The transcription factor aryl hydrocarbon receptor nuclear translocator functions as an estrogen receptor beta-selective coactivator, and its recruitment to alternative pathways mediates antiestrogenic effects of dioxin

Abstract

The biological effects of dioxins are mediated by the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), and include interference with hormonal signaling pathways like the response to estrogens. The effects of estrogens are mediated by two estrogen receptor (ER) isoforms, ERalpha and ERbeta, which belong to the family of nuclear receptors. We have previously shown that ARNT can act as coactivator of the ERs. In this study, we show that recruitment of ARNT to AhR or hypoxia-inducible factor-1alpha signaling pathways as well as small interfering RNA-mediated down-regulation of ARNT levels lead to a reduction in ER transcriptional activity. Using chromatin immunoprecipitation assays, we demonstrate that this decrease coincides with reduced recruitment of ARNT to estradiol-regulated promoters. We show further that coactivation by ARNT as well as inhibition by dioxin acts stronger on ERbeta than on ERalpha activity. Additionally, we demonstrate that the effects of ARNT are dependent on the A/B domain of the ERs with the A/B domain of ERbeta being considerably stronger in mediating the coactivating effects of ARNT. Taken together, our studies show that recruitment of ARNT to the AhR after dioxin treatment can account for the antiestrogenic effect of dioxins. Moreover, we show for the first time that the inhibitory effects of dioxin are more pronounced on ERbeta than on ERalpha.

Fig. 1.

Recruitment of ARNT to AhR or HIF-1α Decreases ER Function A, Activation of dioxin and/or hypoxia signaling inhibits ER transcriptional activity. H-ERE cells were treated with 10 nm E₂ or 1 μm 4-OHT alone or combination with 10 nm TCDD, in hypoxic (<1% O₂) or normoxic conditions, as indicated. After 24 h, luciferase activity was determined and adjusted to protein concentration in each sample. Treatment with E₂ alone was set to 100%. Shown are means and sd for three independent experiments. B, Dioxin does not inhibit transcription by the ER-LBD. HC11 cells were transiently cotransfected with Gal4-ERα LBD or Gal4-ERβ LBD together with a Gal4-regulated luciferase reporter gene construct. The cells were treated with 10 nm E₂ alone or in combination with 10 nm TCDD. After 48 h, reporter gene activity was determined. Luciferase activity without treatment with Gal4-vector only was set to 1. Shown are means and se for three independent experiments. C, Exposure to TCDD or hypoxia does not affect ERα and ERβ protein levels. H-ERE cells were incubated for 12 h with 10 nm E₂ and/or 10 nm TCDD, under normal or hypoxic conditions, as indicated. Representative Western blots on whole-cell extracts with antibodies recognizing ERα, ERβ, ARNT, and actin are shown. D, ARNT c-Fos promoter occupancy is decreased by TCDD. HC11 cells were incubated for 45 min with 10 nm E₂ alone or in combination with 10 nm TCDD and subsequently subjected to ChIP with the indicated antibodies. Shown here are representative agarose gels with PCR products for an ER binding site on the c-fos promoter.

Fig. 2.

Reduction of Intracellular ARNT Levels Decreases ER-Dependent Transcription A, siRNA against ARNT reduces ARNT protein levels. HeLa cells were transfected with siRNA oligonucleotides targeting ARNT (si) and as control, a scramble siRNA (scr), together with 3xERE-TATA-Luc reporter and expression plasmids for ERα and ERβ gene constructs and treated with 10 nm E₂ for 48 h. Cells were lysed, and resulting whole-cell extracts were used for Western blots and luciferase assays shown in B. Shown are representative Western blots using antibodies against ARNT and hsp90 as control comparing nontransfected cells with scr- or si-transfected cells. B, siRNA against ARNT inhibits ER activity. Luciferase activities were measured in the same cell lysates and normalized to β-galactosidase activity. Shown are means and sd of three independent experiments; 100% reflects activity of ERα or ERβ, in the presence of E₂ and scrambled RNA.

Fig. 3.

ARNT Coactivates ERα-ERβ Heterodimers to a Similar Extent as ERα Homodimers Hela cells were transiently transfected with single-chain ER constructs together with a 3xERE-TATA-Luc reporter and an ARNT expression vector or empty plasmid. Cells were treated with 10 nm E₂ for 48 h, and reporter gene activity was determined and normalized to β-galactosidase activity. Luciferase activity in the presence of E₂ and empty vector was set to 100%. Shown are means and se of three independent experiments.

Fig. 4.

Analysis of the Effect of ARNT on N-Terminal ER Mutants A, Schematic representation of the mutants used for the experiments in B and C. ERβ 530 serves as a reference for the numbering of the deleted amino acids. A/B, A/B domain; DBD, DNA-binding domain. B, The A/B domain of the ERs is important for ARNT coactivation. Hela cells were transiently transfected with ERα, ERβ, ERβABα, or ERαABβ together with a 3xERE-TATA-Luc reporter and increasing amounts of ARNT expression vector. Cells were treated with 10 nm E₂ for 48 h, and reporter gene activity was determined and normalized to β-galactosidase activity. Luciferase activity in the presence of E₂ and empty vector was set to 100%. Shown are means and sd of three independent experiments. C, Deletion of the very N terminus of ERβ abolishes its responsiveness to ARNT coactivation. Experiments were carried out as in B but with the ERβ constructs shown in the figure. Luciferase activity in the presence of E₂ and empty vector was set to 100%. Shown are means and sd of three independent experiments.

Fig. 5.

Exposure to TCDD Results in Reduced Recruitment of ARNT to E₂-Responsive Promoters in ERβ-Expressing Cells A, MCF-7 wild type or ERβ cells were incubated for 45 min with 10 nm E₂ or DPN, respectively, alone or in combination with 10 nm TCDD. Subsequently, cells were subjected to ChIP analysis with the indicated antibodies. Shown here are representative agarose gels with PCR products for an ER binding site on the GREB-1 promoter. B, Quantification of the ChIP experiments in A. Real-time PCR was performed on precipitated DNA of at least three independent experiments. The results were normalized to input DNA, and vehicle treatment was set to 1.

Fig. 6.

ARNT Forms Complexes with ERβ and RNA Polymerase II on E₂-Responsive Promoters after E₂ Treatment MCF-7 ERβ cells were incubated for 45 min with 10 nm DPN alone or in combination with 10 nm TCDD. Subsequently, re-ChIP analysis was performed with the indicated antibodies as described in Materials and Methods. Shown here are representative agarose gels with PCR products for an ER binding site on the GREB-1 promoter.

Fig. 7.

TCDD Shows Selectivity for the ERβ Isoform and Acts on the A/B Domain on the ERs A, Differential effects of TCDD on ERα and ERβ transcriptional activity in HC11 cells. H-ERE cells were treated with 10 nm E₂, 10 nm PPT, or 10 nm DPN alone or combination with 10 nm TCDD. After 24 h, luciferase activity was determined and adjusted to protein concentration in each sample. Treatment with the respective agonist alone was set to 100%. Shown are means and sd of three independent experiments. B, Effect of TCDD in HELN cell lines measured by the stably integrated ERE-β glob-luciferase activity. HELN cells were treated with 10 nm E₂ alone or combination with 10 nm TCDD. After 24 h, luciferase activity was determined and adjusted to protein concentration in each sample. Treatment with E₂ alone was set to 100%. Shown are means and sd of three independent experiments. C, Effect of TCDD in HELN cell lines measured by induction of the endogenous pS2 gene. HELN cells were treated with 10 nm E₂ alone or combination with 10 nm TCDD. After 6 h, cells were harvested, total RNA extracted, and mRNA transcribed into cDNA. Amounts of pS2 cDNA was measured by real-time PCR and normalized to amount of 18S RNA. Treatment with E₂ alone was set to 100%. Shown are means and sd of four independent experiments.

Fig. 8.

Model for the Proposed Mechanism of the Antiestrogenic Effects of TCDD and Hypoxia Transcriptional activity of the ERs, particularly of ERβ, is enhanced by the presence of ARNT. Activation of the AhR or HIF-1α signaling pathway leads to reduction of ARNT available for ER and thus to decreased ER function.