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

  • Jinping An
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences and
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  • Christina Tzagarakis-Foster
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences and
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  • Tiffany C. Scharschmidt
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences and
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  • Noureddine Lomri
    Affiliations
    Department of Medicine, Gastroenterology Division and Liver Center, University of California, San Francisco, California 94143
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  • Dale C. Leitman
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences and
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  • Author 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.
Open AccessPublished:May 25, 2001DOI:https://doi.org/10.1074/jbc.M100953200
      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.
      HRT
      hormone replacement therapy
      SERM
      selective estrogen receptor modulator
      E2
      17β-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
      Estrogens are used in hormone replacement therapy (HRT)1 to prevent hot flashes, urogenital atrophy, and osteoporosis in postmenopausal women (
      • Johnson S.R.
      ,
      • Kenny A.M.
      • Prestwood K.M.
      ). HRT also may prevent heart disease (
      • Bush T.L.
      ), Alzheimer's disease (
      • van Duijn C.M.
      ), and colon cancer (
      • Grodstein F.
      • Newcomb P.A.
      • Stampfer M.J.
      ). 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 (

      Collaborative Group on Hormonal Factors in Breast Cancer, Lancet, 350, 1997, 1047, 1059

      ,
      • Colditz G.A.
      ) and endometrial cancer (
      • Pickar J.H.
      • Thorneycroft I.
      • Whitehead M.
      ). 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 (
      • Baker V.L.
      • Leitman D.
      • Jaffe R.B.
      ,
      • McDonnell D.P.
      ). Recently, raloxifene has been approved for the prevention and treatment of osteoporosis (
      • Clemett D.
      • Spencer C.M.
      ). Raloxifene is classified as a SERM because it exhibits agonist activity in some tissues such as the bone (
      • Delmas P.
      • Bjarnason N.
      • Mitlak B.
      • Ravoux A.-C.
      • Shah A.
      • Huster W.
      • Draper M.
      • Christiansen C.
      ,
      • Ettinger B.
      • Black D.
      • Mitlak B.
      • Knickerbocker R.
      • Nickelsen T.
      • Genant H.
      • Christiansen C.
      • Delmas P.
      ) and acts as an antagonist in other tissues including the breast (
      • Cummings S.R.
      • Eckert S.
      • Krueger K.A.
      • Grady D.
      • Powles T.J.
      • Cauley J.A.
      • Norton L.
      • Nickelsen T.
      • Bjarnason N.H.
      • Morrow M.
      • Lippman M.E.
      • Black D.
      • Glusman J.E.
      • Costa A.
      • Jordan V.C.
      ). Although these effects are extremely desirable, raloxifene also increases hot flashes (
      • Davies G.
      • Huster W.
      • Lu Y.
      • Plouffe Jr., L.
      • Lakshmanan M.
      ), is weaker than estrogens at increasing bone mineral density (
      • Sato M.
      • Rippy M.K.
      • Bryant H.U.
      ), and does not improve cognitive function (
      • Nickelson T.
      • Lufkin E.
      • Riggs B.
      • Cox D.
      • Crook T.
      ) or prevent hip fracture (
      • Ettinger B.
      • Black D.
      • Mitlak B.
      • Knickerbocker R.
      • Nickelsen T.
      • Genant H.
      • Christiansen C.
      • Delmas P.
      ). 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 (
      • Clarkson T.
      • Anthony M.
      • Williams J.
      • Honoré E.
      • Cline J.
      ). 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 (
      • Ingram D.
      • Sanders K.
      • Kolybaba M.
      • Lopez D.
      ,
      • Goodman M.T.
      • Wilkens L.R.
      • Hankin J.H.
      • Lyu L.C.
      • Wu A.H.
      • Kolonel L.N.
      ,
      • Adlercreutz H.
      ,
      • Murkies A.
      • Wilcox G.
      • Davis S.
      ,
      • Tham D.
      • Gardner C.
      • Haskell W.
      ,
      • Davis S.R.
      • Dalais F.S.
      • Simpson E.R.
      • Murkies A.L.
      ). Consistent with epidemiological studies are the findings that soy phytoestrogens prevent mammary tumors (
      • Barnes S.
      ,
      • Lamartiniere C.A.
      • Murrill W.B.
      • Manzolillo P.A.
      • Zhang J.X.
      • Barnes S.
      • Zhang X.
      • Wei H.
      • Brown N.M.
      ) and bone loss (
      • Arjmandi B.
      • Alekel L.
      • Hollis B.
      • Amin D.
      • Stacewica-Sapuntzaki M.
      • Guo P.
      • Kukreja S.
      ,
      • Picherit C.
      • Coxam V.
      • Bennetau-Pelissero C.
      • Kati-Coulibaly S.
      • Davicco M.J.
      • Lebecque P.
      • Barlet J.P.
      ) in rodents and atherosclerosis of coronary arteries in monkeys (
      • Anthony M.
      • Clarkson T.
      • Bullock B.
      • Wagner J.
      ). Soy protein relieves hot flashes in postmenopausal women (
      • Albertazzi P.
      • Pansini F.
      • Bonaccorsi G.
      • Zanotti L.
      • Forini E.
      • De Aloysio D.
      ) and attenuates bone loss in the lumbar spine of perimenopausal women (
      • Alekel D.L.
      • Germain A.S.
      • Peterson C.T.
      • Hanson K.B.
      • Stewart J.W.
      • Toda T.
      ). Furthermore, a high intake of dietary phytoestrogens is associated with a lower incidence of breast cancer in women (
      • Ingram D.
      • Sanders K.
      • Kolybaba M.
      • Lopez D.
      ). 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 (
      • Lee M.M.
      • Lin S.S.
      • Wrensch M.R.
      • Adler S.R.
      • Eisenberg D.
      ,
      • Morris K.T.
      • Johnson N.
      • Homer L.
      • Walts D.
      ) because estrogens are contraindicated.
      The isoflavones, genistein, daidzein, and biochanin A, which are abundant in soybeans (
      • Setchell K.D.
      ) 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 (
      • Green S.
      • Walter P.
      • Greene G.
      • Krust A.
      • Goffin C.
      • Jensen E.
      • Scrace G.
      • Waterfield M.
      • Chambon P.
      ,
      • Kuiper G.G.
      • Enmark E.
      • Pelto-Huikko M.
      • Nilsson S.
      • Gustafsson J.A.
      ,
      • Mosselman S.
      • Polman J.
      • Dijkema R.
      ). Compared with ERα, ERβ exhibits a 7–30-fold greater binding affinity for genistein, whereas E2 binds to ERα and ERβ with equal affinity (
      • Kuiper G.
      • Carlsson B.
      • Grandien K.
      • Enmark E.
      • Haggblad J.
      • Nilsson S.
      • Gustafsson J.
      ,
      • Barkhem T.
      • Carlsson B.
      • Nilsson Y.
      • Enmark E.
      • Gustafsson J.
      • Nilsson S.
      ). 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) (
      • Hong H.
      • Kohli K.
      • Trivedi A.
      • Johnson D.L.
      • Stallcup M.R.
      ) compared with ERα. Phytoestrogens may act as natural SERMs by selectively recruiting coregulators that trigger ERβ-mediated transcriptional pathways.

      DISCUSSION

      Estrogens in HRT improve menopausal symptoms but are associated with an increased risk of breast (

      Collaborative Group on Hormonal Factors in Breast Cancer, Lancet, 350, 1997, 1047, 1059

      ,
      • Colditz G.A.
      ) and endometrial cancer (
      • Pickar J.H.
      • Thorneycroft I.
      • Whitehead M.
      ). 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 (
      • Hofseth L.J.
      • Raafat A.M.
      • Osuch J.R.
      • Pathak D.R.
      • Slomski C.A.
      • Haslam S.Z.
      ,
      • Schairer C.
      • Lubin J.
      • Troisi R.
      • Sturgeon S.
      • Brinton L.
      • Hoover R.
      ) and attenuate potential benefits of estrogens on the cardiovascular system (). 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 (
      • Brzozowski A.M.
      • Pike A.C.
      • Dauter Z.
      • Hubbard R.E.
      • Bonn T.
      • Engstrom O.
      • Ohman L.
      • Greene G.L.
      • Gustafsson J.A.
      • Carlquist M.
      ,
      • Shiau A.K.
      • Barstad D.
      • Loria P.M.
      • Cheng L.
      • Kushner P.J.
      • Agard D.A.
      • Greene G.L.
      ,
      • Pike A.C.
      • Brzozowski A.M.
      • Hubbard R.E.
      • Bonn T.
      • Thorsell A.G.
      • Engstrom O.
      • Ljunggren J.
      • Gustafsson J.A.
      • Carlquist M.
      )? One possibility is that isoflavones bind to ERβ more effectively than to ERα. In fact, ERβ has a 30-fold greater affinity for genistein compared with ERα (
      • Barkhem T.
      • Carlsson B.
      • Nilsson Y.
      • Enmark E.
      • Gustafsson J.
      • Nilsson S.
      ). 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 (
      • McKenna N.J.
      • Lanz R.B.
      • O'Malley B.W.
      ,
      • Glass C.K.
      • Rosenfeld M.G.
      ) are required for both transcriptional activation and repression by ERs (
      • An J.
      • Ribeiro R.C.
      • Webb P.
      • Gustafsson J.A.
      • Kushner P.J.
      • Baxter J.D.
      • Leitman D.C.
      ,
      • Katzenellenbogen B.S.
      • Montano M.M.
      • Ediger T.R.
      • Sun J.
      • Ekena K.
      • Lazennec G.
      • Martini P.G.
      • McInerney E.M.
      • Delage-Mourroux R.
      • Weis K.
      • Katzenellenbogen J.A.
      ,
      • Klinge C.M.
      ).
      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) (
      • Barnes S.
      • Sfakianos J.
      • Coward L.
      • Kirk M.
      ) 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 (
      • Gee A.C.
      • Carlson K.E.
      • Martini P.G.
      • Katzenellenbogen B.S.
      • Katzenellenbogen J.A.
      ).
      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 (
      • Ingram D.
      • Sanders K.
      • Kolybaba M.
      • Lopez D.
      ,
      • Adlercreutz H.
      ,
      • Murkies A.
      • Wilcox G.
      • Davis S.
      ,
      • Tham D.
      • Gardner C.
      • Haskell W.
      ,
      • Davis S.R.
      • Dalais F.S.
      • Simpson E.R.
      • Murkies A.L.
      ). 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 (
      • Pacifici R.
      ). Genistein also protects against vascular injury in ovariectomized female rats through ERβ (
      • Makela S.
      • Savolainen H.
      • Aavik E.
      • Myllarniemi M.
      • Strauss L.
      • Taskinen E.
      • Gustafsson J.
      • Hayry P.
      ). 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 (
      • Taylor A.H.
      • Al-Azzawi F.
      ). 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 (
      • Albertazzi P.
      • Pansini F.
      • Bonaccorsi G.
      • Zanotti L.
      • Forini E.
      • De Aloysio D.
      ,
      • Upmalis D.H.
      • Lobo R.
      • Bradley L.
      • Warren M.
      • Cone F.L.
      • Lamia C.A.
      ) but does not exert estrogenic effects on the endometrium in postmenopausal women (
      • Upmalis D.H.
      • Lobo R.
      • Bradley L.
      • Warren M.
      • Cone F.L.
      • Lamia C.A.
      ,
      • Duncan A.M.
      • Underhill K.E.
      • Xu X.
      • Lavalleur J.
      • Phipps W.R.
      • Kurzer M.S.
      ). Furthermore, isoflavone-rich soy protein does not induce proliferation in endometrial and mammary tissue in postmenopausal female macaques (
      • Foth D.
      • Cline J.M.
      ).
      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 (
      • Pike A.C.
      • Brzozowski A.M.
      • Hubbard R.E.
      • Bonn T.
      • Thorsell A.G.
      • Engstrom O.
      • Ljunggren J.
      • Gustafsson J.A.
      • Carlquist M.
      ) compared with E2-bound ERα (
      • Brzozowski A.M.
      • Pike A.C.
      • Dauter Z.
      • Hubbard R.E.
      • Bonn T.
      • Engstrom O.
      • Ohman L.
      • Greene G.L.
      • Gustafsson J.A.
      • Carlquist M.
      ) or ERβ (
      • Pike A.C.
      • Brzozowski A.M.
      • Hubbard R.E.
      • Bonn T.
      • Thorsell A.G.
      • Engstrom O.
      • Ljunggren J.
      • Gustafsson J.A.
      • Carlquist M.
      ). 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.

      Acknowledgments

      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.

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