DHEA metabolites activate estrogen receptors alpha and beta

Abstract

Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ)-regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβin vitro, modulating estrogen target genes in vivo.

Figure 1. E₂ activates ERE-luciferase activity in transiently transfected cells

HEK-293 cells were transfected with ERα and HepG2 were transfected with ERβ expression plasmids Both cell lines were cotransfected with ERELUC (estrogen-response element luciferase) and pCMV-β-gal reporters and were treated for 24 h with EtOH (vehicle) or A) the indicated concentrations of E₂ or B) 10 nM E₂, 5 µM DHEA or 5 µM of the indicated DHEA metabolites. The cells were harvested and the lysates were assayed for β-galactosidase and luciferase activities. Data represent the mean ± S.D. of three separate experiments. Statistical significance is indicated versus vehicle (EtOH control), * p<0.05, ** p<0.01.

Figure 2. Concentration-dependent activation of ERα by DHEA, DHEA-S, and ADIOL in ER transfected cells

HEK-293 and HepG2 cells were transfected with ERELUC reporter plasmid and expression vector for either human ERα (A) or ERβ (B), respectively. Cells were treated for 24 h with varying concentrations of E2, DHEA, DHEA-S, 7-oxo-DHEA, ADIONE and ADIOL. Cells were harvested and lysates were assayed for β-galactosidase and luciferase activities. Data represent the mean ± S.D. of three separate experiments. *, significantly different from vehicle-treated cells, p < 0.05, **,p<0.01. E2, ●; DHEA, ▲; DHEA-S, ○; ADIOL, ■; ADIONE, ♦; and 7-oxo-DHEA, □.

Figure 3. Inhibition of ERELUC reporter activity in the presence of cotransfected ERα in ER transfected cells

HEK-293 and HepG2 cells were transfected with an ERELUC reporter plasmid and an expression vector for either human ERα or ERβ, respectively. Cells were treated for 24 h with 5 µM DHEA metabolite in the absence or presence of either 1 µM 182,780 ICI, 100 nM 4-hydroxytamoxifen (4-OHT), 1 µM R,R-THC, or 50 nM 17β-estradiol (E2). Cells were then harvested and lysates assayed for β-galactosidase and luciferase activities. Data represent the mean ± S.D. of three wells. Experiments were repeated three times with similar results. A. ERα-mediated transactivation; B. ERβ-mediated transactivation. Statistical significance was determined using analysis of variance followed by Student's t tests. *, significantly different from treated cells, p < 0.05 *, or **p, 0.01.

Figure 4. Effect of non-selective P450 inhibitor miconazole or aromatase inhibitor (AI) exemestane on ER-mediated transcriptional activation by DHEA and metabolites

A) HEK-293 cells were transfected with ERα (A) and HepG2 cells were transfected with ERβ (B). All cells were co-transfected with ERELUC reporter plasmid and pRL-tk. After 24 h, cells were incubated for 24 h in phenol red-free MEM supplemented with 5% DCC-FBS plus DMSO, 10 nM E2, 5 µM DHEA, 5 µM ADIOL, 5 µM ADIONE, or 5 µM 7-oxo-DHEA, as indicated in each panel. Where indicated, cells were preincubated with 5 µM miconazole or 100 nM Exemestane (AI) for 6 h prior to addition of the hormone treatment. Values are the average of 3 separate determinations +/− SEM * Significantly different from DMSO (vehicle control), p < 0.05. ^ Significantly different from DHEA alone, p < 0.05.

Figure 5. DHEA and metabolites increase endogenous gene transcription

HEK-293 cells were transfected with ERα (A) and HepG2 were transfected with ERβ (B) expression plasmid for 24 h and then treated with the indicated hormones (10 nM E₂ or 5 µM sterols) or 100 nM ICI 182,780 (ER antagonist) for 24 h. QPCR for A) TFF1 (pS2), PGR (PR), CTSD (cathepsin D) mRNA was normalized to 18S. B) NRF1 (NRF-1) was normalized to GAPDH. Values are the avg. ± SEM of triplicate determinations. * Significantly different from DMSO (vehicle control), p < 0.05. ^ Significantly different from DHEA alone, p < 0.05.

Figure 6. Competitive binding of DHEA metabolite to ERα and ERβ

An ligand binding competition assay using [³H] E₂ was performed using baculovirus expressed ERα (A) or ERβ (B) with increasing concentrations of nonradiolabeled E₂, DHEA or DHEA metabolites (DHEA-S, 7-oxo-DHEA, ADIONE and ADIOL). The values on the Y-axes are expressed as the percentage of [³H] E₂ bound and each data point represents the mean of two independent binding assays. The competitor concentration causing 50% reduction in [³H] E₂ binding (IC₅₀) is found at the intersection of the binding curves with the 50% binding line (---------). E2, ●; DHEA, ▲; DHEA-S, ♦; ADIOL, ■; ADIONE, ♦; and 7-oxo-DHEA, □.

Figure 7. DHEA metabolites stimulate the proliferation of MCF-7 breast cancer cells

The effect of E₂ (10 nM), and 5 µM of either DHEA, DHEA-S, 7-oxo-DHEA, ADIOL or ADIONE alone or in combination with 100 nM ICI 182,780 (fulvestrant, a pure ER antagonist, pretreatment for 6 h), 100 nM Exemestane (AI), or 5 µM miconzaole (A) or in the presence of 10 µM bicalutamide (Casodex, pretreatment for 4 h) on the proliferation of MCF-7 cells was measured by BrdU assay after 48 h treatment as described in Experimental. A) The absorbance values were converted to percent of E₂ values for relative proliferation values. B) Values were normalized to DMSO vehicle control. The data are the mean ± S.E.M. of 3–4 independent experiments in which each treatment was performed in quadruplicate. *, Statistically different from that particular indicated ligand treatment alone; p < 0.05 (one-way ANOVA followed by Student, Newman, Keuls post-hoc testing).

Figure 8. Model of how DHEA metabolites activate ERα and ERβ

DHEA is converted to ADIOL, ADIONE, and mono-hydroxylated metabolites capable of activating the estrogen receptors. ADIOL and ADIONE are high affinity ligand activators of ER similar to the 3β-diol derived from DHT as demonstrated by Muthusamy et al [46]. Other DHEA metabolites, 7-hydroxy- and 7-keto-DHEA are low affinity ligand activators of ER, relative to ADIOL or ADIONE. Further information on the synthesis and metabolism of DHEA is reviewed in [, –92].