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Molecular Endocrinology 13 (2): 320-329
Copyright © 1999 by The Endocrine Society

Transcriptional Regulation by a Naturally Occurring Truncated Rat Estrogen Receptor (ER), Truncated ER Product-1 (TERP-1)

Derek A. Schreihofer, Eileen M. Resnick, Ann Y. Soh and Margaret A. Shupnik

Department of Internal Medicine Division of Endocrinology and Metabolism University of Virginia Health Sciences Center Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Truncated estrogen receptor product-1 (TERP-1) is a naturally occurring rat estrogen receptor (ER) variant transcribed from a unique start site and containing a unique 5'-untranslated region fused to exons 5–8 of ER{alpha}. TERP-1 is detected only in the pituitary, and TERP-1 mRNA levels are highly regulated during the estrous cycle, exceeding those of the full-length ER{alpha} on proestrus. These data suggest that TERP-1 may play a role in estrogen- regulated feedback in the pituitary. We examined the ability of TERP-1 to modulate gene transcription in transiently transfected ER-negative (Cos-1) and ER-positive pituitary ({alpha}T3 and GH3) cell lines. In Cos-1 cells transiently cotransfected with TERP-1 and either ER{alpha} or ERß, low levels of TERP-1 (ratios of < 1:1 with ER) enhanced transcription of model promoters containing estrogen response elements by an average of 3- to 4-fold above that seen with ER alone. At higher concentrations of TERP-1 (> 1:1 with ER) transcription was inhibited. TERP-1 also had a biphasic action on transcription in the {alpha}T3 and GH3 pituitary cell lines, although the stimulatory action was less pronounced. TERP-1 actions were dependent on ligand-activated ER as TERP-1 did not bind estradiol in transfected Cos-1 cells or in vitro, and estrogen antagonists prevented the stimulatory effects of TERP-1. Coimmunoprecipitation studies suggest that TERP-1 does not bind with high affinity to the full-length ER{alpha}. However, TERP-1 may compete with ER for binding sites of receptor cofactors because steroid receptor coactivator-1 (SRC-1) rescued the inhibitory actions of TERP-1. The ability of TERP-1 to both enhance and inhibit ER-dependent promoter activity suggests that TERP-1 may play a physiological role in estrogen feedback in the rat pituitary.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen acts in many tissues to alter gene expression through specific nuclear receptors. These receptors bind estrogen, dimerize, and modulate gene transcription through interactions with estrogen response elements (EREs) in the promoter regions of estrogen-regulated genes (1). As a member of the steroid/thyroid hormone superfamily of nuclear transcription factors, the estrogen receptor (ER) is composed of several distinct functional domains. These include a ligand-independent transactivation function at the N terminus (AF-1), a DNA-binding domain (DBD) that allows dimerization of ERs and binding to EREs in estrogen-regulated genes, and a ligand-binding domain (LBD) near the C terminus (1, 2, 3). The LBD is important for both ligand-activated transactivation through the AF-2 region and dimerization of ERs (1, 2). The DBD and LBD both play a role in nuclear localization of ERs (1, 2).

In addition to the recently described ER isoform ERß (4), several variants of the ER{alpha} have been identified in human breast cancer (5, 6, 7) and tumor cell lines (7, 8). These variants have contained point mutations (5, 9), exon deletions (7, 8, 10), and C terminus truncations (6). Some of these variants can have transcriptional effects (9, 10). However, except in rare cases these variant ERs represent only a small proportion of ER present in cells (8). Further, there have been no reports of specific modulation of the levels of these splice variants or mutant receptors in normal tissue. In rats, a number of ER variants have been identified in reproductive tissues (11, 12, 13). At least some of these rat ERs are highly regulated throughout the estrous cycle and can exceed the levels of full-length ER{alpha} under physiological conditions (11, 12, 13).

We identified one such ER{alpha} variant mRNA and protein in female rat pituitaries (11). This variant, truncated ER product-1 (TERP-1), encodes a protein that is truncated at the N terminus. The transcribed mRNA contains exons 5–8 of the full-length ER{alpha} and a 31-bp unique sequence upstream of exon 5, indicating a unique transcriptional start site and promoter. Two in-phase translational start codons are present in exon 5, and immunoblot analysis confirms that TERP-1 protein is translated in vivo in the pituitary (13), in transfected cells, and in vitro. TERP-1 mRNA expression is also highly regulated by estrogen levels in vivo (11). In male rats and in ovariectomized female rats, pituitary TERP-1 mRNA levels are very low to undetectable as determined by Northern analysis and ribonuclease (RNase) protection, but treatment with estradiol for 1–3 days increases TERP-1 mRNA up to 7- to 10-fold (11, 13, 14). A unique property of TERP-1 is its regulation during the rat estrous cycle, when it is stimulated at least 50-fold, which suggests possible important regulatory actions for this ER receptor variant under physiological conditions. Although TERP-1 is essentially undetectable during early diestrus, mRNA levels exceed ER{alpha} on proestrus (13). This timing of expression suggests that TERP-1 could modulate estrogen actions around the proestrus gonadotropin/PRL surge. In the pituitary, ER{alpha} is present primarily in lactotropes and gonadotropes (14, 15, 16). Recent evidence that TERP-1 is present in ER-containing lactotropes (12, 17), and possibly gonadotropes (18), further supports a role in estrous cyclicity in the pituitary. This tissue-specific expression and regulation of TERP-1 distinguishes it from other ER splice variants or mutants.

Although the physiological role of TERP-1 is unknown at the present time, its regulation by estrogen in the pituitary and timing of expression during the estrous cycle suggests that it may play a role in estrogen feedback in pituitary cells. One possible mechanism for such feedback effects would be that TERP-1 could alter expression of estrogen-regulated genes either alone or in concert with ER{alpha} or ERß. Because TERP-1 lacks a DBD and a portion of the LBD (based on the predicted translational start site and protein size), it was unclear whether TERP-1 could affect the transcription of estrogen-regulated genes. If TERP-1 is able to influence transcription of estrogen-regulated genes through EREs, it is likely that full-length ERs are a necessary component in TERP-1 actions. In the present study we examined the ability of TERP-1 to alter the transcription of model and natural estrogen-responsive promoters in cell culture and the necessity of full-length ERs and estrogen for TERP-1’s actions. We found that TERP-1 could influence the transcription of estrogen-regulated genes. Although TERP-1 alone had no effect on transcription, it had a biphasic action on ERE-containing promoters in the presence of full-length ERs. Low ratios of TERP-1 to ER (<1:1) enhanced transcription, whereas higher ratios were inhibitory. The stimulatory effects were both promoter and cell type dependent, whereas the inhibitory actions of TERP-1 were observed in all cell types.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TERP-1 Protein Expression and Estrogen Binding
Protein levels of ER{alpha} and TERP-1 were assessed in calcium phosphate-transfected Cos-1 cells by immunoblotting. For comparison, in vitro translated ER{alpha} and TERP-1 are also shown (Fig. 1Go). ER{alpha} was clearly detected as a 64- to 66-kDa protein, and TERP-1 was detected as a pair of doublets at 22–24 kDa, which probably represent translation from two methionines (amino acids 393 and 408) in exon 5. In transfected Cos-1 cells, ER{alpha} and TERP-1 proteins of this size were also detected (Fig. 1Go). The larger protein, corresponding to the 5'-translational start site, is preferred in both transfected cells and normal pituitary cells (13). TERP-1 protein was clearly detectable in cell lysates when concentrations of 500 ng or greater were transfected. The inability to detect TERP-1 protein at lower transfection concentrations may be due to high turnover of the protein or the sensitivity of our antibody at low molar concentrations of TERP-1. However, several such experiments consistently demonstrated that levels of full-length ER{alpha} (1 µg) were relatively constant, even with increasing amounts of TERP-1 protein. Based on immunoblotting analysis, both full-length ER{alpha} and transfected TERP-1 were at much lower levels in GH3 cells as compared with Cos-1 cells, even with high transfection concentrations (data not shown).



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  		Figure 1. ER Protein Expression in Transfected Cos-1 Cells

ER{alpha} (1 µg) was transfected into Cos-1 cells in DMEM/10% NCS alone or with 0.1, 1, or 4 µg of TERP-1 by calcium phosphate. Cell lysates were collected 48 h later, and 100 µg of protein were subjected to electrophoresis on a 12% SDS-containing polyacrylamide gel. Western analysis was performed with enhanced chemiluminescence using the C1355 rat C terminus ER antibody. The first two lanes contain in vitro translated (IVT) ER{alpha} and TERP-1 for comparison. The upper panel shows a 10-min exposure, and the lower panel shows a 2-h exposure of TERP-1 from the same blot. Migration positions of mol wt markers are shown on the left.

 
The predicted protein sequence of TERP-1 contains most of the ligand- binding domain of ER{alpha} (predicted amino acids 393–600 of rat ER{alpha}), and thus TERP-1 might exert some transcriptional effects through estrogen binding. We assessed estrogen binding in vitro and in transfected Cos-1 cells using either 1 µg of ER{alpha} or 1 µg of TERP-1. ER{alpha} and TERP-1 were in vitro translated using coupled transcription-translation from rabbit reticulocyte lysate. Lysates (10 µl) were incubated with 0.05 pmol [3H]-estradiol (E2) and 5 pmol of unlabeled diethylstilbestrol (DES), and binding was determined using the hydroxylapatite method (19). E2 binding by ER{alpha} was readily displaced by DES, whereas TERP-1 showed no specific binding (Fig. 2AGo). TERP-1 did not alter the ability of ER{alpha} to bind E2 when they were coincubated with [3H]E2 and DES. Binding was determined in transfected Cos-1 cells with a competition assay using [3H]E2 and varying amounts of unlabeled E2. Parallel experiments with ER{alpha} and TERP-1 were performed in duplicate, and analysis of binding curves generated in two separate experiments showed that ER{alpha}-transfected cells specifically bound [3H]E2 with an affinity of 0.18 nM (Fig. 2BGo). In agreement with the in vitro experiments, TERP-1 did not bind E2 (Fig. 2BGo).



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  		Figure 2. Estrogen Binding by ER{alpha} and TERP-1

A, In vitro translated ER{alpha} and TERP-1 alone and in combination were assessed for [3H]E2 (0.05 pmol) binding in the absence and presence of 100-fold excess DES. Bound and unbound hormone were separated by the hydroxylapatite method. Data represent the mean ± SD of triplicate measurements. B, ER{alpha} (1 µg) or TERP-1 (1 µg) was transfected into Cos-1 cells in DMEM/3.3% charcoal-stripped NCS. Media were changed daily for 3 days before cells were treated with 1 nM [3H]E2 and various concentrations of unlabeled E2. Cells were collected and sonicated, and lysate radioactivity was assessed by scintillation counting. Points represent percentage of binding of ER{alpha} in the presence of 1 pmol unlabeled E2. Open circles represent the binding of E2 in TERP-1 transfected cells, while closed circles represent the binding in cells transfected with ER{alpha}. Points represent the mean of triplicate measures from two independent experiments.

 
TERP-1 Effects on Transcription in Cell Lines
We first tested TERP-1 transcriptional effects in the ER-negative Cos-1 cell line using model promoters containing two consensus vitellogenin EREs upstream of either a TATA box (2EREpGL2) or the thymidine kinase promoter (Vit6luc) fused to a luciferase reporter. As shown in Fig. 3AGo, cotransfection of 1 µg of an expression vector for full-length rat ER{alpha} and treatment of transfected cells with 10 nM E2 stimulated 2EREpGL2 approximately 7-fold and Vit6luc approximately 2.5-fold. Cotransfection of 1 µg ER{alpha} and 100 ng of TERP-1 increased expression of 2EREpGL2 and Vit6luc above that seen with ER{alpha} alone. The effect is specific to ER-mediated transcription, as TERP-1 did not alter the expression of a promoter lacking an ERE, the rat glycoprotein {alpha}-subunit (Fig. 3AGo). However, the response was influenced by the promoter context, as TERP-1 enhancement of E2/ER{alpha}-stimulated transcription was greater for the Vit6luc reporter than the 2EREpGL2 (Fig. 3AGo). TERP-1 also enhanced ERß-stimulated transcription of Vit6luc in Cos-1 cells, but this response was less than for ER{alpha} (Fig. 3AGo). The effect of TERP-1 on transcription of both model promoters was biphasic. Figure 3BGo shows an example of this response with the 2EREpGL2 promoter in Cos-1 cells. Stimulatory responses with TERP-1 occurred at transfected TERP-1 to ER ratios of <1:1. The amount of transfected TERP-1 needed for optimal stimulation of ER-mediated transcription was not absolute, possibly due to efficiency of transfection or resulting levels of TERP-1 protein translated. However, the amount of TERP-1 that enhanced transcription was directly related to the amount of ER{alpha}, increasing with higher levels of ER{alpha} transfected (data not shown). TERP-1 to ER ratios of 1:1 had either no net effect on E2/ER-stimulated transcription or were inhibitory (Fig. 3BGo), and TERP-1 to ER ratios >1:1 inhibited ER-induced transcription (Fig. 3BGo).



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  		Figure 3. Effect of TERP-1 on Transcription in Transfected Cells

A, Representative experiments showing the effect of 100 ng TERP-1 on ER{alpha} - and ERß-stimulated transcription of reporter genes in transiently transfected Cos-1 cells. Open bars represent transcriptional responses of ERE-containing reporters (1 µg of 2EREpGL2 or Vit6luc) and a reporter lacking an ERE ({alpha}3luc) to 1 µg transfected ER{alpha} or ERß in the absence of E2. Hatched bars represent the same conditions after 6 h of treatment with 10 nM E2. Solid bars represent 6 h of E2 treatment in cells cotransfected with reporter, ER, and 100 ng TERP-1. In each case, E2 treatments were normalized to the untreated condition and represent the mean ± SD of triplicate measurements. B, TERP-1 dose response in transfected Cos-1 cells using the 2EREpGL2 reporter (1 µg) cotransfected with 1 µg ER{alpha} and increasing concentrations of TERP-1 (1 ng to 10 µg). The open bar represents transfection in the absence of E2, and solid bars represent 6 h treatment with 10 nM E2 normalized to the untreated condition. Each bar represents the mean ± SEM of three independent experiments with individual experiments performed in triplicate. C, Representative experiments showing the effect of 100 ng or 1 µg TERP-1 on ERE-containing reporters (1 µg Vit6luc or PRLluc) in {alpha}T3 and GH3 pituitary cell lines. Bars are the same as in panel A, with the addition of stippled bars to represent the effect of 1 µg of TERP-1. {alpha}T3 cells were treated with or without 10 nM E2 for 6 h, GH3 cells for 24 h. In all cases, total transfected DNA was normalized with the pcDNA empty vector. In this and subsequent figures rALU refers to luciferase activity in arbitrary light units corrected for total lysate protein and normalized to the untreated condition (nt).

 
Physiologically, TERP-1 is highly expressed in the pituitary, and given its induction by E2 we expect TERP-1 protein to be present in lactotropes and gonadotropes. Recent experiments confirm that TERP-1 mRNA is present in lactotropes (12, 17). Therefore, we examined the ability of TERP-1 to alter transcription in pituitary cell lines that contain endogenous ER. In the presence of 10 nM E2, the endogenous ER modestly stimulated transcription of model promoters in the mouse {alpha}T3 gonadotrope cell line, and TERP-1 enhanced E2/ER-stimulated transcription of both Vit6luc and 2EREpGL2 above levels achieved with the endogenous ER alone (Fig. 3CGo). Transfection of 1 µg or more of TERP-1 was inhibitory (Fig. 3CGo). In the presence of 10 nM E2, the endogenous ER stimulated transcription of the model promoters in the rat somatolactotrope GH3 cell line less than 2-fold (Fig. 3CGo). Transfection of 100 ng of TERP-1 failed to enhance this effect (Fig. 3CGo), although higher concentrations of TERP-1 were inhibitory (Fig. 3CGo). Therefore, we also tested TERP-1 effects on a natural pituitary promoter in a pituitary cell line, and we used a rat PRL reporter (PRLluc) for this purpose. This PRL promoter construct contains 2.5 kb of the physiological promoter with a composite ERE that requires the transcription factor Pit-1 for optimal E2 induction (20). The addition of 10 nM E2 increased expression of PRLluc 5-fold (Fig. 3CGo). As we observed in Cos-1 cells using model promoters, TERP-1 enhanced this estrogen-induced effect at low concentrations of transfection (Fig. 3CGo). However, the stimulatory effect of TERP-1 on PRLluc was much smaller, averaging 50% (Fig. 3CGo). Transfection of 1 µg of TERP-1 failed to enhance E2/ER-stimulated transcription (Fig. 3CGo), and higher doses of TERP-1 were inhibitory (data not shown).

Role of Hormone and ER in the TERP-1 Transcriptional Response
To test the requirement for full-length ER{alpha} and hormone in mediating the effects of TERP-1, stimulation of the Vit6luc promoter was measured in Cos-1 cells. Transfection of TERP-1 at concentrations up to 2 µg in the absence of ER{alpha} had no influence on promoter activity, demonstrating a dependence on ER-induced transcription (Fig. 4Go). TERP-1’s stimulatory actions were also dependent on estrogen. Both the selective ER antagonist 4-hydroxy-tamoxifen and the antiestrogen ICI 182,780 antagonized TERP-1-enhanced E2-stimulated transcription in Cos-1 cells (Fig. 5Go). These data demonstrate a dependence on ligand-activated ER for TERP-1’s stimulatory actions.



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  		Figure 4. TERP-1-Induced Transcription in the Presence and Absence of Full- Length ER{alpha}

The effect of TERP-1 on transcription of the Vit6luc reporter gene in transiently transfected Cos-1 cells. Solid bars represent transcriptional response in the presence of cotransfected ER{alpha} (1 µg), and open bars represent responses in the absence of cotransfected ER{alpha}. All cells were treated with 10 nM E2. Bars represent the means ± SEM of four independent experiments performed in triplicate and are normalized to the ER{alpha}-only condition. In all cases total transfected DNA was normalized with the pcDNA empty vector.

 


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  		Figure 5. Effect of Estrogen Antagonists on TERP-1-Induced Transcription

Representative experiment showing the effect of antiestrogens on TERP-1-induced enhancement of E2/ER-stimulated transcription in transiently transfected Cos-1 cells. Cells were transfected with 1 µg Vit6luc reporter and 1 µg ER{alpha} with or without 10 ng TERP-1. After 16 h, cells were washed and placed in serumless DMEM. Cells were treated for 18 h with 1 nM E2 (solid bars), E2 and 1 µM 4-hydroxytamoxifen (hatched bar), or E2 and 1 µM ICI 182,780 (stippled bar). Values represent means ± SD of triplicate measures normalized to the untreated condition (open bar). In all cases total transfected DNA was normalized with the pcDNA empty vector.

 
Based on its predicted structure, TERP-1 contains one of two heterodimerization regions of the full-length receptor and may interact directly with full-length ER{alpha}. Coimmunoprecipitation studies of ER{alpha} and TERP-1 were performed with both in vitro translated proteins and transfected Cos-1 cells with essentially the same results. Because TERP-1 contains identical sequences to the C terminus of ER{alpha}, the proteins were distinguished by using the hinge antibody ER715, which recognized only the full-length ER{alpha}, and an epitope-tagged construct of TERP-1 (TERP-FLAG). TERP-FLAG contains the eight-amino acid FLAG epitope at the C terminus of TERP-1 and can modulate ER transcriptional activity in Cos-1 cells (not shown). As shown in Fig. 6Go, immunoprecipitation studies failed to show a direct interaction between ER{alpha} and TERP-FLAG (upper panel), although coprecipitation of ER{alpha} and ERß could be demonstrated under these conditions (bottom panel). Thus, under these stringent conditions, TERP-1 does not appear to interact directly with ER{alpha}.



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  		Figure 6. Coimmunoprecipitation of ER Isoforms

Upper panel, Autoradiogram of immunoprecipitated in vitro translated full-length ER{alpha}, TERP-1 (TERP), or epitope-tagged TERP-1 (TERP-FLAG), alone or in combination, by antibodies to the ER{alpha} hinge region (ER715) or to the epitope tag (FLAG). Migration of mol wt markers is shown on the right. Lower panel, Autoradiogram of immunoprecipitated in vitro translated full-length ER{alpha} and ERß, alone and in combination, with an antibody to ER{alpha} (ER{alpha} IP). The solid arrow indicates the position of full-length ER{alpha}, and the dotted arrow indicates the location of ERß. The two lanes on the far left contain 2 µl each of total in vitro translated protein.

 
Interaction of TERP-1 with Steroid Receptor Coactivator-1 (SRC-1)
Although TERP-1 does not bind estrogen and does not appear to have a stable direct interaction with the full-length ER{alpha}, it only has transcriptional effects in the presence of a ligand-activated full-length ER. Therefore, it probably acts through other protein-protein interactions. One possible mechanism is titration of inhibitory cofactors such as steroid corepressors or stimulatory coactivators such as SRC-1 (21) receptor interacting protein 140 [RIP140 (22)]. Experiments with the corepressors N-COR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) suggest that they do not affect estrogen-stimulated transcription by ER{alpha} (Ref. 23 and our unpublished results), a requirement for TERP-1 actions. However, TERP-1 may interact with a coactivator such as SRC-1, either directly or by competing with SRC-1 for binding partners. We tested the ability of SRC-1 to influence TERP-1 effects on ER{alpha}-mediated transcription. SRC-1 enhanced E2/ER{alpha}-stimulated transcription of Vit6luc in transfected Cos-1 cells in a dose-dependent manner (Fig. 7AGo). Cotransfection of low levels of TERP-1 (100 ng) did not further enhance SRC-1’s effects on E2/ER{alpha}-stimulated transcription (Fig. 7AGo). However, increasing levels of SRC-1 overcame the inhibitory action of higher levels (2–5 µg) of cotransfected TERP-1 (Fig. 7BGo). These data suggest that TERP-1 may compete with ER{alpha} for binding sites on SRC-1 or similar cofactors, thus limiting the extent of ER{alpha} activation.



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  		Figure 7. Effect of SRC-1 on TERP-1-Induced Stimulation and Inhibition of ER{alpha}-Stimulated Transcription

A, The effect of 100 ng TERP-1 on SRC-1-enhanced E2/ER{alpha}-stimulated transcription of Vit6luc in transiently transfected Cos-1 cells. All cells were transfected with 1 µg Vit6luc and 1 µg ER{alpha}. Open bars represent transcriptional responses of Vit6luc in the absence of E2. Solid bars represent the same conditions after 6 h of treatment with 10 nM E2. Hatched bars represent cells transfected with 1 µg Vit6luc, 1 µg ER{alpha}, and increasing concentrations of SRC-1 (0.1, 0.25, and 1 µg) treated for 6 h with 10 nM E2. Stippled bars represent cells transfected with reporter, ER{alpha}, SRC-1, and 100 ng TERP-1. In each case, E2 treatments were normalized to the untreated condition and represent the mean ± SEM of four experiments performed in duplicate. B, The effect of SRC-1 on TERP-1 inhibition of E2/ER{alpha}-stimulated transcription of Vit6luc in transiently transfected Cos-1 cells. All cells were transfected with 1 µg Vit6luc and 1 µg ER{alpha}. Open bars represent transcriptional responses of Vit6luc in the absence of E2. Solid bars represent the same conditions after 6 h of treatment with 10 nM E2. Hatched bars represent cells transfected with 1 µg Vit6luc, 1 µg ER{alpha}, 2 µg TERP-1, and increasing concentrations of SRC-1 (0, 0.1, 0.25, and 1 µg) treated for 6 h with 10 nM E2. In all cases the SRC-1 parental pBKcmv empty vector was used to normalize for changes in SRC-1, and pcDNA was used to normalize for total DNA transfected.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previously, we identified a truncated ER mRNA and protein from female rat pituitaries (11). This truncated ER product-1 (TERP-1) cDNA encodes a unique 31-bp 5'-end and the intact exons 5–8 of the rat ER{alpha}. Thus, TERP-1 contains most of domain E and all of domain F but has no AF-1 region or DBD. Although two potential in-phase start codons are present near the N terminus of exon 5 and result in two transcripts in vitro, our studies in transfected cells suggest that the first ATG (residue 393) is used preferentially in Cos-1 cells (Fig. 1Go), in agreement with our observations in normal pituitary cells (13).

TERP-1 expression in vivo is localized to the pituitary, where it may play a role in estrogen-regulated gene expression (11). High TERP-1 mRNA levels are induced by estrogen in castrate male and female rats and during the estrous cycle under conditions where the full-length ER{alpha} is modulated only slightly (13, 14). Recent reports and our unpublished observations demonstrate that TERP-1 is localized to pituitary cells that express ER{alpha}, namely lactotropes and gonadotropes (12, 17, 18). This suggests that TERP-1 is dependent on ligand-bound ER for expression as well as biological activity. Thus, TERP-1 could provide a level of cellular feedback to estrogen-sensitive genes in the pituitary. Our observation that TERP-1 modulated transcription from both heterologous and physiological promoters in pituitary cell lines supports this hypothesis.

Transcriptional activation by naturally occurring truncated or mutant steroid receptors is not uncommon, and the interaction of receptor isoforms may provide an important level of gene regulation. Interestingly, many of these interactions do not require an intact DBD or binding to a hormone response element. Recently a third PR isoform, PR-C, has been identified (24). Like TERP-1, PR-C is amino truncated and lacks the first zinc finger of the DBD. PR-C is also able to enhance PR-A- and PR-B-induced transcription, although it has no transactivating capacity alone. Other superfamily members also have actions that do not require the complete receptor. For example, the LBD of the 9-cis-retinoic acid receptor (RXR) alone can enhance transcriptional activation by the orphan receptor Nurr1 (25). Furthermore, although it has no DBD, the orphan receptor SHP has an LBD and dimerizes with other superfamily members, including ER{alpha} and ERß, to inhibit transcription (26, 27). Mutations of the first zinc finger in the glucocorticoid receptor (GR) DBD inhibit DNA binding to glucocorticoid response elements but allow ligand-dependent activation of interleukin-6, a promoter the intact GR suppresses (28). In addition, ER{alpha} can activate transcription from elements other than EREs by interacting with other transcription factors, and variant ER forms may play a role in this response. For example, tamoxifen-bound ER{alpha} can interact with Jun/Jun or Fos/Jun dimers to enhance transcription driven by AP1 (29), and this effect does require an intact DBD. The estrogen antagonist raloxifene also stimulates ERß-induced transcription through interactions with Fos and Jun at AP1 sites (30). These mechanisms of transcriptional activation by steroid receptors demonstrate that various unique promoter sequences may be identified by the combination of receptor isoforms and ligands present in the cell.

The mechanisms by which TERP-1 influences estrogen-regulated gene transcription clearly require a ligand-activated ER. TERP-1 does not bind estrogen itself, but modulates ER{alpha} or ERß stimulated transcription in the presence of estrogen, while antiestrogens prevent TERP-1 effects. TERP-1 effects are not a general transcriptional phenomenon, as a promoter that does not contain an ERE was not affected by the addition of TERP-1. TERP-1 effects on ER{alpha} and ERß might occur through direct interactions with the activated full-length receptors, or by interactions with coactivator or corepressor proteins that themselves influence ER activity. The inhibition of TERP-1-enhanced E2-stimulated gene activation by antiestrogens is consistent with either or both possibilities.

Fawell et al. (31) localized the dimerization domain of the mouse ER{alpha} in the C terminus. Specifically, amino acids 507–518 are necessary for ligand-induced dimerization, and the homologous region is present in TERP-1. Heterodimerization between receptor isoforms has been noted for several nuclear receptors. The resultant dimers can have transcriptional properties in which the activity of one isoform predominates or may result in modified or enhanced activities. For example, the progesterone receptor isoforms PR-A and PR-B both stimulate transcription from PREs, but have different agonist/antagonist profiles (32). When PR-A/PR-B heterodimers form, PR-A effects predominate on some promoters, but not others (32). Similarly, ER{alpha} and ERß form heterodimers, and ER{alpha} activity can predominate in a promoter- dependent manner (33, 34). A C-terminal truncated form of ER{alpha}, resulting from an exon 5 deletion, was recently reported to stimulate activated ER-dependent promoter activity in osteosarcoma cells (35). This variant can bind DNA, and may form heterodimers on DNA with full-length ER{alpha}, but cannot bind hormone itself. Thus, the enhanced activity of the heterodimer in the osteosarcoma context may occur through enhanced receptor interactions with the transcriptional machinery or altered interactions with other accessory proteins. Our immunoprecipitation studies do not demonstrate direct interactions between TERP-1 and ER{alpha}, even though ER{alpha} and ERß formed heterodimers under these conditions. This result suggests two possibilities. TERP-1 may interact with ER{alpha}, but under less stringent conditions than tested here, or only as part of a multiprotein complex that is not preserved under immunoprecipitation conditions. Alternatively, TERP-1 may act as an intracellular buffer by binding to cofactors that interact with the C-terminal portion of ER, thus altering the levels of proteins available to interact with full-length ER.

Several steroid receptor cofactors, including those with positive (coactivators) and inhibitory (corepressors) effects on transcription have been identified (36). ER-stimulated transcription may be enhanced by several coactivators such as steroid-receptor coactivator-1 [SRC-1, (21)], the closely related ER-associated protein, ERAP160 (37, 38), and receptor-interacting protein 140, RIP140 (39). Such proteins interact with the ER LBD in a ligand-dependent manner, but do not enhance transcription without ER. Our studies demonstrate that transfected human SRC-1 can enhance rat ER{alpha} activity, as has been noted for the human (40) and mouse (34) ER. Low levels of TERP-1 did not increase ligand-activated ER{alpha}-stimulated transcription above levels observed with SRC-1 alone. However, the enhancement of ER activity by TERP-1 may occur by titration of corepressor proteins. Such proteins have been identified for ER{alpha} (41), PR (41), and TR (42, 43). A potential novel ER-specific repressor, which binds to the LBD of the ER without the hinge region, has also recently been described from MCF7 breast cancer cells (44) and could represent one of many possible cell-specific cofactors influencing ER action. The interaction of TERP-1 with corepressor proteins is consistent with our results in that binding of estrogen and SRC-1 alone may reduce the influence of corepressors on ER-mediated transcription to the point that TERP-1’s effects are negligible.

Increasing concentrations of SRC-1 can overcome the inhibitory effect of TERP-1. One potential mechanism is a direct interaction between TERP-1 and SRC-1. Evidence for such an interaction comes primarily from studies of TRß. Recent data from Feng et al. (45) shows that mutations in amphipathic helices 3, 5, 6, and 12 of TRß diminish the binding of glucocorticoid receptor-interacting protein 1 (GRIP1) and SRC-1. The same mutations reduced transactivation by TRß (45). Three similar mutations in helices 3, 5, and 12 of human ER{alpha} decreased GRIP1 binding and hormone-dependent transactivation (45). The authors suggest that all three regions are needed for coactivator binding and full receptor activity. TERP-1 contains only one of these regions, helix 12. However, Jeyakumar et al. (46) recently demonstrated that a 20-amino acid peptide (a.a. 437–456) of TRß reduced SRC-1 binding to TRß by 80%. Thus, TERP-1 might be able to serve an analogous inhibitory role in certain cell contexts. Cell and promoter-dependent responses we observed could result from the relative contribution of such cofactors to ER-mediated transcription of individual promoters and the relative concentration of specific cofactors, ER and TERP-1, in different cell lines.

The expression and regulation of TERP-1 in vivo suggests that it may play a role in estrogen feedback in the female rat pituitary during the estrous cycle. The present study demonstrates that low ratios of TERP-1:ER enhance E2-stimulated gene expression in cell lines transiently transfected with ER{alpha} or ERß and in cell lines expressing endogenous ER{alpha}. However, TERP-1 inhibits E2-stimulated gene expression at higher ratios. TERP-1 appears to modulate transcription of model promoters and a physiological promoter in an estrogen- and ER-dependent manner. The ability of TERP-1 to both enhance and inhibit transcription allows for the possibility that the dynamic regulation of TERP-1 during the estrous cycle leads to both enhancement and inhibition of estrogen actions in the pituitary. It is important to note that in E2-treated rats TERP-1 mRNA levels are similar to those observed during proestrus, and TERP-1 protein is readily detected in the pituitaries of E2-treated rats at near- molar equivalence with ER{alpha} (13). In the present study, TERP-1 protein levels in transiently transfected Cos-1 cells were well below the levels of ER{alpha}, even when greater amounts of TERP-1 plasmid were transfected (Fig. 1Go). These observations suggest that in vivo TERP-1 may play a predominantly inhibitory role. The results of the present study suggest that TERP-1 may act via protein-protein interactions rather than direct high-affinity interactions with ER. Thus, the truncated receptor could act as a buffer to modulate the levels of coactivators and corepressors available for interaction with the full-length ER, and thereby modify its transcriptional activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Transfections
The monkey kidney Cos-1, ER-negative cell line was maintained in DMEM supplemented with 10% newborn calf serum (NCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated in phenol-red free DMEM/3% charcoal- stripped NCS in 30-mm wells and grown to 60–80% confluence before transfection by the calcium phosphate method (47). Each well was transfected with a total of 5 µg DNA for 14–17 h, washed with PBS, and treated with hormone or drugs for 6–8 h before being lysed and collected for assessment of luciferase activity. The ER-containing GH3 rat somatolactotrope cell line was maintained in DMEM/10% NCS and transfected by the diethylaminoethyl (DEAE)-Dextran method (48). Cells were plated in phenol-red free DMEM/10% stripped NCS in 60-mm wells and allowed to grow to 40–50% confluence before transfection. Media were removed, and cells were washed with PBS before 2 h of treatment with DNA in DEAE-dextran. After removal of DEAE-dextran, cells were treated for 2 min with 10% dimethylsulfoxide in HEPES-buffered saline, washed with PBS, and treated with hormones or drugs in media for 24 h. AlphaT3 ({alpha}T3) cells (49), an ER-expressing gonadotropin cell line of murine origin, were maintained in DMEM/10% FCS and plated and transfected with calcium phosphate as described for Cos cells. Cell culture media and sera were purchased from GIBCO-BRL (Gaithersburg, MD). E2 and 4-hydroxytamoxifen were obtained from Sigma Chemical Co. (St. Louis, MO). The antiestrogen ICI 182,780 was kindly provided by Dr. Richard Santen (gift to Dr. Santen from Dr. Alan Wakeling, Zeneca Pharmaceuticals, Macclesfield, England). Details of specific experiments are provided in Results and in figure legends.

Plasmids
TERP-1 and full-length rat ER{alpha} cDNAs were isolated from female rat pituitaries by 5'-rapid amplification of cDNA ends (11) and cloned into a pcDNA expression vector (Invitrogen Corp., San Diego, CA) under the control of the cytomegalovirus (CMV) promoter. The full-length rat ERß clone in the pCMV5 expression vector was obtained from Dr. Deborah Lannigan. The SRC-1 expression vector was obtained as a gift from Dr. Bert O’Malley. All reporter plasmids contained the luciferase reporter gene. Two simple reporter plasmids containing model estrogen-responsive promoters were used in these studies. (ERE)2-vit-TK-luc (Vit6luc) contained two vitellogenin consensus EREs and the thymidine kinase promoter. 2EREpGL2 was kindly provided by Dr. Deborah Lannigan and contained two consensus EREs upstream of a TATA box minimal promoter. One physiological estrogen-responsive reporter plasmid, PRL luc Link V2 (PRLluc, gift of Dr. Richard N. Day), containing 2.5 kb of the rat PRL promoter was also used. A rat glycoprotein {alpha}-subunit promoter construct (-411 to +77 bp) cloned in our laboratory [{alpha}3-luc (50)] and lacking EREs was used as a control for ERE dependence. To normalize the total plasmid DNA concentrations used in transfections, the empty pcDNA vector was used. In experiments with SRC-1, the pBKcmv empty vector was used to normalize total DNA (21). Luciferase activity was assessed on a Turner-20E luminometer using a Promega (Madison, WI) luciferase assay kit, and samples were normalized by assessing lysate protein with Bio-Rad (Hercules, CA) protein dye. Data are presented as relative arbitrary light units, normalized to the estrogen-negative condition. Values represent the mean ± SD of triplicate samples, unless otherwise noted.

Estrogen Binding Assay
Estrogen binding of ER{alpha} and TERP-1 was assessed in vitro and in transfected Cos-1 cells. ER{alpha} and TERP-1 were in vitro translated using coupled transcription-translation from rabbit reticulocyte lysate (TNT kit, Promega). Lysates (10 µl) were incubated with 0.05 pmol [3H]E2 and 5 pmol of unlabeled DES in triplicate at 4 C for 4 h. Bound and free E2 were separated using the hydroxylapatite method (19), and 200-µl aliquots in 10 ml of ReadySafe scintillation fluid (Beckman Instruments, Fullerton, CA) were counted in an LKB Rackbeta scintillation counter (LKB Instruments, Rockville, MD). We assessed estrogen binding in calcium phosphate-transfected Cos-1 cells using either 1 µg of ER{alpha} or 1 µg of TERP-1. Cells were plated in DMEM/3.3% charcoal-stripped NCS in 30-mm wells. Media were changed daily for 3 days before transfection. Cells were transfected for 16 h. Media were removed, the cells washed with PBS, and wells filled with serumless, phenol-red free DMEM. Cells were incubated for an additional 24 h before estrogen binding was assessed.

Estrogen binding was determined with a competition assay. Cells were treated with 1 nM [2,4,5,6-3H]E2 (New England Nuclear, specific activity = 114 Ci/mmol) and varying amounts of unlabeled E2. Cells were incubated for 1 h at 37 C, rinsed twice with PBS/0.1% BSA and twice with PBS, and collected in 10:1 Tris-EDTA (pH 8). Cells were then sonicated for 20 sec with a Fisher sonic dismembrator, and 200 µl of each extract were added to 10 ml of scintillation fluid (ReadySafe; Beckman) and counted in an LKB Rackbeta scintillation counter. Specific binding (labeled E2 - 1 µM unlabeled E2) was calculated, corrected for total protein, and analyzed by Scatchard plots.

Immunoblot Analysis
TERP-1 and ER{alpha} protein expression in transfected cells was determined by immunoblot analysis using a rabbit polyclonal antibody (C1355) generated against the final 14 C-terminal amino acids of the rat ER{alpha}. The antibody and conditions for Western analysis were determined using in vitro translated TERP-1 and ER{alpha} and have been described previously (13). For analysis of cellular expression, Cos-1 cells were transfected with 1 µg of ER{alpha} and varying concentrations of TERP-1 by calcium phosphate for 4 h and allowed to incubate 48 h in DMEM/10% NCS. In some experiments GH3 cells were also analyzed after transfection with TERP-1. Western analysis was performed with enhanced chemiluminescence (Amersham, Arlington heights, IL) using the C1355 antibody at 1:7500 and an horseradish peroxidase-conjugated donkey antirabbit IgG secondary antibody (1:500; Amersham).

Immunoprecipitations
Because TERP-1 contains the identical amino acid sequence as the C terminus of ER{alpha}, it was necessary to differentiate TERP-1 from ER{alpha} by epitope tagging. PCR was used to add the eight-amino acid FLAG epitope (Eastman-Kodak, Rochester, NY) to the TERP-1 sequence. The resulting PCR product was ligated into the PCR2.1 cloning vector (Invitrogen), sequenced, and then subcloned into the pcDNA expression vector. Proteins were synthesized from ER{alpha}, ERß, TERP-1, and TERP-FLAG plasmids (1 µg each construct alone, or in combination) by in vitro translation using coupled transcription-translation from rabbit reticulocyte lysate (TNT kit, Promega). Each reaction also contained 40 µCi [35S]methionine (New England Nuclear; 1175 Ci/mmol), and 10 nM E2. In some experiments, different ER protein isoforms were cotranslated in the same reactions. Results were similar or identical to results with individually translated proteins. Each immunoprecipitation reaction contained 6 µl of translated proteins, 1% BSA, and 50 µl of a 2x slurry of either anti-FLAG M2 affinity gel (VWR), or protein A Sepharose (Pharmacia, Piscataway, NJ) linked to either ER C1355 (C-terminal antibody) or ER715 (gift of Dr. Jack Gorski). ER715 recognizes the hinge region of ER{alpha} and will recognize only full-length receptor. Beads and antibody were first brought to a 2x slurry with 1.5% Nonidet-P40 (NP-40) in PBS, pH 7.6. The proteins, beads, and antibody were incubated at 4 C for 1 h. After incubation, beads were washed four times with 1.5% NP-40 in PBS and resuspended in 25 µl of 1.5% NP-40 in PBS and 25 µl of loading buffer (100 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 0.2% bromphenol blue). Samples were boiled for 3 min and subjected to electrophoresis on a 12% acrylamide gel for 4 h at 150 V. Gels were dried and exposed to film overnight.


   ACKNOWLEDGMENTS
 
The authors wish to acknowledge the excellent technical assistance of Alice Anderson and Anthony Lim at the Core for Studies in Molecular and Cellular Research at the University of Virginia.


   FOOTNOTES
 
Address requests for reprints to: Margaret A. Shupnik, Department of Internal Medicine, Division of Endocrinology and Metabolism, Box 578, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: MAS3X{at}VIRGINIA.EDU

This work was supported by NIH Grants RO1 HD-25719 (M.A.S.), F32 HD-08219 (D.A.S.), a training grant for Diabetes and Hormone Action at the University of Virginia (T32 DK-07320; D.A.S.), training grant T32 GM-07055 (E.M.R.) and the Center for Research in Reproduction (U54-HD28934).

Received for publication October 27, 1997. Revision received October 8, 1998. Accepted for publication October 21, 1998.


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 DISCUSSION
 MATERIALS AND METHODS
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