Mouse Estrogen Receptor ß Forms Estrogen Response Element-Binding Heterodimers with Estrogen Receptor {alpha}

Katarina Pettersson, Kaj Grandien, George G. J. M. Kuiper and Jan-Åke Gustafsson

Department of Medical Nutrition and Center for Biotechnology, Karolinska Institute, S-14186 Huddinge, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-ß (ERß) and the functional characterization of mouse ERß protein. ERß is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-{alpha} (ER{alpha}) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ERß and ER{alpha} subtypes was demonstrated. Furthermore, ERß and ER{alpha} form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ERß/ER{alpha} heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ERß in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors represent a large family of transcription factors that regulate the activity of target genes by direct binding to specific DNA recognition elements located in the vicinity of the transcription start site of genes. Members of this gene family have an evolutionary and functionally conserved structure with a hypervariable N-terminal region that contributes to the transactivation function, a centrally located, well conserved DNA-binding domain (DBD), and a C-terminal domain involved in ligand binding, dimerization and transactivation functions (1).

Steroid hormone receptors constitute a distinct subgroup within the nuclear receptor family (2), which includes receptors for glucocorticoids, mineralocorticoids, androgens, progestins, and estrogens [glucocorticoid receptor, mineralocorticoid receptor, androgen receptor, progesterone receptor, and estrogen receptor (ER), respectively]. In addition two orphan nuclear receptors, the ERR-1 and 2 (ER-related receptors) (3) have been referred to this group (4). The steroid hormone receptors bind as homodimers to palindromic DNA response elements (2). Another important feature of steroid hormone receptors is the interaction with the molecular chaperone hsp 90 (5).

Estrogens influence growth, differentiation, and function of many target tissues, including tissues of the female and male reproductive tract (6). Estrogens also play an important role in the maintenance of bone mass and in the cardiovascular system where estrogens have certain protective effects (7, 8). The ER-encoding cDNAs have been cloned from several species (9, 10, 11, 12). Important examples of genes regulated by estrogens are the PR, epidermal growth factor receptor, certain growth factors (insulin-like growth factor-I, transforming growth factor-{alpha} and -ß) and several protooncogenes (c-fos, c-myc, c-jun) (13). Loss of ER function has long been postulated to be incompatible with life, and therefore the successful generation of ER-deficient mice came as a surprise (14). These mice are viable but display severe dysfunction of the reproductive organs, and both sexes are sterile. The females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. The fact that disruption of the ER gene did not completely eliminate the ability of small follicles to grow, as was evident from the presence of secondary and antral follicles in the knock-out mouse ovary, pointed to the possible existence of alternative ER mediating the intraovarian effects of estradiol. In some tissues from the ER knock-out mice residual binding of estradiol with an affinity and specificity reminiscent of an ER protein could be measured (14, 15). We have recently cloned a novel ER cDNA from rat prostate (16), which was suggested to be named rat ERß subtype to distinguish it from the previously cloned ER cDNA (consequently ER{alpha} subtype). The rat ERß protein was found to be highly homologous to the rat ER{alpha} protein, particularly in the DBD (>95% amino acid identity) and in the C-terminal ligand-binding domain (55% amino acid identity). In ligand-binding assays rat ERß binds estrogens with an affinity and specificity resembling that of ER{alpha}, and ERß is able to activate transcription of an estrogen-response element containing reporter gene construct (16, 17). In subsequent studies it was shown that ERß is the primary ER subtype expressed in rat ovary and that ERß message is down-regulated by gonadotropins in granulosa cells, suggesting that the functional significance of estrogen action in the rat ovary may be mediated primarily by ERß (18).

The detailed biological significance of the existence of two ERs is presently unclear. Perhaps the existence of two ER subtypes may provide, at least in part, an explanation for the selective actions of estrogens and certain antiestrogens in different target tissues (19, 20).

In this paper we describe the cloning of the mouse ovary ERß-cDNA and the characterization of the mouse ERß protein with respect to DNA binding, homo- and heterodimerization, and transactivational functions. Finally, cotransfection of both ER subtypes with an estrogen response element (ERE) containing reporter gene construct showed that the formation of heterodimeric ER{alpha}/ERß complexes may indeed constitute a novel estrogenic gene-regulatory pathway.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Homolog of Rat ERß Is Expressed in Mouse Ovary
The recent discovery of a novel ER protein present in rat prostate and ovary (16) has given a new perspective to studies of estrogen action. The mouse represents an important model system for studies of gene function in mammals. We therefore investigated whether a homolog to the previously cloned rat ERß (rERß) is present in the mouse. Oligonucleotides, constructed to encompass the coding sequence of the rERß cDNA, were used in an RT-PCR amplification of total RNA isolated from mouse ovaries. Amplification products with the expected size (~1.5 kbp) were subcloned and sequenced. The open reading frame of these clones displayed a high degree of amino acid identity with the rERß protein and were therefore recognized as the mouse homolog of rERß and will hereafter be referred to as the mouse ERß (mERß). As shown in Fig. 1Go, the mERß amino acid sequence also manifests considerable similarities to mouse and rat ER{alpha} in the DNA- and ligand-binding domains (Fig. 1Go). Several amino acid residues that have been demonstrated to be required for high-affinity binding of estradiol by ER{alpha} (21) were found to be conserved in rat ERß. The rat ERß binds estradiol (E2) with an affinity very comparable to that of ER{alpha} (17). Since the same amino acid residues are also conserved in the ligand-binding domain of mERß, we concluded that mERß should bind E2 in a similar manner as the rat ERß.



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Figure 1. The Putative Deduced Amino Acid Sequence of the Mouse ERß (mERß) Aligned to the Rat ERß (rERß) and Mouse and Rat ER{alpha} (mER{alpha} and rER{alpha}) Deduced Amino Acid Sequences, Respectively

The DBD is boxed, the two zinc fingers over-lined (CI, CII), and the ligand-binding domain is boxed and shaded.

 
mERß Protein Binds to an ERE
Members of the nuclear receptor superfamily bind to specific DNA motifs, generally palindromic or direct repeats of the sequence AG(A/G)(A/T)CA (22) located in the vicinity of the promoter of target genes. The specificity of DNA recognition by nuclear receptors is mediated essentially through the so called P-box, a short stretch of amino acids located in the N-terminal zinc finger in the DNA binding domain (DBD) (Ref. 1 and references therein). Amino acids in the P-box have been demonstrated to make direct contacts with bases in the DNA response elements, thus participating in dictating the DNA binding specificity (23). The P-box of ER{alpha} (EGCKA) differs from the P-box of other members of the steroid receptor subgroup, such as glucocorticoid receptor and progesterone receptor, with the result that ER{alpha} recognizes DNA elements contrasting from the sequences recognized by GR and PR. The consensus ERE consists of a palindromic repeat of the core sequence AGGTCA spaced by three nucleotides (24). The high degree of conservation in the DBD of the {alpha} and ß ER subtypes (~96%, Fig. 1Go) and the absolute identity of the P-box sequences strongly suggest a shared DNA recognition specificity between the two ER subtypes. We consequently performed DNA binding studies with mERß using radiolabeled consensus ERE oligonucleotides.

Mouse ERß and human ER{alpha} protein were synthesized in vitro in a rabbit reticulocyte lysate (RRL) system before incubation with E2 and a radiolabeled double-stranded ERE oligonucleotide. The resulting DNA-protein complexes were analyzed by electrophoretic gel mobility shift assay (Fig. 2Go). ERß binds to the wild type consensus ERE both in the absence or presence of E2 (lanes 2 and 3), but not to an ERE mutated in both half-sites (Fig. 2Go, lanes 7 and 8). This coincides with the binding specificity of ER{alpha} (Fig. 2Go, lanes 4 and 5 and lanes 9 and 10, respectively). In contrast to results obtained in a recent study by Tremblay and co-workers (25), we did not observe a reduced affinity for the ERE by ERß when equal amounts of ER{alpha} and ERß protein were used (as quantitated by [35S]methionine labeling). The reason for this discrepancy is unclear.



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Figure 2. In Vitro Synthesized Mouse ERß Binds to an ERE

32P-labeled DNA fragments corresponding to a consensus ERE (lanes 1–5) or a derivative ERE mutated in both half-sites (lanes 6–10) were incubated with unprogrammed lysate (lanes 1 and 6) or ERß (lanes 2–3 and 7–8) or ER{alpha} (lanes 4–5 and 9–10) synthesized in the RRL system. DNA-protein complexes were fractionated on a nondenaturing acrylamide gel. The gel was dried and autoradiographed at -70 C.

 
ERß Binds to the ERE as a Homodimer
Efficient DNA binding and transactivation function of nuclear receptors are often dependent on the formation of dimers. Members of the steroid receptor subgroup have been shown to interact with DNA predominantly as homodimers, whereas the receptors for retinoic acid, thyroid hormone, and vitamin D, as well as several orphan nuclear receptors, form heterodimeric complexes with the ubiquitous partner RXR (retinoid X receptor) (26). Importantly, some orphan nuclear receptors have been demonstrated to bind to DNA as monomers (27). To establish whether mERß binds to DNA as a homodimer or as a monomer, we generated a deletion mutant construct in which the 91 most N-terminal amino acids of the A/B domain of ERß were removed (Fig. 3AGo). This region of steroid receptors has not been shown to participate in DNA recognition, and we therefore anticipated that the truncated mERß would be able to bind to DNA as efficiently as the wild type receptor. The mERß construct was also tagged with a nine-amino acid HA1-epitope, which is recognized by the monoclonal 12CA5 antibody (28). The truncated epitope-tagged mERß (ERß-TAG) in electrophoretic mobility shift assays gave rise to a DNA-protein complex clearly distinguishable from the complex formed by the wild type ERß (ERß-wt), (Fig. 3BGo). After mixed synthesis of ERß-TAG and ERß-wt in the RRL system, a third complex of intermediate mobility was visible (Fig. 3BGo, lanes 2–4) representing the dimer between wtERß and ERß-TAG. This experiment clearly shows that ERß binds to DNA as a homodimer, similar to the binding of ER{alpha} (Refs. 1 and 2 and references therein).



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Figure 3. ERß Binds to the ERE as a Dimer

A, Schematic presentation of the wild type ERß (ERß-wt) and the truncated receptor (ERß-TAG). The DBD and the LBD are overlined in the wild type receptor. The inserted hemaglutenine (HA) epitope of the ERß-TAG is indicated (dotted) and overlined. B, 32P-labeled consensus ERE was incubated with wild type ERß alone (ERß-wt, lane 1), or ERß-TAG alone (lane 5), or both proteins together in 3:1, 1:1, and 1:3 ratios (lanes 2, 3, and 4, respectively) after synthesis in the RRL system. DNA-protein complexes were analyzed as described in the legend to Fig. 2Go. Arrows point to complexes formed by ERß-wt and ERß-TAG as indicated. The arrow without label indicates the band with intermediate migration, which represents the heterodimer of wild type and truncated receptors (lanes 2–4).

 
Coexpression of ERß and ER{alpha} Does Not Inhibit ER{alpha}-Stimulated Activity of an ERE-Reporter Gene Construct
Having established the ability of mERß to bind to an ERE, we proceeded to characterize the capacity of mERß to activate transcription of an ERE-containing reporter gene in transient cotransfections of mammalian cells. We also wanted to study whether cotransfection of both ER subtypes would alter the activity of the ERE reporter construct. Human fetal kidney 293 cells were cotransfected with a luciferase enzyme reporter gene construct containing two copies of a consensus ERE in front of a thymidine kinase promoter, together with human ER{alpha}- or mERß-expressing plasmids, or both. As shown in Fig. 4Go, the E2-stimulated activity of the reporter gene construct by mERß was lower when compared with the activity obtained with ER{alpha} when studied under the same transfection conditions. The 2-fold lower E2-stimulated activity was not due to squelching of mERß, since the reporter activity was dose-dependent with regard to the amount of cotransfected mERß-expression plasmid (data not shown). Although ERß and ER{alpha} are highly homologous in the DBD and in parts of the ligand-binding domain (LBD), there remain substantial differences, particularly in the N-terminal A/B domain (Fig. 1Go). This domain contains the AF-1 transcriptional activity function of ER{alpha} (29) and may have a similar function in ERß. The diverging A/B domains and/or dissimilarities in the LBD of ERß and ER{alpha} may result in differences in maximal transactivational activity of both ER subtypes. The slightly lower maximal transactivational activity of ERß compared with ER{alpha} has also been observed by other investigators (25, 30). When ER{alpha} and ERß were cotransfected, however, the reporter activity did not change significantly, when compared with the activity observed with ER{alpha} alone (Fig. 4Go). Based on these observations we conclude that ERß does not repress the activity of ER{alpha}. Furthermore, if the two receptors were competing as homodimers for the ERE-binding sites, higher concentrations of ERß would be expected to result in a decrease in reporter activity, toward the activity pattern of ERß alone. Since no sign of such a competition was observed, we speculated that an interaction was taking place between the two ER subtypes (although we cannot rule out that ER{alpha} alone is responsible for the transcriptional activity of the reporter gene). To be able to monitor a possible interaction between ERß and ER{alpha}, we used a mammalian cell two-hybrid system.



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Figure 4. ERß and ER{alpha} Activate Transcription from an ERE-Containing Reporter Construct

Human fetal kidney 293 cells were transfected with a luciferase reporter construct containing a tandem ERE and ERß or ER{alpha} expression plasmids as indicated (described in Materials and Methods). Cells were treated with vehicle (-, gray bars) or 10 nM 17ß-estradiol (10 nM E2, black bars). The results are presented as fold induction over values obtained from cells transfected with only the luciferase reporter and treated with vehicle, which were arbitrarily set to 1. Values represent the mean ± SD of three independent experiments.

 
ERß Interacts with ER{alpha} in Vivo and in Vitro
The two-hybrid system provides a powerful technique for studying potential interactions between two proteins within a cell. The principle is, in short, to fuse protein A to an autonomous DBD and protein B to a strong transactivation domain. The hybrid protein constructs are cotransfected into cells together with a reporter gene construct containing the cognate response element. The activity of the reporter gene will depend on an interaction between the fusion proteins, which will direct the transactivation domain to the promoter. The system has been widely used in yeast, but is also applicable in mammalian cells. For our studies we chose to use the DBD of the yeast protein Gal4 and the transactivation domain of the viral factor VP16. The Gal4-DBD was fused to the full-length mER{alpha}, and the VP16 transactivation domain was coupled to full-length mER{alpha} and mERß, respectively (Fig. 5AGo). The chimera constructs were then cotransfected together with a Gal4-luciferase reporter construct into COS 7 cells. Comparisons were made between cells with or without E2 incubation. Reporter activity remained low when cells were transfected with the vectors expressing only the Gal4-DBD or the VP16 transactivation domain, or when either of these constructs was transfected together with any of the hybrid constructs (Fig. 5BGo). In contrast, when the Gal4-mER{alpha} construct was transfected together with the VP16-mER{alpha} construct or the VP16-mERß construct, the activity of the reporter was induced approximately 3- to 4-fold, respectively (Fig. 5BGo, two most right-hand panels, white bars), compared with reporter transfected alone, indicating an interaction between the chimeric proteins. In the presence of E2 the induction levels rose to about 18-fold (Fig. 5BGo, two most right-hand panels, black bars). These results clearly demonstrate an interaction between the mERß and mER{alpha} proteins in vivo, suggesting that the transcriptional activity observed during coexpression of ER{alpha} and ERß (Fig. 4Go) is, at least in part, due to the formation of ER{alpha}/ERß heterodimers. The rise in reporter activity observed, especially in the presence of E2 (~5-fold), with the Gal4-mER{alpha} hybrid and the original VP-16 construct (Fig. 5BGo, second panel) is in all likelihood due to the ligand-inducible transactivation function of ER{alpha} itself, when directed to the promoter of the Gal4 reporter gene construct by binding via the Gal4 DBD.



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Figure 5. ERß Interacts with ER{alpha} in a Mammalian Two-Hybrid Protein System

A, Schematic description of the fusion proteins used in the two-hybrid assay. The Gal4-DBD was fused to the full-length ER{alpha}, creating Gal4-ER{alpha}, and the transactivating domain of VP16 (VP16-TA) was linked to full-length ER{alpha} and ERß, creating VP16-ER{alpha} and VP16-ERß, respectively. B, COS-7 cells were transfected with a luciferase reporter plasmid containing Gal4-binding sites and expression plasmids for Gal4 (Gal4-), VP16 (VP16-), Gal4-ER{alpha}, VP16-ER{alpha}, and VP16-ERß as indicated. The cells were treated with vehicle (-, white bars) or 1 µM 17ß-estradiol (1 µM E2, black bars). Data are presented as fold induction and represent the mean ± SD of three separate experiments performed in duplicate. The values obtained from cells transfected with reporter alone and treated with vehicle were arbitrarily set to 1. C, ER{alpha} was labeled with [35S]methionine by translation in RRL and incubated with GST-mERß or GST-protein. Samples were subsequently incubated with GST-Sepharose, washed, eluted in SDS buffer, and separated on 10% SDS-PAGE gels.

 
We also performed glutathione S-transferase (GST) pulldown experiments with [35S]methionine-labeled ER{alpha} and a GST-mERß fusion protein, in order to detect a direct interaction between the two proteins in vitro. As shown in Fig. 5CGo, ER{alpha} could be successfully coprecipitated with the GST-mERß fusion protein but not with the GST alone (compare lane 2 to lane 3, lane 1 = input of ER{alpha}, 20%), demonstrating a direct interaction between both ER subtypes.

ERß and ER{alpha} Form DNA-Binding Heterodimers
The results from the two-hybrid assay and from the pulldown experiment suggested that ER{alpha} and ERß are able to form heterodimers. In combination with the results from the cotransfection experiments (Fig. 4Go), it appeared likely that the putative ERß/ER{alpha} heterodimer would be able to bind to an ERE.

We performed electrophoretic mobility shift assay with in vitro synthesized ER{alpha} and ERß to examine this possibility. Because the wild type ERß migrates closely with ER{alpha} on native gels (see Fig. 2Go), we decided to use the truncated ERß-TAG (described above and in Materials and Methods) in order to identify DNA-protein complexes. ERß-TAG and ER{alpha} were synthesized in vitro and then mixed at increasing and decreasing amounts, respectively, incubated on ice, followed by incubation with the 32P-labeled ERE. An ERE-protein complex of intermediate mobility was formed in samples in which the two ER subtypes were coincubated (Fig. 6AGo, lanes 2–5), probably representing a heterodimeric complex between ERß-TAG and ER{alpha}. This putative heterodimerization was also evident when full-length wt ERß was used instead of ERß-TAG, but the complexes were not as easily distinguished (not shown).



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Figure 6. ERß Forms DNA-Binding Heterodimers with ER{alpha}

A, ER{alpha} or ERß-TAG alone (lanes 1 and 7, respectively) or together as indicated (lanes 2–6) were incubated with a 32P-labeled consensus ERE, and DNA-protein complexes were separated on a nondenaturing acrylamide gel. The arrow indicates the intermediate complex formed in the presence of both ER{alpha} and ERß. B, ER{alpha} or ERß-TAG alone or together were incubated with a mouse monoclonal ER{alpha}-specific antibody (ER{alpha} ab, lanes 3, 5–8, and 10) or with no antibody (lanes 2 and 9) together with 32P-labeled ERE. Complexes were analyzed as above. C, Essentially the same experiment as in B except that the 12CA5-TAG antibody recognizing the epitope-tagged ERß (TAG ab, lanes 2, 4–7 and 9), or no antibody was used (lanes 1, 3, and 8). D, 32P-labeled DNA fragments corresponding to either a consensus ERE (lanes 1–4) or a derivative ERE mutated in one of the half-sites (lanes 5–8) were incubated with unprogrammed lysate (lanes 1 and 5) or ER{alpha} (lanes 2 and 6) or ERß (lanes 3 and 7) or a mixture of both ER{alpha} and ERß (lanes 4 and 8). DNA-protein complexes were analyzed as described above.

 
To confirm the presence of both ER subtypes in the heterodimeric complex, the DNA-binding assay was also performed in the presence of an ER{alpha} antibody and the 12CA5 antibody. Figure 6BGo shows the result of incubation with the monoclonal ER{alpha} antibody 1D5. The ER{alpha} homodimer is efficiently supershifted with this antibody as expected (Fig. 6BGo, lane 3 compared with lane 2), whereas the ERß homodimer is not affected (lanes 9 and 10). The intermediate complex formed in the presence of both ER{alpha} and ERß-TAG is also supershifted with the ER{alpha} antibody (Fig. 6BGo, lanes 5–8 compared with lane 4), thus demonstrating the presence of ER{alpha} within the heterodimeric complex. In Fig. 6CGo essentially the same experiment has been repeated using the 12CA5 monoclonal antibody directed against the HA-epitope of mERß-TAG. The 12CA5 antibody successfully interacts with both the ERß homodimer and the intermediate complex previously shown to contain also ER{alpha} (Fig. 6CGo, lanes 10 and 4–7). The 12CA5 antibody does not cross-react with the ER{alpha} homodimer (lane 2). These results clearly demonstrate that the intermediate complex that is formed when ER{alpha} and ERß are coincubated contains both receptors and is a true heterodimeric complex.

To determine whether both partners of the heterodimer participate in DNA binding, experiments were performed with an ERE mutated in one of the half-sites at a position previously demonstrated to be crucial for efficient binding by the ER{alpha} protein (31). In crystallographic studies of the DBD of ER{alpha} bound to the ERE (31), it was established that a mutation in one of the half-sites resulted in reduced cooperativity in binding to the second half-site, due to lack of proper interaction between the dimer interfaces present in the DBD. Binding by ER{alpha} to such a mutated ERE was therefore less efficient. We found that ERß did not bind to this mutated ERE in analogy to the ER{alpha} (Fig. 6DGo, lanes 7 and 6, respectively). In addition, no protein-DNA complex was formed with the heterodimer (lane 8), indicating that cooperativity in DNA binding is also required for efficient DNA binding by ERß/ER{alpha} heterodimers.

Furthermore, the ERß as well as ER{alpha} were unable to bind to oligonucleotides containing direct repeats of the core sequence AGGTCA spaced by one or four nucleotides (DR1 or DR4), irrespective of the presence or absence of retinoid X receptor (data not shown).

Our findings on ER{alpha}/ERß heterodimerization and the recent demonstration of GR/MR heterodimerization (32, 33) challenge the commonly held view that steroid receptors form only homodimers. Previous biochemical and structural evidence indicated that steroid receptors form homodimers through a dimerization interface within their zinc finger DNA binding domain, and a generally much stronger dimerization interface within the ligand binding domain (Refs. 1 and 22 and references therein). Further studies will be required to localize the dimerization interfaces involved in the formation of ER{alpha}/ERß heterodimers.

The rat tissue distribution and/or relative level of ER{alpha} and ERß mRNA seems to be quite different; that is, moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER{alpha} and prostate, ovary, uterus, lung, bladder, brain, and testis for ERß (17). This may imply that in testis and ovary both subtypes are expressed to some extent. In the mouse, both ER mRNAs can be found in ovary and uterus (not shown). In the rat, hypothalamus ER{alpha} and ERß are coexpressed in certain regions, most likely in the same neurons (34). The coexpression of ER{alpha} and ERß in the same tissue and/or cells suggests the interesting possibility that ER{alpha} and ERß proteins may interact with each other. In this study we have indeed shown that the two ER subtypes have the ability to form heterodimers. The discovery of an ERß protein and the ability of ER{alpha} and ERß to form heterodimers strongly suggest the existence of two previously unrecognized pathways of estrogen signaling: via ERß homodimers in cells exclusively expressing this subtype and via ER{alpha}/ERß heterodimers in cells expressing both subtypes (Fig. 7Go). The ERß homodimers and the ER{alpha}/ERß heterodimers may possibly interact with novel response elements, different from the known EREs. By such a mechanism the physiological regulatory potential of estrogenic hormones may be greatly expanded. Different target tissues may respond differently to the same hormonal stimulus due to alternative composition of receptors. Varying ratios of ER{alpha} and ERß in different cells, resulting in different populations of homo- and heterodimers, could constitute a hitherto unrecognized mechanism involved in the tissue- and cell type-specific effects of estrogens and certain antiestrogens (19, 20).



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Figure 7. Alternative Estrogen-Signaling Pathways

The existence of two ER subtypes and their ability to form DNA-binding heterodimers suggests the existence of three potential alternative pathways of estrogen signaling. In cells expressing only the ER{alpha} or ERß subtype, homodimers of either subtype can interact with response elements in target gene promoters and influence transcription levels. In cells expressing both subtypes, heterodimers can be formed depending on the ratio of the subtypes. Although we have shown that ERß homodimers and ER{alpha}/ERß-heterodimers interact with consensus EREs, it cannot be excluded at this stage that unique response elements exist within the context of target gene promoters that interact preferentially with the ERß homodimer or the ER{alpha}/ERß-heterodimer. In that way, the regulatory potential of the liganded ER protein could be greatly expanded. An open question in this regard is the existence of estrogen target genes that are exclusively regulated by either of the homodimers or the heterodimer.

 
Future studies will be required to determine the physiological significance of the existence of more than one ER protein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Mouse ERß cDNA
Total RNA from ovaries dissected from 5-week-old mice was prepared as described (35). Complementary DNA was synthesized using Super-Script reverse transcriptase (GIBCO BRL, Paisley, Scotland) as described previously (36) using 1 µg of total RNA. PCR amplification of the cDNA was carried out with 35 cycles of repeated denaturation for 15 sec at 95 C, 15 sec of annealing at 57 C, and 60 sec of extension at 72 C with Taq-polymerase (Pharmacia, Uppsala, Sweden) under the conditions described by the manufacturer with 1:20 of synthesized cDNA and oligonucleotides Erbkg3 5'-ATGAGTATTCAGCCATGGCATTCTACAG and Erbkg4 5'-CAGGCCTGGCCATCACTGAGACTG, which were constructed to encompass the entire coding region of rat ERß cDNA. To facilitate subsequent subcloning, an NcoI restriction enzyme recognition site was introduced over the start codon (bases -2 to +4). The PCR product was visualised on a 1% agarose gel, revealing a single band of approximately 1450 bp, corresponding in size to the rat ERß open reading frame. The DNA band was excised and the DNA was purified using the QIAEX gel purification kit (QIAGEN, Hilden, Germany). The resulting DNA was phosphorylated with T4 polynucleotide kinase (Amersham, Solna, Sweden) and cloned into the T-overhang vector pTKS (37). The insert of pTKS-mERß was sequenced by the dideoxy method (38) with T7 DNA-polymerase (Pharmacia).

Plasmid Constructs
For in vitro transcription/translation in RRL, pTKS-mERß was digested with NcoI and EcoRI, which yielded a fragment encompassing the entire coding sequence (cds), which was inserted Sp6-sense into NcoI/EcoRI-digested pSP72 (Promega, Madison, WI), thus generating pSP72-mERß. pSP72-mERß-TAG was made by replacement of nucleotides 1–273 of the mERß cds with oligonucleotides 5'-CATGGGCTACCCCTACGACGTGCCCGACTACGCCGTGAACA and 5'-CTAGTGTTCACGGCGTAGTCGGGCACGTCGTAGGGGTAGCC, which encode the HA1 epitope recognized by the 12CA5 monoclonal antibody. The plasmid pSP72-hER{alpha} has been described elsewhere (36). For transfections of mammalian cells the XhoI/BglII-fragment from pSP72-mERß was inserted into the pSG5 expression vector (Stratagene, La Jolla, CA) digested with EcoRI/BglII; the XhoI-site of the mERß-fragment and the EcoRI-site of pSG5 were filled in with Klenow fragment to allow blunt-ended ligation. The vector pSG5-hER{alpha} was made by excising hER{alpha} cDNA from pSP72-hER{alpha} with EcoRI and SacI and inserting it into pSG5 digested with EcoRI and BglII. To enable this ligation, the SacI site was filled in with T4 DNA-polymerase and BglII with Klenow fragment. The reporter construct 2xERE-TK-Luc was constructed by subcloning of a tandem ERE (39) with XhoI overhangs into the SalI site of the p19-TK-Luc reporter plasmid (40). The Gal4-mER{alpha} and VP16-mER{alpha} two-hybrid constructs were made through PCR amplification of the plasmid MOR101 (containing the mouse ER{alpha} cDNA) (41) to introduce a KpnI site upstream of the start codon of the mER{alpha}, with the use of oligonucleotides mER-ATG (5'-GCCAGGTACCATGGCCATGACC) and mER-EagI (5'-CCCAGGCTGTTGGCACTGAAGGC). The 275-bp long PCR product was cut with KpnI and EagI, MOR101 was digested with EagI (nucleotide 460 in the mouse ER{alpha} cDNA) and BamHI 3' of the mER{alpha} cDNA), and both fragments were subcloned into the KpnI and BamHI sites of pCMX-Gal4 or pCMX-VP16 (42). VP16-ERß was made by in-frame insertion of the NcoI/EcoRI fragment of pSP72-mERß into the EcoRV/EcoRI sites of pCMX-VP16, and the NcoI site of mERß was filled in with Klenow fragment to enable the ligation. The Gal4-luciferase reporter construct used in the two-hybrid assay has been described elsewhere (43). The pGST-mERß was generated by in-frame ligation of the NcoI/EcoRI fragment from pSP72-mERß into the corresponding sites of pGST (I. Pongratz and F. Delauney, unpublished data) creating a GST-mERß fusion that could be translated in the RRL system.

Cell Culture and Transient Transfections
Cells from the human fetal kidney cell line 293 were routinely cultured in a 1:1 mixture of Ham’s Nutrient mixture F12 (F12, GIBCO BRL) and DMEM (GIBCO-BRL) supplemented with 7.5% FBS, 0.5% nonessential amino acids (NEA, GIBCO BRL) and 1% PEST (100 U penicillin/ml and 100 µg streptomycin/ml). Cells were seeded in six-well plates 24 h before transfection. Transfections using the Lipofectin (GIBCO BRL) reagent were performed as described by the manufacturer in a serum- and antibiotic-free mixture of 1:1 of F12 and phenol-red free DMEM with 0.75 µg of the 2xERE-TK-Luc reporter and 0.1–0.4 µg of pSG5-ER{alpha} or pSG5-ERß as indicated. The pSG5 vector was used to equalize plasmid concentrations, and 0.1 µg of a placental alkaline phosphatase (AP) expression plasmid (44) was included to control for differences in transfection efficiences. Medium was changed to a phenol red-free mixture of F12 and DMEM containing 7.5% dextran-coated charcoal-treated FBS, 0.5% NEA, and 1% PEST after 24 h. Hormone or vehicle (0.1% ethanol) was added simultaneously. Cells were allowed to stand for 48 h with a renewed change of media and hormone after 24 h. Media were collected for assaying of AP activity. The cells were harvested in 10 mM Tris-HCl/10 mM EDTA/150 mM NaCl and centrifuged for 4 min at 4000 rpm, supernatant was removed, and cell pellets were lysed in Lysis Buffer 2 (Bio-Orbit, Turku, Finland). Luciferase activity was measured using the GenGlow system (Bio Orbit). The results are presented as the mean ± SD of fold induction of three separate transfections performed in duplicate.

COS 7 cells were routinely maintained in DMEM (GIBCO BRL) supplemented with 5% FBS and 1% PEST. For transient transfections, cells were seeded in six-well plates 24 h prior to transfection. Transfections were carried out with Lipofectin reagent in phenol red- free DMEM without serum and antibiotics, using 0.5 µg of the GAL4-luciferase reporter construct and 0.1 µg of each of the two-hybrid expression plasmids as indicated in the legend to Fig. 5BGo. Expression vector concentrations were kept constant in all transfections by addition of the original pCMX-Gal4 or pCMX-VP16 plasmids, and 0.2 µg of the AP expression vector was included in all transfections as an internal control for transfection efficiency. Cells were left in the Lipofectin-DNA mixture for 24 h after which the medium was changed to phenol red-free DMEM supplemented with 5% dextran-coated charcoal-treated FBS and 1% PEST. Hormone (1 µM E2) or vehicle (0.1% ethanol) was added. After 24 h cells were harvested as described previously, and luciferase activity was measured. All samples were normalized against the activity of the AP internal standard. Transfections were carried out in duplicate, and the results are presented as fold induction and represent the mean value ± SD of three separate experiments.

In Vitro Translation, GST Pulldown, and DNA-Binding Assays
For the ERE-binding studies, 1 µg pSP72-mERß or 1 µg pSP72-hER{alpha} was transcribed/translated in the TNT-coupled RRL system (Promega) with Sp6 RNA polymerase, according to the manufacturer’s instructions, in the presence of 100 nM 17ß-estradiol or vehicle (0.01% ethanol). Five microliters of the lysate were used in each DNA-binding reaction with a 32P end-labeled wild type or double-mutated ERE as indicated in the legend to Fig. 2Go. Protein-DNA complexes were separated on 5% polyacrylamide/0.25x Tris-borate-EDTA gels at ~10 V/cm, followed by drying and autoradiography at -70 C.

In the homodimerization experiments, increasing amounts of pSP72-mERß-TAG (0, 0.1, 0.2, 0.3, and 0.4 µg) were translated in the RRL together with decreasing amounts of pSP72-mERß (0.4, 0.3, 0.2, 0.1, and 0 µg). Five microliters of programmed lysate were used in each DNA-binding reaction with radiolabeled ERE.

For the heterodimerization studies, 1 µg pSP72-hER{alpha} or pSP72-mERß was translated in RRL. In the experiment of Fig. 6AGo, 5Go, 4Go, 3Go, 2Go, 1Go or 0 µl of ER{alpha}-containing lysate were mixed with 0, 1, 2, 3, 4, or 5 µl of ERß lysate and incubated 15 min on ice before the DNA-binding reaction with radiolabeled ERE.

Translations were carried out in the same manner for the antibody-upshift experiments, and 4 µl ER{alpha} or ERß lysate, respectively, or a mixture of 2 µl of each was incubated for 15 min on ice. Thereafter, 1.5 µl monoclonal ER{alpha} antibody 1D5 (Dako, Carpinteria, CA) or 12CA5 TAG antibody (BAbCOf) were added to the respective homodimers and 0.5, 1, 2, or 3 µl of each antibody were added to the heterodimer reactions. The DNA-binding reaction was started immediately. The single-mutated ERE used in the band shift assay in Fig. 6DGo has been described previously (36). Four microliters of ER{alpha} or ERß protein containing RRL or a mix of 2 µl of each were used in the DNA-binding assay.

For the GST pulldown experiments, pSP72-hER{alpha} was translated in the presence of [35S]methionine in RRL and pGST-mERß or the original pGST was translated in RRL in the absence of radiolabeled amino acids. Five microliters of ER{alpha}-containing lysate were mixed with 5 µl lysate containing GST-mERß or GST-protein. Samples were incubated for 15 min on ice before 50 µl GST-Sepharose diluted in PBS were added to each sample followed by 30 min of incubation on ice. The Sepharose beads were washed four times in PBS/0.1% Triton X-100, and bound proteins were eluted by incubation in 2x SDS-buffer for 5 min at 100 C. ER{alpha} lysate (=20%) was loaded as input together with the eluted samples on a 10% SDS-PAGE and run at 150 V. The gel was immersed in 1 M salicylic acid for 20 min, dried, and autoradiographed at -70 C.


    ACKNOWLEDGMENTS
 
The authors wish to thank Drs. Ingemar Pongratz and Franck Delauney for the generous gift of the pGST plasmid, Göran Bertilsson for technical assistance, and Jane Thomsen for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Katarina Pettersson, Department of Medical Nutrition, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden.

This work was supported by a grant from the Swedish Cancer Society to J-ÅG and GGJMK was supported by a visiting scientist fellowship from the Karolinska Institute.

The sequence reported in this paper has been deposited in the GenBank database (AJ000220).

Received for publication December 18, 1996. Revision received May 7, 1997. Accepted for publication June 2, 1997.


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