Identification of a functional variant of estrogen receptor beta in an African population
Chunyan Zhao1,
Li Xu1,
Michio Otsuki1,
Gudrun Toresson1,
Konrad Koehler2,
Qiang Pan-Hammarström1,
Lennart Hammarström1,
Stefan Nilsson2,
Jan-Åke Gustafsson1 and
Karin Dahlman-Wright1,3
1 Karolinska Institute, Department of Biosciences at Novum, S-141 57 Huddinge, Sweden and 2 KaroBio AB, Novum, S-141 57 Huddinge, Sweden
3 To whom correspondence should be addressed Email: kada{at}cbt.ki.se
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Abstract
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In this study, we identified five novel polymorphisms in the estrogen receptor beta (ERß) gene in an African population. Interestingly, two of these variants are expected to change the amino acid sequence of the ERß protein. These changes correspond to an isoleucine to valine substitution at amino acid position 3 (I3V) and a valine to glycine substitution at position 320 (V320G), respectively. The functional consequences of these amino acid substitutions were determined in different in vitro assays. The I3V mutation displayed no differences with regard to transcriptional activity in a reporter assay, as compared with the wild-type receptor. The V320G mutation, however, showed significantly decreased maximal transcriptional activity in a reporter assay, although its binding affinity for 17ß-estradiol was not affected. A pull-down assay indicated that the interaction of full-length TIF2 with hERßV320G was weaker than with hERßwt. Moreover, surface plasmon resonance analysis revealed reduced interaction of the V320G ERß variant with the NR box I and II modules of TIF2. To our knowledge, this represents the first identification of a functional polymorphism in the ERß gene. This novel polymorphism provides a tool for human genetic studies of diseases in the African population.
Abbreviations: ER, estrogen receptor; ERE, estrogen response element; LBD, ligand-binding domain; SPR, surface plasmon resonance
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Introduction
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Estrogen receptors (ERs) belong to the steroid/retinoid receptor gene superfamily, which contains the receptors for glucocorticoids, mineralocorticoids, progesterone, androgen, thyroid hormone, vitamin D and retinoic acid. As a family, its members share some structural and functional similarities including four functional domains. From the N-terminus to the C-terminus of the receptor molecule, these are: the A/B region that contributes to the transcriptional activation function; the C-region, or the DNA-binding domain, that harbors the DNA-binding function mediating specific DNA binding; the hinge region followed by the ligand-binding domain (the LBD or the E/F domain). The LBD harbors the ligand-binding pocket as well as sites for co-factor binding, transactivation, nuclear localization and interactions with heat shock proteins (1). Upon ligand-dependent or -independent activation, these receptors form dimers and modulate transcription by binding to their corresponding hormone response elements (for example ERE, estrogen response element) in the promoter region of target genes (2). There are two estrogen receptors, ER
and ERß. These two receptors show high homology, particularly in the DNA-binding domain. The receptors are expressed in a distinct but sometimes over-lapping mode and display functional similarities as well as differences, sometimes even opposite actions (3).
Polymorphisms in ER genes, the major mediators of estrogen signaling, are associated with some endocrine related disorders. Polymorphisms in ER
are associated with breast cancer (46), endometrial cancer (7), lupus nephritis (8), menstrual disorder (9), Alzheimer's disease (10), osteoporosis (11) and coronary artery disease (12). Polymorphisms in the ERß gene have been correlated to other pathological states as compared with ER
polymorphisms, such as ovulatory dysfunctions (9), hypertension (13), bone mineral density (14) and androgen levels (15). No data are available regarding polymorphisms in ER genes in African populations.
Several genetic differences have been described between African Americans and white Americans, which may account for the higher incidence of certain diseases in the former population. For example, estrogen metabolism appears to vary according to race, with a higher ratio of inactive:active metabolites in whites compared with blacks (16). Polymorphisms in some steroid hormone nuclear receptors have been shown to correlate with race related endocrine diseases. For example, short CAG repeat lengths in the androgen receptor gene were found in African Americans and possibly associated with a higher stage of prostate cancer (17). Polymorphisms in the vitamin D receptor gene are associated with bone mass differences between African Americans and white Americans (18).
In this investigation we screened the ERß gene in an African population for polymorphisms. Any identified polymorphism, particularly a functional polymorphism, would constitute important tools for further association with diseases.
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Materials and methods
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Samples
Nigerian healthy blood donors (n = 96 from Banjul, Gambian) were included in the study. Genomic DNA was isolated using standard phenolchloroform extraction followed by ethanol precipitation. The studies were approved by the ethical committee of the Karolinska Institute.
PCR
PCR amplifications were performed in a total volume of 25 µl containing 200 µM dNTPs, 1050 ng of template DNA, 0.4 µM each of primers, 1.25 U Taq DNA polymerase (Roche, Mannheim, Germany), in 1x reaction buffer (10 mM TrisHCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2). Primer sequences to amplify the ERß gene were designed based on the published GenBank ERß gene sequence NT_025892 (Table I). PCR amplification was carried out at 94°C for 10 min followed by 35 cycles (94°C for 30 s, 5760°C for 30 s and 72°C for 45 s), and finally 10 min at 72°C.
Denaturing high-performance liquid chromatography (DHPLC)
PCR products from amplification of genomic DNA from all individuals were analysed using DHPLC on a WAVE DNA Fragment Analysis System (Transgenomic, Cheshire, UK) and DNASep Column as described (19) using the temperature suggested by the WAVEMAKERTM Software package (Transgenomic, Crewe, UK). Prior to DHPLC analysis, PCR products were denatured at 95°C for 5 min and then cooled to 25°C over 45 min to enable the formation of heteroduplexes. Aliquots of 5 µl were automatically loaded on the DNAsep column for heteroduplex analysis.
Samples with aberrant HPLC profiles were subjected to DNA sequencing using ABI Prism® BigDyeTM Terminator Cycle Sequence Ready Reaction Kit (Applied Biosystems) and compared with the published genomic sequence of the ERß gene. In certain cases, Restriction Fragment Length Polymorphism analysis was used to confirm polymorphisms. FokI was used to score 105A
G, RsaI was used to score 1082G
A, AluI was used to score1730G
A and Van91I was used to score 1057T
G.
Generation of human ERß plasmids containing the 105A
G or 1057T
G mutations
A wild-type pSG5-hERß plasmid was a gift from Dr Michel Tujague at the Department of Biosciences, Karolinska Institute. hERß plasmids that incorporate the identified amino acid changes of the ERß gene were created from the wild-type hERß530 plasmid using the QuickChangeTM XL Site-Directed mutagenesis kit (Stratagene, La Jolla, CA) according to the instruction manual. DNA sequencing confirmed the sequences of the mutant clones. The resulting plasmids are named hERß105A
G and hERß1057T
G, respectively.
Transient transfection assays and western blot analysis
HEK293 cells were cultured in a 1:1 mixture of Ham's Nutrient mixture F12 (Invitrogen) and DMEM (Invitrogen) supplemented with 5% FBS and 100 U penicillin/ml and 100 mg streptomycin/ml. For transfection, the cells were seeded at a density of 1 x 104 cells/well in 96-well plates and co-transfected with 2x ERE TK luciferase reporter plasmid (0.4 µg) (20) and the respective ER expression vectors (0.016 µg). A pRL-TK control plasmid, which contains a Renilla luciferase gene, was included to control for differences in transfection efficiencies. The medium was replaced with a phenol red-free mixture of F12 and DMEM containing 5% dextran-coated charcoal-treated FBS and 100 U penicillin/ml and 100 µg streptomycin/ml upon transfection. 17ß-Estradiol (0.1, 1, 10, 100 nM) or vehicle (in 0.1% ethanol) was added just after transfection. The cells were harvested 24 h after transfection and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
Western blotting was done according to the protocol as described (21). ERß was detected with an ERß LBD rabbit polyclonal antibody produced by us as described previously (22). As a positive control, the recombinant human ERß530 protein purchased from Panvera (Madison, WI) was used.
Cloning and expression of hERßwt and hERß1057T
G LBDs
The LBDs (R254 to Q530) were obtained by PCR using full-length cDNAs as templates and primers that contained appropriate restriction sites. hERßwt and hERß1057T
G were cloned into pET15b (Novagen, Madison, WI) to generate proteins with N-terminal His-tags. The sequences of all constructs were verified by DNA sequencing.
Cultures (500 ml) of the Escherichia coli strain BL21, transformed with the appropriate expression plasmids, were cultivated overnight in LB supplemented with 100 µg/ml of ampicillin at 37°C. When the OD600 reached 1.0, IPTG was added to a final concentration of 1 mM and incubation continued for 3 h at 25°C. The cells were pelleted and the supernatant was discarded. The pellet was suspended in 5 ml (one-tenth of the culture volume) of extraction buffer [complete EDTA-free, Roche Diagnostics, Germany, 0.3 M NaCl, 20 mM Tris (pH 8.0), 0.01 mg/ml DNase, 0.01 mg/ml RNase, 10 mM MgCl2, 0.25 mg/ml lysozyme, 1 mM ß-ME and protease inhibitor cocktail tablet]. The samples were sonicated for 4 min at 50% duty (total sonication time 2 min). The homogenate was centrifuged at 13 000 g for 20 min at 4°C. The supernatant was applied to a TALON metal affinity column (Clontech Laboratories, Palo Alto, CA). Fractions containing the purified protein were dialyzed against 20 mM Tris (pH 8), 150 mM NaCl, 1 mM DTT and frozen at 80°C. The purified protein was >95% pure as determined from Coomassie stained SDSPAGE gels. The protein concentrations were measured using the Coomassie Protein Assay Kit (Pierce, IL) according to the manufacturer's instructions.
Scintillation proximity assay
The assay was performed in 96-well microplates (PerkinElmer Life Sciences, MA). Polyvinyltoluene copper-loaded his-tag beads were purchased from Amersham. The reaction mixture (60 µl/well) containing assay buffer (1 mM EDTA, 0.9 M KH2PO4, 0.1 M K2HPO4, 20 mM Na2MoO4 and 0.05% monothioglycerol), beads (30 µg/well) and purified ERß LBD (final concentration of 20 nM) was incubated at 4°C for at least 1 h. For saturation ligand-binding analysis, a sample of various concentrations of [3H]17ß-estradiol (S.A. = 95 Ci/mmol) in the presence or absence of a 300-fold excess of unlabeled 17ß-estradiol was then added. The assay plates were sealed, allowed to settle overnight and subsequently counted on a Wallac 1450 micro-ß counter. The dissociation constant (Kd) was calculated as the free concentration of radioligand at half-maximal specific binding by fitting data to the Hill equation and by linear Scatchard transformation (23). Curve fitting was done in Prism (GraphPad Software).
For ligand competition studies, purified ERß LBDs (20 nM) were incubated overnight at 4°C with a range of test compound concentrations. A final concentration of 1.5 nM [3H]17ß-estradiol (30 µl/well) was used. The ligands were tested three times with similar results. Curve fitting was performed using Prism (GraphPad Software) and the IC50s determined. IC50 values were converted to Ki using the Cheng-Prusoff equation, Ki = IC50/(1 + D/Kd), where D is the concentration of the radioligand (24).
Pull-down assay
For pull-down assays, purified His-tagged ERß LBDs (100 µg) were bound to 60 µl of Talon resin and then equilibrated in 50 mM TrisHCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 10% glycerol and 0.5% NP-40 (equilibration buffer). The gel slurry was then divided into two equal aliquots and to each tube, 2.5 ml of in vitro translated, 35S-labeled (TNT coupled reticulocyte lysate system, Promega), full-length TIF2 was added in a total volume of 150 µl equilibration buffer containing 1.5% BSA. Estradiol or vehicle (ethanol) was added as indicated. As control, TIF2 was mixed with Talon gel without bound ERß. All samples were incubated for 2 h with gentle shaking at 4°C. After washing three times with equilibration buffer, bound proteins were eluted with SDSPAGE sample buffer and separated on a 12% polyacrylamide gel. The gel was stained with Coomassie Blue, followed by determination of 35S using a PhosphoImager instrument.
Surface plasmon resonance (SPR) analysis
All SPR measurements were performed on a BIAcore 2000 instrument (BIAcore AB, Uppsala, Sweden). All experiments were performed at 25°C, and at a flow rate of 5 µl/min. Research grade streptavidin sensor chips were obtained from BIAcore AB. The streptavidin chips were first treated with three 1-min pulses of 50 mM NaOH and 1 M NaCl at a flow rate of 5 µl/min. N-terminally biotinylated peptides (>95% purity) were purchased from Interactiva (Germany). The human TIF2 LXXLL peptide sequences were as follows: Box 1 (residues 636649), KGQTKLLQLLTTKS and Box 2 (residues 685698), EKHKILHRLLQDSS. Peptides were immobilized on individual surfaces to 200 RU responses. Samples of purified hERßwt-LBD or hERßV320G-LBD in the presence or absence of estradiol were then injected over each surface. After injection stop, the surfaces were washed with buffer to monitor the dissociation phase. The buffer used was 50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20. For the kinetic measurements, various concentrations of hERßwt-LBD and hERßV320G-LBD (from 0.25 to 2 µM) were injected over the chip surfaces. The BIAevaluation software version 3.1 was used for evaluation. Different binding models (different rate equations) were tested in the global curve fitting procedure, and the model best describing the experimental data was a 1:1 binding with drifting baseline model. The apparent Kd values are calculated as described (25).
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Results
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ERß polymorphism screening in an African population
We screened genomic DNA from 96 Nigerians to identify polymorphisms in the ERß gene in an African population. Analysis of the coding exons and flanking intron sequences revealed several known but also novel variants. The results are summarized in Table II. Three variants (1082G
A, 1505-4A
G and 1730A
G) that have been reported previously in Caucasian populations (26,27) were also found in the African population but with different frequencies. None of these polymorphisms change the amino acid sequence of the ERß protein. Five novel polymorphisms in the coding region of the ERß gene were found in the African population. Interestingly, as shown in Table II, two of these novel polymorphisms change the amino acid sequence of the ERß protein. The novel amino acid changes are 105A
G [changing amino acid 3, isoleucine (I)
valine (V)] in exon 1 and 1057T
G [changing amino acid 320, valine (V)
glycine (G)] in exon 5. These two ERß variants are, in the following, referred to as hERßI3V and hERßV320G, respectively.
hERßV320G shows reduced transcriptional activation in a transactivation assay
To test if the identified receptor variants displayed any differences compared with the wild-type receptor with regard to transcriptional activation, a reporter assay was used. We generated plasmids expressing either hERßI3V or hERßV320G under control of the SV40 promoter. The transcriptional activities of the variants were compared with that of the wild-type ERß using an ERE-luciferase reporter system. No differences with regard to transcriptional activation were observed for the hERßI3V variant (Figure 1A). However, hERßV320G showed significantly decreased maximal transcriptional activity compared with wild-type ERß (Figure 1A). To confirm equivalent ERß expression, extracts from HEK293 cells transfected with equal amounts of the expression vectors for hERßwt, hERßV320G, or hERßI3V were separated by SDSPAGE and analysed for ERß expression by western blot (Figure 1B). This analysis shows that the different ERß derivatives are expressed at similar levels. The observed reduction in maximal transcriptional activity could imply that hERßV320G is defective for interactions with co-factors. Interestingly, as shown in Figure 2, the amino acid changed in hERßV320G is located on the surface of the receptor protein, quite distant from the ligand-binding pocket.

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Fig. 2. Modeled structure of human ER-ß LBD complexed with 17ß-estradiol and the TIF2 co-activator peptide [PBD accession number 1QKM (29) and human ER- complexed with 17ß-estradiol and the TIF2 co-activator peptide (1GWR) (50)]. The protein is depicted by a green ribbon/tube while the ligand estradiol is represented by white and red spheres (carbon and oxygen atoms, respectively). Helix-12 and the TIF2 co-activator peptide are depicted in cyan and red, respectively. The positions of the Lys-314 and Val-320 sidechains are shown as magenta tubes. This figure was produced using the PyMOL program (http://www.pymol.org).
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hERßwt and hERßV320G bind ligands with similar affinity
To facilitate a biochemical characterization of hERßV320G, and particularly to compare it to hERßwt, the LBDs of these two proteins were expressed in E.coli and the recombinant proteins were purified. In Figure 3A and B, the results of saturation ligand-binding assays with tritiated estradiol are shown. The measured Kd values were 1.50 nM for hERßwt and 1.79 nM for hERßV320G (Figure 3). Affinities for estradiol, tamoxifen and raloxifen, as determined in a competition assay, were also similar for hERßwt and hERßV320G. Thus, the Kis of estradiol, tamoxifen and raloxifen for hERßwt were 0.25, 5.45 and 9.45 nM and for hERßV320G they were 0.33, 5.33 and 10.11 nM, respectively. This is consistent with the position of the variant amino acid distant from the ligand-binding pocket (Figure 2).

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Fig. 3. Saturation and Scatchard analyses for [3H]17ß-estradiol binding to hERßwt and hERßV320G. hERßwt and hERßV320G LBDs (20 nM) were incubated with increasing concentrations (020 nM) of [3H]17ß-estradiol at 4°C overnight. The inset shows linear transformation of the data by Scatchard analysis.
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hERßV320G shows reduced co-factor interaction
Having demonstrated that hERßV320G binds ligands with similar affinity as hERßwt, yet shows significantly reduced maximal transactivation in a reporter assay, we hypothesized that hERßV320G is defective for co-factor interactions. This is consistent with the position of the V320G amino acid change, on the surface of the receptor protein in a region postulated to be involved in co-factor interactions (Figure 2). To test this hypothesis, we performed pull-down assays using purified His-tagged ERß LBDs and 35S-labeled full-length TIF2 in the presence or absence of estradiol. As shown in Figure 4, TIF2 bound to hERßwt and hERßV320G in a ligand-dependent manner. Furthermore, the interaction of TIF2 with hERßV320G was weaker than with hERßwt.

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Fig. 4. Analysis of TIF2 intercation with hERßwt and hERßV320G by pull-down assay. In vitro translated TIF2 was incubated with hERßwt (lanes 4 and 5) and hERßV320G (lanes 6 and 7), with or without estradiol (E2) as indicated. Non-specific binding of TIF2 to Talon beads is shown in lanes 2 and 3. Input corresponds to 0.5 µl of the in vitro translation mixture. The experiment was repeated three times with similar results.
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To more quantitatively examine TIF2 interaction to hERßV320G and hERßwt, we used the SPR analysis, where biotinylated 14mer peptides containing NR box motifs (LXXLL) from TIF2 were captured via streptavidin to the chip surface. Figure 5 illustrates the binding of hERßV320G and hERßwt in the presence or absence of estradiol to the NR box of TIF2. The result showed that binding of estradiol to ERß enhances the affinity of ERß-peptide interaction. Binding studies were made for the different TIF2 NR box peptides using five to six different concentrations of hERßwt-LBD or hERßV320G-LBD unliganded or liganded with estradiol ranging from 0.25 to 2 µM. Affinity determination analysis was then performed using BIAevaluation software. The curve fitting analysis showed that the model best describing the experimental data was a 1:1 binding with drifting baseline model. The Kd for binding to TIF2 box1 was 0.23 ± 0.04 µM (mean ± SD) and 0.52 ± 0.05 µM and Kd for binding to TIF2 box2 was 0.39 ± 0.1 µM and 0.79 ± 0.1 µM for hERßwt-LBD and hERßV320G-LBD liganded with estradiol, respectively. The determined affinities show that hERßV320G displays reduced affinity for TIF2 as compared with that of wild-type ERß. Reduced affinity for TIF2 was also seen with unliganded hERßV320G-LBD as compared with unliganded hERßwt-LBD (data not shown).

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Fig. 5. SPR analyses of hERßwt and hERßV320G binding to TIF2. The real-time binding sensogram shows injection of 1 µM hERßwt and 1 µM hERßV320G in the presence or absence of estradiol over a surface immobilized with 200 RU of TIF2 NR box1.
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Discussion
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In this paper we identify five novel polymorphisms in the hERß gene in an African population. Interestingly, two of these polymorphisms are expected to change the corresponding amino acid of the encoded ERß protein. These polymorphisms have not been identified in any of the published reports that describe the characterization of polymorphisms in the ERß gene in both Caucasians (n = 502) (27) and Asians (n = 184) (28). In Africans, the variant protein hERßV320G, encoded by one of these polymorphic ERß genes, displays lower maximal transcriptional activity compared with wild-type ERß. To our knowledge, this represents the first example of a functional polymorphism in the ERß gene.
The hERßV320G change results in the amino acid substitution of valine for glycine in helix 4 of the LBD. Helix 4 does not participate directly in binding of the ligand (29). However, it forms part of the surface that interacts with co-activators (30). The hERßV320G side chain is in close proximity to K314, which forms half of the charge-clamp of the co-activator-binding pocket. Therefore, the hERßV320G may perturb the conformation of K314, which in turn perturbs cofactor interactions. Consistent with this, we did not find any changes in ligand-binding affinity of hERßV320G. However, this variant displays decreased interaction with the co-activator TIF2. We propose that reduced co-factor interaction is the cause for the observed reduced maximal transactivation.
Several endocrine-related diseases show population-based differences in terms of disease incidence and/or disease progression but the reason for this racial difference is unclear. When compared with white American women, African American women have a higher incidence of breast cancer before the age of 40 years, and the prognosis after a diagnosed breast cancer is reported to be poorer at all ages (31). Studies have suggested that higher plasma levels of insulin-like growth factor-1 (IGF-1) may account for African American women having higher risk of premenopausal breast cancer than white women and may also be an important determinant of breast cancer risk in postmenopausal women (3234). A prolonged exposure to estrogens has been repeatedly found to be associated with an increased risk for breast cancer. Racial disparities in conditions such as postmenopausal obesity, early menarche and late menopause that all increase endogenous levels of estrogens may put an African American woman at a greater risk for breast cancer (35). African American men present a higher stage of prostate cancer and a worse outcome of this disease than non-African American men (36). The increased frequency of prostate cancer among African American men has been attributed to either greater plasma levels of testosterone (37) or an increased dihydrotestosterone/testosterone ratio (38) in this population. Previous studies also revealed that the plasma level of IGF-1 might be a predictor of prostate cancer risk (39,40). Some of these racial differences could be attributed to population differences in genetic polymorphisms. Polymorphisms involving genes coding for the androgen receptor, phase I/II enzymes and vitamin D receptor have been associated with an increased risk of prostate cancer (41). Polymorphisms that are associated with breast cancer risk have been identified in the genes involved in a wide variety of functions including steroid hormone metabolism, detoxification of environmental carcinogens and tumor suppressor genes (42).
In this study, we have identified one novel polymorphism in the ERß gene in an African population that showed decreased transcriptional activity. Dysregulation of ERß expression has been reported to be associated with progressions of breast and prostate cancer (4346), indicating that ERß functions as a tumor suppressor gene. The hyperplastic prostate (47) and dedifferentiated mammary gland (48) phenotypes displayed by ERß knockout mice support a role for ERß in control of mammary and prostate growth. Further association studies are required to determine whether this polymorphism is involved in the increased incidence of prostate and breast cancers in Africans.
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Acknowledgments
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We greatly appreciate the contributions of Xiaolei Zhou at the Centre for Molecular Medicine, Karolinska Institute for technical support and advices on DHPLC. We are grateful to Dr Damian Labuda, Pediatrics Department, Montreal University for providing valuable samples for this study. This study was supported by grants from the Swedish Cancer Fund and from KaroBio AB.
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References
|
---|
- Nilsson,S., Makela,S., Treuter,E., Tujague,M., Thomsen,J., Andersson,G., Enmark,E., Pettersson,K., Warner,M. and Gustafsson,J.-Å. (2001) Mechanisms of estrogen action. Physiol. Rev., 81, 15351565.[Abstract/Free Full Text]
- Mangelsdorf,D.J., Thummel,C., Beato,M. et al. (1995) The nuclear receptor superfamily: the second decade. Cell, 83, 835839.[ISI][Medline]
- Nilsson,S. and Gustafsson,J.-Å. (2000) Estrogen receptor transcription and transactivation: Basic aspects of estrogen action. Breast Cancer Res., 2, 360366.[CrossRef][ISI][Medline]
- Andersen,T.I., Heimdal,K.R., Skrede,M., Tveit,K., Berg,K. and Borresen,A.L. (1994) Oestrogen receptor (ESR) polymorphisms and breast cancer susceptibility. Hum. Genet., 94, 665670.[ISI][Medline]
- Kang,H.J., Kim,S.W., Kim,H.J., Ahn,S.J., Bae,J.Y., Park,S.K., Kang,D., Hirvonen,A., Choe,K.J. and Noh,D.Y. (2002) Polymorphisms in the estrogen receptor-alpha gene and breast cancer risk. Cancer Lett., 178, 175180.[CrossRef][ISI][Medline]
- Schubert,E.L., Lee,M.K., Newman,B. and King,M.C. (1999) Single nucleotide polymorphisms (SNPs) in the estrogen receptor gene and breast cancer susceptibility. J. Steroid Biochem. Mol. Biol., 71, 2127.[CrossRef][ISI][Medline]
- Weiderpass,E., Persson,I., Melhus,H., Wedren,S., Kindmark,A. and Baron,J.A. (2000) Estrogen receptor alpha gene polymorphisms and endometrial cancer risk. Carcinogenesis, 21, 623627.[Abstract/Free Full Text]
- Liu,Z.H., Cheng,Z.H., Gong,R.J., Liu,H., Liu,D. and Li,L.S. (2002) Sex differences in estrogen receptor gene polymorphism and its association with lupus nephritis in Chinese. Nephron, 90, 174180.[CrossRef][ISI][Medline]
- Sundarrajan,C., Liao,W.X., Roy,A.C. and Ng,S.C. (2001) Association between estrogen receptor-beta gene polymorphisms and ovulatory dysfunctions in patients with menstrual disorders. J. Clin. Endocrinol. Metab., 86, 135139.[Abstract/Free Full Text]
- Brandi,M.L., Becherini,L., Gennari,L., Racchi,M., Bianchetti,A., Nacmias,B., Sorbi,S., Mecocci,P., Senin,U. and Govoni,S. (1999) Association of the estrogen receptor alpha gene polymorphisms with sporadic Alzheimer's disease. Biochem. Biophys. Res. Commun., 265, 335338.[CrossRef][ISI][Medline]
- Ongphiphadhanakul,B., Chanprasertyothin,S., Payattikul,P., Saetung,S., Piaseu,N., Chailurkit,L. and Rajatanavin,R. (2001) Association of a G2014A transition in exon 8 of the estrogen receptor-alpha gene with postmenopausal osteoporosis. Osteoporos. Int., 12, 10151019.[CrossRef][ISI][Medline]
- Lu,H., Higashikata,T., Inazu,A., Nohara,A., Yu,W., Shimizu,M. and Mabuchi,H. (2002) Association of estrogen receptor-alpha gene polymorphisms with coronary artery disease in patients with familial hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol., 22, 817823.[Abstract/Free Full Text]
- Ogawa,S., Emi,M., Shiraki,M., Hosoi,T., Ouchi,Y. and Inoue,S. (2000) Association of estrogen receptor beta (ESR2) gene polymorphism with blood pressure. J. Hum. Genet., 45, 327330.[CrossRef][ISI][Medline]
- Ogawa,S., Hosoi,T., Shiraki,M., Orimo,H., Emi,M., Muramatsu,M., Ouchi,Y. and Inoue,S. (2000) Association of estrogen receptor beta gene polymorphism with bone mineral density. Biochem. Biophys. Res. Commun., 269, 537541.[CrossRef][ISI][Medline]
- Westberg,L., Baghaei,F., Rosmond,R., Hellstrand,M., Landen,M., Jansson,M., Holm,G., Bjorntorp,P. and Eriksson,E. (2001) Polymorphisms of the androgen receptor gene and the estrogen receptor beta gene are associated with androgen levels in women. J. Clin. Endocrinol. Metab., 86, 25622568.[Abstract/Free Full Text]
- Taioli,E., Garte,S.J., Trachman,J., Garbers,S., Sepkovic,D.W., Osborne,M.P., Mehl,S. and Bradlow,H.L. (1996) Ethnic differences in estrogen metabolism in healthy women. J. Natl Cancer Inst., 88, 617.[Free Full Text]
- Bennett,C.L., Price,D.K., Kim,S. et al. (2002) Racial variation in CAG repeat lengths within the androgen receptor gene among prostate cancer patients of lower socioeconomic status. J. Clin. Oncol., 20, 35993604.[Abstract/Free Full Text]
- Nelson,D.A., Vande Vord,P.J. and Wooley,P.H. (2000) Polymorphism in the vitamin D receptor gene and bone mass in African-American and white mothers and children: a preliminary report. Ann. Rheum. Dis., 59, 626630.[Abstract/Free Full Text]
- Kuklin,A., Davis,A.P., Hecker,K.H., Gjerde,D.T. and Taylor,P.D. (1999) A novel technique for rapid automated genotyping of DNA polymorphisms in the mouse. Mol. Cell Probes, 13, 239242.[CrossRef][ISI][Medline]
- Pettersson,K., Grandien,K., Kuiper,G.G. and Gustafsson,J.-Å. (1997) Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha. Mol. Endocrinol., 11, 14861496.[Abstract/Free Full Text]
- Saji,S., Sakaguchi,H., Andersson,S., Warner,M. and Gustafsson,J. (2001) Quantitative analysis of estrogen receptor proteins in rat mammary gland. Endocrinology, 142, 31773186.[Abstract/Free Full Text]
- Saji,S., Jensen,E.V., Nilsson,S., Rylander,T., Warner,M. and Gustafsson,J.-Å. (2000) Estrogen receptors alpha and beta in the rodent mammary gland. Proc. Natl Acad. Sci. USA, 97, 337342.[Abstract/Free Full Text]
- 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.-Å. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology, 139, 42524263.[Abstract/Free Full Text]
- Cheng,Y. and Prusoff,W.H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol., 22, 30993108.[CrossRef][ISI][Medline]
- Warnmark,A., Almlof,T., Leers,J., Gustafsson,J.-Å. and Treuter,E. (2001) Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha and ERbeta. J. Biol. Chem., 276, 2339723404.[Abstract/Free Full Text]
- Forsti,A., Zhao,C., Israelsson,E., Dahlman-Wright,K., Gustafsson,J.-Å. and Hemminki,K. (2003) Polymorphisms in the estrogen receptor beta gene and risk of breast cancer: no association. Breast Cancer Res. Treat., 79, 409413.[CrossRef][ISI][Medline]
- Rosenkranz,K., Hinney,A., Ziegler,A., Hermann,H., Fichter,M., Mayer,H., Siegfried,W., Young,J.K., Remschmidt,H. and Hebebrand,J. (1998) Systematic mutation screening of the estrogen receptor beta gene in probands of different weight extremes: identification of several genetic variants. J. Clin. Endocrinol. Metab., 83, 45244527.[Abstract/Free Full Text]
- Hasegawa,S., Miyoshi,Y., Ikeda,N., Egawa,C., Tamaki,Y., Monden,M. and Noguchi,S. (2003) Mutational analysis of estrogen receptor-beta gene in human breast cancers. Breast Cancer Res. Treat., 78, 133134.[CrossRef][ISI][Medline]
- Pike,A.C., Brzozowski,A.M., Hubbard,R.E., Bonn,T., Thorsell,A.G., Engstrom,O., Ljunggren,J., Gustafsson,J.-Å. and Carlquist,M. (1999) Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J., 18, 46084618.[Abstract/Free Full Text]
- Kong,E.H., Pike,A.C. and Hubbard,R.E. (2003) Structure and mechanism of the oestrogen receptor. Biochem. Soc. Trans., 31, 5659.[ISI][Medline]
- Elmore,J.G., Moceri,V.M., Carter,D. and Larson,E.B. (1998) Breast carcinoma tumor characteristics in black and white women. Cancer, 83, 25092515.[CrossRef][ISI][Medline]
- Agurs-Collins,T., Adams-Campbell,L.L., Kim,K.S. and Cullen,K.J. (2000) Insulin-like growth factor-1 and breast cancer risk in postmenopausal African-American women. Cancer Detect. Prev., 24, 199206.[ISI][Medline]
- DeLellis,K., Ingles,S., Kolonel,L., McKean-Cowdin,R., Henderson,B., Stanczyk,F. and Probst-Hensch,N.M. (2003) IGF1 genotype, mean plasma level and breast cancer risk in the Hawaii/Los Angeles multiethnic cohort. Br. J. Cancer, 88, 277282.[CrossRef][ISI][Medline]
- Jernstrom,H., Chu,W., Vesprini,D., Tao,Y., Majeed,N., Deal,C., Pollak,M. and Narod,S.A. (2001) Genetic factors related to racial variation in plasma levels of insulin-like growth factor-1: implications for premenopausal breast cancer risk. Mol. Genet. Metab., 72, 144154.[CrossRef][ISI][Medline]
- Ademuyiwa,F.O. and Olopade,O.I. (2003) Racial differences in genetic factors associated with breast cancer. Cancer Metastasis Rev., 22, 4753.[CrossRef][ISI][Medline]
- Burks,D.A. and Littleton,R.H. (1992) The epidemiology of prostate cancer in black men. Henry Ford Hosp. Med. J., 40, 8992.[Medline]
- Ross,R., Bernstein,L., Judd,H., Hanisch,R., Pike,M. and Henderson,B. (1986) Serum testosterone levels in healthy young black and white men. J. Natl Cancer Inst., 76, 4548.[ISI][Medline]
- Wu,A.H., Whittemore,A.S., Kolonel,L.N., John,E.M., Gallagher,R.P., West,D.W., Hankin,J., Teh,C.Z., Dreon,D.M. and Paffenbarger,R.S.,Jr (1995) Serum androgens and sex hormone-binding globulins in relation to lifestyle factors in older African-American, white and Asian men in the United States and Canada. Cancer Epidemiol. Biomarkers Prev., 4, 735741.[Abstract]
- Wolk,A., Mantzoros,C.S., Andersson,S.O., Bergstrom,R., Signorello,L.B., Lagiou,P., Adami,H.O. and Trichopoulos,D. (1998) Insulin-like growth factor 1 and prostate cancer risk: a population-based, case-control study. J. Natl Cancer Inst., 90, 911915.[Abstract/Free Full Text]
- Chan,J.M., Stampfer,M.J., Giovannucci,E., Gann,P.H., Ma,J., Wilkinson,P., Hennekens,C.H. and Pollak,M. (1998) Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science, 279, 563566.[Abstract/Free Full Text]
- Gonzalgo,M.L. and Isaacs,W.B. (2003) Molecular pathways to prostate cancer. J. Urol., 170, 24442452.[CrossRef][ISI][Medline]
- Miyoshi,Y. and Noguchi,S. (2003) Polymorphisms of estrogen synthesizing and metabolizing genes and breast cancer risk in Japanese women. Biomed. Pharmacother., 57, 471481.[CrossRef][ISI][Medline]
- Fixemer,T., Remberger,K. and Bonkhoff,H. (2003) Differential expression of the estrogen receptor beta (ERbeta) in human prostate tissue, premalignant changes and in primary, metastatic and recurrent prostatic adenocarcinoma. Prostate, 54, 7987.[CrossRef][ISI][Medline]
- Ito,T., Tachibana,M., Yamamoto,S., Nakashima,J. and Murai,M. (2001) Expression of estrogen receptor (ER-alpha and ER-beta) mRNA in human prostate cancer. Eur. Urol., 40, 557563.[CrossRef][ISI][Medline]
- Poola,I., Abraham,J. and Liu,A. (2002) Estrogen receptor beta splice variant mRNAs are differentially altered during breast carcinogenesis. J. Steroid Biochem. Mol. Biol., 82, 169179.[CrossRef][ISI][Medline]
- Roger,P., Sahla,M.E., Makela,S., Gustafsson,J.-Å., Baldet,P. and Rochefort,H. (2001) Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res., 61, 25372541.[Abstract/Free Full Text]
- Weihua,Z., Makela,S., Andersson,L.C., Salmi,S., Saji,S., Webster,J.I., Jensen,E.V., Nilsson,S., Warner,M. and Gustafsson,J.-Å. (2001) A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc. Natl Acad. Sci. USA, 98, 63306335.[Abstract/Free Full Text]
- Forster,C., Makela,S., Warri,A., Kietz,S., Becker,D., Hultenby,K., Warner,M. and Gustafsson,J.-Å. (2002) Involvement of estrogen receptor beta in terminal differentiation of mammary gland epithelium. Proc. Natl Acad. Sci. USA, 99, 1557815583.[Abstract/Free Full Text]
- Ogawa,S., Inoue,S., Watanabe,T., Hiroi,H., Orimo,A., Hosoi,T., Ouchi,Y. and Muramatsu,M. (1998) The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro. Biochem. Biophys. Res. Commun., 243, 122126.[CrossRef][ISI][Medline]
- Warnmark,A., Treuter,E., Gustafsson,J.-Å., Hubbard,R.E., Brzozowski,A.M. and Pike,A.C. (2002) Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha. J. Biol. Chem., 277, 2186221868.[Abstract/Free Full Text]
Received February 2, 2004;
revised May 30, 2004;
accepted June 10, 2004.