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* This work was supported by a National Institutes of Health postdoctoral training grant and a Bank of America Giannini postdoctoral fellowship (to C. T.-F.) and grants from the Paul Beeson Physician Faculty Scholars in Aging Research Program (funded by the Alliance for Aging Research, John A. Hartford Foundation, Commonwealth Fund and Starr Foundation), NICHD Women's Reproductive Health Research Program, National Institutes of Health, and the Susan B. Komen Foundation (to D. C. L.).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.
Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potential different clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens. Our results demonstrate that the estrogen 17β-estradiol effectively triggers the transcriptional activation and repression pathways with both estrogen receptors (ERs) ERα and ERβ. In contrast, soybean isoflavones (genistein, daidzein, and biochanin A) are ERβ-selective agonists of transcriptional repression and activation at physiological levels. The molecular mechanism for ERβ selectivity by isoflavones involves their capacity to create an activation function-2 surface of ERβ that has a greater affinity for coregulators than ERα. Phytoestrogens may act as natural selective estrogen receptor modulators that elicit distinct clinical effects from estrogens used for hormone replacement by selectively recruiting coregulatory proteins to ERβ that trigger transcriptional pathways.
hormone replacement therapy
selective estrogen receptor modulator
tumor necrosis factor
polymerase chain reaction
TNF responsive element
estrogen response element
Estrogens are used in hormone replacement therapy (HRT)1 to prevent hot flashes, urogenital atrophy, and osteoporosis in postmenopausal women (
). Interest in phytoestrogens has been fueled by observational studies showing a lower incidence of menopausal symptoms, osteoporosis, cardiovascular disease, and breast and endometrial cancers in Asian women who have a diet rich in soy products (
). Many postmenopausal women are taking phytoestrogens in an effort to alleviate menopausal symptoms without increasing their risk of developing breast cancer. Moreover, many women with a history of breast cancer take phytoestrogens to control menopausal symptoms (
) and available widely as herbal tablets, are especially popular among postmenopausal women. Despite their popularity and putative health benefits it is clear that we need to know much more about the molecular mechanisms, safety, and efficacy of isoflavones before they can be recommended to postmenopausal women as an alternative to estrogens for HRT. However, it is clearly important to elucidate the molecular mechanisms whereby isoflavones may elicit distinct clinical actions from estrogens used in HRT. Isoflavones have a structure similar to that of 17β-estradiol (E2) and are capable of binding to the two known estrogen receptors, ERα and ERβ (
). The relatively selective binding of genistein to ERβ indicates that isoflavones may produce distinct clinical effects from estrogens by selectively triggering ERβ-mediated transcriptional pathways or differentially triggering transcriptional activation or repression pathways by ERβ.
To test this hypothesis, we compared the effects of isoflavones and E2 on transcriptional repression and activation in the presence of ERα or ERβ. Our data demonstrate that isoflavones selectively trigger the transcriptional pathways of ERβ, particularly transcriptional repression. In addition to selectively binding to ERβ, our results suggest that the ERβ selectivity of isoflavones involves their capacity to induce an activation function-2 (AF-2) surface of ERβ that has greater affinity for coregulators such as glucocorticoid interacting receptor protein 1 (GRIP1) (
) or one copy of the ERE from the frog vitellogenin A2 gene (5′-TCAGGTCACAGTGACCTGA-3′; vitA2-ERE) were ligated into the polylinker upstream of −32 to +45 herpes simplex thymidine kinase (tk) promoter linked to luciferase (TNF-RE tkLuc and ERE tkLuc, respectively). A synthetic oligonucleotide containing the 17-nucleotide Gal-responsive element (5′-CGGAGTACTGTCCTCCG-3′) was inserted in between the G and C of the AP-1-like site (5′-TGAGCTCA-3′) at the −105 to −95 region of the TNF-RE and cloned upstream of the −32 to + 45 tk promoter (Gal-TNF-RE tkLuc).
Cell Culture, Transfection, and Luciferase Assays
U937, U2OS, MDA-MB-435, and MCF-7 cells were obtained from the cell culture facility at the University of California, San Francisco. U937 cells were maintained as described previously (
), whereas U2OS, MDA-MB-435, and MCF-7 cells were maintained and subcultured in phenol red-free Dulbecco's modified Eagle's medium/F-12 media containing 5% fetal bovine serum, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. For experiments, cells were collected, transferred to a cuvette, and then electroporated with a Bio-Rad gene pulser as described previously (
) using 3 μg of reporter plasmid and 1 μg of ERα or ERβ expression vectors. After electroporation, the cells were resuspended in media and plated at 1 ml/dish in 12-well multiplates. The cells were treated with E2, genistein, daidzein, or biochanin A (Sigma-Aldrich) 3 h prior to exposure to 5 ng/ml TNF-α (R & D Systems) for 24 h at 37 °C. Cells were solubilized with 200 μl of 1× lysis buffer, and luciferase activity was determined using a commercially available kit (Promega). The concentration of hormone required to produce a half-maximal induction (EC50) or inhibition (IC50) of luciferase activity was calculated with the Prism curve-fitting program (Graph Pad Software, version 2.0b). For proliferation studies, parental MCF-7 cells were subcloned at 1 cell/well in the presence of 0.1 nm E2, and the fastest growing clone was selected for experiments. These cells expressed exclusively ERα as determined by reverse transcription polymerase chain reaction (RT-PCR). The cells were plated in duplicate at a density of 25,000 cells/35-mm plate in tissue culture medium containing 3% stripped fetal bovine serum. One day after plating they were treated with increasing concentrations of E2 or genistein. The medium was changed every other day, and E2 or genistein was added to the medium. After 8 days the cells were counted with a Coulter counter. All experiments presented in the figures were performed at least three times, and the data were similar between experiments.
U20S cells were grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 3% stripped fetal bovine serum. Cells were treated for 24 h with 10 nm E2or 1 μm genistein and then exposed to 5 ng/ml TNF-α for 1 h at 37 °C and 5% CO2. Total RNA was prepared using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. First-strand cDNA synthesis was performed using oligo(dT) primers (Life Technologies, Inc.) and Maloney murine leukemia virus(H−) reverse transcriptase (Promega) as recommended by the manufacturer. PCR amplification using primers for the human TNF-α (sense 5′-GAGTGACAAGCCTGTAGCCCATGTTGTAGCA-3′ and antisense 5′-GCAATGATCCCAAAGTAGACCTGCCCAGACT-3′) or glyceraldehyde-3-phosphate dehydrogenase gene (sense 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ and antisense 5′-TCCTTGGAGG CCATGTGGGCCAT-3′) was performed using the HotStarTaq PCR kit (Qiagen) or Ready-To-Go PCR beads (Amersham Pharmacia Biotech). The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide.
MDA-MB-453 Stable Cell Line
The ER-negative human breast cancer cell line, MDA-MB-453 (
), was transfected by electroporation with pcDNA 6/V5-His (Invitrogen) vector containing human ERα. The cells were maintained in 10 μg/ml blastocidin (Invitrogen) until resistant colonies formed. Individual clones were obtained after the cells were plated into 96-well dishes at 1 cell/well in the presence of blastocidin. The expression of ERα and blastocidin-S deaminase, which confers resistance, was confirmed by RT-PCR in the clonal stable cell line.
Glutathione S-Transferase Pull-down Assays
GST pull-down assays were performed as described previously (
). Briefly, human ERα and ERβ were transcribed and translated in vitrousing the TNT T7 Quick Coupled Transcription/Translation system (Promega) and [35S]methionine. For each binding reaction, a 2-μl aliquot of translation product was incubated withEscherichia coli-expressed GST-GRIP1 immobilized to glutathione-Sepharose beads (Amersham Pharmacia Biotech) in the presence of vehicle control (0.1% ethanol), E2, or genistein. The samples were rocked gently at 4 °C for 2 h. After extensive washing of the beads, the labeled proteins were eluted with SDS-polyacrylamide gel electrophoresis loading buffer and separated on a 12% SDS-polyacrylamide gel. The radiolabeled bound ERs were detected by autoradiography and analyzed using the Storm phosphorimaging system and ImageQuant software (Molecular Dynamics).
Estrogens Selectively Repress the TNF-α Promoter through ERβ
To investigate the effects of isoflavones on transcriptional repression, we used the −125 to −82 region (
). E2 produced a profound dose-dependent repression of TNF-α activation of the TNF-RE upstream of a minimal tk promoter (TNF-RE tkLuc) with either transfected ERα (Fig.1A) or ERβ (Fig.1B) in U937 cells. Daidzein and biochanin A had no effect on TNF-α activation of the TNF-RE with ERα, whereas genistein produced a minor repression at 1 μm (Fig. 1A). In contrast, all three isoflavones produced a large repression (30–60%) of TNF-α activation of TNF-RE in the presence of ERβ (Fig.1B). Genistein is the most potent isoflavone and is about 65-fold weaker than E2 at repression (IC50 = 8.5 versus 0.13 nm). The isoflavones are more effective also at triggering transcriptional activation of a classical estrogen response element (ERE) in U937 cells with ERβ (Fig.2B) compared with ERα (Fig.2A). However, isoflavones are about 10–300-fold more potent at triggering transcriptional repression compared with transcriptional activation with ERβ (genistein, IC50 = 8.5 nm, EC50 = 55 nm; daidzein, IC50 = 0.072 μm, EC50 = 1.2 μm; biochanin A, IC50 = 0.17 μm, EC50 = 50 μm).
Genistein Decreases TNF-α mRNA in Bone Cells
The effect of genistein on endogenous TNF-α gene expression was investigated in a human osteosarcoma cell line (U20S) because these cells express ERα and ERβ, as demonstrated by RT-PCR (data not shown), and TNF-α is involved in the pathogenesis of osteoporosis (
). U20S cells were treated with E2 or genistein for 24 h and then exposed to TNF-α for 1 h. TNF-α produced a profound induction of TNF-α mRNA as determined by RT-PCR that was repressed markedly by E2 or genistein (Fig.3A). The observation that genistein inhibits endogenous TNF-α mRNA in untransfected cells demonstrates that repression of TNF-α transcription by genistein is physiological and not caused by nonspecific squelching of transcriptional factors by transfected ERs. To determine which ER isoform is responsible for repressing the endogenous TNF-α gene, we transfected U2OS cells with ERα or ERβ. Although the endogenous ERs are capable of repressing the native TNF-α gene, they are not present in high enough levels to repress the large number of transfected plasmids containing the TNF-RE. Fig. 3B shows that genistein is very effective at repressing the TNF-RE in cells transfected with ERβ but not ERα. These results indicate that genistein represses the endogenous TNF-α gene through ERβ even though U2OS cells also express ERα.
Isoflavones Are Weak ERα Agonists in Breast Cancer Cells
Our results indicate that isoflavones selectively promote ERβ-mediated transcription. To explore the activity of genistein on ERα in breast cancer cell lines, we compared the effects of E2 and genistein on ERα activation of ERE tkLuc in an ER-negative breast cancer cell line (MDA-MB-453) stably transfected with ERα and on the proliferation of MCF-7 cells, which express endogenous ERα but not ERβ as determined by RT-PCR (data not shown). Similar to transiently transfected U937 and U20S cells, genistein is much weaker than E2 at activating an ERE in the ERα MDA-MB-453 stable cells (Fig.4A) and stimulating the proliferation of MCF-7 cells (Fig. 4B). Thus, genistein is a weak ERα agonist in cells transiently (U937 and U20S) or stably (MDA-MB-453) transfected with ERα and in cells that express endogenous ERα (MCF-7).
Isoflavones Selectively Recruit GRIP1 to ERβ
A potential explanation for ERβ-selective activity is that isoflavones induce a functional AF-2 surface in ERβ but not ERα because we showed previously that the AF-2 surface is required for repression (
). Consistent with this hypothesis is the observation that an ERβ with a mutation in helix 3 (K314A) of the AF-2 surface failed to promote repression in response to genistein (Fig.5). Because binding of coregulatory proteins (
) but may be tethered to the TNF-RE through coregulators such as GRIP1. Gal-GRIP1 activated Gal-TNF-RE tkLuc ∼20-fold (data not shown). E2 is extremely potent at inhibiting Gal-GRIP1 activation of Gal-TNF-RE tkLuc in the presence of either ERα (Fig.6A) or ERβ (Fig.6B) (IC50 = 28.5 pm for ERα, and IC50 = 1.5 pm for ERβ). In contrast, genistein is much more potent at repressing Gal-GRIP1 activation with ERβ (IC50 = 49 pm) compared with ERα (IC50 = 1.8 μm). Furthermore, at saturating levels (10 μm), genistein produced a 33% repression with ERα compared with a maximal 72% repression with ERβ at only 10 nm.
These results suggest that genistein creates an AF-2 surface in ERβ that permits the binding of GRIP1 more efficiently compared with ERα. To investigate this hypothesis directly, glutathioneS-transferase-GRIP1 pull-down assays were performed with either 35S-labeled ERα or ERβ in the presence of E2 or genistein. A similar dose-dependent increase in binding of ERα or ERβ to GRIP1 was observed with E2 (Fig. 7A). In contrast, genistein is more effective at enhancing the interaction between GRIP1 and ERβ (Fig. 7B). At 10 μm, binding of ERβ to GRIP1 is 2-fold greater than with ERα. These findings demonstrate that genistein creates an AF-2 surface in ERβ that has a higher affinity for GRIP1 than that in ERα.
Estrogens in HRT improve menopausal symptoms but are associated with an increased risk of breast (
). To overcome the uterotropic effects of estrogens, women with a uterus are treated also with progesterone in HRT regimens. Unfortunately, the addition of progesterone may increase the risk of breast cancer further (
). The current challenge is to discover estrogens that retain their ability to prevent menopausal symptoms without promoting breast cancer or requiring progesterone for endometrium protection. The development of more ideal estrogens for HRT requires a greater understanding of how different estrogenic compounds differentially regulate gene activation and repression by ERα and ERβ.
We have shown that isoflavones elicit distinct transcriptional actions from estrogens. E2 effectively triggers both ERα- and ERβ-mediated transcriptional activation or repression pathways. In contrast, our results demonstrate that isoflavones are weak ERα agonists and potent ERβ agonists because they are effective only at triggering transcriptional activation or repression with ERβ. The key question is how do isoflavones elicit distinct transcriptional actions from estrogens despite the fact they both bind to the same binding pocket of ERα and ERβ (
). However, this difference in binding affinity is unlikely to account entirely for the distinct transcriptional actions of isoflavones because we observed that isoflavones were over a 1,000-fold more potent at triggering transcriptional activity with ERβ compared with ERα. Furthermore, at saturating levels (10 μm), genistein was less effective at repressing GRIP1 activation of Gal-TNF-RE tkLuc with ERα and recruiting GRIP1 to ERα, compared with ERβ. These studies indicate that the divergent transcriptional actions of estrogens and isoflavones probably also result from differences in their ability to recruit coregulators and trigger transcriptional functions of ERα or ERβ. These data are consistent with the discoveries that coregulator proteins (
E2 nonselectively recruits coregulators to ERα and ERβ, whereas isoflavones selectively recruit coregulators to ERβ. By recruiting coregulators such as GRIP1 to both ERs, E2effectively triggers transcriptional activation and repression pathways for both ERα and ERβ. Undoubtedly, E2 elicits its full spectrum of beneficial and adverse effects by triggering all transcriptional pathways of ERs. In contrast, at physiological levels (0.55–0.86 μm) (
) genistein is very weak at recruiting GRIP1 to ERα, but it is potent at recruiting GRIP1 to ERβ. By selectively recruiting coregulators to ERβ, isoflavones would only trigger ERβ-mediated transcriptional pathways. These results suggest that isoflavones should be effective at eliciting the clinical effects that are mediated by ERβ but not ERα. Moreover, isoflavones are 10–300-fold more potent at triggering transcriptional repression compared with activation. These results indicate that it may be possible to develop transcriptional activation or repression-selective estrogens for HRT. It is unclear why genistein recruits GRIP1 more effectively to ERβ than to ERα. However, the binding of GRIP1 may stabilize the genistein-ERβ complex more effectively than the genistein-ERα complex because the binding of coregulators has been shown to slow the rate of dissociation of an agonist from the ER-coregulator complex (
The lack of regulation of ERα-mediated genes and the potent repression of ERβ-mediated genes by isoflavones may account for the low incidence of menopausal symptoms, osteoporosis, cardiovascular disease, and breast and endometrial cancer in Asian countries (
). For example, our studies suggest that ERβ-mediated repression of the TNF-α gene may be an important mechanism whereby isoflavones may prevent osteoporosis because excessive production of TNF-α after menopause is thought to lead to osteoporosis (
). We have shown also that E2produces a robust stimulation of proliferation of breast cancer (MCF-7) cells. ERα undoubtedly mediates this effect because these cells only express ERα. Furthermore, it is likely that ERα mediates the proliferative effects on endometrial cells because these cells do not express ERβ (
). Based on these findings, we hypothesize that ERβ-selective estrogens such as isoflavones may prevent some menopausal symptoms and conditions but will be less likely to elicit stimulatory effects on breast and endometrial cells compared with estrogens present in current HRT regimens that also trigger ERα-transcriptional pathways. Consistent with this hypothesis are the observations that isoflavone-rich soy protein relieves menopausal symptoms (
Understanding how natural estrogens and synthetic SERMs elicit selective clinical effects is a key to the development of safer estrogens for HRT. We have shown that isoflavones elicit distinct transcriptional actions from estrogens by selectively recruiting coregulators to ERβ. These data are consistent with the observation that helix 12 of the AF-2 surface exists in a different position when genistein is bound to ERβ (
). Our results suggest that isoflavones may act as natural SERMs, which may be safer than estrogens in current HRT regimens because they selectively trigger the transcriptional pathways of ERβ. Estrogens in HRT also trigger ERα transcriptional pathways, which may promote the proliferation of breast and endometrial cells.
We thank P. Chambon, J.-A. Gustafsson, and M. Stallcup for providing plasmids and Keith Yamamoto and Paul Webb for critical review of the manuscript.