Estrogen receptor α and aryl hydrocarbon receptor cross-talk in a transfected hepatoma cell line (HepG2) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Abstract

The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR) by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα) signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2) in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP) induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.

Keywords: 17β-estradiol; AhR; AhR, aryl hydrocarbon receptor; COMT, catechol-O-methyltransferase; CPRG, chlorophenol red β-d-galactopyranoside; CYP, cytochrome P450; Ct, cycle threshold; DMSO, dimethyl sulfoxide; Dioxin; E, strogen receptor; E2, 17β-estradiol; ERE, estrogen response element; Estrogen receptor; Gene reporter assay; Human hepatoma cell line HepG2; TCDD; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element; α-NF, α-naphthoflavone.

Fig. 1

Effects of ERα on ER-mediated transcription. HepG2 cells were transiently transfected with ERE-TK-LUC and co-transfected with human ERα expression plasmid as described in Section 2. Cells were treated with TCDD and/or E2 over a period of 20 h. An induction equal to 100% was attributed to the luciferase activity of treatment with E2 and all results were expressed as a percentage of the luciferase activity of this positive control (corresponding to 111,133 ± 7564 cps). Mean ± SD (n ≥ 3); One-way ANOVA followed by Tukey's post test: significantly different from solvent control (DMSO 0.2% max.): ***p ≤ 0.0001; significant difference between respective E2 and E2/TCDD treatments as indicated; n.s.: not significant.

Fig. 2

Effects of ERα on AhR-mediated transcription. HepG2 cells were transiently transfected with pGL3-XRE and co-transfected or not with human ERα expression plasmid as described in Section 2. Cells were treated with TCDD and/or E2 over a period of 20 h. An induction equal to 100% was attributed to the luciferase activity of treatment with TCDD in ERα-transfected cells and all results were expressed as a percentage of the luciferase activity of this positive control (corresponding to 1,740,574 ± 484,008 cps). Mean ± SD (n ≥ 3); One-way ANOVA followed by Tukey's post test: significantly different from solvent control (DMSO 0.2% max.): *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001; significant difference between respective TCDD and E2/TCDD treatments as indicated; n.s.: not significant.

Fig. 3

Real-time RT PCR analysis of ERα, AhR, CYP1A1, CYP1B1, and COMT mRNA in non-transfected and ERα-transfected HepG2. Effects of TCDD 1 nM and/or E2 10 nM were assayed after 20 h exposure. Mean ± SD (n ≥ 3); Two-way ANOVA followed by the Bonferroni post test: statistically significant from respective transfected or untransfected solvent control (DMSO 0.25%): ^*p ≤ 0.05, ^***p ≤ 0.001; significant difference between respective treatment of transfected and non-transfected cells: ^$p ≤ 0.05; n.d.: not detected.