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BOR - Papers in Press, published online ahead of print June 16, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029421
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BIOLOGY OF REPRODUCTION 71, 1252–1261 (2004)
DOI: 10.1095/biolreprod.104.029421
© 2004 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Independent Downstream Gene Expression Profiles in the Presence of Estrogen Receptor {alpha} or ß1

A.G.B. Hurst, D.W. Goad, M. Mohan, and J.R. Malayer2

Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The two known forms of estrogen receptor (ER), {alpha} and ß, exhibit differences in structure, affinity for certain ligands, and tissue distribution, suggesting differential roles. It is of interest from several perspectives to determine whether the two receptors elicit similar or differing responses within the same cell type in the presence of the same ligand. To evaluate roles of ER, we have examined responses to estrogen in a rat embryonic fibroblast cell line model, normally naive to ER, engineered to stably express ER{alpha} or ERß. Rat1+ER{alpha}, Rat1+ERß, and precursor Rat1 cell lines were treated with estradiol-17ß (E2; 1 nM) or an ethanol vehicle for 24 h. Total RNA was extracted, and cDNA generated and subjected to suppression subtractive hybridization (SSH), followed by differential screening using dot blot hybridization. In the presence of ER{alpha}, products were identified that represent classic responses to E2, including markers for cell proliferation. In the presence of ERß, an alternate transcription profile was observed, including upregulation of pro-alpha-2(I) collagen. These data support a model in which ER{alpha} and ERß regulate unique subsets of downstream genes within a given cell type.

estradiol receptor, mechanisms of hormone action, steroid hormone receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen signaling system plays a critical role in the physiology of the reproductive organs, as well as the cardiovascular, skeletal and central nervous systems, and in carcinogenesis. Estrogen receptors (ER) are members of the steroid receptor family and function as ligand-inducible transcription factors. Upon binding of the ligand, ER within the nucleus undergo phosphorylation [1, 2], dimerization [3], and binding to DNA at specific cis-acting sites, termed estrogen-response elements (ERE) [4, 5]. The receptor protein is next involved in the recruitment of coregulator proteins and other protein-protein interactions, resulting ultimately in modification of the rate of transcription from the target promoter [4, 6, 7]. In some instances, the capability exists for ligand-independent activation through phosphorylation pathways [8, 9], including MAP-kinases [10]. Nuclear ER mediate most of the actions of estrogens resulting in transcriptional activation and repression, control of cell cycle progression, and integration of intracellular signaling pathways [11].

Toft and Gorski [12] first isolated and began characterizing a receptor protein for estrogen in 1966, and through 1994, only one receptor form had been identified. In 1995, the second estrogen receptor, ERß, was identified from rat prostate [13], and since that time, ERß has been characterized in the mouse [14], human [15], and numerous other species. Variations exist in the tissue distribution of ER{alpha} and ERß, though they appear to have similar gene expression levels in the testis, epididymis, bone, and adrenal gland [16]. ER{alpha} predominates in the proliferative cells of the mammary, pituitary, and thyroid glands, as well as in uterus, skeletal muscle, and the smooth muscle of the coronary arteries, while ERß is predominant in the prostate [13], granulosa cells of the ovary, and the lung, bladder, brain, and hypothalamus [17]. In tissues where both receptors are present, it has been shown that the receptors may form either homodimers or heterodimers [18–20].

While there is great similarity in their DNA- and ligand-binding domains, the two ER forms exhibit significant structural differences, especially in the NH2-terminal A/B domain and the COOH-terminal F domain, and there is some variation in affinity for certain ligands [13, 17], suggesting differential physiological roles. Several complementary approaches have been used to verify and characterize these differences. Phenotypic variation between mouse knockout models for ER{alpha} [21, 22] and ERß [23] suggest that, in addition to differences in tissue distribution, the receptors exert different effects in the same tissue. These models have provided valuable insights into the differential roles of the estrogen receptors. It is difficult, however, with knockout models to separate developmental effects from functional effects in adult tissue. Ligands designed to activate only ER{alpha} or ERß have been used to show differential effects of the two receptors [24, 25] and have contributed significantly to understanding estrogen signaling, although limitations include the degree of selectivity by the ligand for one receptor over the other. Stable expression of the receptors in a naïve cell line, such as fetal osteoblasts [26–28], in culture has resulted in the demonstration of responses specific to ER{alpha}- and ERß-mediated signaling. This approach allows for characterization of the roles of the two receptors independent of one another in the presence of the same natural ligands. Although there are limitations with this approach as well, we have similarly examined gene expression specifically regulated by ER{alpha} or ERß in a set of engineered rat embryonic fibroblast cell lines [29–31]. Utilizing the technique of suppression subtractive hybridization (SSH), we have attempted to identify examples of unique downstream genes autonomously activated by each receptor within the same cell type in response to the same ligand.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reporter Assay

Cell culture Rat1 fibroblast cell lines [29] stably expressing either ER{alpha} (Rat1+ER{alpha}) [30] or ERß (Rat1+ERß) [31] were used as a model system for examining unique subsets of downstream genes regulated by each receptor type. Cell types originated as previously reported [31]. Rat1+ER{alpha} cells [30] express a mutant form of the human ER{alpha} (HEG0), which has a single amino acid substitution in the hormone binding domain resulting in a 10-fold lowering of the affinity of the receptor for E2 (Kd = 1.0 nM). Rat1+ERß cells [31] express the normal rat ERß isolated by Kuiper et al. [13]. Cells were grown in sterile, filtered (0.22 µM) phenol red-free Dulbecco modified Eagle medium (DMEM; Gibco-BRL, Grand Island, NY), with NaHCO3 (3.7 g/L). The cells were supplemented with bovine insulin (0.6 µg/ml) in HEPES (25 µM) (Sigma, St. Louis, MO), 1x antibiotic-antimycotic (Sigma), and 10% charcoal-stripped/dextran-treated fetal bovine serum (Hyclone, Logan, UT). Cells were maintained in approximately 0.133 ml/cm2 medium at 37°C with a humidified atmosphere of 5% CO2 gas and 95% air, and media was replaced every 48 h. Additionally, Rat1+ER{alpha} cells were supplemented with Hygromyocin B (100 µg/ml) in PBS (Gibco-BRL) beginning 24 h after plating [30]. Rat1+ERß cells were supplemented with Geneticin (50 µg/ml) (Gibco-BRL) [31].

Chloramphenicol acetyltransferase reporter assay A chloramphenicol acetyltransferase (CAT) reporter assay was used to verify a functional response to E2 by ER in each cell line and to verify that the precursor Rat1 cell line lacked ER expression and response to E2. Cells were plated at 400 000 cells per well in a 6-well cluster (35-mm diameter) tissue culture plate (Costar, Cambridge, MA) and transfected in triplicate with pERE15 [32] construct to determine E2 responsiveness as described by Cheng and Malayer [31]. After a 24-h recovery period in DMEM, transfected cells were treated with either E2 [1 nM] (98% 17-ß estradiol; Sigma) or an equal volume of ethanol vehicle for 24 h. Protein lysates were prepared and incubated with [3H]chloramphenicol and n-butyrl-coenzyme A. Following extraction, acetylated [3H]chloramphenicol levels in the organic phase were measured via scintillation spectroscopy [31].

Treatments and RNA extraction Cells were grown in 225-cm2 cell culture flasks (Corning, Corning, NY) to 80% confluency and then incubated with E2 [1 nM] for 24 h. After 24 h, cells were washed three times in PBS and total RNA extracted as described by Chomczynski and Sacchi [33].

Detection of Unique Gene Expression

SSH RNA was subjected to SSH [34] modified to a kit available from BD Biosciences (BD Biosciences Clontech, Palo Alto, CA). Total RNA (3–5 µg) extracted from cell culture was used to generate cDNA from Rat1+ER{alpha}, Rat1+ERß, and precursor Rat1. The cDNA was cut with RsaI (BD Biosciences Clontech), and two different adaptors [34] were ligated onto individual aliquots of the tester population. Hybridizations were carried out as described by Mohan et al. [35] with modifications. The first hybridization was carried out on the tester with either Adaptor 1 or 2R, and these were heat denatured and hybridized in the presence of excess denatured driver. A second hybridization was then performed where the two tester populations were combined in the presence of fresh denatured driver. Due to the presence of the adaptors, only those genes unique to the tester population were enriched and amplified in the polymerase chain reaction (PCR) [35]. PCR products from SSH were then compared against unsubtracted products. To further enrich the products, a secondary PCR was performed using nested primers to the adaptors.

Analysis of Unique Gene Expression

Cloning and differential analysis—qualitative Secondary PCR products were cloned into a pCRII Topo T/A vector (Invitrogen, Carlsbad, CA) and resulting colonies screened by differential analysis using digioxigenin (DIG)-labeled probes (Roche Molecular Biochemicals, Indianapolis, IN) to verify subtractions. Due to the large number of inserts being transfected into the Topo vector, an extended incubation (1 h) was used. Following chemical transfection, colonies were grown on selective agar plates at 37°C overnight. Colonies from the Rat1+ERß tester population (96 clones) and from the Rat1+ER{alpha} tester population (120 clones) were then picked and grown in Terrific Broth (Fisher Biotech, Fair Lawn, NJ) supplemented with carbanocillin (100 µg/ml; ICN, Costa Mesa, CA) in 96-well culture plates (Promega, Madison WI). DNA purification was carried out using Promega SV96 DNA purification system, and plasmid DNAs were spotted onto a series of 8.5- x 12-cm positively charged nylon membranes (Roche Molecular Biochemicals) using a BioDot apparatus (BioRad, Hercules, CA). Purified DNA was chemically denatured using denaturing solution (0.5 M NaOH, 1.5 M NaCl]) for 5–10 min in a 96-well plate and equally distributed to the BioDot apparatus followed by gentle vacuum for 1 min. Neutralization solution (0.7 M Tris-HCl, pH 8.0, 1.5 M NaCl) was then added to wells for 5–10 min. Membranes were rinsed in 2x SSC for 1–2 min, ultraviolet cross-linked, and stored at 4°C.

Probes were prepared from the secondary PCR products generated by SSH. These were digested with RsaI (Gibco-BRL) to remove adaptors, which cause background during hybridization. Excess inactivated enzyme, buffer, and adaptors were removed using Qiagen PCR purification columns (Qiagen, Valencia, CA). After denaturation (95°C, 7 min) to generate ssDNA, SSH secondary PCR products (1 µg) were added to a premixed DIG-dUTP (Roche Molecular Biochemicals) and incubated for 20 h at 37°C. The reaction was stopped by heating to 65°C for 10 min, and the labeled products were stored at –20°C. Labeling efficiency was performed according to the manufacture's instructions and compared with standard labeled product (Roche Molecular Biochemicals) based on chemiluminescence (CSPD) detection. Labeled PCR probes were used at a concentration of 25 ng/ml in DIG EasyHyb hybridization solution (Roche Molecular Biochemicals).

Membranes were prehybridized in DIG Easy-Hyb solution for 15 min (42°C; 10 ml/100 cm2 membrane) and probed in DIG EasyHyb solution with 25 ng/ml probe overnight (42°C; 3.5 ml/100 cm2). Membranes were then washed as described by the manufacturer to remove excess probe. Bound probe was detected using an anti-DIG conjugate for 30 min (75 mU/ml in 40 ml) at room temperature under constant agitation. Detection by CSPD was carried out via enzyme immunoassay (Roche Molecular Biochemicals). Spots were qualitatively analyzed for signal intensity after exposure to Kodak X-OMAT LS 8 x 10 x-ray film (Kodak, Rochester, NY). Comparison of subtracted and unsubtracted probes was conducted using criteria detailed in the Clontech Differential Screening manual (BD Biosciences Clontech). Primary candidates for sequencing (Fig. 2C; denoted +, +, –, –) hybridized to both subtracted and unsubtracted tester probes. Low-abundance (Fig. 2C; denoted +, –, –, –) products that hybridized only to subtracted tester probes were also sequenced. Colonies that were positive to the subtracted probe for the tester and driver and had fivefold intensity in the tester were also picked and sequenced (Fig. 2C; denoted +>5, +, +, –). Colonies positive in the unsubtracted driver were sequenced (Fig. 2C; denoted +, +, –, +). Though present in the driver population, the products were in such low abundance as to not be detected through SSH, and therefore, the differences between populations are still significant. Colonies positive on all four membranes were not considered.



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  		FIG. 2. Qualitative differential analysis of dot blot membranes probed using DIG-labeled probes: (A) Differentially screened nylon membranes spotted with purified plasmid DNAs (96 of 120 shown) from subtracted population when Rat1+ER{alpha} E2 was the tester and Rat1+ERß E2 was the driver. Membranes were probed with 1) tester subtracted from driver (forward subtracted), 2) tester unsubtracted with both adaptors present but no driver present, 3) Rat1+ERß E2 as tester and Rat1+ER{alpha} E2 as driver (reverse subtracted), 4) reverse unsubtracted (absence of Rat1+ER{alpha} E2 driver added and both adaptors present). DIG-labeled probes (25 ng/ml) hybridized (42°C overnight), detected through CSPD, and exposed to X-OMAT blue film for approximately 30 sec. B) Differentially screened nylon membranes spotted with purified plasmid DNAs [96] resulting from subtracted Rat1+ERß E2 tester with Rat1+ER{alpha} E2 driver. Membranes were probed with 1) Rat1+ERß E2 tester subtracted from Rat1+ER{alpha} E2 driver (forward subtraction), 2) Rat1+ERß E2 unsubtracted (absence of ER{alpha} E2 driver and both adaptors present), 3) Rat1+ER{alpha} E2 tester subtracted from Rat1+ERß E2 driver (reverse subtracted), 4) Rat1+ER{alpha} E2 unsubtracted (absence of Rat1+ERß E2 driver added and both adaptors present). C) Definition of parameters considered for differential screening and sequencing of colonies

Automated sequencing Once differential analysis of the subtracted products was verified, automated sequencing was carried out by the OSU Recombinant DNA/Protein Resource Facility and results analyzed using MacVector 7.0 in conjunction with NCBI Basic Local Alignment Search Tool [36] to identify homologous sequences in the GenBank database.

Real-time PCR—quantitative Taqman primers and probes were generated using Primer Express software (PE Applied Biosystems, Foster City, CA) to gene targets of interest defined by SSH, qualitative analysis, and sequence information. Quantitative reverse transcription PCR (qRT-PCR) was then carried out as described by Hettinger et al. [37] with modifications [38–40]. ABI primer probe sets were generated from sequences deduced from SSH products. Probes contained a 3' fluorescent TAMRA reporter dye and a 5' FAM quencher dye. Expression was examined for four individual targets in triplicate using total RNA (10 ng) from each of the treatment schemes for the cell lines (Rat1+ER{alpha}, Rat1+ERß, Rat1 treated with E2 or vehicle) by means of primer (300 nM) and probe (200 nM) sets shown in Table 1. Each population of total RNA (50 pg) was normalized in duplicate using 18S ribosomal RNA (Ribosomal RNA control kit; PE Biosystems) at a (200 nM) primer (100 nM) probe concentration, and the efficiency was checked via a standard curve of serial dilutions of Rat1+ER{alpha} E2 treated. For individual targets, 10 pg, 100 pg, 1 ng, 10 ng, and 100 ng were used. For 18S ribosomal RNA, 500 pg, 50 pg, 10 pg, 5 pg, and 1 pg were used. Real-time PCR was carried out in the ABI PRISM 7700 (PE Applied Biosystems) under the following thermal cycler conditions: 48°C for 30 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60 °C for 1 min, in a 25-µl reaction. Analysis and fold differences were determined using the comparative CT method as described in the ABI technical bulletin #2 for the ABI PRISM 7700 [38– 40], where the Rat1 vehicle-treated cell line was used as a calibrator.


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  		TABLE 1. Primers and probe sequences for Taqman real-time quantitative PCR

Standard reverse transcription-PCR—semiquantitative Reverse transcription (RT) followed by PCR was carried out for additional putative gene products identified through SSH and differential screening, to further check for the presence of false positives in our data set. Annexin 1 (935 base pairs [bp]) identified in the Rat1 + ER{alpha} E2 tester population and Nuclear factor I/B (963 bp) identified in the Rat1+ERß E2 tester population were selected and primers generated via MacVector software (Table 2). Primers were synthesized by Integrated DNA Technologies (Coralville, IA), and G3PDH (500 bp) was run simultaneously as a PCR loading control. Conditions were carried out as described in Table 2.


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  		TABLE 2. Primers for standard RT-PCR and conditions

Statistical analysis Results for CAT assay were analyzed with the Statistical Analysis System (SAS; Package 8.0) by constructing a 3 cell type x 2 treatment factorial ANOVA table and using least square differences in PROC-GLM to determine significant differences. Statistical comparison of real-time qRT-PCR values with means ± SD were reported, where n = 3 and results were tested using least square differences, reported as PROC-MIXED in a 3 cell type x 2 treatment factorial ANOVA table constructed using the SAS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of E2 Stimulation

Similar to previously reported results, chloramphenicol acetyltransferase reporter assay confirmed an increase of pERE15 reporter gene activity in the presence of estradiol-17ß (E2) (1 nM) after 24 h in cell lines expressing ER{alpha} and ERß [31] (Fig. 1). In the presence of ER{alpha} or ERß, there was a significant increase in chloramphenicol acetyltransferase activity (P < 0.01), as determined by the amount of acetylated [3H] chloramphenicol present following E2 treatment, compared with either ethanol vehicle treatment or the parental cell line, which does not express ER{alpha} or ERß. There was no significant difference in chloramphenicol acetyltransferase activity between Rat1+ER{alpha} and Rat1+ERß cells following E2 treatment. This apparent similarity in transcriptional activation by the two receptors was not unexpected here and is in accordance with previously published data on the 10-fold higher Kd of the HEG0 mutant [41] present in the Rat1+ER{alpha} cell line. While the use of this mutant form of the receptor may seem at first problematic, in fact, it proved very useful in the current study, in which we are interested in comparison of downstream responses to the ligand-activated receptor under similar conditions of transcriptional activation.



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  		FIG. 1. Chloramphenicol acetyltransferase reporter assay was carried out to test responsiveness of estrogen receptor to E2 using a pERE15 construct. Responsiveness was determined by levels of acetylated [3H]chloramphenicol levels in the organic phase determined by scintillation spectrometry. For CAT assay, analysis of variance was carried out using PROC-GLM least square difference in SAS. E2 treatment resulted in elevated reporter gene expression in ER{alpha}- and ERß-expressing cell lines (P ≤ 0.05) compared with vehicle control treatments, and E2 or vehicle treatment of the parental Rat-1 cell line. There was no difference between ER{alpha}- and ERß-expressing cell lines treated with E2

Suppression Subtractive Hybridization

Profiles of unique gene products were evaluated through comparisons of subtracted SSH products to unsubtracted controls [34]. Four experiments were carried out, each consisting of a forward and reverse reaction: 1) comparison of Rat1+ER{alpha} and Rat1+ERß following E2 stimulation, 2) comparison of Rat1+ER{alpha} and Rat1+ERß following vehicle treatment, 3) comparison of Rat1+ER{alpha} and Rat1 following E2 stimulation, and 4) comparison of Rat1+ERß and Rat1 following E2 stimulation. G3PDH (500 bp) efficiency controls were carried out to confirm that there was a decrease in levels of G3PDH in subtracted products. This was demonstrated by an increase in number of PCR cycles required to amplify G3PDH in comparison with the unsubtracted control.

Qualitative Analysis of Differential Screening Following SSH

A total of 814 SSH products were screened in four forward and reverse experiments outlined above, and of these, 208 were identified as differentially expressed through dot-blot hybridization assays using DIG-labeled probes (Fig. 2). These candidates were subjected to single-pass sequencing, and BLAST analysis was performed [33]. When clones from the four experiments were examined, 150/208 returned quality sequence data and 107/150 showed identity to known sequences with a 44% rate of redundancy, while 43/150 had no significant match to any known sequence with a 30% rate of redundancy (Tables 3–5). In the presence of E2, Rat1+ER{alpha} appeared more robust in terms of gene activation and approximately 26 unique products exhibited identity to known genes (Table 3), while 6 showed no homology to GenBank sequences. Within the homologous putative genes, classic E2 responsive gene products involved in cell growth, transcriptional activity, and signal transduction were found. When Rat1+ERß with E2 stimulation was utilized as tester, four unique gene homologs were identified (Table 4), and one showed no homology. In vehicle-treated experiments, most results aligned with ribosomal RNA or mitochondrion products (Table 3 and 4). However, within the Rat1+ER{alpha} vehicle-treated tester population, a match occurred to procollagen C-proteinase enhancer protein (PCOLCE) (Table 3). When the parental cell line was used as the tester, the majority of products showed no homology to known sequences or aligned to ribosomal or mitochondrion products (Table 5). These data support the concept that, in addition to differences in tissue distribution, ER{alpha} and ERß regulate both overlapping and unique subsets of downstream genes in the same genetic background.


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  		TABLE 3. GenBank dbEST submissions for putative cDNA clones identified through SSH with Rat1+ER{alpha} cell lines as tester


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  		TABLE 5. GenBank dbEST submissions for putative cDNA clones identified through SSH with Rat1 parental cell line as tester


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  		TABLE 4. GenBank dbEST submissions for putative cDNA clones identified through SSH with Rat1+ERß cell lines as tester

Quantitative RT-PCR Analysis of Target Genes of Interest Identified Through SSH

Targets of particular interest, especially genes considered to be involved in inflammatory responses or MAP-kinase related pathways were selected for qRT-PCR [38–40] and normalized to 18S rRNA expression. Pro-alpha-2(I) collagen (COL1A2), procollagen C-proteinase enhancer protein (PCOLCE), cathepsin L (CtsL), and receptor for activated protein kinase C (RACK1) were selected for qRT-PCR and analyzed using the comparative cycle threshold (CT) method (Table 6). Validation of qRT-PCR efficiency was determined by the evaluation of the R2-value of the standard curve for 18S ribosomal RNA, which was 0.9818, with the R2-values of the individual targets varying by less than ±0.01 from this value. Efficiency of the PCR was further measured by the equation ([101/–s]–1) and found to be ≥90% [40].


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  		TABLE 6. Quantitative analysis of real-time PCR to targets of interest

Pro-alpha-2(I) collagen was identified from the Rat1+ERß E2 tester/Rat1+ER{alpha} E2 driver comparison and increased fourfold in the Rat1+ERß cell line versus the Rat1+ER{alpha} following E2 treatment (P ≤ 0.05). An increase in COL1A2 of 14-fold was observed in the Rat1+ERß E2-treated cell line over the predetermined calibrator Rat1 vehicle treated cells (Fig. 3A).



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  		FIG. 3. Fold differences in target gene expression were determined by the comparative CT method. The difference between the CT values of the target genes and the 18s rRNA ({Delta}CT) were calibrated to an index value by subtracting all individual {Delta}CT's from that of the Rat1 cell line receiving vehicle treatment to derive the {Delta}{Delta}CT. Fold differences were then calculated by the equation . Statistical analysis for fold differences are those determined for the {Delta}{Delta}CT using SAS PROC-MIXED. Fold differences shown for (A) COL1A2 detected in the Rat1+ERß stimulated tester/Rat1+ER{alpha} stimulated driver experiment, (B) RACK1, and (C) CtsL, both detected in Rat1+ER{alpha} stimulated tester/Rat1+ERß stimulated driver, and (D) PCOLCE detected in the Rat1+ER{alpha} vehicle tester/Rat1+ERß vehicle driver SSH

Cathepsin L and RACK1 were identified from Rat1+ER{alpha} E2 tester/Rat1+ERß E2 driver comparisons. The RACK1 target had a twofold increase in Rat1+ER{alpha} E2-stimulated cells compared with Rat1+ERß E2 cells (P ≤ 0.05) (Fig. 3B), and CtsL increased twofold in Rat1+ER{alpha} cells when compared with Rat1+ERß E2 cells (P ≤ 0.05) (Fig. 3C).

Procollagen C-proteinase enhancer protein was identified in the Rat1+ER{alpha} vehicle-treated tester subtracted from Rat1+ERß vehicle-treated driver comparison. However, PCOLCE turned out to be a false positive in the population in which it was first identified, as Rat1+ER{alpha} vehicle-treated cells showed significantly lower expression levels than Rat1+ERß vehicle-treated cells (P ≤ 0.05). Interestingly, qRT-PCR detected a threefold increase in Rat1+ER{alpha} E2 versus Rat1+ERß E2 (P ≤ 0.05) (Fig. 3D).

Semiquantitative Analysis of Additional Genes of Interest Using RT-PCR

Semiquantitative reverse transcription-PCR analysis of gene targets was also used to confirm the validity of targets detected through SSH. A 935-bp product for annexin 1 was detected in Rat1+ER{alpha} E2-stimulated cDNA, as expected (Fig. 4A). A 963-bp product for nuclear factor I/B was detected in Rat1+ERß E2-stimulated as well as in Rat1+ER{alpha} vehicle-treated cells (Fig. 4B). These data are in agreement with the profiles predicted through SSH and differential screening.



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  		FIG. 4. Amplification of target genes annexin 1 and nuclear factor I/B identified through SSH: gel electrophoresis in 2% agarose followed by ethidium bromide (5 µg/µl) staining was used to detect putative gene products identified through SSH. A) Annexin 1 (935 bp) and (B) nuclear factor I/B (963-bp) amplification within the cell lines at 24 h E2 (1 nM) was analyzed beside G3PDH (500-bp) positive controls and bands extracted and sequenced to verify gene identification


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The study of ER{alpha} and ERß are of principal interest in relation to the critical role of the estrogen signaling system in the physiology of the reproductive organs and the cardiovascular, skeletal, and central nervous systems, and in carcinogenesis. There is particular interest in the selective activation of tissue-specific responses within the context of hormone replacement therapy and treatment of disease [42, 43]. There exists a need to better determine each receptors involvement in eliciting downstream gene expression both in tissues where the receptors function autonomously and especially where both are present. For these reasons, we have examined the nature of unique gene regulation by ER{alpha} and ERß in this undifferentiated model system designed to isolate each receptor protein from the influence of the other.

The known structural differences in ERs suggest unique independent downstream gene function. The amino and carboxyl terminal domains of the ER{alpha} protein are significantly longer than those of ERß [13, 14]. These domains interact with the various coregulator proteins crucial for transcriptional activation by the receptor to elicit cell-specific responses [14]. Furthermore, differences in amino acid composition and conformational differences within the binding pocket exist between ER{alpha} and ERß [44, 45], and these likely account for differences in affinity for various ligands. It is these differences that are at the heart of such pharmaceuticals as selective estrogen response modulators.

Using SSH, we have identified a profile of products differentially regulated by ER{alpha} and ERß in response to exposure to a single dose of E2 for 24 h. This represents a small subset of potential target genes, as any response that would have occurred prior to 24 h or at an alternative dose level, would not be recognized. Further, the experimental protocol has limited the number of products identified by limiting the number of colonies examined to 814 overall. Although, following sequencing of the cDNAs in the experimental set involving Rat1+ERß tester population, only five gene products were found and, among these, redundancy was as high as 47% for human gastric-associated differentially expressed protein. Conversely, when Rat1+ER{alpha} was the tester population, there was low redundancy in the population of sequenced cDNA clones, no greater than 14% redundancy for one unidentified product. Therefore, it is expected that cloning and sequencing of more Rat1+ER{alpha} colonies would result in identification of an increased number of target gene products, including other known targets for E2 modulation, such as the progesterone receptor.

Among downstream genes identified in the present study, several were of particular interest due to potential involvement in processes associated with reproduction, diseases of aging, and carcinogenesis. Their quantitative expression following treatment was characterized to verify responses seen in the SSH analysis. In relation to bone and cartilage remodeling, type 1 collagen accounts for the majority of total collagens and is most abundant in bone [46]. Additionally, COL1A2 is one of two alpha chains that comprise one third of the type I collagen heterotrimer [47]. PCOLCE is a glycoprotein enhancer element that binds the C-terminus of the type I procollagen propeptide and, as such, enhances the enzymatic ability of procollagen C-proteinase [48]. It is present in high levels in the uterus [49] and plays a role in intracellular collagen formation and extracellular cell differentiation and proliferation as well as possible stabilization of COL1A2 mRNA [50].

RACK1 binds the isozyme protein kinase C (PKC) and acts to stabilize the active conformation of PKC, which is necessary for subcellular translocation [51]. RACK 1 is a homologue of guanine nucleotide-binding protein (G-protein) ß subunit [51], and ER{alpha} studies have noted a relationship between E2 and G-protein coupled receptors to affect PKC [52]. Cathepsin L is a lysosomal cysteine protease implicated in human trophoblast invasiveness [53], bone resorption [54], and degradation of extracellular matrix [55], to a name few cellular functions that tie into reproduction, inflammatory responses, and bone remodeling. It also has implications in oncogenesis and tumor invasiveness.

Additionally, to further verify our SSH profiles, annexin 1 and nuclear factor I/B were selected for RT-PCR due to their role in the anti-inflammatory response and transcriptional regulation, respectively. Annexin 1 interacts with glucocorticoids to act in suppressing inflammation [56], which is an important component of implantation. Additionally, annexin 1 plays a role in cell growth and regulation [56] as well as in DNA unwinding [57]. Nuclear factor I/B is a member of the nuclear factor family and has a role in regulation of gene transcription through promoter interactions [58].

Due to the structural similarities in the ligand-binding domains of ER{alpha} and ERß and the ability to form heterodimers in vivo, it becomes important to definitively separate the two ERs. An engineered cell line that truly expresses only one subtype offers numerous avenues for examining independent ER function. As an undifferentiated fibroblast cell line, this model offers insight into the function of these ER in naïve developing systems as well as in a more global expression profile due to decreased complexity in comparison with highly differentiated cell lines. Previous studies have verified that the basic architecture is still present for the engineered cells to function in a physiologically relevant manner, such as the ability to activate progesterone receptor, which is silent in the parental Rat1 cells [30–31]. While not an alternative to in vivo studies, the Rat1, Rat1+ER{alpha}, and Rat1+ERß cell lines can indicate initial areas of interest at the gene level, which can in turn lead to directed, in-depth, focused physiological studies.

While there is overlap in the downstream targets for genomic effects in response to E2, it is evident that ER{alpha} and ERß are responsible for regulation of expression of unique subsets of downstream genes in response to the same ligand in the same cell type. This is in agreement with recent microarray studies at similar time points that have examined the differing global profiles of ER{alpha} or ERß in differentiated cell types [59]. However, the genes identified by Monroe et al. 2003 [59] do not overlap with those profiles found in the present study. This is most likely due to the nature of the varying levels of differentiation and complexity between models used as well as the unique properties of SSH in comparison with microarray. In this study, ER{alpha} appeared to be responsible for regulating a larger number of products, of which a majority was involved in general cell housekeeping and proliferation. ERß activation resulted in fewer detectable products in comparison with ER{alpha} and most of these had specific cellular functions, although some general regulatory products were also detected. Differential activity of these two receptors in response to the same ligand has important implications for understanding cell regulatory functions and inflammatory responses, which are integral to reproductive processes as well as oncogenesis, bone remodeling, and aging.


   ACKNOWLEDGMENTS
 
The authors would like to thank Leanne Weir and Jason Ross for technical assistance as well as Frankie White for assistance with statistical analysis. We would also like to thank the staff of the OSU Recombinant DNA/Protein Resource Facility CORE facility for providing DNA sequencing.


   FOOTNOTES
 
1 Supported by the Oklahoma Agricultural Experiment Station OKL02277. Back

2 Correspondence: J.R. Malayer, Department of Physiological Sciences, 264 McElroy Hall, Stillwater, OK 74078. FAX: 405 744 8263; malayer{at}okstate.edu Back

Received: 9 March 2004.

First decision: 9 April 2004.

Accepted: 1 June 2004.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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