Molecular Determinants of the Stereoselectivity of Agonist Activity of Estrogen Receptors (ER) alpha  and beta *

Stefan O. MuellerDagger §, Julie M. HallDagger , Deborah L. SwopeDagger , Lars C. PedersenDagger , and Kenneth S. KorachDagger ||

From the Laboratories of Dagger  Reproductive and Developmental Toxicology and  Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, April 14, 2002, and in revised form, December 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The two known estrogen receptors, ERalpha and ERbeta , are hormone-inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. Previously, our laboratory demonstrated that ERalpha exhibits stereoselective ligand binding and transactivation for several structural derivatives and metabolites of the synthetic estrogen diethylstilbestrol. We have previously described the properties of indenestrol A (IA) enantiomers on ERalpha . In the study presented here, the estrogenic properties of the S and R enantiomers of IA, IA-S and IA-R, respectively, were expanded to examine the activity in different cell and promoter contexts using ERalpha and ERbeta . Using human cell lines stably expressing either ERalpha or ERbeta , we found that IA-S was a more potent activator of transcription than IA-R through ERalpha in human endometrial Ishikawa and breast MDA-MB 231 (MDA) cells. Interestingly, IA-R was more potent on ERbeta when compared with ERalpha in MDA, but not in Ishikawa cells, and IA-R exhibited equally low binding affinities to ERalpha and ERbeta in vitro in contrast to its cell line-dependent preferential activation of ERbeta . Alignment of the protein structures of the ligand-binding domains of ERalpha and ERbeta revealed one mismatched residue, Leu-384 in ERalpha and Met-283 in ERbeta , which may be responsible for making contact with the methyl substituent at the chiral carbon of IA-S and IA-R. Mutagenesis and exchange of this one residue showed that the binding of IA-R and IA-S was not affected by this mutation in ERalpha and ERbeta . However, in transactivation studies, IA-R showed higher potency in activating L384M-mutated ERalpha and wild-type ERbeta compared with wild-type ERalpha and M283L-mutated ERbeta in all cell and promoter contexts examined. Furthermore, IA-R-bound ERalpha L384M and wild-type ERbeta displayed enhanced interactions with the nuclear receptor interaction domains of the coactivators SRC-1 and GRIP1. These data demonstrate that a single residue in the ligand-binding domain determines the stereoselectivity of ERalpha and ERbeta for indenestrol ligands and that IA-R shows cell type selectivity through ERbeta .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The estrogenic effects of a variety of structurally diverse endogenous and xenobiotic compounds are mediated through the estrogen receptors (ERalpha and ERbeta ),1 which function as ligand-inducible transcription factors for genes involved in cell growth, proliferation, and differentiation (1). Studies using knock-out mice for the two ER subtypes (alpha ERKO and beta ERKO, respectively) have revealed that each receptor plays a unique role in estrogen biology in a wide variety of target tissues (2). Furthermore, in vitro studies indicated that ERalpha and ERbeta display marked differences in binding affinity and activation by natural and synthetic ER ligands (3, 4). Interestingly, although ERbeta shows lower binding affinity for and activation by endogenous estrogens, several xenoestrogens preferentially bind and activate ERbeta (3). These observations have prompted further studies aimed at elucidating the molecular mechanisms of action of ERalpha and ERbeta , and the search for ER subtype-selective agonists that could be used to evaluate the physiological roles of each receptor.

Diethylstilbestrol (DES) is a known carcinogen, which is oxidatively metabolized to a variety of metabolites with varying degrees of hormonal activity (34). Indenestrol A (IA) is a metabolite derived from DES that has high binding affinity for ERalpha but weak biological activity (35). IA exists as a racemic mixture of enantiomers, IA-S and IA-R (36), which have a methyl substitution on the chiral carbon (Fig. 1). Previously, our laboratory demonstrated that IA-R displays a lower binding affinity and transactivation potency on ERalpha than IA-S (5), which may help explain the differential biological activity and allow the study of the stereoselectivity of ligand binding and transcriptional responses of ERs.

The discovery of ERbeta prompted us to investigate whether both ER subtypes would exhibit the same stereoselectivity for the IA enantiomers. Human ERalpha and ERbeta share high sequence homology (97%) in the central DNA-binding domain, but only moderate conservation (60%) in the C-terminal ligand-binding domain (LBD) and activation function-2 (AF-2). Since AF-2 mediates the ligand-dependent transcriptional activities of both ER subtypes (1), it is possible that structural differences within this region could account for differences in the specificities of ERalpha and ERbeta for several estrogenic chemicals (4, 6).

To expand our knowledge about the molecular determinants of the stereoselectivity of ERalpha and ERbeta , we have characterized the ability of IA-S and IA-R to function as agonists/antagonists of the two ER subtypes. For the study of ER subtype-specific activation, stable cell lines were established by transfecting MDA-MB 231 (human breast adenocarcinoma; MDA) and Ishikawa (human uterine adenocarcinoma) cells with human or mouse ERalpha or ERbeta . Using these cell models and in vitro binding assays, the ability of the IA enantiomers to bind ERalpha and ERbeta , activate ERE-mediated transcription, and regulate coactivator recruitment was studied and compared between the two ER subtypes. Alignment of the LBDs of ERalpha and ERbeta revealed one mismatched amino acid (Leu-384 and Met-283, respectively) that may be responsible for making contact with the methyl substituent at the chiral carbon of IA-S and IA-R. This observation suggested that this residue could account for the functional differences observed between the two ER subtypes, a hypothesis that was further investigated in this study.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Biochemicals-- Media, serum, supplements, enzymes, and chemicals were purchased from Sigma.

Cell Culture-- Ishikawa and MDA-MB 231 (MDA) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10 and 5% fetal bovine serum (FBS), respectively, and penicillin/streptomycin. For MDA, 5 µg/ml insulin was added to the culture medium. Human hepatoma HepG2 were maintained in minimum essential medium (MEM) supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM glutamine, and 0.1 mM non-essential amino acids in flasks coated with 0.1% gelatin. Cells were cultured at 37 °C in a 5% CO2-humidified atmosphere.

Expression Vectors and Reporter Constructs-- For the generation of cell lines stably expressing mouse (m) or human (h) ERalpha or ERbeta , the cDNAs of mERalpha (GenBankTM accession M38651), mERbeta (U81451, Ref. 7), hERalpha (M12674, cDNA kindly provided by D. McDonnell, Duke University, Durham, NC) or hERbeta (AF051427, cDNA kindly provided by D. McDonnell, Duke University, Durham, NC) were subcloned into the mammalian expression plasmid pcDNA3 (Invitrogen, San Diego, CA) using standard cloning procedures. The pcDNA3 vector carries the neomycin resistance gene that allows selection of transfected cells with geneticin (G418).

Stable Transfection of Human Cells with the cDNAs of h/mERalpha /beta -- MDA and Ishikawa cells were seeded in 6-well plates for stable transfection. Transfection of cells was performed with the FuGENE 6 reagent according to the manufacturer's protocol with 0.5 µg of DNA and 1 µg of FuGENE 6 (Roche Diagnostics Corp.) per well in DMEM/F12 supplemented with 1% FBS. Three days after transfection with the pcDNA3 vector containing either the mERalpha , mERbeta , hERalpha , or hERbeta cDNA, cells were trypsinized and seeded in 60-mm tissue culture plates. Stable clones were selected by adding 200-400 µg/ml G418 to the culture medium. After 4 weeks of selection, 10-20 clones each for mERalpha , mERbeta , hERalpha , or hERbeta in MDA and Ishikawa were isolated and cultured separately in medium with 100 µg/ml G418. Control cell lines were established by transfecting parental MDA and Ishikawa lines with the empty pcDNA3 vector.

RNase Protection Assay (RPA)-- For the generation of riboprobe templates for hERalpha , a fragment corresponding to base pairs (bp) 1785-2092 of the cDNA and for hERbeta , a fragment corresponding to bp 1820-2011 of the cDNA were subcloned into pDP18 (Ambion, Austin, TX), respectively. A human cyclophilin probe (Ambion) was used as an internal control. Antisense riboprobes were generated from the linearized templates using the Maxiscript kit (Ambion) and [32P]CTP (Amersham Biosciences). RPAs were performed using the HybSpeed kit from Ambion according to the manufacturer's instructions (Ambion). 10 µg of total RNA of each sample, isolated with the Trizol reagent according to the manufacturer's protocol (Invitrogen), was analyzed. 5 µg of RNA isolated from untreated MCF-7 (human breast carcinoma) cells and BG-1 (ovarian carcinoma) cells were used as positive controls for hERalpha and hERbeta , respectively. RPAs were performed exactly as described previously (8).

Analysis of ER Protein Expression Using Western Blotting-- Nuclear extracts were prepared as described previously with modifications (9, 10). Aliquots containing 100 µg of protein were analyzed on 10% Tris-glycine polyacrylamide gels (NOVEX, San Diego, CA) and electroblotted onto nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) as described previously (8). The membrane was probed with either 1 µg/ml H222 ERalpha antibody (kindly provided by G. Greene, University of Chicago, Chicago, IL) or 2 µg/ml anti-ERbeta antibody (06-629, Upstate Biotechnology, Lake Placid, NY). Immunocomplexes were detected with a horseradish peroxidase-conjugated anti-rat (ERalpha ) or anti-rabbit (ERbeta ) IgG secondary antibody at 1:300 dilution (Oncogene, Cambridge, MA) and an ECL chemiluminescence kit (Amersham Biosciences).

Transient Transfection and Transactivation Assay-- Cells were seeded on 24-well plates 15 h prior to transfection in phenol red-free DMEM/F12 supplemented with 5% dextran-coated charcoal stripped FBS (DCC/FBS). The reporter plasmids were transfected in DMEM/F12 supplemented with 1% DCC/FBS using FuGENE 6 according to the manufacturer's protocol. Each well received 0.5 µg of reporter plasmid and 0.01 µg pRL-CMV (Renilla luciferase for normalization; Promega, Madison, WI). A firefly luciferase reporter driven by three copies of the vitellogenin estrogen response element (3×ERE-Luc) and a reporter containing the human complement 3 gene (C3) promoter (C3-Luc; kindly provided by D. McDonnell, Duke University, Durham, NC) were used to measure ER transcriptional activity (11). For cotransfection of wild-type or mutated ER with the luciferase reporter vectors, 0.09 µg of receptor plasmid, 0.4 µg of reporter, and 0.01 µg of Renilla luciferase normalization vector were used per well. After transfection, cells were incubated in medium supplemented with 5% DCC/FBS as described above with DES, 17beta -estradiol, IA-S, or IA-R (final concentration of vehicle ethanol 1%, v/v) for 20 h. Luciferase assays were performed using the Dual-Luciferase reporter assay system according to the manufacturer's protocols (Promega). Each value was normalized to the Renilla luciferase control, and each data point generated is the average of duplicate determinations. All experiments were repeated at least three times with consistent results.

Mammalian Two-hybrid Assays-- For mammalian two-hybrid assays, HepG2 (human hepatoma) cells were plated in 24-well plates (coated with 0.1% gelatin) 24 h prior to transfection. DNA was introduced into cells using FuGENE 6. In standard transfections, 0.5 µg of reporter 5×-Gal4-TATA-Luc, containing 5 binding sites for the yeast Gal4 transcription factor, 0.09 µg of receptor (either pVP16-hERalpha , pVP16-hERalpha L384M, pVP16-hERbeta , or pVP16-hERbeta M283L), 0.5 µg of Gal4DBD-coactivator fusion (12, 13) (pM-SRC-1 (NR-box) or pM-GRIP1 (NR-box); plasmids kindly provided by D. McDonnell, Duke University, Durham, NC), and 0.01 µg of the pRL-CMV Renilla luciferase normalization vector were used for each well. All transfections were performed in triplicate. Prior to transfection, cells were washed with phosphate-buffered saline, and 200 µl of phenol red-free MEM containing 5% DCC/FBS was added to each well. Cells were incubated with the DNA/FuGENE 6 mix for 6 h; receptor ligands in 200 µl of phenol red-free MEM were then added to the cells and incubated for 20 h. Luciferase assays were performed using the Dual-Luciferase reporter assay system according to the manufacturer's protocols. Each value was normalized to the Renilla luciferase control, and each data point generated is the average of triplicate determinations. All experiments were repeated three times.

Ligand Binding Studies-- Ligand binding was analyzed by competition of the test compound against 1 nM 17beta -[3H]estradiol (71 Ci/mmol, PerkinElmer Life Sciences). Reactions were performed with 1 µl of in vitro translated wild-type or mutated ERalpha and ERbeta , respectively, in 100 µl of TEGM buffer (10 mM TRIS, 1.5 mM EDTA, 3 mM EGTA, 3 mM MgCl2, 10% glycerol). ERs were in vitro translated with the TNT kit (Promega) using the pcDNA3 expression plasmids according to the manufacturer's protocol. Samples were incubated for 15 h at 4 °C. Unbound 17beta -[3H]estradiol was removed by adding DCC (0.25% charcoal, 0.025% dextran). Samples were spun at 3000 × g, and remaining radioactivity contained in the supernatant was measured in a scintillation counter. Nonspecific binding was measured in the presence of 500× excess of unlabeled estradiol. Scatchard analyses were performed with 0.1-2.5 nM 17beta -[3H]estradiol.

Site-directed Mutagenesis-- The exchange of one amino acid in the LBD of hERalpha and hERbeta was accomplished using the QuikChange site-directed mutagenesis kit according to the manufacturer's protocol (Stratagene). The expression plasmids pcDNA3-hERalpha and pcDNA3- hERbeta were used to generate point mutations in the LBD. ERalpha L384 M was generated by exchanging the codon for leucine at position 384 for a methionine. The primers used for mutagenesis were: forward primer, 5'-CTAGAATGTGCCTGGATGGAGATCCTGATG (mutated codon in bold) and reverse primer, 5'-CATCAGGATCTCCATCCAGGCACATTCTAG-3' (mutated codon in bold). ERbeta M283L was generated by exchanging the codon for methionine at position 283 for a leucine. The primers used for mutagenesis were: forward primer, 5'-GGAGAGCTGTTGGCTAGAGGTGTTAATGATG-3' (mutated codon in bold) and reverse primer, 5'-CATCATTAACACCTCTAGCCAACAGCTCTCC-3' (mutated codon in bold). The mutations in the plasmids used in the mammalian two-hybrid assay, pVP16-hERalpha L384M and pVP16-hERbeta M283L were created exactly as described above using the pVP16-hERalpha and pVP16-hERbeta plasmids (kindly provided by D. McDonnell, Duke University, Durham, NC) as templates for mutagenesis.

Statistical Analysis-- Statistical analyses were performed using Student's t test (unpaired, one-tailed). Statistical significance is indicated by asterisk (p <=  0.05).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Human Cell Lines with Stable Expression of ERalpha and ERbeta -- ERalpha and ERbeta were introduced into the ER-negative human mammary MDA and endometrial Ishikawa cell lines (14-16) to obtain in vitro models to study the ligand-mediated transcriptional responses of ERalpha and ERbeta . For the generation of ER subtype and cell-specific models, each cell line was transfected with expression plasmids containing either the murine (m) or human (h) ERalpha or ERbeta cDNAs. Clonal cell lines with stable and comparable expression of the respective ER were established. The expression of ERalpha or ERbeta mRNA and protein in each cell line was confirmed by RNase protection assay (RPA) and Western blotting. The mRNA expression levels were between 1 and 4% of the expression of the housekeeping gene cyclophilin for mouse and human ERalpha or ERbeta expressed in MDA and Ishikawa cells (data not shown). The parental cell lines, as well as control cell lines stably transfected with the empty pcDNA3 vector, lacked endogenous ERalpha and ERbeta mRNA expression (data not shown). One cell clone for each cell line expressing functional mouse or human ERalpha or ERbeta was selected by assessing its estrogen responsiveness in transactivation assays using a reporter construct that contained three copies of the vitellogenin consensus estrogen response element (ERE, 3×ERE-Luc) as reporter (data not shown). However, we were not able to establish a cell clone of the MDA line with expression of functional hERalpha .

IA-R and IA-S Show Distinct Abilities to Activate ERalpha and ERbeta in Human Breast and Endometrial Cell Lines-- We first examined the ability of DES, E2, and the IA enantiomers IA-R and IA-S (Fig. 1) to activate transcription through ERalpha and ERbeta in dose response experiments. Transactivation studies were performed in human MDA breast and Ishikawa endometrial cell lines using the 3×ERE-Luc (containing 3 copies of the vitellogenin A2 ERE) and C3-Luc (containing the natural complement 3 (C3)) reporter constructs (11, 17). The parental MDA and Ishikawa cell lines, as well as cells transfected with empty pcDNA3 vector, lacked any ligand-inducible transactivation of the 3×ERE or C3 reporters, confirming the absence of endogenous functional ERalpha and ERbeta in these cell lines (data not shown). IA-S and IA-R, like E2 and DES, were agonists of ERalpha and ERbeta in all cell and promoter contexts examined. In contrast to the weak agonism of IA-R on ERalpha , this compound showed a high potency to activate both murine and hERbeta in MDA cells (Table I). In Ishikawa cells, IA-R was less potent than IA-S in transactivation of ERbeta (EC50: 4.0-8.0 nM for IA-R versus 0.2-0.5 nM for IA-S; see Table II), but showed a slight, statistically significant preference for hERbeta over hERalpha on the 3×ERE reporter (EC50: 4.0 ± 3.6 nM for ERbeta versus 9.3 ± 1.1 nM for ERalpha ; see Table II). Interestingly, IA-R showed no marked difference in activation of ERbeta versus ERalpha in Ishikawa cells on the C3 promoter, whereas IA-R was a significantly more potent activator of transcription through ERbeta compared with ERalpha on both the 3×ERE and C3 reporters in MDA cells, indicating a degree of cell type specificity influencing gene expression by the different ER subtypes (see Tables I and II).


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Fig. 1.   Chemical structures of diethylstilbestrol (DES), 17beta -estradiol (E2), and the indenestrol A (IA) enantiomers IA-S and IA-R.


                              
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Table I
Potency of ER ligands in MDA-MB 213 (MDA) cells stably expressing mERalpha , mERbeta , or hERbeta


                              
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Table II
Potency of ER ligands in Ishikawa cells stably expressing mERalpha , mERbeta , hERalpha , or hERbeta

IA-R Has a Low Binding Affinity to ERbeta Although It Is a Potent ERbeta Agonist-- The finding that IA-R was a less potent activator of ERalpha -mediated transcription compared with IA-S in MDA and Ishikawa cells (Tables I and II) confirmed data previously obtained in a yeast-based ER system (5). In addition, IA-R was a more potent activator of ERbeta in MDA and partly in Ishikawa cells compared with ERalpha , which suggests that IA-R might also display a higher binding affinity for ERbeta as compared with ERalpha . Competition binding studies with in vitro transcribed and translated hERalpha or hERbeta and 3H-labeled E2 confirmed our previous report (5) that IA-R has a low binding affinity for ERalpha (Table III). Inconsistent with the transactivation data, however, IA-R also showed low binding affinity for ERbeta , although both IA-S and IA-R exhibited slightly higher binding affinities for ERbeta than for ERalpha (Table III).


                              
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Table III
Relative binding affinities (RBA) of ER ligands to ERalpha and ERbeta

Mutagenesis Studies Reveal That One Residue in the LBD Is Critical for the Stereoselective Ligand Activation of ERalpha and ERbeta -- The differences in IA-R transactivation and binding of ERbeta as compared with ERalpha prompted us to examine differences in the LBD of the two ER subtypes that could contribute to the distinct pharmacology of IA-R on ERalpha and ERbeta . The structures predicted by x-ray crystallographic studies of the LBDs of ERalpha and ERbeta provide a potential answer to this question (18, 19). A structural model of the superimposition of the IA isomers with DES and genistein bound to the LBD of ERalpha and ERbeta , respectively, predicts that several residues within the LBD of ERalpha and ERbeta could make contact with the methyl substituent at the chiral carbon of IA (Fig. 2) (18, 19). From these selected residues, only leucine 384 (Leu-384) in ERalpha and the analogous methionine 336 (Met-336) in ERbeta are mismatched based on the alignment of the reported protein sequences of rodent and human ERalpha and ERbeta (18) (Fig. 2). The ERbeta cDNA used in this study represents the short form of human ERbeta (GenBankTM accession no. AF051427) and position 283 is identical to position 336 of the full-length hERbeta cDNA used for the alignment in Fig. 2. Furthermore, the complete sequence of the ligand-binding domain of the "short" hERbeta is identical to the full-length hERbeta cDNA (20, 21). Notably, the residues Leu-384 and Met-283 are among those that may be involved in contact with the IA ligand when bound to the LBD.


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Fig. 2.   Superimposition of IA-S (navy) and IA-R (green) molecules with genistein (red) bound to the LBD of hERbeta . Structural model of a part of the aligned LBD of hERalpha (blue) and hERbeta (red) drawn according to data presented in Refs. 18 and 19 using Molscript with the coordinates from the Brookhaven protein data bank (accession 2ERD and 2ERT). Superimposition of the ligands and numbering of residues was done based on the crystal structural data of genistein bound to the LBD of hERbeta (18).

To determine whether this one residue might be responsible for differences in the IA-R activation profile of ERalpha compared with ERbeta , the mismatched residues in ERalpha and ERbeta were exchanged, i.e. Leu-384 in hERalpha was mutated to a methionine (ERalpha L384M) and Met-283 in hERbeta to a leucine (ERbeta M283L). We first determined whether the resulting ER mutants bound the IA enantiomers with different affinities than the wild-type ERs. Scatchard analysis of competition binding studies with in vitro transcribed and translated receptors revealed that wild-type and mutated ERs have comparable binding constants (Table IV). Using competition binding studies we then determined the affinities of DES, E2, IA-S, and IA-R for wild-type and mutated ERalpha and ERbeta (Table V). Relative binding affinities (RBA) compared with DES (set to 100) revealed no significant differences between the mutated and the wild-type ERs (Table V, ERalpha versus ERalpha L384M and ERbeta versus ERbeta M283L) for any ligand tested, indicating that the receptor binding affinity of the IA enantiomers was not affected by these mutations.


                              
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Table IV
Binding affinities of radioactively labeled 17beta -estradiol to wild-type and mutated ERalpha and ERbeta


                              
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Table V
RBA of investigated compounds to wild-type and mutated ERalpha and ERbeta

We then tested the ability of DES, E2, IA-S, and IA-R to activate wild-type and mutated ERalpha and ERbeta . MDA and Ishikawa cells were transiently transfected with the cDNAs of wild-type or mutated hERalpha or hERbeta . Transactivation assays were used as described above to measure receptor activity on the 3×ERE and the C3 promoters (Tables VI and VII, Fig. 3). The cell line-dependent activation profile of IA-R observed in the stably ER expressing cell lines (Tables I and II) was confirmed by results obtained in cells transiently expressing ER (Tables VI and VII). Fig. 3 shows the dose-response curves obtained on the C3 promoter in MDA cells. A compilation of all data obtained from transactivation assays performed in MDA and Ishikawa cells is given in Tables VI and VII, respectively. Specifically, these data confirmed that IA-R was a more potent activator of ERbeta than of ERalpha in MDA cells on both promoters examined (Table VI). However, in Ishikawa cells IA-R was only slightly more potent in activating ERbeta compared with ERalpha on the 3×ERE, but displayed no difference in potency between ERalpha and ERbeta on the C3 promoter (Table VII). Consistent with their binding affinities, E2, DES, and IA-S showed no significant differences in their abilities to activate wild-type or mutated ERalpha and ERbeta in MDA and Ishikawa cells (Fig. 3 and Tables VI and VII). In contrast, the L384M mutation of ERalpha rendered IA-R more active compared with wild-type ERalpha (note the shift in the IA-R induced transcriptional potency of wild-type ERalpha compared with mutated ERalpha in Fig. 3B). Consistent with this observation, the ERbeta mutant M283L exhibited lower potency than wild-type ERbeta when treated with IA-R (note the shift in the IA-R induced transcriptional activity of wild-type ERbeta compared with mutated ERbeta in Fig. 3D). Furthermore, these effects were statistically significant in MDA cells on both promoters examined and in Ishikawa cells on the 3×ERE reporter (Tables VI and VII). Also, IA-R was a significantly less potent activator of the ERbeta mutant M283L than wild-type ERbeta on the C3 promoter in Ishikawa cells. However, although the L384M mutation of ERalpha rendered IA-R consistently more potent compared with wild-type ERalpha in Ishikawa cells in all performed experiments, this effect failed to reach statistical significance (EC50: 1.7 ± 1.2 nM for ERalpha L384M versus 5.0 ± 4.4 nM for ERalpha , p = 0.1; see Table VII). The nature of these results suggests that these alterations were not apparently dependent on the gene regulatory sequence nor the cell type, but most likely a result of an intramolecular effect on the ER protein.


                              
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Table VI
Potency of ER ligands in MDA cells transiently transfected with wild-type and mutated ERalpha and ERbeta


                              
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Table VII
Potency of ER ligands in Ishikawa cells transiently transfected with wild-type and mutated ERalpha and ERbeta


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Fig. 3.   Comparison of transactivation of wild-type versus mutated ER in MDA cells. Cells were transiently transfected with the cDNA of wild-type hERalpha (solid diamonds and squares), wild-type hERbeta (solid diamonds and squares), hERalpha L384M mutants (open diamonds and squares), or hERbeta M283L mutants (open diamonds and squares) together with the C3-Luc reporter and pRL-CMV Renilla luciferase normalization vector. Transcriptional activity of wild-type versus mutant ERalpha (A and B) or wild-type versus mutant ERbeta (C and D) was compared. Following transfection, cells were treated with E2 or DES (A and C) or with the IA enantiomers IA-S and IA-R (B and D) at the indicated concentrations for 20 h. After 20 h, cells were harvested and dual luciferase assays were performed. Each value was normalized to the internal luciferase control. Note that IA-R (B and D) is a more potent activator of mutated ERalpha and wild-type ERbeta compared with wild-type ERalpha and mutated ERbeta . Shown is a representative experiment, with each data point being the average of duplicate determinations. Experiments were repeated at least three times with consistent results.

Mutagenesis of the Mismatched Residue in the hERalpha and hERbeta LBD Influences the Interaction of the IA-R-bound Receptors with Coactivators-- The observation that methionine 283 in hERbeta enhanced activation by IA-R suggested that this residue could be important in coactivator recruitment by the IA-R-liganded receptor. To test this hypothesis, mammalian two-hybrid assays were performed to demonstrate the interaction of hERalpha , hERalpha L384M, hERbeta , and hERbeta M283L with the nuclear receptor-interacting regions (NR-boxes) of the coactivators SRC-1 and GRIP1. Previously, GAL4-DBD fusions of the SRC-1 and GRIP1 NR-boxes were shown to interact with ERalpha and ERbeta in an agonist-dependent manner, consistent with known receptor-coactivator interactions (25). In the current study, as expected, the interactions between the Gal4DBD-SRC-1 and Gal4DBD-GRIP-1 NR-box fusions, and each receptor were enhanced by the addition of the agonists DES, E2, IA-S, and IA-R (Fig. 4, A-D). The interaction of SRC-1 with wild-type and mutant hERalpha or wild-type and mutant hERbeta was similar for the DES, E2, and IA-S bound receptors (Fig. 4, A and B), in correlation with the transactivation data (Tables VI and VII, Fig. 3). Notably, the interaction of SRC-1 in the presence of IA-R, which displayed a higher potency on hERalpha L384M and hERbeta compared with hERalpha and hERbeta M283L in transactivation studies (Tables VI and VII, Fig. 3), was enhanced when the methionine residue was present in hERalpha (hERalpha L384M) and in hERbeta (wild-type). GRIP1 binding to the IA-R-bound hERalpha L384M and hERbeta receptors was also clearly increased (Fig. 4, C and D). None of the receptor or coactivator fusion proteins were capable of activating the 5×-Gal4-TATA-Luc reporter vector when tested together with the empty Gal4-DBD or pVP16 vectors (data not shown). Taken together, the results from these studies indicate that methionine 283 in hERbeta plays an important role in IA-R activation through facilitating the recruitment of coactivators to the ligand-bound receptor, as illustrated for these types of ligand agonists.


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Fig. 4.   Mutagenesis of the one mismatched residue in the hERalpha and hERbeta LBD influences the interaction of the IA-R-bound receptors with coactivators. Mammalian two-hybrid assays were used to quantitate the interaction of hERalpha , hERalpha L384M, hERbeta , and hERbeta M283L with the coactivators SRC-1 and GRIP1. For these experiments, constructs containing the SRC-1 and GRIP receptor interaction domains fused to the Gal4 transcription factor DNA-binding domain (pM-SRC-1 (NR) and pM-GRIP1 (NR)) were used together with constructs containing either the hERalpha , hERalpha L384M, hERbeta , or hERbeta M283L cDNA fused in-frame to the VP16 activation domain. HepG2 cells were transiently transfected with the 5×-Gal4-TATA-Luc reporter, the pRL-CMV normalization plasmid and either pM-SRC-1 (A and B) or pM-GRIP1 (C and D), together with pVP16-hERalpha , pVP16-hERalpha L384M, pVP16-hERbeta , or pVP16-hERbeta M283L. Following transfection, cells were treated with vehicle (veh) or 100 nM of E2, DES, or the indenestrol enantiomers IA-S or IA-R. After 20 h, cells were harvested and dual luciferase assays were performed. Each value was normalized to the internal luciferase control. Shown is a representative experiment with each data point being the average of triplicate determinations. Experiments were repeated three times with consistent results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, our laboratory and others (5, 22-25) determined that ERalpha exhibits stereoselective ligand activation and identified potential molecular determinants for ERalpha ligand-dependent activation and stereoselectivity. With the discovery of ERbeta (3, 4) the question emerged as to whether ERbeta also exhibits stereoselective ligand activation; this may explain the activity of the DES-type compounds as noted in the present studies and may elucidate the functional properties of ERbeta . Complementary to this mechanistic question is the search for ER subtype-specific agonists or antagonists that would permit the identification of distinct activities and roles for ERalpha and ERbeta in estrogen target tissues. Katzenellenbogen and co-workers (26) were the first to determine the stereoselectivity of ERbeta using a chiral tetrahydrocrysene (THC). They found that the enantiomer R,R-THC activates ERalpha but is an antagonist on ERbeta , whereas S,S-THC is an agonist on both ERalpha and ERbeta (26).

We showed in an earlier report that IA-S is a strong ERalpha agonist, whereas IA-R displayed only weak agonistic activity in a reconstituted ER transcription system in yeast (8). In the study described here, we further explored the activity profile of these DES derivatives by determining the potency of the enantiomers of IA to activate ERalpha and ERbeta in different cell and promoter contexts. For analysis of cell type specificity, we generated human mammary and endometrial cell lines that stably express mouse or human ERalpha or ERbeta . In transactivation studies using the cell lines generated, IA-S and IA-R exhibited agonistic and partial agonistic properties, respectively, through ERalpha and ERbeta for all cell and promoter contexts examined. This demonstrated that both IA enantiomers have agonistic properties similar to E2 and DES, but with differing degrees.

However, although both IA enantiomers function as agonists of both ER subtypes, each ligand differed markedly with regard to its potency as an activator of ERalpha or ERbeta . The IA enantiomer IA-R was a more potent activator of ERbeta compared with ERalpha in human mammary MDA cells. In endometrial Ishikawa cells, IA-R showed higher potency of activation on mERbeta than mERalpha . However, both hERalpha and hERbeta showed a similar ability to activate transcription from the C3 reporter in the presence of IA-R, whereas IA-R was a more potent activator of hERbeta than hERalpha on the more sensitive 3×ERE reporter. Taken together, these results indicated that IA-R could function as a potency and cell type-selective ERbeta agonist in MDA cells when compared with the endometrial Ishikawa cell line. Data reported here also indicated that ERbeta bound to IA-S is transcriptionally active to a similar degree in both mammary and endometrial cells. These findings support the notion that the activity of the ER can be dependent on the cellular context, as highlighted by the cell type-specific mixed agonist/antagonist activity of the selective ER modulator tamoxifen through ERalpha (27).

The fact that IA-R is a weak agonist for ERalpha in all cell and promoter contexts examined suggests that either IA-R has a low binding affinity for ERalpha and/or the conformation of IA-R-liganded ERalpha does not favor activation. IA-R exhibited comparably low binding affinities for ERalpha and ERbeta , which might be responsible for the weak activation of ERalpha by IA-R. However, the low binding affinity of IA-R for ERbeta is not predictive of its higher potency to activate ERbeta in MDA cells as compared with ERalpha . Paige et al. (28) used affinity-selected peptides to show that ERalpha and ERbeta assume ligand-specific conformations, which are likely to modulate the activity of the liganded receptors. It is therefore reasonable to assume that a conformational change could cause the differential potencies of the activation of ERalpha versus ERbeta by IA-R, i.e. IA-R induces a conformational change permissive of ligand-dependent activation principally of ERbeta , but only minimally of ERalpha .

Recent studies analyzed the conformational changes that occur in ERalpha and ERbeta upon binding of the ER subtype-specific THC ligands (29, 30). Kraichely et al. (29) showed that the THCs induced a distinct conformation of the receptors concomitant with quantitative differences in coactivator recruitment. They concluded that the ligand-dependent conformational change favors association of the receptor with coactivators and through that interaction causes transactivation by the ER. This mode of action is mechanistically described by the tripartite model of steroid hormone receptor action (31). This model takes into account the ligand binding affinity, the conformation of liganded receptor, and the association of the receptor with coregulator molecules, i.e. coactivators. In line with this model, we hypothesized that IA-R permits transcriptional activation of ERbeta but not of ERalpha , due to induction of a conformation resulting in altered coactivator recruitment.

We performed studies to test this hypothesis through experiments pinpointing the molecular determinants responsible for the differences in transactivation between ERalpha and ERbeta when bound by IA-R. Comparison of the amino acid sequence of the LBD of ERalpha and ERbeta revealed one mismatched residue within the LBD, Leu-384 in ERalpha and Met-283 in ERbeta that is likely to be proximal to the methyl substituent of the chiral carbon in IA based on the crystal structure data of liganded ERalpha and ERbeta (18, 19). Given this information, we speculated that this residue could interfere with the bound IA, inducing a distinct conformational change either facilitating or impeding activation by ER. Indeed, when this residue was exchanged in ERalpha and ERbeta , the potency of activation of mutated ERalpha L384M and ERbeta M283L by IA-R changed accordingly: IA-R was a more potent activator of transcription of ERalpha L384M compared with wild-type ERalpha and also a less potent activator of mutated ERbeta M283L compared with wild-type ERbeta . Importantly, this shift in activation of IA-R was independent of the cellular and promoter context tested and, furthermore, the activities of IA-S, as well as of DES and E2 were not markedly affected by the Leu/Met mutational exchange. This suggests that IA-R has a unique ligand structure that is sensitive to this residue in the binding pocket and subsequently affects receptor conformation. As expected by the low binding affinity of IA-R observed for ERalpha and ERbeta , this mutation did not affect the binding affinities of IA-R or IA-S to ERalpha and ERbeta , supporting the concept of dissociation of ligand binding affinity from biological transcriptional activity. The residues Leu-384 in ERalpha and Met-283 in ERbeta are therefore likely to be important molecular determinants for the ER subtype-specific activity of IA-R.

Several studies have analyzed the molecular determinants of ligand-dependent activation of ERalpha (5, 22-25, 32, 33). In most of these reports, mutations of residues in helices 11 and 12 of the AF-2 domain were described, and results indicated reduced DNA and/or ligand binding accompanied by weaker transactivation. Studies on the enantiomers of indenestrol showed that none of the ERalpha mutants analyzed reversed the stereoselectivity of ERalpha (5, 22). In the latter study and in results presented here, the higher potency of IA-S to induce ERalpha activity was accompanied by a higher binding affinity of IA-S to ERalpha as compared with IA-R (36). Here, we showed that Leu-384 in helix 6 of hERalpha impedes the activation mediated by IA-R, independent of its binding affinity. Feng et al. (33) mutated the residues of lysine 362 in helix 3, valine 376 in helix 5, or glutamic acid 542 in helix 12 of the hERalpha LBD. All three mutations independently resulted in diminished transcriptional activity and coactivator binding (33). Similar results were reported for mERalpha ; Mak et al. (25) mutated residues in the LBD and showed that the hydrophobic surface in the LBD, specifically Leu-376 in mERalpha , which corresponds to leucine 379 in hERalpha , is required for binding of the coactivator SRC-1 and subsequent transactivation. In this report, we examined and compared the putative molecular determinants of the ligand specificity of both ERalpha and ERbeta . The leucine 384 residue of ERalpha impeded transactivation by IA-R, but not by other ligands, and therefore might also impede recruitment of coactivators only when IA-R is bound. In contrast, methionine 283 in the LBD of ERbeta enhanced transactivation induced by IA-R, which might be due to a conformational change leading to increased interactions with coactivators. Indeed, the interaction of SRC-1 and GRIP1 in the presence of IA-R, which was a more potent activator of hERalpha L384M and hERbeta compared with hERalpha and hERbeta M283L, was enhanced when the methionine residue was present in hERalpha (hERalpha L384M) and in wild-type hERbeta . Taken together, this agrees with our suggested mechanism of IA-R activation: that IA-R, albeit with low binding affinity, induces a conformational change in ERbeta that facilitates ligand-dependent activation, whereas IA-R bound to ERalpha is unable to induce a favorable conformation and impedes activation.

In conclusion, this series of studies has revealed that IA-R is a potency-selective agonist for ERbeta in a cell type-specific manner, and a single residue in the LBD of ERalpha and ERbeta modulates their transcriptional activity in a cell type-independent fashion. Analysis of conformational changes and crystallographic and structural analysis of wild-type and mutated ERalpha and ERbeta bound to subtype specific agonists would further extend our understanding of the molecular mechanisms and structural determinants of ligand-specific transcriptional activity of ERalpha and ERbeta .

    ACKNOWLEDGEMENTS

We thank members of the Receptor Biology Section for critical discussion and editing of the manuscript. We gratefully acknowledge Dr. D. P. McDonnell (Duke University, Durham, NC) for kindly providing several plasmids used in these studies.

    FOOTNOTES

* This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (Mu 1490/1) (to S. O. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed: Merck KGaA, Institute of Toxicology, P.O. Box 64271, Darmstadt, Germany. Fax: 49-6151-72-91-8517; E-mail: stefan.o.mueller@merck.de.

|| To whom correspondence may be addressed: NIEHS, MD B3-02, 111 TW Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. Fax: 919-541-0696; E-mail: korach@niehs.nih.gov.

Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M203578200

    ABBREVIATIONS

The abbreviations used are: ER, estrogen receptor; FBS, fetal bovine serum; ERE, estrogen response element; DES, diethylstilbestrol; DCC, dextran-coated charcoal; IA, indenestrol A; THC, tetrahydrocrysene; E2, 17beta -estradiol; m, murine; h, human; MEM, minimal essential medium; NR, nuclear receptor-interacting regions; RPA, RNase protection assay; LBD, ligand-binding domain; DBD, DNA-binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hall, J. M., Couse, J. F., and Korach, K. S. (2001) J. Biol. Chem. 276, 36869-36872[Free Full Text]
2. Couse, J. F., and Korach, K. S. (1999) Endocrinol. Rev. 20, 358-417[Abstract/Free Full Text]
3. Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B., and Gustafsson, J. A. (1998) Endocrinology 139, 4252-4263[Abstract/Free Full Text]
4. Kuiper, G. G. J. M., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J. (1997) Endocrinology 138, 863-870[Abstract/Free Full Text]
5. Kohno, H., Bocchinfuso, W. P., Gandini, O., Curtis, S. W., and Korach, K. S. (1996) J. Mol. Endocrinol. 16, 277-285[Abstract]
6. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J., and Nilsson, S. (1998) Mol. Pharmacol. 54, 105-112[Abstract/Free Full Text]
7. Couse, J. F., Lindzey, J. K., Grandien, K., Gustafsson, J.-A., and Korach, K. S. (1997) Endocrinology 138, 4613-4621[Abstract/Free Full Text]
8. Mueller, S. O., and Korach, K. S. (2001) J. Androl. 22, 652-664[Abstract/Free Full Text]
9. Chaidarun, S. S., and Alexander, J. M. (1998) Mol. Endocrinol. 12, 1355-1366[Abstract/Free Full Text]
10. Schreiber, E., Matthias, P., Mueller, M. H., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve]
11. Norris, J. D., Fan, D., Kerner, S. A., and McDonnell, D. P. (1997) Mol. Endocrinol. 11, 747-754[Abstract/Free Full Text]
12. Hall, J. M., and McDonnell, D. P. (1999) Endocrinology 140, 5566-5578[Abstract/Free Full Text]
13. Chang, W. Y., and Prins, G. S. (1999) Prostate 40, 115-124[CrossRef][Medline] [Order article via Infotrieve]
14. Ignar-Trowbridge, D. M., Teng, C. T., Ross, K. A., Parker, M. G., Korach, K. S., and McLachlan, J. A. (1993) Mol. Endocrinol. 7, 992-998[Abstract]
15. Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson Jr, W. D., Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F., and Brooks, S. C. (1990) Cancer Res. 50, 6075-6086[Abstract]
16. Cailleau, R., Young, R., Olive, M., and Reeves, W. J., Jr. (1974) J. Natl. Cancer Inst. 53, 661-674[Medline] [Order article via Infotrieve]
17. Norris, J. D., Fan, D. J., Wagner, B. L., and McDonnell, D. P. (1996) Mol. Endocrinol. 10, 1605-1616[Abstract]
18. Pike, A. C. W., Brzozowski, A. M., Hubbard, R. E., Bonn, T., Thorsell, A. G., Engstrom, O., Ljunggren, J., Gustafsson, J. K., and Carlquist, M. (1999) EMBO J. 18, 4608-4618[Abstract/Free Full Text]
19. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve]
20. Moore, J. T., McKee, D. D., Slentz Kesler, K., Moore, L. B., Jones, S. A., Horne, E. L., Su, J. L., Kliewer, S. A., Lehmann, J. M., and Willson, T. M. (1998) Biochem. Biophys. Res. Commun. 247, 75-78[CrossRef][Medline] [Order article via Infotrieve]
21. Enmark, E., PeltoHuikko, M., Grandien, K., Lagercrantz, S., Lagercrantz, J., Fried, G., Nordenskjold, M., and Gustafsson, J. A. (1997) J. Clin. Endocrinol. Metab. 82, 4258-4265[Abstract/Free Full Text]
22. Bocchinfuso, W. P., and Korach, K. S. (1997) Mol. Endocrinol. 11, 587-594[Abstract/Free Full Text]
23. Valentine, J. E., Kalkhoven, E., White, R., Hoare, S., and Parker, M. G. (2000) J. Biol. Chem. 275, 25322-25329[Abstract/Free Full Text]
24. Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000) Mol. Cell 5, 939-948[Medline] [Order article via Infotrieve]
25. Mak, H. Y., Hoare, S., Henttu, P. M., and Parker, M. G. (1999) Mol. Cell. Biol. 19, 3895-3903[Abstract/Free Full Text]
26. Sun, J., Meyers, M. J., Fink, B. E., Rajendran, R., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1999) Endocrinology 140, 800-804[Abstract/Free Full Text]
27. McDonnell, D. P. (1999) Trends Endocrinol. Metab. 10, 301-311[CrossRef][Medline] [Order article via Infotrieve]
28. Paige, L. A., Christensen, D. J., Gron, H., Norris, J. D., Gottlin, E. B., Padilla, K. M., Chang, C. Y., Ballas, L. M., Hamilton, P. T., McDonnell, D. P., and Fowlkes, D. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3999-4004[Abstract/Free Full Text]
29. Kraichely, D. M., Sun, J., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (2000) Endocrinology 141, 3534-3545[Abstract/Free Full Text]
30. Tyulmenkov, V. V., and Klinge, C. M. (2000) Arch. Biochem. Biophys. 381, 135-142[CrossRef][Medline] [Order article via Infotrieve]
31. Katzenellenbogen, J. A., O'Malley, B. W., and Katzenellenbogen, B. S. (1996) Mol. Endocrinol. 10, 119-131[Medline] [Order article via Infotrieve]
32. Mahfoudi, A., Roulet, E., Dauvois, S., Parker, M., and Wahli, W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4206-4210[Abstract]
33. Feng, W., Ribeiro, R. C. n. J., Wagner, R. L., Nguyen, H., Apriletti, J. W., Fletterick, R. J., Baxter, J. D., Kushner, P. J., and West, B. L. (1998) Science 280, 1747-1749[Abstract/Free Full Text]
34. Korach, K. S., Metzler, M., and McLachlan, J. A. (1978) Proc. Natl. Acad. Sci. U. S. A.  75, 468-471[Abstract]
35. Korach, K. S., Metzler, M., and McLachlan, J. A. (1979) J. Biol. Chem. 254, 8963-8968[Medline] [Order article via Infotrieve]
36. Korach, K. S., Chae, K., Levy, L. A., Duax, W. L., and Sarver, P. J. (1989) J. Biol. Chem. 264, 5642-5647[Abstract/Free Full Text]


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