Cloning, Chromosomal Localization, and Functional Analysis of the Murine Estrogen Receptor ß

Gilles B. Tremblay1, André Tremblay1, Neal G. Copeland, Debra J. Gilbert, Nancy A. Jenkins, Fernand Labrie and Vincent Giguère

Molecular Oncology Group (G.B.T., A.T., V.G.) Royal Victoria Hospital Montréal, Québec, Canada H3A 1A1
Departments of Biochemistry, Medicine, and Oncology (V.G.) McGill University Montréal, Québec, Canada
Mammalian Genetics Laboratory (N.G.C., D.J.G., N.A.J.) ABL-Basic Research Program National Cancer Institute-Frederick Cancer Research and Development Center Frederick, Maryland 21702
Laboratory of Molecular Endocrinology (F.L.) Centre Hospitalier de l’Université Laval Research Center Ste-Foy, Québec, Canada G1V 4G2


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor ß (ERß) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ERß and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ER{alpha}. Mouse ERß binds to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner. Scatchard analysis indicates that mERß has slightly lower affinity for E2 [dissociation constant (Kd) = 0.5 nM] when compared with mER{alpha} (Kd = 0.2 nM). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ER{alpha} on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mERß. Cotransfection of mERß and H-RasV12 causes enhanced activation in the presence of E2. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mERß is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mERß transactivation both in the absence and presence of E2, and in vitro interaction between SRC-1 and the ERß ligand-binding domain is enhanced by E2. Moreover, the ligand-independent stimulatory effect of SRC-1 on ERß transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ERß shares many of the functional characteristics of ER{alpha}, the molecular mechanisms regulating the transcriptional activity of mERß may be distinct from those of ER{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens regulate female reproductive functions through a nuclear receptor that belongs to a superfamily of ligand-activated transcription factors that includes receptors for steroids, thyroid hormone, retinoids, prostanoids, and vitamin D (1, 2). Like other members of the superfamily, the estrogen receptor (ER) has a modular structure consisting of distinct functional domains (3). The DNA-binding domain (DBD) enables the receptor to bind to its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AGGTCA (or closely related sequences) spaced by 3 bp and referred to as an estrogen response element (ERE). The ligand-binding domain (LBD) also harbors a nuclear localization signal as well as sequences necessary for dimerization and transcriptional activation (AF-2). A second activation function, AF-1, is present in the amino-terminal domain of the receptor. Although transcriptional activation is mediated through both AF-1 and AF-2, only activation by AF-2 requires hormone binding (4, 5). Studies using the estrogen antagonists 4-hydroxytamoxifen (OHT) and ICI 164,384 indicated that although both compounds blocked estrogen effects, their mode of action differed: the mixed agonist/antagonist OHT inhibited only AF-2 function while the pure antiestrogen ICI 164,384 inhibited activation by both AF-1 and AF-2 (6).

Additional signals that modulate nuclear receptor function have been described and commonly involve phosphorylation. Steroid receptors, including the progesterone and glucocorticoid receptors, have an increased state of phosphorylation upon ligand binding (7). Similar results have been described for ER where treatment of cells with peptide growth factors or agents that alter cellular cAMP levels result in up-regulation of the receptor coupled with an increase in its phosphorylation state (8, 9). These reports, as well as subsequent studies (10), demonstrated that antiestrogens also caused an increase in receptor phosphorylation albeit to a lesser extent than estradiol (E2), even though they inhibited transactivation. Deletion mapping and mutagenesis of human ER revealed that phosphorylation at Ser118 was required for full activity of AF-1 (10). Furthermore, this residue was shown to be a direct substrate for mitogen-activated protein (MAP) kinase, providing an in vivo link between estrogen action and the Ras-MAP kinase signaling cascade (11, 12). These results have begun to shed light on the molecular events responsible for regulation and proliferation of different cell types by ER in response to estrogens and growth factors.

Transactivation by nuclear receptors has recently been shown to be modulated by a growing family of coregulators (13). These include corepressors such as N-CoR and silencing mediator for retinoid and thyroid hormone receptors (SMRT) (14, 15, 16), which participate in the ligand-independent silencing functions of TR and retinoic acid receptor, and several coactivators: TRIP1/SUG-1 (17, 18), ERAP-140 (19), RIP-140 and RIP-160 (20, 21), TIF1 (22), and the related TIF2 (23), GRIP1 (24), SRC-1, and ERAP-160 (19, 25, 26). The LBD of ER was demonstrated to specifically interact with many of these coactivators, including steroid receptor coactivator-1 (SRC-1), ERAP-160, and RIP-140, and the strength of interaction was increased by E2 but not by the antiestrogens OHT and ICI 164,384 (20, 25, 27). The significance of these coactivators in nuclear receptor function is underscored by recent results demonstrating that SRC-related proteins interact with the nuclear integrators, CBP and p300, to augment nuclear receptor transactivation in mammalian cells (26, 28, 29). The activation domain of CBP can interact with TFIIB (30), thus providing evidence for a link between nuclear receptors and the basal transcription machinery. This growing network of interacting factors has increased our understanding of how steroids such as E2 are able to alter the expression of specific genes at the molecular level.

Although human and mouse ER cDNAs were cloned several years ago (31, 32), RT-PCR of rat prostate mRNA has revealed the presence of a second ER, referred to as ERß (33). This novel receptor was found to bind E2 with relatively high affinity and to be capable of activating transcription of a reporter gene in the presence of this ligand. In situ hybridization of rat tissues indicated that ERß is expressed in female animals in primary, secondary, and mature follicles as well as granulosa cells in the ovary and in the prostate of male rat. As an initial step toward elucidating the physiological role of this second ER and understanding its functional relationship with ER{alpha}, we report the cloning of the murine homolog of ERß. We show that mERß is able to bind to an ERE in electrophoretic mobility shift assays as well as to transactivate, in an E2-dependent manner, promoters containing either synthetic or natural EREs in Cos-1 and HeLa cells. In addition, E2-induced mERß activity is inhibited by several antiestrogens that have been previously shown to be selective for ER{alpha}. We also demonstrate that SRC-1 interacts in vitro with the mERß LBD in a ligand-dependent manner. However, SRC-1 modulates mERß transcriptional activity in intact cells both in the presence and absence of ligand. Finally, we show that the E2-induced transcriptional activity of mERß is enhanced by cotransfection with Ras. The functional differences observed between the two ER isoforms are discussed.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Mouse ERß cDNA
The mERß cDNA contains an open reading frame of 1455 nucleotides encoding a protein of 485 amino acids (Fig. 1Go). Alignment of mERß with mER{alpha} indicates that the receptors share 97% and 60% amino acid similarity in the DBD and the LBD, respectively (Fig. 2Go). The AF-2 core sequences in each receptor are virtually identical; however, no significant regions of homology could be detected between the two amino-terminal domains, a region of the protein that contains the AF-1 in ER{alpha}. Two potential MAP kinase phosphorylation sites (Ser60 and Ser94) are present in the amino-terminal region of the mERß.



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Figure 1. Nucleotide Sequence of Murine ERß

Amino acid numbers are indicated on the left. The potential MAP kinase phosphorylation sites are underlined. GenBank accession number of the mERß sequence is U81451.

 


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Figure 2. Amino Acid Sequence Comparison between Murine ERß with ER{alpha}

The alignment begins with the first Cys residue of the respective DBDs. Identities are indicated with vertical bars, biochemically similar residues by vertical dots, and gaps by dashed lines. The stop codon in ERß is indicated by an asterisk. The AF-2 domain is underlined.

 
Chromosomal Localization
The mouse chromosomal location of ERß (Estrb) was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice. This interspecific backcross mapping panel has been typed for more than 2200 loci that are well distributed among all the autosomes as well as the X chromosome (34). C57BL/6J x M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse cDNA ERß probe (positions 816-1361). The 8.4- and 5.1-kb EcoRI M. spretus RFLPs (see Materials and Methods) were used to follow the segregation of the Estrb locus in backcross mice. The mapping results indicated that Estrb is located in the central region of mouse chromosome 12 linked to Sos2, Spnb1, and Fos. Although 165 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 3Go), up to 184 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromere - Sos2 - 7/184 - Spnb1 - 0/181 - Estrb 7/173 - Fos. The recombination frequencies [expressed as genetic distances in centiMorgans (cM) ± the SE] are - Sos2 - 3.8 ± 1.4 - [Spnb1, Estrb] - 4.1 ± 1.5 - Fos. No recombinants were detected between Spnb1 and Estrb in 181 animals typed in common, suggesting that the two loci are within 1.7 cM of each other (upper 95% confidence limit).



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Figure 3. Chromosomal Localization of the ERß Gene

The ERß gene, Estrb, maps to the central region of mouse chromosome 12. Estrb was placed on mouse chromosome 12 by interspecific backcross analysis. The segregation patterns of Estrb and flanking genes in 165 backcross animals that were typed for all the loci are shown at the top of the figure. For individual pairs of loci, more than 165 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus) F1 parent. The shaded boxes represent the presence of C57BL/6J allele, and open boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 12 linkage map showing the location of Estrb in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

 
The mERß Gene Expresses Several Transcripts
A Northern analysis was performed to determine the size of the message encoding mERß using the 550-bp PCR fragment that spans the LBD of mERß as a probe. As shown in Fig. 4Go, mouse ovary expressed at least four predominant transcripts of approximately 3.5, 4.6, 7.2, and 9.5 kb and a weaker message of about 4.9 kb. In contrast, only one transcript could be detected in ovary mRNA when probed with the mER{alpha} cDNA. Analysis of total RNA from several other mouse tissues including liver, heart, kidney, skeletal muscle, thymus, spleen, and brain were all negative for presence of the ERß transcripts indicating that expression of ERß was below the level of detection by Northern blotting in these tissues (data not shown).



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Figure 4. Northern Blot Analysis of ERß mRNA

Five micrograms of mouse ovary poly-A+ RNA were probed with a 550-bp cDNA fragment encoding the mERß LBD and a full-length mER{alpha} cDNA. The blot was hybridized with the mERß cDNA fragment, stripped, and subsequently probed with the mER{alpha} cDNA. Exposure was carried out for the same length of time in each case. The position of 18S and 28S rRNAs are indicated on the left.

 
Mouse ERß Binds to an ERE
The homology between the DBDs of mER{alpha} and ß indicated that the receptors most likely bind to the same type of response element, namely an inverted repeat spaced by three nucleotides (IR-3). We tested this by conducting electrophoretic mobility shift assays (EMSA) with both mouse receptors. Figure 5Go demonstrates that both ER isoforms produced in vitro by rabbit reticulocyte lysates were able to bind specifically to a vitellogenin A2 (vitA2)-ERE (35) probe. It was noted that the mERß did not bind as strongly to the element as mER{alpha} (compare lanes 2–5 with lanes 7–10) even though both receptors were present at roughly the same level in the crude lysates as shown by [35S]methionine-labeled proteins (data not shown). In addition, both receptors were able to bind to the pS2-ERE (36) but less efficiently than to the vitA2-ERE (data not shown), which can most likely be accounted for by the fact that the pS2-ERE contains an imperfect consensus sequence in the second half-site.



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Figure 5. Binding of mERß and mER{alpha} to an ERE

Each receptor produced by rabbit reticulocyte lysates (RRL) was incubated at room temperature with approximately 0.1 ng of labeled vitA2-ERE in the absence (lanes 2 and 7) and presence of 100 nM E2 (lanes 3 and 8), 100 nM OHT (lanes 4 and 9), and 100 nM ICI 182,780 (lanes 5 and 10). Unprogrammed RRL was used as a negative control (lane 1) and specificity was determined in the presence of 100x unlabeled vitA2-ERE (compare lanes 6 and 11).

 
Addition of E2 to the binding reactions did not have an effect on binding under the conditions used (Fig. 5Go, lanes 3 and 8) nor was there any effect on binding upon addition of specific antagonists such as OHT (lanes 4 and 9) and ICI 182,780 (lanes 5 and 10), although the migration rate of the protein/DNA complexes were differentially altered by each ligand. However, as previously observed with ER{alpha} (37), a severe decrease in binding by ERß was observed when the preincubation step was conducted at room temperature, 37 C, or for shorter periods of time. The formation of the ERß/DNA complexes could be partially restored in the presence of E2 at these temperatures (data not shown).

Transcriptional Activity of mERß on Synthetic and Natural Promoters
To test whether the vitA2-ERE could mediate mERß transcriptional activity, we linked one copy of the ERE to either a basal promoter containing a TATA box (BLuc) or to the more complex viral thymidine kinase promoter (TKLuc) driving the expression of the luciferase reporter gene for transactivation studies. In Cos-1 cells, mERß induced a 5- to 10-fold response on all the ERE-containing reporters tested when 10 nM E2 was added to the medium (Fig. 6AGo). mER{alpha} produced a slightly larger fold induction of the ERE-containing reporters when studied under the same transfection conditions (Fig. 6BGo). In addition, E2-dependent transcriptional activity of mERß was also observed in HeLa cells (Fig. 6CGo), suggesting a cell type- independent effect. It is noteworthy that, as already observed in Cos-1 cells, the levels of E2-induced activation in HeLa cells were somewhat higher when mER{alpha} was used as compared with mERß (Fig. 6DGo), although it is not known whether similar levels of ER proteins were present in these transient transfections.



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Figure 6. E2-Dependent Transcriptional Activities of mERß vs. mER{alpha}

A, Cos-1 cells were transfected with 2 µg of the various reporter plasmids carrying either one copy of the vitA2-ERE (ERE-BLuc and ERE-TKLuc) or vector only (BLuc and TKLuc), along with 1 µg pCMX-mERß. Cells were treated with or without 10 nM E2 for 12 h before being assayed for luciferase activity. B, Transfection conditions were as in panel A, except that a pCMX-mER{alpha} expression vector was used. C and D, Transfection conditions were as in panels A and B, respectively, except that HeLa cells were used. E, mERß transactivates the pS2 promoter. HeLa cells were transfected with 500 ng pCMX-mERß and 1 µg pS2Luc (pS2) or pS2{Delta}ERELuc (pS2{Delta}ERE) reporter plasmids and incubated with or without 10 nM E2 in the presence or absence of 100 nM of the indicated antagonists, 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI).

 
We next investigated whether mERß could modulate the transcriptional activity of an ERE-containing promoter in its natural context. The pS2 promoter has been shown to respond to hER{alpha} (36). As shown in Fig. 6EGo, mERß was able to transactivate the pS2 promoter in HeLa cells with a 6-fold induction when E2 was added. In addition, OHT and ICI 182,780 efficiently blocked the E2-induced effect of mERß on pS2. This interaction is mediated through the ERE since its deletion abolished the response to E2 (Fig. 6EGo).

Ligand Binding
To ascertain whether there was a dose dependency of E2 for mERß and mER{alpha}, we tested the activity of both receptors in the presence of increasing E2 concentrations using the vitA2-ERE-TKLuc reporter in Cos-1 cells. Comparison of the dose-response curves of Fig. 7AGo shows a shift of approximately 4-fold of E2 concentration required to achieve half of the maximal level of induction between the two receptors. These results suggested that mERß may have lower affinity for E2 than measured for mER{alpha}. To verify if the difference in E2 responsiveness was due to a difference in ligand binding, we performed a binding analysis on both mERß and mER{alpha}. [3H[E2 was used to conduct binding studies with mERß, and results were plotted by the method of Scatchard. As shown in Fig. 7BGo, this analysis yielded an average dissociation constant (Kd) of 0.5 nM for E2 when performed on receptor prepared from rabbit reticulocyte lysates. This value is comparable to that obtained for the rat ERß, which was reported to be 0.6 nM (33). We obtained an average Kd of 0.2 nM for mER{alpha} (Fig. 7CGo), which is well within the range of previously published determinations for the cloned human receptor (38). Therefore, this slightly reduced affinity of mERß for E2 may provide an explanation for the shift in E2 responsiveness indicated by the dose-response curves (Fig. 7AGo).



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Figure 7. Dose-Response and Binding Properties of mERß

A, Cos-1 cells were transfected with 500 ng mERß (open circles) or mER{alpha} (closed circles) expression vectors and 1 µg vitA2-ERE-TKLuc and then incubated for 12 h with increasing concentrations of E2 as indicated. B, Specific binding of [2,4,6,7-3H]-17ß-estradiol ([3H]E2) to mERß was determined using receptors generated from rabbit reticulocyte lysates as described in Materials and Methods. Binding was determined over a concentration range of 0.01–3 nM [3H]E2 in the absence or presence of a 200-fold excess of unlabeled E2. The saturation plot is shown in the inset, and results were plotted by the method of Scatchard. Each point was determined in triplicate in each experiment, and the above results are representative of at least two separate experiments. C, Specific binding to mER{alpha} using the conditions described in panel B.

 
Estrogen Antagonists Block the E2-Dependent Activity of mERß
Estrogen antagonists such as OHT and ICI 164,384 are known to interact with hER{alpha} by blocking its transcriptional activity. More precisely, OHT is a mixed agonist-antagonist and blocks the activity of AF-2 but not of AF-1, whereas ICI 164,384 and 182,780 are pure antagonists that block both AF-1 and AF-2 activities (Refs. 6 and 39 and see below). We tested the effects of OHT and ICI 182,780 along with other antagonist compounds on mERß transactivation, and the results are shown in Fig. 8Go. All antagonists tested, including OHT, ICI 182,780, hydroxy-toremifen, raloxifene, and EM-652 (the active derivative of the novel nonsteroidal antiestrogen EM-800), effectively inhibited E2 activity (Fig. 8AGo). In contrast to its stimulatory effect on mER{alpha}, OHT did not display an agonistic activity on mERß when tested with a vitA2-ERE-BLuc reporter in either Cos-1 (Fig. 8BGo) and HeLa cells (data not shown).



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Figure 8. Effect of Antagonists on mERß-Mediated Transactivation

A, Cos-1 cells cotransfected with pCMX-mERß and vitA2-ERE-TKLuc plasmids were incubated for 12 h in the presence or absence of 10 nM E2 or 100 nM of the indicated antagonists: 4-hydroxitamoxifen (OHT), ICI 182,780 (ICI), hydroxy-toremifen (OH-tor), raloxifen (RLX), and EM652. Results are expressed as fold response over the mERß basal level in the absence of E2; this value was set arbitrarily to 1. B, Cos-1 cells cotransfected with pCMX-mERß or pCMX-mER{alpha} and ERE-BLuc plasmids were incubated for 12 h in the presence or absence of 100 nM OHT. C, Dose response of OHT and ICI in the presence of 10 nM E2 on mERß activity with the ERE-TKLuc in Cos-1 cells. The maximal induction by E2 alone was arbitrarily set at 100%. The untreated mERß basal level is also shown. Compounds within a panel are differentiated by open squares (OHT) and circles (ICI). D, Comparative panel of the dose responses to OHT in the presence of 10 nM E2 between mERß (open squares) and mER{alpha} (filled squares). Results are expressed as in panel B. E, Comparative panel of the dose responses to ICI in the presence of 10 nM E2 between mERß (open circles) and mER{alpha} (filled circles).

 
We further evaluated the specificity and potency of OHT and ICI 182,780 on both ER isoforms. Increasing concentrations of ICI 182,780 and OHT led to a complete inhibition of the E2-induced mERß activity in Cos-1 cells (Fig. 8CGo). Furthermore, when added at 10 nM and above, ICI 182,780 and OHT lowered mERß activity even below its basal level (compared with untreated in Fig. 8CGo). We observed a similar dose response for OHT and ICI 182,780 using either receptor (Fig. 8Go, D and E). However, for OHT, there was an apparent shift in the dose response toward the lower concentrations for mERß as compared with mER{alpha} (Fig. 8DGo).

Ras Enhances E2-Induced Transcriptional Activity of mERß
Phosphorylation of serine residues, in particular Ser118, has been shown to be necessary for maximal activity of AF-1 in the hER{alpha} and to mediate the effect of the Ras-Raf-1-mitogen-activated protein kinase (MAPK) kinase and MAPK pathway on the transcriptional activity of the ER (10, 11, 12, 40, 41). In an attempt to investigate the effect of the activation of this pathway on mERß activity, we used H-RasV12, a dominant active form of H-Ras, in transactivation studies. As shown in Fig. 9AGo, H-RasV12 acted to further increase by a factor of 3 the E2-induced activation of mERß using the vitA2-ERE-TKLuc reporter in Cos-1 cells. ICI 182,780, but not OHT, completely abolished the E2-dependent induction of mERß (Fig. 9AGo) and mER{alpha} (Fig. 9BGo) by Ras, suggesting that the effect of Ras on mERß activity is mediated by a putative AF-1 present in the amino-terminal domain. These results also show that ICI 182,780 suppresses transactivation mediated by both AF-1 and AF-2.



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Figure 9. Effects of H-Ras on E2-Induced mERß Transcriptional Activity

A, Cos-1 cells were cotransfected with 1 µg ERE-TKLuc, 500 ng pCMX-mERß, and with or without H-RasV12 expression plasmid. The cells were then grown in the presence or absence of 10 nM E2 or 100 nM of the indicated antagonists, 4-hydroxytamoxifen (OHT), and ICI 182,780 (ICI). B, Similar to panel A except that mER{alpha} was used. C, Potential MAP kinase phosphorylation sites in the mouse ER{alpha} and ß. D, Mutation of Ser60 abolishes the stimulatory effect of H-RasV12 on E2-induced mERß transcriptional activity. Transfection conditions were as in panel A, except that mERß mutants were used as indicated.

 
Two potential serines at positions 60 and 94 in the mERß amino-terminal domain matched the consensus MAPK phosphorylation site (42) (Fig. 9CGo). To determine whether one or both serine residues were involved in the Ras-mediated induction, we mutated Ser60 to Ala (S60A) and Ser94 to Ala (S94A) in mERß and used both mutants in transactivation studies. Figure 9DGo shows that H-ras had no inducing effect on the S60A mutant in the presence of E2 while the S94A mutant retained its responsiveness to H-ras as compared with the wild type mERß (Fig. 9AGo). This suggests that Ser60 in mERß is a potential target for phosphorylation by the Ras-Raf-1-MAPK kinase-MAPK pathway.

SRC-1 Interacts with and Augments Transcriptional Activity of mERß
The contribution of the AF-2 domain to mERß activity was investigated by examining the effect of the coactivator SRC-1, which has been shown to interact with a number of nuclear receptors including hER{alpha} (25, 43). Glutathione-S-transferase (GST) fusion proteins were generated with both the mERß and mER{alpha} LBDs (Fig. 10AGo) and tested in a GST-pull down experiment (Fig. 10BGo). GST-mERßEF and GST-mER{alpha}DEF were expressed in Escherichia coli, purified with GST-Sepharose, and incubated with [35S]methionine-labeled SRC-1. As shown in Fig. 10BGo, the LBD of mERß interacted weakly with SRC-1 in the absence of E2 (lane 3), whereas addition of E2 caused an increase in interaction between the two proteins (lane 4). As expected, estrogen antagonists that affect the AF-2 of ER, namely OHT (lane 5) and ICI 182,780 (lane 6), do not promote a ligand-dependent SRC-1 interaction. The E2-dependent interaction with SRC-1 was also efficiently blocked in the presence of 10-fold higher concentration of the various antagonists (data not shown). Protein-protein interactions between SRC-1 and the mER{alpha} LBD paralleled those observed with mERß (see Fig. 10BGo, lanes 8–12).



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Figure 10. SRC-1 Interacts with mERß and Induces Its Transcriptional Activity

A, Structure of the GST fusion proteins used in the analysis. B, GST pull-down experiments. The purified fusion proteins were incubated with labeled SRC-1 in the absence (lanes 3 and 9) or presence of 10 nM E2 (lanes 4 and 10), 10 nM each of OHT (lanes 5 and 11) and ICI 182,780 (ICI) (lanes 6 and 12). The input lane represents 10% of the total amount of labeled SRC-1 used in each binding reaction. An equivalent amount of protein extract was used in the samples containing only GST (lanes 2 and 9). C, Cos-1 cells were cotransfected with mERß and vitA2-ERE-TKLuc in the presence or absence of 1 µg CMX-SRC-1. Cells were incubated with or without 10 nM E2 or 100 nM antagonists as indicated. Separate experiments were also done with mER{alpha} as shown. D, Similar to panel A except that pS2Luc was used as the reporter and HeLa cells were transfected.

 
The contribution of SRC-1 on mERß activity was analyzed in vivo by transfection studies. When SRC-1 was cotransfected with mERß and vitA2-ERE-TKLuc in Cos-1 cells, a 2- to 3-fold induction over that of E2 alone was observed (Fig. 10CGo). A similar response was also seen with mER{alpha} in the presence of E2 (Fig. 10CGo). However, SRC-1 alone efficiently increased the basal level of transcriptional activity by mERß, suggesting a ligand-independent effect of SRC-1 on mERß (Fig. 10CGo). The basal transcriptional level of mER{alpha} was not affected by the addition of SRC-1. We also studied the effect of SRC-1 on the pS2 promoter in HeLa cells and found a similar ligand-independent activation of mERß by SRC-1 as well as a potentiation of the E2-induced activity (Fig. 10DGo). These results suggest that SRC-1 does not act on mERß in a promoter- and cell-specific manner. Furthermore, ICI 182,780, but not OHT, was able to abrogate the ligand-independent effect of SRC-1 on mERß transcriptional activity (Fig. 10Go, C and D).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The physiological actions of E2 are mediated through a member of the steroid hormone receptor family, ER{alpha}, which, for many years, has remained the only nuclear receptor known to have E2 as a ligand. Recently, it has been shown that a second receptor, ERß, is also able to respond to this hormone. ERß was cloned by degenerative PCR from rat prostate (33) and more recently from human testes (44), but its physiological role remains to be elucidated. As a first step toward investigating the role of ERß in development and homeostasis, we report the cloning of the murine homolog. The availability of the murine cDNA provides us with the means to characterize this receptor utilizing both biochemical and genetic methodologies and will allow us to study its relationship, if any, with ER{alpha} in normal reproductive physiology.

We first used a fragment of the LBD of the mouse ERß cDNA to establish the localization of the gene to chromosome 12. We have compared our interspecific map of chromosome 12 with a composite mouse linkage map location of many uncloned mouse mutations (provided from Mouse Genome Database, computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Estrb mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). The central region of mouse chromosome 12 shares a region of homology with human chromosome 14q, suggesting that Estrb will reside on 14q as well.

The mERß cDNA was used in a Northern analysis on murine ovary mRNA that revealed that the mERß gene generates several transcripts as compared with a single message for mER{alpha} in this tissue. Partial cDNAs obtained from an ovary library all contained a 3'-untranslated region (UTR) of 657 bp followed by a polyA+ tail, suggesting that the different transcripts seen in the Northern analysis may differ at their 5'-ends or in the coding region. Although we were unable to detect any messages in the total RNA from other mouse tissues, the hERß homolog appears to be expressed in mRNA isolated from testes, spleen, and thymus in addition to ovary (44).

The two mouse ER isoforms are closely related to each other in their LBD and DBD, indicating that these receptors may regulate common gene networks and respond to similar ligands. Using EMSA, a ligand-binding assay, and transient transfection experiments, we showed that mERß has slightly lower affinities than mER{alpha} for ERE and E2 binding in vitro, but that mERß can transactivate reporter genes driven by synthetic and natural E2-responsive promoters in vivo as efficiently as mER{alpha} in both Cos-1 and HeLa cells. However, the two ERs display no sequence homology in their amino-terminal domains, suggesting that each receptor may possess distinct transactivation functions. We made use of the differential mode of action of the estrogen antagonists OHT and ICI 182,780 and activation of receptor activity by the Ras-Raf-1-MAPK pathway to demonstrate functional similarities and differences between mERß and mER{alpha}. We first showed that, when assayed in Cos-1 cells with an E2-responsive reporter gene driven by a basal promoter, OHT does not display an agonistic activity with ERß. It has recently been shown that a specific region of the mER{alpha} AF-1 is required for OHT agonism (45): the absence of a corresponding domain in mERß may explain the present observation. We demonstrated that mERß, as previously shown for ER{alpha} (11, 12), can be activated by the Ras-Raf-1-MAPKK-MAPK pathway. We have identified a serine residue in mERß that could be the target of phosphorylation upon cotransfection with Ras. This was demonstrated by the fact that a mutation at Ser60 in the amino terminus of mERß eliminates the effect seen with Ras. Although another serine residue at position 94 also matches the MAP kinase consensus, mutation at this position has no effect on Ras-mediated activation. Moreover, alignment of the mouse, rat, and human amino acid sequences in this region indicates that Ser60 is conserved in all species whereas Ser94 is replaced by a glycine in the human ERß. Finally, we showed that the induction of the E2-dependent activation of mERß by Ras can be abolished by ICI 182,780, but not OHT, an observation that provides further evidence of the involvement of an AF-1-like domain in regulating mERß functions.

Antiestrogens play an important role in the treatment of breast cancer. We therefore monitored the efficacy of a series of antiestrogens previously shown to be selective for ER{alpha} on mERß and found that all compounds tested, including OHT, ICI 182,780, hydroxy-toremifen, raloxifen, and the novel nonsteroidal antiestrogen EM-800, inhibited E2-dependent activation by mERß. We showed that OHT is also AF-2 selective on mERß, and that ICI 182,780 inhibits both activation functions, displays no estrogenic activity, and thus can be considered as a pure antagonist of mERß activity. However, in dose-response studies, OHT proved to be a more potent inhibitor of mERß than of mER{alpha}. In addition, we show that the mode of action of ICI 182,780, as previously observed for ICI 164,384, involves the inhibition of both AF-1 and AF-2 activity of ER{alpha}.

A growing network of coactivators that interact with ER{alpha} has now been cloned. These include RIP140 (20), TIF2 (23), and SRC-1 (25). However, only SRC-1 has been shown to up-regulate ER-stimulated transcription (25, 43). mERß transcriptional activity could also be stimulated by SRC-1 in a ligand-dependent manner in cotransfection assays as well as in vitro where we observed a very strong ligand-dependent interaction with a GST-mERß LBD fusion protein. Surprisingly, we also observed ligand-independent SRC-1 enhancement of mERß transcriptional activity. These results suggest that SRC-1 may interact with a region, other than the AF-2, of the mERß protein. McInerney et al. (43) have shown that constructs expressing the ABCD (no LBD) and EF (LBD only) domains of hER{alpha} separately are able to transactivate in the presence of E2, and that SRC-1 can enhance the level of activation under these conditions. These results led them to suggest that SRC-1 may act as an adapter to promote AF-1 and AF-2 receptor activities and are in agreement with our suggestion that SRC-1 may interact with several regions of nuclear receptor proteins.

The identification of a second ER in mammals is very exciting, and these and previous studies have only begun to define the putative role that it plays in the mediation of estrogen action. Clearly, further studies into the mechanism of action of ERß are required if we are to understand how it functions in vivo. The results we present in this paper indicate that ERß and {alpha} may have both redundant and distinct functions, as exemplified by the manner with which they respond to OHT, Ras, and SRC-1, and suggest that ERß plays an unique role in the physiological actions of natural estrogens.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the Mouse ERß cDNA
A combination of PCR and cDNA library screening was used to obtain the full-length cDNA encoding the murine ERß. All oligonucleotides used in this study were synthesized at the Sheldon Biotechnology Center, McGill University. Total RNA was prepared from several mouse ovaries and used to isolate poly-A+ RNA over two oligo-dT columns (Pharmacia, Piscataway, NJ). An initial 550-bp fragment was amplified from 100 ng poly-A+ RNA using degenerate primers specific for the rat ERß LBD (33). The 5'- and 3'-sequence of these primers spanned amino acids 269–277 (KKIPGVE) and 443–450 (YDLLLEML, noncoding strand), respectively. The reaction was carried out for 40 cycles using Pfu polymerase (Stratagene, La Jolla, CA) at an annealing temperature of 54 C and for an extension time of 1 min. The PCR product was separated on low melt agarose (Life Technologies, Gaithersburg, MD) and subcloned into pBluescriptKSII (Stratagene). The sequence was determined on both strands using the T3 and T7 primers and found to encode an open reading frame that was highly homologous to the rat ERß. This 550-bp fragment served as a probe to screen a mouse ovary cDNA library constructed using the Superscript cDNA Synthesis System (Life Technologies). Three strong positives were plaque purified, subcloned into pBluescriptKSII, and sequenced on both strands. Each of these clones started in the LBD and ended with a poly-A tail. A PCR primer was designed based on the sequence of the 3'-UTR of the partial mouse ERß cDNA and used to synthesize first-strand cDNA from mouse ovary poly-A+ RNA. The cDNA served as a template in a PCR reaction using a primer that spans the first 21 bases of the 5'-UTR of rat ERß ending with the putative initiator codon and a second specific mouse ERß 3'-UTR primer. This reaction yielded a single product of 2.1 kb that was subcloned into pBluescriptKSII directionally using SalI and EcoRI sites that were designed in the 5'- and 3'-PCR primers, respectively, and sequenced on both strands. Sequencing was conducted at the sequencing facility at the Sheldon Biotechnology Center, McGill University.

Chromosomal Localization of the ERß Gene
Interspecific backcross progeny were generated by mating (C57BL/6J x M. spretus) F1 females and C57BL/6J males as described (34). A total of 205 N2 mice were used to map the Estrb locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (46). All blots were prepared with Hybond N+ nylon membrane (Amersham). The probe, a 550-bp HindIII/EcoRI fragment of the mouse cDNA, was labeled with [32P]-{alpha}-dCTP using a random-primed labeling kit (Stratagene); washing was done to a final stringency of 0.8x SSCP, 0.1% SDS, at 65 C. Fragments of 13.5 and 5.5 kb were detected in EcoRI-digested C57BL/6J DNA, and fragments of 8.4 and 5.1 kb were detected in EcoRI-digested M. spretus DNA. The presence or absence of the 8.4- and 5.1-kb EcoRI M. spretus-specific fragments, which cosegregated, was followed in the backcross mice.

A description of the probes and RFLPs for the loci linked to Estrb including Sos2, Spnb1, and Fos has been reported previously (47). Recombination distances were calculated as described (48) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

Northern Analysis
Five micrograms of mouse ovary poly-A+ RNA were electrophoresed on a 1% formaldehyde-agarose gel and blotted onto Hybond-N+. The entire mouse ER{alpha} cDNA (49) and the 550-bp PCR product encoding mERß were radiolabeled with [32P]-{alpha}-dCTP (Amersham, Arlington Heights, IL) by random priming (Pharmacia). Prehybridization was carried out for 4 h in 50% formamide, 5xSSPE, 5x Denhardt’s solution, 1% glycine, and 100 µg/ml denatured salmon sperm DNA at 42 C. Hybridization was conducted overnight at the same temperature in 50% formamide, 5x SSPE, 1x Denhardt’s solution, 10% dextran sulfate, 0.3% SDS, 100 µg/ml denatured salmon sperm DNA, and 2 x 106 cpm/ml denatured probe. The membrane was washed to high stringency (0.1x SSC, 0.1% SDS at 65 C for 30 min) and exposed to X-OMAT AR film (Kodak) overnight at -85 C. The positions of the 18S and 28S rRNAs were determined by the electrophoresis of total mouse ovary RNA on the same gel.

Plasmids
The expression vector pCMX-mER{alpha} was constructed by ligating a 1.9-kb NaeI-EcoRI fragment of mER{alpha} (49) into the appropriate sites of the eukaryotic expression vector, pCMX (50). pCMX-mERß was constructed in a similar fashion by subcloning the 2.1-kb SalI-EcoRI fragment described above into pCMX. Fusion proteins were generated between the mERß and mER{alpha} LBDs and GST by subcloning fragments of the mouse cDNAs into the pGEX-2T expression vector (Pharmacia). Briefly, a unique PvuII site located at position 678 and an EcoRI site in the vector were used to excise the LBD (domains E and F) from the full-length mERß cDNA. The fragment was then subcloned into the SmaI site of pGEX to produce pGST-mERßEF. For mER{alpha}, the hinge region and the LBD (domains D, E, and F) were amplified from the full length mER{alpha} cDNA using PCR and subcloned directionally into the BamHI and the blunted EcoRI sites of the pGEX vector (pGST-mER{alpha}DEF) (see Fig. 9AGo). Both constructs were verified to be in-frame with GST by sequencing. vitA2-ERE-BLuc and vitA2-ERE-TKLuc were constructed by ligating the vitA2-ERE oligonucleotide (see below for sequence) into SalI-BamHI-digested TKLuc vector. pS2-ERELuc contains the ~1050-bp pS2 promoter (36) preceding the luciferase reporter of pGL3 (Promega, Madison, WI). The pS2{Delta}ERELuc, in which the ERE was replaced by sequences encoding EcoRI-EcoRV sites, was generated by PCR mutagenesis using the ExSite kit from Stratagene as described by the manufacturer. The serine to alanine mutants at positions 60 and 94 of mERß were also generated by PCR mutagenesis. The oligonucleotides used were: S60A, 5'-CTCTATGCAGAACCTCAAAAGGCTCCTTGGTGTGAAGC-3'; S94A, 5'-GGTTGTGCCAGCCCTGTTACTGCTCCAAGCGCCAAGAGG-3'. The H-RasV12 expression plasmid was a generous gift from Dr. Morag Park, McGill University.

Chemicals
E2 was obtained from Sigma Chemical Co. (St. Louis, MO). EM-800, EM-652, ICI-182780, and OH-toremifene were synthesized in the medicinal chemistry division of the Laboratory of Molecular Endocrinology, CHUL Research Center, Québec, Canada. OHT was kindly provided by Dr. D. Salin-Drouin, Besins-Iscovesco, Paris, France.

EMSA
mERß and mER{alpha} proteins were synthesized by in vitro transcription-translation using rabbit reticulocyte lysates (Promega) and pCMX-mERß and pCMX-mER{alpha}, respectively, as templates. Typically 5 µl of programmed lysate were used in each binding reaction. DNA-binding reactions were carried out in binding buffer (5 mM Tris, pH 8.0, 40 mM KCl, 6% glycerol, 1 mM dithiothreitol, 0.05% Nonidet P-40), 2 µg of poly(deoxyinosinic-deoxycytidylic)acid, 0.1 µg of denatured salmon sperm DNA, and 10 µg of BSA with 0.1 ng probe that was labeled by end-filling with Klenow in the presence of [32P]-{alpha}-dCTP. Preincubations containing ligand and/or cold competitor as indicated were conducted on ice for 30 min, after which the probe was added and allowed to bind for 30 min at room temperature. The entire reaction (20 µl) was loaded onto a 4% polyacrylamide gel and electrophoresed at 150 V at room temperature. Gels were dried and exposed overnight at -85 C. The following oligonucleotides and their compliments were used as probes and competitors: vitA2-ERE, 5'-TCGACAAAGTCAGGTCACAG-TGACCTGATCAAG-3' (51); pS2-ERE, 5'TCGACCCTGCAAGGTCACGGTGGCCA-CCCCGTG-3' (36); IR3, 5'-TCGACGTGTAGGTCA-CAGTGACCTCTTCA-3'.

Scatchard Analysis
Ligand binding studies were conducted essentially as previously described (52) with the following modifications. mERß and mER{alpha} were produced using rabbit reticulocyte lysates, diluted 12-fold in TEG buffer (10 mM Tris, pH 7.5, 1.5 mM EDTA, 10% glycerol) and kept on ice until use. One hundred microliters of this dilution were used in each binding reaction at 4 C overnight containing [2,4,6,7-3H]17ß-estradiol concentrations ranging from 0.01–3 nM. Nonspecific binding was assessed by including 200-fold excess E2 in a parallel set of samples. Unbound steroids were removed with dextran-coated charcoal and counts per min were determined by liquid scintillation counting.

Cell Culture, DNA Transfection, and Luciferase Assay
For transfection, Cos-1 and HeLa cells were seeded in six-well plates in phenol red-free DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% charcoal dextran-treated FBS, and 100 µg/ml penicillin and 100 µg/ml streptomycin. At 50–60% confluency, cells were transfected with 1–2 µg of reporter plasmid, 0.5–1 µg receptor expression vector, 1 µg CMX-ßgal or RSV-ßgal, and 6–7.5 µg pBluescriptKSII, using the calcium phosphate/DNA precipitation method (53). After 8–16 h, cells were washed and typically 10 nM E2 or 100 nM antiestrogens, unless otherwise stated, were added to the growth medium for 16 h. For luciferase assay, cells were lysed in potassium phosphate buffer containing 1% Triton X-100, and light emission was detected using a luminometer after addition of luciferin. Values are expressed as arbitrary light units normalized to the ß-galactosidase activity of each sample.

In Vitro Protein-Protein Interactions
Fusion proteins were expressed in E. coli DH5{alpha} as follows. An overnight culture was diluted 10-fold in 500 ml prewarmed LB containing 100 µg/ml ampicillin and incubated for 1 h at 37 C. Isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 1 mM to induce expression, and the culture was allowed to grow for a further 3 h. Cells were cooled on ice for 10 min and centrifuged at 2500 x g for 20 min. The pellet was resuspended in 12 ml ice-cold PBS and sonicated. After the addition of one tenth volume of 10% triton, the extract was centrifuged at 12,000 x g for 20 min at 4 C. Clarified extracts were aliquoted in 1-ml samples and frozen at -85 C.

The 3.3-kb SRC-1 cDNA encoding the predicted 1061 amino acid open reading frame as originally cloned (25) was digested with XbaI and SalI, blunt ended with Klenow, ligated into pCMX, and labeled with [35S]methionine (Amersham) in vitro using rabbit reticulocyte lysates as described above. Approximately 300 µg of total protein from extracts containing the GST fusion proteins were loaded onto glutathione-Sepharose 4B (Pharmacia) for 30 min at 4 C with gentle agitation. After a short centrifugation, the beads were resuspended in 150 µl IPAB buffer (20) containing 10 nM ligand or antagonists and 4 µl of labeled SRC-1 crude lysate and allowed to bind for 90 min at 4 C. Beads were washed twice in the presence of IPAB and twice with IPAB without BSA, dried briefly, and resuspended in 30 µl loading buffer. Bound proteins were analyzed by SDS-PAGE. The gel was treated with Amplify (Amersham), dried, and exposed at -85 C.


    ACKNOWLEDGMENTS
 
We thank M. Parker for the gift of mouse ER{alpha} and human SRC-1 cDNAs and M. Park for the gift of the H-RasV12 expression vector. We are grateful to L. McBroom and R. Sladek for their suggestions and comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, Room H5.21, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.

Financial support was provided by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Cancer Research Society, Inc., to V. Giguère. G. B. Tremblay is a Postdoctoral fellow and V. Giguère a Scientist of the Medical Research Council of Canada. This work was also supported by EndoRecherche Inc.

1 Co-first authors. Back

Received for publication December 6, 1996. Revision received December 23, 1996. Accepted for publication December 30, 1996.


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 DISCUSSION
 MATERIALS AND METHODS
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