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Endocrinology Vol. 139, No. 11 4513-4522
Copyright © 1998 by The Endocrine Society

ARTICLES

Transcription Activation by the Human Estrogen Receptor Subtype ß (ERß) Studied with ERß and ER{alpha} Receptor Chimeras1

Eileen M. McInerney, Karen E. Weis, Jun Sun, Sietse Mosselman and Benita S. Katzenellenbogen

Departments of Molecular and Integrative Physiology (E.M.M., K.E.W., J.S., B.S.K.) and Cell and Structural Biology (B.S.K.), University of Illinois, Urbana, Illinois 61801; and the Target Discovery Unit, N.V. Organon (S.M.), Oss, The Netherlands

Address all correspondence and requests for reprints to: Dr. Benita Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied the two estrogen receptor (ER) subtypes, ER{alpha} and ERß, and chimeric constructs with ER{alpha} and ERß to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ERß is highly dependent on cell/promoter context and on the nature of the ligand. ERß activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER{alpha} in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER{alpha}, exhibit no agonistic activity with ERß. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ERß. Upon replacing the A/B domain of ERß with the A/B domain of ER{alpha}, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ERß and the ERß/{alpha} chimera containing the amino-terminal A/B domain of ERß fused to domains C through F of ER{alpha}, but was restored in an ER{alpha}/ß chimera containing the A/B domain of ER{alpha}, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER{alpha}. Together, these results indicate that the differences in the amino-terminal regions of ER{alpha} and ERß contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID hormones influence a wide variety of cellular processes, such as cell proliferation and differentiation, by regulating the expression of responsive genes (1, 2, 3). The interaction of steroid hormones with specific intracellular receptor proteins allows high affinity binding of the receptor to specific enhancer-like sequences, termed hormone response elements (2). The estrogen receptor (ER) belongs to a large superfamily of nuclear hormone receptors that share a common modular organization. The ER is organized into domains that are responsible for specific functions, such as ligand binding, dimerization, DNA binding, and trans-activation (4, 5, 6, 7). Like other members of this superfamily, ER contains a centrally located C domain, corresponding to the DNA-binding domain, and two transcription activation functions (6, 7, 8, 9). Activation function-1 (AF-1) is located in the amino-terminal A/B domain, and activation function-2 (AF-2) is located within the E domain along with the hormone binding function (10, 11). Although AF-2 is highly conserved (1, 12, 13), AF-1 is less well conserved among species and shows little conservation among other members of the steroid hormone receptor superfamily (1, 13, 14). Furthermore, the activity of each activation function is dependent on cell and promoter context (7, 10, 11, 15, 16).

Transcription of estrogen-responsive genes by ER can be antagonized by antiestrogens, such as trans-hydroxytamoxifen (TOT) and ICI 164,384 (17, 18). Although these antiestrogens promote DNA binding by ER (19, 20), it is thought that antiestrogens such as TOT activate transcription only poorly because they are unable to effectively stimulate AF-2 activity (7, 17). However, antiestrogens such as TOT have been shown to have partial agonistic activity in certain cells, such as chicken embryo fibroblasts, MDA-231 human breast cancer cells, and human endometrial cancer cells (21). In addition, it has been suggested that certain antiestrogens can activate transcription in these cell types, because in them AF-1 acts as a strong transcriptional activator (8, 17, 21). Together, these reports have implied that antiestrogen agonism is AF-1 dependent.

Since the cloning of the ER about 10 yr ago (22, 23), there has been the general acceptance that only one ER existed. This contrasted with other members of the nuclear receptor superfamily, for which multiple forms have been reported (e.g. thyroid receptor {alpha} and ß and retinoic acid receptor {alpha}, ß, and {gamma}) (1, 24). Recently, however, a novel ER has been cloned and characterized (25, 26, 27, 28); it has been termed ERß to distinguish it from the previously identified ER, now called ER{alpha}. There is currently intense interest in understanding its role in estrogen action and how its activity compares and contrasts with that of ER{alpha}. ERß, which is encoded by a different gene, has an overlapping, but nonidentical, tissue distribution as ER{alpha} (27). ERß, like ER{alpha}, binds estrogens and antiestrogens and has been shown to modulate the transcription of estrogen-responsive reporter genes in Chinese hamster ovary (CHO) cells (25, 26). However, it should be noted that in this one cell type examined, the transcriptional activity of ERß was significantly less (~50% of ER{alpha}) than the activity observed with ER{alpha}.

Human ERß is highly homologous to ER{alpha}. The DNA-binding domain is highly conserved (96% identity) between ERß and ER{alpha}, and the hormone-binding domain is also relatively well conserved (58% identity) between these two receptors (25). It is of note, however, that the AF-2 core at the C-terminus of the hormone-binding domain of these receptors differs by one amino acid. Sequence alignment of ER{alpha} and ERß reveals a highly conserved region from amino acids 537–547 in ER{alpha} and 488–498 in ERß, with the only exception being amino acid D545 in ER{alpha} (amino acid N496 in ERß). In addition, the A/B domains of ERß and ER{alpha} are poorly conserved (only ~20%), suggesting that their AF-1 activities might be different and possibly that different coactivators interact with this region.

We were interested in examining the transcriptional activity of ERß and determining how the activity of ERß is influenced by cell and promoter context, and we postulated that the different transcriptional activity of ERß compared with that of ER{alpha} might be attributable to the low conservation of the A/B domain. We also examined the importance of the nonconserved residue within the AF-2 core. In the studies presented, we demonstrate that the ability of ERß to function as an estrogen-dependent transcriptional activator is highly dependent on cell and promoter context. We also found that in certain contexts where antiestrogens are able to activate ER{alpha}, ERß displays no transcriptional response to antiestrogens. However, upon replacing the A/B domain of ERß with the A/B domain of ER{alpha}, this chimeric receptor not only shows a significantly greater transcriptional response to estrogens, but also exhibits a transcriptional response to antiestrogens. Our findings suggest that despite great similarity between the DNA-binding domains of these two receptor subtypes, the transcriptional responses of ERß to different ligands are distinctly different and are strongly influenced both by the nature of the receptor N-terminal A/B domain and by the cellular context.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and materials
Cell culture media were purchased from Life Technologies (Grand Island, NY). Calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT), and FCS was purchased from Atlanta Biologicals (Atlanta, GA). [14C]Chloramphenicol (50–60 Ci/mmol) and [3H]estradiol ([3H]E2) were purchased from DuPont-New England Nuclear Research Products (Boston, MA). The Flag epitope monoclonal antibody M2 was obtained from IBI (Carlsbad, CA), and the anti-ER{alpha} monoclonal antibodies H222 and H226 were provided by Dr. G. Greene (University of Chicago, Chicago, IL). The antiestrogen TOT was provided by Dr. A. Wakeling, Zeneca Pharmaceuticals (Macclesfield, UK), and the 2-phenylbenzofuran (BF) antiestrogen was provided by Dr. E. von Angerer, University of Regensburg (Regensburg, Germany) (29). The estrogen P1496 was described previously (21).

Plasmid constructions
The human ER expression vector for ER{alpha} (pCMV5-hER) was constructed as previously described (15, 30). The expression vector pCMV5-ERß was constructed by inserting the full-length complementary DNA encoding the human ERß (530 residues, pNGV1-ERß), described previously (25), and including the additional 53 N-terminal amino acids shown in Fig. 1AGo (same as GenBank accession no. AF051427) into the BamHI site of pCMV5. The expression vector for the chimeric ER{alpha}/ß receptor was constructed to contain the A/B domain of ER{alpha} and domains C, D, E, and F of ERß. First, an MluI site was created at residue 145 of pCMV5-ERß by site-directed mutagenesis (31) to create pCMV5-ERß-(Mlu145). The MluI fragment of pCMV5-ERß-(Mlu145) was replaced by a PCR-generated insert of the A/B domain of ER{alpha} containing an MluI site created at residue 181 of ER{alpha}. The ERß(N496D) point mutant was created by site-directed mutagenesis by replacing nucleotides AAT, representing amino acid 496 of pCMV5-ERß, with nucleotides GAT. The expression vector for the chimeric receptor ERß/{alpha} was constructed to contain the A/B domain of ERß and domains C, D, E, and F of ER{alpha}. Using site-directed mutagenesis, we incorporated an EcoRV site at amino acid 195 of ER{alpha}. Because of high sequence homology between ER{alpha} and ERß, there is an EcoRV site in ERß at amino acid 158 that is in the equivalent location and reading frame as the EcoRV site created in ER{alpha}. Consequently, the A/B domain of ER{alpha} was excised by EcoRV digestion and replaced by the EcoRV fragment containing the A/B domain of ERß. The sequences of all mutants were confirmed by dideoxy sequencing methods to assure accuracy.



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  		Figure 1. A, Alignment of the A/B domains of human ER{alpha} and ERß. Residues 1–180 of ER{alpha} and residues 1–144 of ERß are shown and aligned for sequence homology. Vertical lines indicate identical amino acids. B, Structure of ER derivatives. The functional domains (A/B, C, D, E, and F) and activation functions (AF-1 and AF-2) of ER are shown at the top along with schematics for ER{alpha}, ERß, the point mutant ERß(N496D), and the chimeric proteins we have made, denoted ER{alpha}/ß and ERß/{alpha}. Note that ERß contains 53 additional N-terminal amino acids not identified in the original report (25 ) on human ERß.

 
To construct the Flag epitope-tagged receptors, the following oligonucleotides that encode the Flag epitope sequence (IBI) were annealed and inserted into the MluI/ClaI sites of pCMV5-ERß or pCMV5-ER{alpha}/ß: forward oligo, 5'-CG CGT CCA CGG ACC ATG GAC TAC AAG GAC GAC GAT GAC AAG TCT AGA AT-3'; and reverse oligo, 5'-C GAT TCT AGA CTT GTC ATC GTC GTC CTT GTA GTC CAT GGT CCG TGG A-3'. The Flag epitope-tagged receptors contain 29 additional amino acids at the amino-terminus of ER{alpha}/ß and 28 additional amino acids at the amino-terminus of ERß.

The estrogen response element (ERE)-containing reporter plasmids were (ERE)3-pS2-chloramphenicol acetyltransferase (CAT), constructed as described previously (15), and (ERE)2-TATA-CAT (32), provided by D. J. Shapiro of the University of Illinois (Urbana, IL). The plasmid pCH110 (Pharmacia Biotech, Piscataway, NJ) or pCMVß (Clontech, Palo Alto, CA), which contains the ß-galactosidase gene, or pSV40-luciferase was used as an internal control for transfection efficiency.

Cell culture and transient transfections
ER-negative CHO cells were maintained in phenol red-free DMEM-Ham’s F-12 tissue culture medium supplemented with 5% charcoal dextran-treated calf serum (CDCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated at 1.8 x 105 cells/60-mm plate, maintained in phenol red-free DMEM-Ham’s F-12 medium with 5% CDCS, and given fresh medium 5 h before transfection. Cells were transiently transfected by the CaPO4 coprecipitation method (33), and all cells for transfection were maintained at 37 C in a humidified CO2 atmosphere. Cells were given 400 µl precipitate containing 2 µg (ERE)2-TATA-CAT reporter plasmid, 0.3 µg pCH110 internal control ß-galactosidase plasmid, 6 µg pTZ19R carrier DNA, and 10 ng ER expression vector, or 400 µl precipitate containing 0.5 µg (ERE)3-pS2-CAT reporter plasmid, 0.5 µg pSV40-LUC internal control plasmid, 10 ng ER expression plasmid, and 7 µg pTZ19R carrier DNA, as indicated. After 12–16 h, cells were shocked with 20% glycerol-HBSS for 1.5 min and rinsed with HBSS. Cells were then given fresh medium and hormone treatment as indicated.

Human endometrial cancer (HEC-1) cells were maintained in MEM plus phenol red (21) supplemented with 5% calf serum and 5% FCS. MDA-MB-231 human breast cancer (231) cells were maintained in Leibovitz’s L-15 medium (21) supplemented with 5% calf serum. MDA-231 cells or HEC-1 cells were grown in MEM plus phenol red supplemented with 5% CDCS for 2 days before transfection. Cells were plated at a density of 3 x 106 cells/100-mm dish in phenol red-free improved MEM and 5% CDCS and were given fresh medium 24 h before transfection. One milliliter of precipitate contained 0.8 µg pCMVß as an internal control, 5 µg of an ERE-containing reporter plasmid (ERE)3-pS2-CAT, 200 ng ER expression vector, and pTZ19R carrier DNA to a total of 15 µg DNA. Cells remained in contact with the precipitate for 4 h and were then subjected to a 2.5-min glycerol shock (20% in transfection medium). Cells were rinsed with HBSS and given fresh medium with hormone treatment as indicated.

After transfection, cells were harvested 24 h after glycerol shock and hormone treatment, and extracts were prepared in 200 µl 250 mM Tris, pH 7.5, using three freeze-thaw cycles. ß-Galactosidase activity was measured to normalize for transfection efficiency in all experiments, and CAT assays were performed as previously described (34).

Immunoblots
Protein expression for the receptors and receptor chimeras and for Flag epitope-tagged receptors and receptor chimeras was monitored in CHO, 231, and HEC-1 cells after transfection with each cytomegalovirus promoter-based expression plasmid. Cells were transfected in 100-mm dishes with 10 µg ER expression plasmid and 5 µg pTZ19R using either the calcium phosphate method (30, 35, 36) or the transferrin-lipofectin method (37). The transferrin-lipofectin method gave increased transfection efficiency, but similar findings were observed using both methods. For the transferrin-lipofectin method, we used 250 µg transferrin (Sigma) in 1 ml HBSS complexed with 45 µg lipofectin reagent (Life Technologies). After 15-min incubation, 15 µg DNA were added to the mixture and incubated for an additional 15 min before dispensing to the 100-mm plate of cells. Whole cell extracts were prepared, and immunoblots were performed as previously described (35, 36), using ER{alpha} monoclonal antibody H222 or H226 (2 µg/ml) or anti-Flag M2 monoclonal antibody (4 µg/ml; IBI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcriptional activity of ERß, ER{alpha}, and ER chimeras in CHO cells
Previous reports have shown that ERß is a transcriptional activator in CHO cells (25, 26); however, the transcriptional activity of ERß was significantly less than that of ER{alpha}. In addition, although the DNA-binding and hormone-binding domains show considerable homology between ER{alpha} and ERß, the A/B domains of these two receptors are poorly conserved (Fig. 1AGo). To determine whether the reduction in transcriptional activity of ERß (entry 2, Fig. 1BGo) compared with that of ER{alpha} (entry 1, Fig. 1BGo) was due to the differences in the amino-terminal A/B domain, we replaced the A/B domain of ERß with the A/B domain of ER{alpha} to generate a chimeric receptor ER{alpha}/ß (entry 4, Fig. 1BGo). We also created an ERß point mutant, ERß(N496D), containing an N to D amino acid substitution at residue 496 to determine whether the differences in transcriptional activity of ER{alpha} compared with ERß could be partially attributed to this nonconserved residue in the AF-2 activation helix (entry 3, Fig. 1BGo). We also made a chimeric receptor, ERß/{alpha}, containing the amino-terminal A and B domains of ERß and the carboxyl-terminal domains C, D, E, and F of ER{alpha} (entry 5, Fig. 1BGo). The functional domains (A/B, C, D, E, and F) and activation functions (AF-1 and AF-2) of ER{alpha} are shown at the top of Fig. 1BGo along with schematics for ERß and the receptors we constructed and tested.

We first examined the E2-dependent transcriptional activity of ER{alpha} and ERß over a range of expression vector amounts in CHO cells. Maximal transcriptional activity for both ER{alpha} and ERß was obtained in response to E2 within the range of 1.5–15 ng expression vector (Fig. 2Go). In these cells, the trans-activation by ERß was only about half of that achieved by ER{alpha} regardless of the amount of expression vector employed. In human endometrial cancer (HEC-1) cells and MDA-231 human breast cancer (231) cells, 50–500 ng expression vector resulted in maximal E2-stimulated activity for ER{alpha}, ERß, and the chimeric receptors (data not shown). Therefore, for our studies to be within this range of maximal trans-activation for the receptors, we used 10 ng ER expression vectors for studies in CHO cells and 200 ng for studies in HEC-1 and MDA-231 cells.



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  		Figure 2. Transcription activation by ER{alpha} or ERß over a range of expression vector concentrations. CHO cells were transfected with increasing amounts of expression vector for ER{alpha} or ERß as indicated and a (ERE)2-TATA-CAT reporter gene. Cells were treated with 10-9 M E2 for 24 h. CAT activity was normalized for ß-galactosidase activity from an internal control plasmid. Values represent the mean ± SEM for three or more determinations and are expressed as a percentage of the ER{alpha} response with 10-9 M E2.

 
As shown in Fig. 3AGo, we examined the transcriptional responses of ER{alpha}, ERß, and the chimeric ER{alpha}/ß or ERß/{alpha} to increasing concentrations of E2. In the CHO cells, ER{alpha} maximal activity was stimulated approximately 10-fold at 10-8 M E2. ERß activated maximal transcription that was about 50% of that seen with ER{alpha}, consistent with previous reports (25, 26) as well as with the data in Fig. 2Go. ERß(N496D) showed no change in transcriptional activity from that observed with the wild-type ERß in CHO cells or in another cell type examined [note the point labeled ERß(N496D) in Figs. 3AGo and 7AGo], suggesting that although this is the only single amino acid difference in the AF-2 region between ER{alpha} and ERß, it does not play a critical role in the activity of ERß. In addition, replacing the A/B domain of ERß with the A/B domain of ER{alpha} (ER{alpha}/ß) resulted in a modest, but significant, increase in E2-dependent activity compared with that of the wild-type ERß. Interestingly, ERß/{alpha} showed transcriptional activity similar to that of ER{alpha}.



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  		Figure 3. Transcription activation by ER{alpha}, ERß, or chimeric ER{alpha}/ß or ERß/{alpha} in response to two different estrogens in CHO cells. ER-negative CHO cells were transfected with expression vector for ER{alpha}, ERß, ER{alpha}/ß, or ERß/{alpha} as indicated, and a (ERE)2-TATA-CAT reporter gene. The transcriptional activity of ERß(N496D) is also shown. Cells were treated with increasing concentrations of E2 (A) or the zearalanol estrogen P1496 (B) for 24 h. CAT activity was determined as described in Fig. 2Go. Values represent the mean ± SEM for three or more determinations and are expressed as a percentage of the ER{alpha} response with 10-8 M E2 or 10-8 M P1496.

 


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  		Figure 7. Transcriptional response of ER{alpha}, ERß, ER{alpha}/ß, or ERß/{alpha} to estrogens in MDA-231 breast cancer cells. ER-negative MDA-231 human breast cancer cells were transfected with ER expression vectors as indicated and a (ERE)3-pS2-CAT reporter gene. Cells were treated for 24 h with increasing concentrations of E2 (A) or the zearalanol estrogen P1496 (B). CAT activity was determined as described in Fig. 2Go. Values are the mean ± SEM for three or more determinations from separate experiments.

 
We also examined these receptors for transcriptional response to the resorcylic acid lactone estrogen compound P1496 (21). P1496 stimulated a pattern of activity similar to that observed with E2 (Fig. 3BGo). Both ER subtypes activated transcription in response to P1496 in a dose-dependent manner, reaching maximum levels at 10-8 M. Again, as was the case with E2, the highest activity of ERß with P1496 was only approximately 40% the activity of ER{alpha}. In addition, replacing the A/B domain of ERß with the ER{alpha} A/B domain (ER{alpha}/ß) resulted in a slight increase in the P1496-dependent activity compared with that of wild-type ERß. No significant transcriptional activity was observed for any of these receptors upon treatment with the antiestrogens TOT or BF in this cell context [with either the (ERE)2-TATA-CAT or (ERE)3-pS2-CAT promoter reporter gene constructs; data not shown]. Together, these results demonstrate that ERß is transcriptionally active in CHO cells in response to estrogens, albeit to a lesser extent than ER{alpha}, and that the presence of the C-terminal or N-terminal region of ER{alpha} improves trans-activation by ERß in this cell context.

We also examined the responses of ER{alpha}, ERß, and the receptor chimeras using a different estrogen-responsive promoter construct in CHO cells (Fig. 4Go). The general pattern of activity observed for the receptor constructs was similar to that seen with the more simple TATA promoter. ERß activity was about 40% of that seen with ER{alpha}, and the presence of either the C- or N-terminal region of ER{alpha} improved trans-activation by ERß.



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  		Figure 4. Transcription activation by ER{alpha}, ERß, or chimeric ER{alpha}/ß or ERß/{alpha} in response to E2 in CHO cells. Cells were transfected with expression vector for ER{alpha}, ERß, ER{alpha}/ß or ERß/{alpha}, and a (ERE)3-pS2-CAT reporter gene and were treated with control 0.1% ethanol vehicle (cont) or 10-8 M E2 for 24 h. CAT activity, normalized for internal reference transfection efficiency, was determined. Values are the mean ± SEM for three or more determinations and are expressed as a percentage of the ER{alpha} response with E2.

 
ERß is transcriptionally active in human endometrial cancer (HEC-1) cells in response to estrogen, but not antiestrogens, and alteration of the A/B domain allows ERß to activate transcription in response to antiestrogens
We next examined ERß activity in another cell type, the human endometrial cancer (HEC-1) cell line, where we have shown previously that the antiestrogens TOT and BF are partial agonists on ER{alpha} (21). We first characterized the transcriptional response to E2 for ER{alpha}, ERß, and the chimeric receptors ER{alpha}/ß and ERß/{alpha} (Fig. 5Go).



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  		Figure 5. E2-dependent transcription activation by ER{alpha}, ERß, and the chimeric receptors ER{alpha}/ß and ERß/{alpha}. Human endometrial cancer (HEC-1) cells were transfected with ER expression vectors as indicated and a (ERE)3-pS2-CAT reporter gene and were treated with the indicated concentrations of E2. CAT activity was determined as described in Fig. 2Go. Values are the mean ± SEM for three or more determinations from separate experiments and are expressed as a percentage of the ER{alpha} response with 10-8 M E2.

 
ER{alpha} evoked maximal levels of transcription at 10-9 M E2 in HEC-1 cells. Although ERß was transcriptionally active, its E2-dependent activity was significantly less than that of ER{alpha}, as observed in CHO cells, and using greater amounts of ERß expression plasmid did not increase this activity further (data not shown). ER{alpha}/ß activity in the presence of E2 was greater than the activity of the wild-type ERß. Therefore, the patterns of E2 response of ER{alpha}, ERß, and ER{alpha}/ß in the HEC-1 cells were similar to the pattern of activity observed in CHO cells. Interestingly, however, ERß/{alpha}, which showed very good transcriptional activity in CHO cells, was a poorer transcriptional activator in HEC-1 cells (Fig. 5Go).

Of note, ERß was not activated by the antiestrogens TOT or BF, although ER{alpha} and the ER{alpha}/ß chimera showed a substantial response to these antiestrogen ligands (Fig. 6Go). In fact, the ER{alpha}/ß chimera displayed a relatively strong transcriptional response to these antiestrogens, which was about 50–60% of the ER{alpha} response. Therefore, replacement of the A/B domain of ERß with the A/B domain of ER{alpha} allows this chimeric receptor to activate transcription upon antiestrogen binding. That the activation function-1-mediated agonism of antiestrogens works through ER{alpha} activation function-1 is supported by the observations presented in Fig. 6Go that in the ERß/{alpha} chimera, the ERß activation function-1 region (domains A and B) is ineffective in supporting antiestrogen agonism. These findings highlight the fact that the A/B domain of ER{alpha} plays an important role in antiestrogen-dependent transcription, and that the nature of the A/B domain can determine the ligand specificity of transcription activation by ERß.



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  		Figure 6. Transcription activation by ER{alpha}, ERß, ER{alpha}/ß, or ERß/{alpha} in response to E2 or antiestrogens in HEC-1 cells. ER-negative human endometrial cancer (HEC-1) cells were transfected with ER expression vectors as indicated and a (ERE)3-pS2-CAT reporter gene and treated with 10-8 M E2, 10-7 M TOT, or 10-7 M BF. CAT activity was determined as described in Fig. 2Go. Values are the mean ± SEM for three or more determinations from separate experiments and are expressed as a percentage of the ER{alpha} response. Numbers at the top of the leftmost set of bars show the fold induction in CAT activity for ER{alpha} treated with the ligand E2, TOT, or BF (100-, 25-, or 25-fold, respectively).

 
Examination of the transcriptional activity of ERß and chimeric ERs in MDA-231 human breast cancer cells
We also examined the transcriptional activity of these receptors in the ER negative MDA-231 human breast cancer (231) cell line. Upon treatment with E2 in increasing concentrations (Fig. 7AGo), ER{alpha} reached maximal activity at 10-8 M E2; however, ERß appeared to be almost transcriptionally inactive, even at high (10-7 M) or very high (1 x 10-6 or 3 x 10-6 M; not shown) levels of E2. In addition, almost no activity was observed with ERß when using higher amounts of ERß, up to 1 µg ERß expression plasmid (data not shown). Likewise, the ERß point mutant (N496D) and the chimeric ERß/{alpha} containing domains A and B from ERß and domains C–F of ER{alpha} showed no transcriptional response to E2. Results similar to those seen with E2 were obtained with the estrogen compound P1496 (Fig. 7BGo). With P1496, ER{alpha} activated a high level of transcription, yet no stimulation of activity was observed with transfected ERß (even with 10-5 M P1496; not shown).

As the transcriptional activity of AF-1 in the A/B domain of ER{alpha} is known to vary in different cell contexts (7, 21), we examined the effects of replacing the A/B domain of ERß with the A/B domain of ER{alpha}. Interestingly, this ER{alpha}/ß chimera showed a dramatic increase in transcriptional response to E2 (Fig. 7AGo) to nearly 60% of ER{alpha} activity in the 231 cells. Similarly, with the estrogen P1496, the chimeric ER{alpha}/ß activated transcription to approximately 50% of that observed with ER{alpha} (Fig. 7BGo).

Using the same cell type, we examined the transcriptional response of these receptors to the antiestrogen TOT and the antiestrogen BF. In the 231 cells, these antiestrogens are known to function as partial agonists, activating transcription by ER{alpha} to levels approximately 25% of that achieved by ER{alpha} with E2 (21).

The transcriptional response of ER{alpha} to the antiestrogen TOT reached maximal levels at 10-9 M in 231 cells (Fig. 8AGo). In these cells, where ERß appeared transcriptionally inactive in response to estrogens, ERß also exhibited no transcriptional response to the antiestrogen TOT. However, when the A/B domain of ERß was replaced with the A/B domain of ER{alpha}, this chimeric receptor (ER{alpha}/ß) activated some transcription upon treatment with TOT, to a level about 20% of that observed with ER{alpha}.



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  		Figure 8. Transcription activation by ER{alpha}, ERß, or ER{alpha}/ß in response to antiestrogens in MDA-231 breast cancer cells. ER-negative MDA-231 human breast cancer cells were transfected with ER expression vectors as indicated and a (ERE)3-pS2-CAT reporter gene. Cells were treated for 24 h with increasing concentrations of TOT (A) or BF (B) as indicated. CAT activity was determined as described in Fig. 2Go. Values are the mean ± SEM for three or more determinations from separate experiments and are expressed as a percentage of the ER{alpha} response to 10-9 M TOT or 10-7 M BF.

 
Transcriptional activity was also examined upon treatment with the antiestrogen BF (Fig. 8BGo). ER{alpha}, but not ERß, transfected cells showed a transcriptional response to BF. The chimeric ER{alpha}/ß was able to activate significant levels of transcription upon treatment with BF (~60% of ER{alpha}). Interestingly, we observed a large difference in the magnitude of transcriptional activity of ER{alpha}/ß with these two antiestrogens, as ER{alpha}/ß exhibited a significantly greater response to the antiestrogen BF than to TOT.

Expression of ER{alpha}, ERß, and ER chimeric proteins in the three cell types
ER{alpha} and ERß and the ER{alpha}/ß and ERß/{alpha} chimeric constructs were expressed in the three cell types (CHO cells, HEC-1 cells, and human breast cancer MDA-231 cells) after transient transfection. The immunoblot data in Fig. 9Go show that proteins of the correct size were produced in the cells: approximately 66 kDa for ER{alpha}, 58 kDa for ERß, and chimeric receptor proteins of about 62 kDa. (Note that the Flag epitope adds an additional 3 kDa to the epitope-tagged constructs.) As ER{alpha} antibodies do not detect ERß (27) and antibodies are not currently available that detect human ERß, we detected these proteins in two different ways. Flag epitope-tagged versions of these proteins were produced and detected with a Flag monoclonal M2 antibody, and chimeric proteins were detected with monoclonal antibodies to human ER{alpha} using either H222 that recognizes an epitope in the carboxyl-terminal domain E of ER{alpha} or H226 that recognizes an epitope in the amino-terminal B domain of ER{alpha}. These studies revealed that all receptors were expressed at high and approximately similar levels (Fig. 9Go and data not shown), except in the case of ERß and ERß/{alpha} in MDA-231 cells, where only low levels of these two receptors were observed. Hence, the very poor transcriptional response to ERß and ERß/{alpha} in these cells may in part be due to the low level of these proteins in the transfected 231 cells. In studies not shown, we also compared the transcriptional activity of ER{alpha} and ERß with the activity of Flag-ER{alpha} and Flag-ERß and found their activities to be very similar, indicating that the epitope tag did not alter the character of the ER{alpha} or ERß protein.



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  		Figure 9. Expression of ER{alpha}, ERß, and chimeric ER{alpha}/ß and ERß/{alpha} proteins. The expression of ER{alpha}, ERß, ER{alpha}/ß, and ERß/{alpha} proteins was monitored after transient transfection into CHO, MDA-231, or HEC-1 cells, followed by immunoblotting with anti-Flag M2 monoclonal antibody or anti-ER{alpha} monoclonal antibody H222 or H226. A, Expression of ER{alpha} and ERß/{alpha} in the three cell types was monitored by immunoblotting with the anti-ER{alpha} monoclonal antibody H222. B, Expression of Flag epitope-tagged ER{alpha}/ß and ERß in the three cell types was detected with Flag M2 antibody. C, Expression of ER{alpha} and ER{alpha}/ß in CHO cells was monitored with the anti-ER{alpha} monoclonal antibody H226.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized the transcriptional activity of a new ER subtype, ERß, in several different cell lines with estrogen and antiestrogen ligands and have investigated the involvement of the N- and C-terminal portions of ER{alpha} and ERß and their two major activation functions (AF-1 and AF-2) in transcriptional effectiveness. Intriguingly, we found that the transcriptional activity of ERß and that of ERß and ER{alpha} receptor chimeras were highly dependent on cell type. In addition, changing the amino-terminal A/B domain of the receptor can result in a dramatic change in both cell type- and ligand-specific transcriptional activity. Upon replacing the A/B domain of ERß with the A/B domain of ER{alpha}, this receptor not only exhibited an improved transcriptional response in certain cell types, but was able to activate transcription upon treatment with tamoxifen and phenylbenzofuran antiestrogens.

In the cell and promoter contexts studied, ER{alpha} showed the greatest transcriptional effectiveness, but the degree to which receptor activity depended on the type of AF-1 or AF-2 (i.e. an ER{alpha} or ERß type), as studied in our chimeras, differed in different cells and with the different ligands. This is summarized in Table 1Go. In CHO cells, the activity observed with E2 with the TATA promoter was determined by the type of AF-2, being maximal with an ER{alpha}-type AF-2 and only approximately half-maximal with ERß-type AF-2. As long as the AF-2 was of the ER{alpha} type, either an ER{alpha} or ERß AF-1 gave maximal activity. The activity dependence on the AFs seen in CHO cells with the TATA promoter appeared to be largely maintained in CHO cells with the more complex pS2 promoter. A somewhat different pattern was seen in HEC-1 cells with the pS2 promoter. Maximal activity with E2 required both an ER{alpha}-type AF-1 and AF-2. If either or both AFs were of the ERß type, activity was reduced.


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  		Table 1. Transcriptional activity of estrogen receptor subtypes and chimeras

 
In studies with ER{alpha} (17, 21), it is known that mixed agonist/antagonist antiestrogens, such as tamoxifen, derive their agonistic activity principally through the AF-1 of ER{alpha}. Consistent with this, we have found the agonistic activity of TOT and BF to have an absolute requirement for an ER{alpha}-type AF-1. AF-2 appears to play an enhancing role, in that activity levels of TOT and BF were twice as high with both {alpha}-type AFs than in the {alpha}/ß chimera containing a ß-type AF-2. Thus, the activities of different ligand classes show distinctly different reliance on the AF types.

The AF-1/AF-2 dependence of different ligands in the MDA-231 cell/pS2 promoter context appeared quite similar to that observed in the HEC-1/pS2 system as far as we could determine in these experiments. Maximal activity for all ligands was observed with ER{alpha}-type AF-1 and AF-2, with the activities of E2, P1496, and BF reduced to approximately 50% with an ERß-type AF-2. For TOT, the decrease in agonism with the ERß-type AF-2 was even greater. Interpretation of the transcriptional effectiveness of ERß and the ß/{alpha} chimera, the two species with a ß-type AF-1, is unfortunately limited by the low expression levels observed for these proteins in 231 cells. The role of an ERß-type AF-1 could thus not be unequivocally determined in these cells. Still, as was the case in HEC-1 cells, we saw in 231 cells that the agonism of all four compounds with an ER{alpha}-type AF-1 is enhanced by an ER{alpha}-type AF-2.

A biologically important finding was that antiestrogens are not agonists with human ERß. Similar findings have been reported recently for mouse ERß (38), suggesting that the inability of antiestrogens to activate ERß may be general for ERßs of different species. Our studies further amplify these observations by showing that antiestrogen agonism via the ER is largely mediated via the A/B region of ER{alpha} and is not supported by the AF-1 (A/B domain) of ERß. We previously identified a distinct region of 24 residues within the A/B domain of ER{alpha} that is required for antiestrogen agonism, but not for transcription stimulated by E2 (21). Interestingly, this region is not found within the A/B domain of ERß, and ERß exhibits no transcriptional activity with antiestrogens. Our findings suggest that the differences in sequence between the amino-terminal domains of ER{alpha} and ERß contribute to the cell- and promoter-specific transcriptional activity of these receptors and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

In contrast to the major effect that the nonconserved N-terminal A/B domains of the ER{alpha} and ERß receptors have on the transcriptional activity, the single, nonconserved amino acid in the AF-2 activation helix region [ER{alpha} (D545) vs. ERß(N496)] does not appear to account for any of these differences. Indeed, it is likely that other regions in the ER ligand-binding domain contribute to forming a composite protein surface important for ligand binding and coregulator interactions (12, 16, 39, 40, 41).

The cell/promoter differences in the activities of our ER constructs are intriguing. In each of these cell types, we performed plasmid titration experiments to ensure that our studies used receptor levels at which we obtained maximal, i.e. plateau, trans-activation activity levels (as shown in Fig. 2Go), and transfection of higher amounts of expression vector failed to increase transcriptional activity. We therefore believe that we are observing the intrinsic trans-activation activities of the different ER subtypes and chimeras. However, in MDA-231 cells, in which the expression levels of ERß and the ERß/{alpha} chimera appeared to be very low, the lack of response could be due in part to deficient protein levels as well as to the low intrinsic activity of these receptors in these cells. However, it is intriguing that we found ERß RNA levels to be similar in CHO, HEC-1 and MDA-231 cells after transfection (data not shown), indicating that the ERß expression plasmid is transcribed well in all of these cells. This aspect merits further examination.

The cell type dependence of the transcriptional activity of ERß and ER chimeras may be related to cell-specific differences in phosphorylation patterns of the different ER subtypes and/or to differences in the levels of various coregulator proteins. It is known that the hormone-dependent phosphorylation of ER{alpha} enhances receptor activity (42, 43, 44). Significantly, the major sites of phosphorylation of ER{alpha} are located in the A/B domain and appear to be critical for full ER{alpha} transcriptional activity (43, 44). In view of the differences in the A/B domain between ER{alpha} and ERß as well as the different kinase pathways that are activated in different cell types, it is perhaps not surprising that ERß exhibits variable transcriptional activity in different cell backgrounds. There is also now substantial evidence for multiple coregulators that interact with both the A/B and E domains of nuclear receptors (3, 16, 39, 45, 46, 47, 48) and promote a transcriptionally functional association of AF-1 and AF-2 (15), required for high levels of transcription activation. It will be of interest to determine whether the cell- and promoter-specific factors required for ERß function are similar to or distinct from those used by ER{alpha}. Continued analysis of the functional domains and coactivator interactions of these two ERs will be important for a better understanding of ERß function and how it compares with ER{alpha} in the diverse target cells known to be responsive to estrogens.


   Acknowledgments
 
We are grateful to Drs. Edwin von Angerer and Alan Wakeling for providing the antiestrogens. We thank Ramji Rajendran for his early contributions to these studies.


   Footnotes
 
1 This work was supported by NIH Grants CA-18119 and CA-60514 (to B.S.K.). Back

Received March 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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