The FASEB Journal • Research Communication Mechanisms enforcing the estrogen receptor ␤ selectivity of botanical estrogens Yan Jiang,* Ping Gong,* Zeynep Madak-Erdogan,* Teresa Martin,† Muthu Jeyakumar,† Kathryn Carlson,† Ikhlas Khan,§ Troy J. Smillie,§ Amar G. Chittiboyina,§ Sateesh C. K. Rotte,§ William G. Helferich,‡ John A. Katzenellenbogen,† and Benita S. Katzenellenbogen*,1 *Department of Molecular and Integrative Physiology, †Department of Chemistry, and ‡Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; and §National Center for Natural Products Research, University of Mississippi, Oxford, Mississippi, USA ABSTRACT Because little is known about the ac- Key Words: chromatin binding ⅐ gene regulation ⅐ proliferation ⅐ tions of botanical estrogens (BEs), widely consumed breast cancer cells by menopausal women, we investigated the mecha- nistic and cellular activities of some major BEs. We As the average age of the U.S. population rises, an examined the interactions of genistein, daidzein, increasing number of women are postmenopausal for equol, and liquiritigenin with estrogen receptors ER␣ many years, and due to the dramatic reduction in levels of and ER␤, with key coregulators (SRC3 and RIP140) and chromatin binding sites, and the regulation of estrogens, they often experience hot flashes, night sweats, gene expression and proliferation in MCF-7 breast and mood changes, and they suffer from urogenital cancer cells containing ER␣ and/or ER␤. Unlike the atrophy and loss of bone density. The traditional treat- endogenous estrogen, estradiol (E2), BEs preferen- ment for menopausal symptoms is to restore estrogen tially bind to ER␤, but their ER␤-potency selectivity levels by hormone replacement therapy (HRT). Despite in gene stimulation (340- to 830-fold vs. E2) is the well-known advantages of HRT, studies from the enhanced at several levels (coregulator recruitment, Women’s Health Initiative revealed several adverse effects chromatin binding); nevertheless, at high (0.1 or 1 of estrogen alone or estrogen plus progestin, such as ␮M) concentrations, BEs also fully activate ER␣. increased risk of heart disease (1), stroke (2), breast Because ER␣ drives breast cancer cell proliferation cancer (3), and dementia (4, 5). Consequently, there is and ER␤ dampens this, the relative levels of these great interest from researchers, clinicians, and the public two ERs in target cells and the BE dose greatly affect in the development of new treatment strategies to avoid gene expression and proliferative response and will the adverse effects of HRT. be crucial determinants of the potential benefits vs. Botanicals containing estrogenic compounds are widely risks of BEs. Our findings reveal key and novel available and are consumed by women, in particular by mechanistic differences in the estrogenic activities of older women seeking relief from menopausal symptoms, BEs vs. E2, with BEs displaying patterns of activity dis- with the expectation that these botanical estrogens (BEs) tinctly different from those seen with E2 and provide may provide a safe, natural source of estrogens to replace valuable information to inform future studies.—Jiang, Y., the loss of endogenous estrogens in menopause. Soy- Gong, P., Madak-Erdogan, Z., Martin, T., Jeyakumar, M., based products have drawn increasing attention lately as Carlson, K., Khan, I., Smillie, T. J., Chittiboyina, A. G., alternative treatments for relief of menopausal symptoms Rotte, S. C. K., Helferich, W. G., Katzenellenbogen, J. A., because their major isoflavone components, genistein Katzenellenbogen, B. S. Mechanisms enforcing the estro- ␤ and daidzein, are known to have estrogenic effects (6). gen receptor selectivity of botanical estrogens. FASEB J. However, preclinical studies (7, 8) and studies in humans 27, 4406–4418 (2013). www.fasebj.org have given inconclusive results regarding the efficacy of soy for this purpose (9–11). Because estrogens have stimulatory effects on many tissues, including increasing Abbreviations: BE, botanical estrogen; ChIP, chromatin the growth of some breast cancers, the unregulated immunoprecipitation; ChIP-seq, chromatin immunoprecipi- tation-DNA sequencing; E2, estradiol; ER, estrogen receptor; FRET, fluorescence resonance energy transfer; HRT, hor- mone replacement therapy; OTUB2, otubain 2; PgR, proges- 1 Correspondence: Department of Molecular and Inte- terone receptor; RBA, relative binding affinity; RCA, relative grative Physiology, University of Illinois and College of coactivator binding affinity; RIP140, receptor interacting pro- Medicine at Urbana-Champaign, Urbana, IL 61801, USA. tein 140; SRC, steroid receptor coactivator; TR-FRET, time- E-mail: [email protected] resolved fluorescence resonance energy transfer doi: 10.1096/fj.13-234617 4406 0892-6638/13/0027-4406 © FASEB consumption of BEs might not be contributing uniformly Technologies, Grand Island, NY, USA), supplemented with to healthy aging in women (12–16). 5% calf serum (HyClone, Logan, UT, USA) and 100 ␮g/ml Estrogens exert effects on diverse target tissues and penicillin/streptomycin (Invitrogen, Carlsbad, CA). For es- cells, and they act through two different estrogen recep- trogen-free experiments, the cells were seeded in phenol ␣ ␤ ␣ ␤ red-free DMEM (Gibco/Life Technologies) plus 5%charcoal- tors (ERs), ER and ER (17–20). ER and ER are dextran-treated calf serum at a density of 2.5 ϫ 105 cells/well encoded by different genes and have different tissue of 6-well plate (for mRNA studies) or 1 ϫ 106 cells/10-cm distributions and different ligand binding specificities. tissue culture dish (for ChIP assays) for 3 d before siRNA ER␣ is generally the more potent regulator of gene transfection and adenovirus infection. Recombinant adenovi- expression and appears responsible for mediating the ruses were constructed and prepared as described (22, 24). proliferative drive of estrogens in some target tissues, such Cells were infected with either control adenovirus (Ad) ␤ expressing ␤-galactosidase or adenovirus expressing ER␤ as the uterus and some breast cancers, whereas ER when ␤ present together with ER␣ has a generally restraining (AdER ) for 72 h. Conditions used were those described ␣ previously (24, 29) to generate MCF-7 cells expressing levels effect on ER activities (21–25). Because we and others of ER␤ equal to that of the endogenously expressed ER␣. The have shown that many estrogens isolated from botanicals siRNA experiments for knockdown of the endogenous ER␣ in (e.g., genistein, daidzein, equol, liquiritigenin) are prefer- MCF-7 cells were performed as described previously and ential ligands for binding to ER␤ (6, 17, 26–28), they resulted in knockdown of ER␣ mRNA and protein by greater differ in this respect from endogenous estrogens and than 95% (24). siER␣ sequences (Dharmacon, Lafayette, CO, most estrogen pharmaceutical agents, which are ER␣- USA) were forward, 5=-UCAUCGCAUUCCUUGCAAAdTdT- binding preferential. Thus, one might expect the ER␤- 3=, and reverse, 5=-UUUGCAAGGAAUGCGAUGAdTdT-3= ␣ ␤ preferential botanicals to have different biological activi- (24). Because ER knockdown did not affect ER levels, the level of ER␤ obtained in the ER␤-only cells (24) was similar to ties than estradiol (E2). that of ER␣ in the original MCF-7 cells. E2, genistein, and To gain mechanistic information and to examine dose- formononetin were from Sigma-Aldrich (St. Louis, MO, dependent effects of BEs that might provide a new USA), liquiritigenin from Tocris Bioscience (Bristol, UK), conceptual framework for understanding whether BEs daidzein from Indofine Chemical Co. (Hillsborough, NJ, have similar or unique activities compared to those of USA), R-equol from Cayman Chemical Co. (Ann Arbor, MI, other estrogens, such as E2, we have studied in detail 4 USA). Racemic and S-equol were prepared as described (26, BEs, genistein, daidzein, S-equol, and liquiritigenin. We 30). All compounds were checked for identity and purity by measured their binding affinities to ER␣ and ER␤, and mass spectrometry and NMR. the affinity with which their complexes with ER recruit the key steroid receptor coactivator (SRC), SRC3; we exam- Relative binding affinity assay ined the chromatin binding of these ligand-receptor complexes along with that of the coregulators SRC3 and Relative binding affinities were determined by a competitive receptor interacting protein 140 (RIP140) at estrogen- radiometric binding assay as described previously (31, 32) using regulated genes by chromatin immunoprecipitation 2nM[3H]-E2 as tracer ([2,4,6,7-3H]-estra-1,3,5(10)-triene- ␤ (ChIP) assays, and we assessed their potency and efficacy 3,17 -diol, 70–115 Ci/mmol; Perkin Elmer, Waltham, MA, ␣ ␤ in regulating gene expression and their effects on prolif- USA), and purified, full-length, human ER and ER purchased from PanVera/Invitrogen. Incubations were for 18–24 h at 0°C. eration of human breast cancer cells (MCF-7) containing ␣ ␤ ␣ ␤ Hydroxyapatite (Bio-Rad, Hercules, CA, USA) was used to only ER , only ER , or both ER and ER , thereby absorb the receptor-ligand complexes, and free ligand was mimicking the different ratios of these two ERs present in washed away. The binding affinities are expressed as relative different ER target tissues and human breast tumors. Our binding affinity (RBA) values with the RBA of E2 set to 100%. studies highlight that BEs bind, induce coactivator recruit- The values given are the average Ϯ range or sd of2to3 ␣ ment, and stimulate chromatin binding preferentially