Estrogen receptor-α and aryl hydrocarbon receptor involvement in the actions of botanical estrogens in target cells

Abstract

Botanical estrogen (BE) dietary supplements are consumed by women as substitutes for loss of endogenous estrogens at menopause. To examine the roles of estrogen receptor α (ERα) and aryl hydrocarbon receptor (AhR) and their crosstalk in the actions of BEs, we studied gene regulation and proliferation responses to four widely used BEs, genistein, daidzein, and S-equol from soy, and liquiritigen from licorice root in breast cancer and liver cells. BEs and estradiol (E2), acting through ERα, stimulated proliferation, ERα chromatin binding and target-gene expression. BEs but not E2, acting through AhR, bound to xenobiotic response element-containing chromatin sites and enhanced AhR target-gene expression (CYP1A1, CYP1B1). While E2 and TCDD acted quite selectively through their respective receptors, BEs acted via both receptors, with their AhR activity moderated by negative crosstalk through ERα. Both ERα and AhR should be considered as mediators of the biology and pharmacology of BEs.

Keywords: Aryl hydrocarbon receptor; Botanical estrogens; Cell proliferation; Estrogen receptor; Gene regulation; Xenobiotic metabolism.

Fig. 1. Comparison of the ability of E2, TCDD, and botanical estrogens (BEs) to up-regulate and/or down-regulate ERα and AhR target genes

Progesterone receptor (PgR), pS2, and CYP1A1 and CYP1B1 are differently regulated by these compounds. MCF-7 cells were treated with 10⁻⁸ M E2 or 10⁻⁹ M TCDD or 10⁻⁶ M BE for 4 h and mRNA levels of the ERα target genes, PgR and pS2 (panel A) and the AhR target genes CYP1A1 and CYP1B1 (panel B) were monitored by qPCR mRNA levels were normalized relative to the housekeeping gene 36B4, and fold change calculated relative to the control vehicle treated samples. Results are the average ±SD from at least two independent experiments. *, p < 0.05; **, p < 0.01.

Fig. 2. Dose-response relationships for regulation of the ERα target gene, PgR, and the AhR target gene CYP1A1 by E2, TCDD and four botanical estrogens

MCF-7 cells were treated with the indicated concentrations of compounds for 24 h and gene expression was monitored by qPCR. Note that the CYP1A1 mRNA scale (at right) differs in the panels to highlight the different up- and down-regulations by the different compounds. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 3. Use of ERα and AhR inhibitors and the siRNA knockdown of ERα or AhR to analyze receptor mediation of the effects of BEs, E2, and TCDD on gene regulation

A. MCF-7 cells were treated with the indicated compounds or control vehicle for 4 h. For treatment with inhibitors, cells were pretreated with 10⁻⁶ MICI 182,780 or 10⁻⁶ M CH 223191 for 45min before cotreatment with 10⁻⁸ M E2 or 10⁻⁶ M BE + ICI or + CH for an additional 4 h prior to RNA harvest and gene expression determination by qPCR. B. MCF-7 cells were transfected with 25 nM of control or ERα siRNA or AhR siRNA for 48 h and were then treated with the indicated compounds for 4 h prior to RNA harvest. Western blot inset in lowest right panel shows selective knockdown of ERα or AhR by treatment with their respective siRNAs. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 4. Assessment of the activation of AhR and ERα target genes, by BEs, E2, and TCDD in primary hepatocytes from wild type mice or AhR-knockout mice and in HepG2 cells stably expressing ERα

A. Mouse primary hepatocytes were prepared and pooled from livers of at least two littermate mice with the same genotype. Cells were cultured for 24 h and then treated with vehicle, 10⁻⁸ M EE2, or 10⁻⁹ M TCDD or BE for an additional 24 h prior for determination of gene expression by qPCR. B. HepG2 cells stably expressing ERα (HepG2-ERα) were cultured with 0.1% ethanol vehicle; 10⁻⁹ M EE2; 10⁻⁹ M TCDD; or 10⁻⁶ M Genistein, Daidzein, S-equol or liquiritigenin alone or in the copresence of 10⁻⁶ M antiestrogen ICI 182,780 or AhR antagonist CH 223,191 for 24 h prior to determination of expression of the ERα target gene PI9, and the AhR target gene, CYP1A1 by qPCR. Values are mean ± SD of 3 determinations. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5. Impact of E2, TCDD, and botanical estrogens on MCF-7 cell proliferation

A. Ki67 mRNA level monitored after 24 h of control vehicle or compound (10⁻⁸ M E2, 10⁻⁹ M TCDD or 10⁻⁶ M BE) treatment in the absence or presence of 10⁻⁶ M ICI 182,780 or 10⁻⁶ M CH 223191. B. Cell proliferation monitored after 6 days of treatment with the indicated concentrations of E2 or TCDD. C. Cell proliferation monitored at 6 days after treatment with veh, 10⁻⁸ M E2, 10⁻⁹ M TCDD or 10⁻⁶ M BE alone or with 10⁻⁶ M ICI 182,780 or 10⁻⁶ M CH 223191. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 6. Recruitment of ERα vs. AhR to (A) estrogen responsive (estrogen response element-containing) regions of the TFF1/pS2 and LRRC54 estrogen target genes, and (B) to xenobiotic response element-containing regions of the CYP1A1 or CYP1B1 genes

MCF-7 cells were treated with 10⁻⁸ M E2, 10⁻⁹ M TCDD, or 10⁻⁶ M liquiritigenin or 10⁻⁶ M S-equol for 45 min prior to chromatin crosslinking and chromatin immunoprecipitation (ChIP) using control IgG antibody, ERα antibody, or AhR antibody. Receptor recruitment is presented as percent of input. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 7. The effect of co-treatment with E2 or BEs plus TCDD on the expression of ERα target genes, PgR and pS2, and AhR target genes, CYP1A1 and CYP1B1

MCF-7 cells were treated with Veh, 10⁻⁸ M E2 or 10⁻⁶ M BE alone (open bars) or with 10⁻⁹ M TCDD (grey bars) for 24 h and mRNA levels of the ERα target genes, PgR and pS2 (Panel A) and the AhR target genes CYP1A1 and CYP1B1 (Panel B) were monitored by qPCR. Student’s t-test was performed on the data in Panel A to compare E2 or BE treatment alone vs. treatment plus TCDD. One-way ANOVA was performed on the data in Panel B to compare cotreatment with E2 or BE plus TCDD vs. TCDD treatment alone. *, p< 0.05; **, p< 0.01; ***, p< 0.001; NS, not significant.

Fig. 8. Schematic model depicting the specificity and crosstalk between ERα and AhR in mediating the differential actions of the ERα ligand E2, and the AhR ligand TCDD, and the dual acting botanical estrogens (BEs)

E2 and TCDD largely activate only their own receptors, ERα and AhR, respectively, with crosstalk by E2-ERα suppressing the activity of TCDD-AhR. By contrast, BEs have dual specificity, activating estrogen target genes via ERα and also dioxin/xenobiotic target genes through AhR, with their AhR activity being modulated by negative crosstalk via ERs. Thus, treatment of cells with ERα antagonist ICI 182,780 or depletion of cellular ERα by siRNA-mediated knockdown enhanced the magnitude of stimulation of AhR target genes by BEs. The suppressive effect of the estrogens acting through ERα on the expression of AhR target genes could be due to either a direct competitive binding effect between ERα and AhR at the chromatin level or could be due to more indirect effects via ERα.