Estrogen Receptor beta -Selective Transcriptional Activity and Recruitment of Coregulators by Phytoestrogens*

Jinping AnDagger , Christina Tzagarakis-FosterDagger , Tiffany C. ScharschmidtDagger , Noureddine Lomri§, and Dale C. LeitmanDagger

From the Dagger  Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences and § Department of Medicine, Gastroenterology Division and Liver Center, University of California, San Francisco, California 94143

Received for publication, February 1, 2001, and in revised form, February 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -estradiol effectively triggers the transcriptional activation and repression pathways with both estrogen receptors (ERs) ERalpha and ERbeta . In contrast, soybean isoflavones (genistein, daidzein, and biochanin A) are ERbeta -selective agonists of transcriptional repression and activation at physiological levels. The molecular mechanism for ERbeta selectivity by isoflavones involves their capacity to create an activation function-2 surface of ERbeta that has a greater affinity for coregulators than ERalpha . 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 ERbeta that trigger transcriptional pathways.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogens are used in hormone replacement therapy (HRT)1 to prevent hot flashes, urogenital atrophy, and osteoporosis in postmenopausal women (1, 2). HRT also may prevent heart disease (3), Alzheimer's disease (4), and colon cancer (5). Unfortunately, HRT has not lived up to its potential to improve the health of women, because estrogens have been associated with an increased incidence of breast (6, 7) and endometrial cancer (8). This relationship has hampered compliance with HRT severely and has sparked an intense pursuit for selective estrogen receptor modulators (SERMs) that have a safer profile (9, 10). Recently, raloxifene has been approved for the prevention and treatment of osteoporosis (11). Raloxifene is classified as a SERM because it exhibits agonist activity in some tissues such as the bone (12, 13) and acts as an antagonist in other tissues including the breast (14). Although these effects are extremely desirable, raloxifene also increases hot flashes (15), is weaker than estrogens at increasing bone mineral density (16), and does not improve cognitive function (17) or prevent hip fracture (13). Thus, the quest for superior SERMs for HRT continues to be intense.

There also is a growing interest in using dietary natural plant estrogens (phytoestrogens), particularly those found in soy products, as a potential alternative to the estrogens in HRT (18). 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 (19-24). Consistent with epidemiological studies are the findings that soy phytoestrogens prevent mammary tumors (25, 26) and bone loss (27, 28) in rodents and atherosclerosis of coronary arteries in monkeys (29). Soy protein relieves hot flashes in postmenopausal women (30) and attenuates bone loss in the lumbar spine of perimenopausal women (31). Furthermore, a high intake of dietary phytoestrogens is associated with a lower incidence of breast cancer in women (19). 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 (32, 33) because estrogens are contraindicated.

The isoflavones, genistein, daidzein, and biochanin A, which are abundant in soybeans (34) 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 17beta -estradiol (E2) and are capable of binding to the two known estrogen receptors, ERalpha and ERbeta (35-37). Compared with ERalpha , ERbeta exhibits a 7-30-fold greater binding affinity for genistein, whereas E2 binds to ERalpha and ERbeta with equal affinity (38, 39). The relatively selective binding of genistein to ERbeta indicates that isoflavones may produce distinct clinical effects from estrogens by selectively triggering ERbeta -mediated transcriptional pathways or differentially triggering transcriptional activation or repression pathways by ERbeta .

To test this hypothesis, we compared the effects of isoflavones and E2 on transcriptional repression and activation in the presence of ERalpha or ERbeta . Our data demonstrate that isoflavones selectively trigger the transcriptional pathways of ERbeta , particularly transcriptional repression. In addition to selectively binding to ERbeta , our results suggest that the ERbeta selectivity of isoflavones involves their capacity to induce an activation function-2 (AF-2) surface of ERbeta that has greater affinity for coregulators such as glucocorticoid interacting receptor protein 1 (GRIP1) (40) compared with ERalpha . Phytoestrogens may act as natural SERMs by selectively recruiting coregulators that trigger ERbeta -mediated transcriptional pathways.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Human ERalpha and ERbeta were provided by P. Chambon and J.-A. Gustafsson, respectively (41). Gal-GRIP1 and GST-GRIP1 were provided by M. Stallcup (42). Three copies of the -125 to -82 human TNF-alpha promoter fragment (43) 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 (44), 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 (41) using 3 µg of reporter plasmid and 1 µg of ERalpha or ERbeta 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-alpha (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 ERalpha 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.

RT-PCR-- 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 E2 or 1 µM genistein and then exposed to 5 ng/ml TNF-alpha 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-alpha (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 (45), was transfected by electroporation with pcDNA 6/V5-His (Invitrogen) vector containing human ERalpha . 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 ERalpha 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 (46). Briefly, human ERalpha and ERbeta were transcribed and translated in vitro using 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 with Escherichia 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogens Selectively Repress the TNF-alpha Promoter through ERbeta -- To investigate the effects of isoflavones on transcriptional repression, we used the -125 to -82 region (43) of the TNF-alpha promoter (TNF-alpha -responsive element, (TNF-RE)) because this region mediates TNF-alpha activation and E2 repression (41). E2 produced a profound dose-dependent repression of TNF-alpha activation of the TNF-RE upstream of a minimal tk promoter (TNF-RE tkLuc) with either transfected ERalpha (Fig. 1A) or ERbeta (Fig. 1B) in U937 cells. Daidzein and biochanin A had no effect on TNF-alpha activation of the TNF-RE with ERalpha , whereas genistein produced a minor repression at 1 µM (Fig. 1A). In contrast, all three isoflavones produced a large repression (30-60%) of TNF-alpha activation of TNF-RE in the presence of ERbeta (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 ERbeta (Fig. 2B) compared with ERalpha (Fig. 2A). However, isoflavones are about 10-300-fold more potent at triggering transcriptional repression compared with transcriptional activation with ERbeta (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).


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Fig. 1.   Isoflavones selectively repress transcription of the TNF-RE through ERbeta . Three copies of the -125 to -82 region of the TNF-alpha promoter were cloned upstream of the minimal tk promoter (TNF-RE tkLuc). U937 cells were transfected with 3 µg of TNF-RE tkLuc and 1 µg of expression vector for human ERalpha (A) or ERbeta (B). After transfection, the cells were treated for 24 h with TNF-alpha (5 ng/ml) in the presence of increasing concentrations of E2, genistein, daidzein, or biochanin A, and luciferase activity was measured. TNF-alpha activated the TNF-RE tkLuc by 5-10-fold in the absence of drugs. Each data point represents the mean of triplicate samples ± S.E. RLU, relative luciferase units.


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Fig. 2.   Isoflavones selectively activate transcription of an ERE through ERbeta . A single copy of the vitellogenin A2 ERE upstream of the minimal tk promoter (ERE tkLuc) was transfected into U937 cells with either 1 µg of expression vector for human ERalpha (A) or ERbeta (B). After transfection, the cells were treated for 24 h with increasing concentrations of E2, genistein, daidzein, or biochanin A, and luciferase activity was determined. Each data point represents the mean of triplicate samples ± S.E. RLU, relative luciferase units.

Genistein Decreases TNF-alpha mRNA in Bone Cells-- The effect of genistein on endogenous TNF-alpha gene expression was investigated in a human osteosarcoma cell line (U20S) because these cells express ERalpha and ERbeta , as demonstrated by RT-PCR (data not shown), and TNF-alpha is involved in the pathogenesis of osteoporosis (47). U20S cells were treated with E2 or genistein for 24 h and then exposed to TNF-alpha for 1 h. TNF-alpha produced a profound induction of TNF-alpha mRNA as determined by RT-PCR that was repressed markedly by E2 or genistein (Fig. 3A). The observation that genistein inhibits endogenous TNF-alpha mRNA in untransfected cells demonstrates that repression of TNF-alpha 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-alpha gene, we transfected U2OS cells with ERalpha or ERbeta . Although the endogenous ERs are capable of repressing the native TNF-alpha 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 ERbeta but not ERalpha . These results indicate that genistein represses the endogenous TNF-alpha gene through ERbeta even though U2OS cells also express ERalpha .


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Fig. 3.   A, genistein inhibits endogenous TNF-alpha gene expression in human osteosarcoma cells. U20S cells were treated with 10 nM E2, 1 µM genistein (Gen) or ethanol (Cont) for 24 h and then exposed to TNF-alpha (5 ng/ml) for 1 h. Total RNA was isolated, and the expression of TNF-alpha mRNA was determined by RT-PCR. Ethidium bromide staining shows a 444-base pair PCR product of the TNF-alpha gene and a 217-base pair product for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as internal control for the quality of RNA prepared. B, genistein selectively represses transcription of the TNF-RE in U2OS cells through ERbeta . U2OS cells were transfected with TNF-RE tkLuc (3 µg) and 1 µg of expression vector for either ERalpha (black-square) or ERbeta (black-triangle). The cells were treated for 24 h with increasing concentrations of genistein and then assayed for luciferase activity. Each data point represents the mean of triplicate samples. The S.E. was < 10%.

Isoflavones Are Weak ERalpha Agonists in Breast Cancer Cells-- Our results indicate that isoflavones selectively promote ERbeta -mediated transcription. To explore the activity of genistein on ERalpha in breast cancer cell lines, we compared the effects of E2 and genistein on ERalpha activation of ERE tkLuc in an ER-negative breast cancer cell line (MDA-MB-453) stably transfected with ERalpha and on the proliferation of MCF-7 cells, which express endogenous ERalpha but not ERbeta 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 ERalpha MDA-MB-453 stable cells (Fig. 4A) and stimulating the proliferation of MCF-7 cells (Fig. 4B). Thus, genistein is a weak ERalpha agonist in cells transiently (U937 and U20S) or stably (MDA-MB-453) transfected with ERalpha and in cells that express endogenous ERalpha (MCF-7).


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Fig. 4.   A, genistein is weak at activating an ERE in ERalpha -stable MDA-MB-453 cells. ERE tkLuc (3 µg) was transfected into MDA-MB-453 stably transfected with ERalpha . Cells were treated for 24 h with increasing concentrations of E2 or genistein, and luciferase activity was measured. B, genistein is weak at stimulating the growth of MCF-7 breast cancer cells. MCF-7 cells were plated 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. After 8 days the cells were counted with a Coulter counter. Each data point represents the mean of duplicate samples. The S.E. was < 10%. RLU, relative luciferase units.

Isoflavones Selectively Recruit GRIP1 to ERbeta -- A potential explanation for ERbeta -selective activity is that isoflavones induce a functional AF-2 surface in ERbeta but not ERalpha because we showed previously that the AF-2 surface is required for repression (41). Consistent with this hypothesis is the observation that an ERbeta 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 (48, 49) to the AF-2 surface is required for repression by E2 (41), we compared the effects of E2 and genistein on functional interactions that occur at the TNF-RE between the coregulator, GRIP1 (40), and ERalpha or ERbeta . For these studies, a Gal response element was inserted in the center of an AP-1-like site in the TNF-RE (Gal-TNF-RE), which is essential for TNF-alpha activation and E2 repression (41). Gal-GRIP1 was used for these studies instead of Gal-ER because ERs do not bind directly to the TNF-RE (41) 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 ERalpha (Fig. 6A) or ERbeta (Fig. 6B) (IC50 = 28.5 pM for ERalpha , and IC50 = 1.5 pM for ERbeta ). In contrast, genistein is much more potent at repressing Gal-GRIP1 activation with ERbeta (IC50 = 49 pM) compared with ERalpha (IC50 = 1.8 µM). Furthermore, at saturating levels (10 µM), genistein produced a 33% repression with ERalpha compared with a maximal 72% repression with ERbeta at only 10 nM.


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Fig. 5.   Genistein requires a functional AF-2 surface for repression by ERbeta . U937 cells were transfected with 3 µg of TNF-RE tkLuc and 1 µg of human wild-type ERbeta or a helix 3 activation function-2 mutant (K314A in human ERbeta 530). Cells were treated for 24 h with TNF-alpha (5 ng/ml) in the absence or presence of 100 nM genistein, and luciferase activity was measured. Each data point represents the mean of triplicate samples ± S.E. RLU, relative luciferase units.


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Fig. 6.   Genistein selectively represses Gal-GRIP1 activation of Gal-TNF-RE through ERbeta . A synthetic oligonucleotide that contained the 17-nucleotide Gal-responsive element 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. Because the AP-1-like site is destroyed, this construct is inactive in the absence of Gal-GRIP1. Cells were transfected with Gal-GRIP1, Gal-TNF-RE tkLuc, and ERalpha (A) or ERbeta (B) and then treated with increasing concentrations of E2 or genistein for 24 h, and luciferase activity was determined. Gal-GRIP1 activated Gal-TNF-RE tkLuc by about 20-fold in the absence of E2 or genistein. Each data point represents the mean of triplicate samples ± S.E. RLU, relative luciferase units.

These results suggest that genistein creates an AF-2 surface in ERbeta that permits the binding of GRIP1 more efficiently compared with ERalpha . To investigate this hypothesis directly, glutathione S-transferase-GRIP1 pull-down assays were performed with either 35S-labeled ERalpha or ERbeta in the presence of E2 or genistein. A similar dose-dependent increase in binding of ERalpha or ERbeta to GRIP1 was observed with E2 (Fig. 7A). In contrast, genistein is more effective at enhancing the interaction between GRIP1 and ERbeta (Fig. 7B). At 10 µM, binding of ERbeta to GRIP1 is 2-fold greater than with ERalpha . These findings demonstrate that genistein creates an AF-2 surface in ERbeta that has a higher affinity for GRIP1 than that in ERalpha .


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Fig. 7.   Genistein is more effective at recruiting GRIP1 to ERbeta . [35S]Methionine-labeled ERalpha or ERbeta was synthesized in an in vitro transcription/translation system and then incubated with E. coli-expressed glutathione S-transferase-GRIP1 in the presence of increasing concentrations of E2 (A) or genistein (B). [35S]methionine-labeled ERalpha or ERbeta bound to glutathione S-transferase-GRIP1 was separated by SDS-polyacrylamide gel electrophoresis. The dried gels were exposed to x-ray film (top) and a phosphorscreen and then scanned with a PhosphorImager (bottom). The first lane on the autoradiograms represents the input of [35S]methionine-labeled ERalpha or ERbeta in the binding reaction. This is a single experiment that is representative of six experiments that produced similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogens in HRT improve menopausal symptoms but are associated with an increased risk of breast (6, 7) and endometrial cancer (8). 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 (50, 51) and attenuate potential benefits of estrogens on the cardiovascular system (52). 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 ERalpha and ERbeta .

We have shown that isoflavones elicit distinct transcriptional actions from estrogens. E2 effectively triggers both ERalpha - and ERbeta -mediated transcriptional activation or repression pathways. In contrast, our results demonstrate that isoflavones are weak ERalpha agonists and potent ERbeta agonists because they are effective only at triggering transcriptional activation or repression with ERbeta . 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 ERalpha and ERbeta (53-55)? One possibility is that isoflavones bind to ERbeta more effectively than to ERalpha . In fact, ERbeta has a 30-fold greater affinity for genistein compared with ERalpha (39). 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 ERbeta compared with ERalpha . Furthermore, at saturating levels (10 µM), genistein was less effective at repressing GRIP1 activation of Gal-TNF-RE tkLuc with ERalpha and recruiting GRIP1 to ERalpha , compared with ERbeta . 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 ERalpha or ERbeta . These data are consistent with the discoveries that coregulator proteins (48, 49) are required for both transcriptional activation and repression by ERs (41, 56, 57).

E2 nonselectively recruits coregulators to ERalpha and ERbeta , whereas isoflavones selectively recruit coregulators to ERbeta . By recruiting coregulators such as GRIP1 to both ERs, E2 effectively triggers transcriptional activation and repression pathways for both ERalpha and ERbeta . 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) (58) genistein is very weak at recruiting GRIP1 to ERalpha , but it is potent at recruiting GRIP1 to ERbeta . By selectively recruiting coregulators to ERbeta , isoflavones would only trigger ERbeta -mediated transcriptional pathways. These results suggest that isoflavones should be effective at eliciting the clinical effects that are mediated by ERbeta but not ERalpha . 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 ERbeta than to ERalpha . However, the binding of GRIP1 may stabilize the genistein-ERbeta complex more effectively than the genistein-ERalpha complex because the binding of coregulators has been shown to slow the rate of dissociation of an agonist from the ER-coregulator complex (59).

The lack of regulation of ERalpha -mediated genes and the potent repression of ERbeta -mediated genes by isoflavones may account for the low incidence of menopausal symptoms, osteoporosis, cardiovascular disease, and breast and endometrial cancer in Asian countries (19, 21-24). For example, our studies suggest that ERbeta -mediated repression of the TNF-alpha gene may be an important mechanism whereby isoflavones may prevent osteoporosis because excessive production of TNF-alpha after menopause is thought to lead to osteoporosis (47). Genistein also protects against vascular injury in ovariectomized female rats through ERbeta (60). We have shown also that E2 produces a robust stimulation of proliferation of breast cancer (MCF-7) cells. ERalpha undoubtedly mediates this effect because these cells only express ERalpha . Furthermore, it is likely that ERalpha mediates the proliferative effects on endometrial cells because these cells do not express ERbeta (61). Based on these findings, we hypothesize that ERbeta -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 ERalpha -transcriptional pathways. Consistent with this hypothesis are the observations that isoflavone-rich soy protein relieves menopausal symptoms (30, 62) but does not exert estrogenic effects on the endometrium in postmenopausal women (62, 63). Furthermore, isoflavone-rich soy protein does not induce proliferation in endometrial and mammary tissue in postmenopausal female macaques (64).

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 ERbeta . 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 ERbeta (55) compared with E2-bound ERalpha (53) or ERbeta (55). 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 ERbeta . Estrogens in HRT also trigger ERalpha transcriptional pathways, which may promote the proliferation of breast and endometrial cells.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

To whom correspondence should be addressed: University of California, San Francisco, Center for Reproductive Sciences, HSE 1619 P. O. Box 0556, San Francisco, CA 94143-0556. Tel.: 415-502-5261; Fax: 415-753-3271; E-mail: leitmand@obgyn.ucsf.edu.

Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100953200

    ABBREVIATIONS

The abbreviations used are: HRT, hormone replacement therapy; SERM, selective estrogen receptor modulator; E2, 17beta -estradiol; ER, estrogen receptor; AF-2, activation function-2; GST, glutathione S-transferase; TNF, tumor necrosis factor; tk, thymidine kinase; RT, reverse transcription; PCR, polymerase chain reaction; TNF-RE, TNF responsive element; ERE, estrogen response element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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