three replicates of the experiment. All cultures of UBF cells were used during passages 2 through 4. On the basis of the rate of expansion of a fixed number of cells to confluency in 60-mm2 culture wells (Becton-Dickinson Labware), each passage was estimated to contain five doublings of the cell population, or five generation times.

CAT Assay

CAT enzyme activity was assayed in duplicate in cell lysates by incubating in the presence of [3H]chloramphenicol and n-butyryl-coenzyme A, then measuring the acetylated [3H]chloramphenicol present in the organic (xylene) phase after two-phase partitioning (CAT Enzyme Assay System Kit; Promega). The radioactivity present in the acetylated chloramphenicol was determined by scintillation spectroscopy. Enzyme activity was compared to that in cells not transfected as a negative control; [3H]chloramphenicol was also measured in the xylene phase after extraction in the absence of cell lysates to determine background radioactivity following two-phase partitioning.

Control assays for ß-galactosidase activity were carried out using the same cell extracts as for the CAT assays. The ß-galactosidase enzyme assay system (Promega) was based on spectrophotometric detection of the cleavage product of the substrate o-nitrophenyl-ß-D-galactopyranoside.

Statistical Analysis

Values obtained in the assay of CAT enzyme activity were normalized to ß-galactosidase activity to correct for variation in transfection efficiency and cell numbers. The resulting ratios were used to calculate means within each cell type representing duplicate sampling of cell cultures prepared and subjected to each treatment in duplicate within each experiment. Data were collected from similar experiments replicated from three to six times and were expressed in arbitrary units relative to controls to allow statistical analysis of data from separate experiments. The relative number, given as fold induction, was calculated as the ratio of treatment with hormone or hormone plus ICI 182,780 to the vehicle treatment data point. Overall treatment means were calculated based on data from three to six separate experiments as described in each figure legend. SE was calculated for each averaged point except the vehicle control, which was set to 1. Fold induction data were subjected to one-way ANOVA using the data analysis package of Microsoft Excel 97 (Redmond, WA) to test effects of hormone treatment on each enhancer-element type. Comparisons were made of the effects of E2 or EGF treatment versus control, of E2 or EGF plus ICI versus control, and of E2 or EGF treatment versus E2 or EGF plus ICI.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As determined by RT-PCR, ERß mRNA was present in fetal uterine cells collected at both 120 and 180 days of gestation (Fig. 2). We previously showed that ER{alpha} mRNA was present in the fetal uterus during this same time frame but that ligand binding and a functional hormone response were present only after Day 160 of pregnancy [6]. In addition, ER{alpha} mRNA and a functional hormone response were present in UBF180 cells under these conditions [23], but there was no estrogen response in UBF120 cells. Rat-1 cells do not exhibit estrogen ligand binding [31].



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  		FIG. 2. Agarose gel electrophoresis of RT-PCR of ERß mRNA in fetal uterine cells collected at 120 and 180 days of gestation. The 262-bp product of RT-PCR was isolated and sequenced for identification. No product was observed in the blank control reaction. The positive control was the same 262-bp fragment of bovine ERß previously subcloned into the Bluescript cloning vector (Stratagene) and verified by sequence analysis.

When Rat-1 cells were transiently transfected with ER{alpha}, treatment with E2 for 12 h resulted in an increase to 2 fold in CAT reporter activation (p < 0.001) when an ERE enhancer element was present (pERE-CAT; Fig. 3). Treatment with E2 plus ICI 182,780 effectively antagonized this estradiol-induced rise. There was no effect of E2 on CAT reporter activation when an AP-1 enhancer element was present (pAP-1-CAT). Similarly, there was no effect in the absence of an enhancer element (pBLCAT), or in the absence of the promoter (pBLCAT3).



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  		FIG. 3. Mean values ± SE of acetylated [3H]chloramphenicol expressed as fold increase over control in Rat-1 cells. Cells were transiently transfected with a vector expressing ER{alpha} and a reporter plasmid having an ERE (pERE-CAT), an AP-1 element (pAP-1-CAT), no enhancer (pBLCAT), or no promoter or enhancer (pBLCAT3). Cultures were incubated for 12 h with vehicle, E2, or E2 plus ICI 182,780. Each mean represents duplicate assay of cell cultures prepared in duplicate in each experiment, replicated 3–6 times. Means within each reporter plasmid classification marked with different letters (a, b) were different (p < 0.001).

When Rat-1 cells were transiently transfected with ERß, treatment with E2 for 12 h resulted in an increase to 1.7 fold in CAT reporter activation (p < 0.06) when an ERE enhancer element was present (pERE-CAT; Fig. 4). After treatment with E2 plus ICI 182,780, mean CAT reporter activity was not different from the control value but also not different from the E2 treatment mean. There was no effect of E2 treatment on CAT reporter activation when an AP-1 enhancer element was present (pAP-1-CAT). Similarly, there was no effect in the absence of an enhancer element (pBLCAT), or in the absence of the promoter (pBLCAT3).



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  		FIG. 4. Mean values ± SE of acetylated [3H]chloramphenicol expressed as fold increase over control in Rat-1 cells. Cells were transiently transfected with a vector expressing ERß and a reporter plasmid having an ERE (pERE-CAT), an AP-1 element (pAP-1-CAT), no enhancer (pBLCAT), or no promoter or enhancer (pBLCAT3). Cultures were incubated for 12 h with vehicle, E2, or E2 plus ICI 182,780. Each mean represents duplicate assay of cell cultures prepared in duplicate in each experiment, replicated 3 times. Means within each reporter plasmid classification with different letters (a, b) were different (p < 0.06).

When UBF120 cells were transiently transfected with ER{alpha}, treatment with E2 for 12 h resulted in an increase to 2.5 fold in CAT reporter activation (p < 0.05) when an ERE enhancer element was present (pERE-CAT; Fig. 5, left). Treatment with E2 plus ICI 182,780 antagonized this estradiol-induced rise, although activation remained elevated compared to control values. There was no effect of E2 treatment on CAT reporter activation when an AP-1 enhancer element was present (pAP-1-CAT; Fig. 5, right). When UBF120 cells were transiently transfected with ERß, treatment with E2 for 12 h resulted in an increase to 1.8 fold in CAT reporter activation (p < 0.05) when an ERE enhancer element was present (pERE-CAT; Fig. 5, left). Treatment with E2 plus ICI 182,780 antagonized this E2-induced rise; similarly, activation remained elevated compared to control values. There was no effect of E2 treatment on CAT reporter activation when an AP-1 enhancer element was present (pAP-1-CAT; Fig. 5, right).



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  		FIG. 5. Mean values ± SE of acetylated [3H]chloramphenicol expressed as fold increase over control in UBF120 cells. Cells were transiently transfected with a vector expressing ER{alpha} or ERß and a reporter plasmid having an ERE (pERE-CAT; left) or an AP-1 element (pAP-1-CAT, right). Cultures were incubated for 12 h with vehicle (control), E2, or E2 plus ICI 182,780. Each mean represents duplicate assay of cell cultures prepared in duplicate in each experiment, replicated 3 times. Means within each treatment classification with different letters (a, b, c) were different (p < 0.05).

Exposure of UBF180 cells to E2 for 12 h resulted in an increase to 4.7 fold in CAT reporter activation (p < 0.01) when an ERE enhancer element was present (pERE-CAT; Fig. 6). When an AP-1 enhancer element was present (pAP-1-CAT), E2 treatment resulted in an rise to 1.7 fold (p < 0.05) in CAT reporter activation. Similar to observations in Rat-1 cells, there was no effect in the absence of an enhancer element (pBLCAT), or in the absence of the promoter (pBLCAT3).



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  		FIG. 6. Mean values ± SE of acetylated [3H]chloramphenicol expressed as fold increase over control in UBF180 cells. Cells were transiently transfected with a reporter plasmid having an ERE (pERE-CAT), an AP-1 element (pAP-1-CAT), no enhancer (pBLCAT), or no promoter or enhancer (pBLCAT3). Cultures were incubated for 12 h with vehicle or E2. Each mean represents duplicate assay of cell cultures prepared in duplicate in each experiment, replicated 3 times. Means with different letters (a, b, c) were different (p < 0.05).

Exposure of UBF180 cells to EGF for 2 h resulted in an increase to 1.75 fold in CAT reporter activation (p < 0.01) when an ERE enhancer element was present (pERE-CAT; Fig. 7). Treatment with EGF plus ICI 182,780 effectively antagonized this EGF-induced rise, suggesting reporter gene activation mediated through the ER.



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  		FIG. 7. Mean values ± SE of acetylated [3H]chloramphenicol expressed as fold increase over control in UBF180 cells. Cells were transiently transfected with a reporter plasmid having an ERE (pERE-CAT). Cultures were incubated for 2 h with vehicle (control), EGF, or EGF plus ICI 182,780. Each mean represents duplicate assay of cell cultures prepared in duplicate in each experiment, replicated 3 times. Means with different letters (a, b) were different (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Rat-1 cells and in cells from the bovine fetal uterus at Days 110–120 of gestation, after transfection with either ER{alpha} or ERß, E2 treatment resulted in increased reporter gene activity in the presence of an ERE but not in the presence of an AP-1 response element. These data suggest that in both of these cell types, all components were present for estrogen-dependent gene activation from an ERE except ER. The cells were not competent for estrogen-dependent gene activation mediated through AP-1, suggesting that components of the AP-1 heterodimer were lacking. The antiestrogen ICI 182,780 acted as an estrogen antagonist in Rat-1 cells.

Cells from the bovine fetal uterus at Days 180–200 of gestation express endogenous ER [6]. In these cells, E2 treatment resulted in increased reporter gene activity in the presence of an ERE or an AP-1 response element. This suggests that in UBF180 cells all components were present for estrogen-dependent gene activation from an ERE, including the ER. The cells were also competent for estrogen-dependent gene activation mediated through AP-1, suggesting that components of the AP-1 heterodimer begin to be expressed between 120 and 180 days of gestational age, possibly in response to expression of the ER itself. Further, treatment with EGF stimulated gene activation from an ERE, demonstrating the phenomenon of ligand-independent activation of the ER. It is noteworthy that ICI 182,780 exhibited mixed agonist-antagonist activity in the presence of estradiol in some experiments. At the time points reported in this study, antagonist activity was consistent. At longer incubation times up to 24 h, agonist activity was often observed. In general, agonist activity of ICI plus estradiol treatment was coincident with reduction of estradiol stimulation back to baseline values when estradiol alone was used, suggesting a differential time course for estradiol action in the presence and absence of ICI.

The ERß was first identified in the rat [8], and homologues in the mouse [32] and human [33] have been cloned; there is recent evidence for expression of an ERß in the bovine uterus (Fig. 2; [9, 10]). Sequence alignment analysis shows an expected high degree of homology in ERß sequence among cattle, rats, mice, and humans. Comparison of the ERß with ER{alpha} sequence revealed a high degree of conservation in the C-domain (95%) and E-domain (60%), suggesting that the two ER forms would bind to DNA and ligand in a similar manner. This has been established [13, 14], although several ligands exhibit significantly different binding characteristics with the two receptors, including estriol, tamoxifen, and the phytoestrogens coumestrol and genistein [13]. The ER{alpha} and ERß sequences diverge significantly in the regions of the receptors involved with transactivation (TAF-1 and TAF-2). Specific interactions between the ER and these coactivator proteins occur at the amino-terminal transactivator functional domain, TAF-1, and the C-terminal transactivator domain, TAF-2 [4, 34–38]. These interactions are critical to ER transactivation, and steroid receptors may compete for binding to these coactivation factors [39]. This divergence suggests that interactions with coactivator proteins would differ at the enhancer or promoter site during modification of gene expression and, as a result, that ER{alpha} and ERß play different roles in gene regulation. If this is true, then both sets of interacting proteins were already present in UBF120 and Rat-1 cells, suggesting a low degree of specialization. Further complexity arises from the findings that ER{alpha} and ERß may operate together as heterodimers [15]. Additionally, more than one ERß transcript may be present in target tissues [40–42].

ER{alpha} and ERß act through AP-1 [14, 19] via a protein-protein interaction between the ligand-bound receptor and the DNA-bound AP-1 heterodimer. The AP-1 heterodimer consists of members of the fos and jun gene families. In general, fos family members combine with jun proteins to form heterodimers; jun family members can also form heterodimers or homodimers with other jun proteins. Transcriptional activity and target gene specificity depend upon the composition of the protein complex [43]. Estrogen induced immediate and transient activation of several proto-oncogenes, including c-jun and c-fos, in uteri of the mouse [44] and rat [45, 46], and these play important roles in cellular differentiation and proliferation. Activation of c-jun was limited to stromal and myometrial cells, while activation of c-fos was specific to luminal and glandular epithelium in both the mouse [44] and rat [47, 48]. It is reasonable to suggest that an early response to ER expression at the end of the sixth month of pregnancy in the bovine fetal uterus is cell type-specific activation of c-fos and c-jun.

The ER undergoes phosphorylation and dephosphorylation as part of the process of transition from a hormone-bound to hormone-unbound state. This process involves specific kinase and phosphatase enzymes and is mediated by phosphorylation at a tyrosine residue [49–51]. The receptor also contains several other target sites for protein kinases and appears to be phosphorylated at specific serine residues in the presence of activated protein kinase A or protein kinase C signal transduction pathways [52]. In the rat uterus, stimulation of ER-mediated transcription and serine phosphorylation on the receptor were caused by estrogen, by cAMP, and by insulin-like growth factor-1 (IGF-1) [20]. Phosphorylation of ER in the absence of estrogen resulting in ligand-free activation of the receptor was likely mediated through mitogen-activated protein kinase [53]. IGF-1 was present in the bovine fetal blood circulation in increasing concentration throughout the last third of gestation [54]. The bovine fetal uterus contains mRNA for EGF receptor [6], and EGF stimulation of ER-mediated transcriptional activity was present in UBF180 cells. These data suggest a role for ligand-independent activation of ER activity mediated by posttranslational modification of the receptor protein in late pregnancy in the developing bovine uterus.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. George Kuiper of the Karolinska Institute, Stockholm, for providing the rat ERß cDNA, and Dr. Jack Gorski for providing human ER{alpha} cDNA. We thank the staff of Wellington Quality Meats, Wellington, KS, for their assistance and donation of materials used in the study. The authors would also like to acknowledge the Oklahoma State University Recombinant DNA/Protein Resource Facility for the synthesis of synthetic oligonucleotides and the sequencing of cloned cDNA.


   FOOTNOTES
 
1 This study was supported by NIH NICHHD 34260 and USDA NRICGP 94–37206–0937. Back

2 Correspondence: FAX: 405 744 7110; malayer{at}okway.okstate.edu Back

Accepted: December 22, 1998.

Received: September 30, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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