Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

doi:10.1016/S0960-0760(98)00067-3

The Journal of Steroid Biochemistry and Molecular Biology

Volume 67, Issue 1, October 1998, Pages 17-24

https://doi.org/10.1016/S0960-0760(98)00067-3 Get rights and content

Abstract

TCDD, the most potent congener of the polychlorinated dioxins, has been shown to be an antiestrogen. The mechanisms of TCDD-induced antiestrogenicity are still under investigation. In this study, we investigated the effects of TCDD on the expression of the estrogen receptor (ER) gene. We studied the levels of un-spliced ER transcript (hnRNA) as well as the ER mRNA in ovary, uterus and liver of TCDD-treated mice with different genetic backgrounds. To quantitate the ER hnRNA levels, the intron and exon boundary of ER hnRNA was amplified by competitive RT-PCR. The ER mRNA from these mice was quantitated by competitive RT-PCR amplifying exons separated by an intron. ER hnRNA and ER mRNA levels were quantitated 4 days after a single i.p. dose of TCDD (5 μg/kg) in female C57BL/6J (B6) mice, which carry the responsive allele to TCDD. TCDD treatment significantly (p<0.05) suppressed the levels of ER hnRNA in the ovary (27.4%) and uterus (21.9%). The decreases in ER hnRNA were coordinated with significant (p<0.01) decreases in ER mRNA in ovary (57.7%) and uterus (37.6%). There was a significant decrease (20.3%, p<0.05) in liver ER mRNA, however, the changes of ER hnRNA in liver were not significant. The coordinated decreases in ER hnRNA and mRNA in TCDD-treated mice suggest a suppression of transcription of the ER gene. We performed the same study on DBA/2J (D2) mice, which possess the “non-responsive” allele of the aryl hydrocarbon receptor (AhR). These mice demonstrated no significant decrease in either the ER mRNA or hnRNA after TCDD treatment. Overall, these results suggest that TCDD suppresses the gene expression of the ER receptor by decreasing its transcription, and the AhR plays an important role in mediating this response.

Introduction

TCDD is an ubiquitous environmental contaminant of significant public concern. It is a prototype of polyhalogenated aromatic hydrocarbons and the most potent congener of these compounds. Many of these compounds are persistent contaminants in the environment and in the mammalian body, and have been shown to cause various toxic responses in humans and animals. TCDD causes toxic responses mainly by altering normal expression of many critical genes. The commonly recognized model proposes that TCDD exerts its toxic effects by first binding specifically to a cytosolic aryl hydrocarbon receptor (AhR)1, 2, which is a basic helix–loop–helix (bHLH) transcription factor3, 4. Upon binding to TCDD, the AhR translocates into the nucleus and dimerizes with another bHLH protein, the aryl hydrocarbon receptor nuclear translocator (ARNT)5, 6. The heterodimer then binds to a consensus sequence (T/ANCGTG) known as the xenobiotic response element (XRE) in the promoter regions of genes (notably P450 1A1), thereby altering gene expression[7]. The toxic effects caused by TCDD include chloracne, wasting syndrome, birth defects, immune suppression and endocrine disruptions, such as the antiestrogenic effects8, 9, 10, 11, 12, 13.

The antiestrogenicity of TCDD has been studied by several investigators and the following lines of evidence have been considered to be important: (1) TCDD-activated AhR interferes with the binding of ER to its cognate enhancer sequences, i.e. estrogen response element (ERE)[14]; (2) TCDD treatment reduces the nuclear translocation of the ligand-activated ER12, 15; (3) TCDD treatment decreases ER protein and ligand binding16, 17, which may result from a reduced ER mRNA level[18]; (4) TCDD-activated AhR interferes with the transcription of the ER gene by binding to its promoter resulting in steric hindrance of transcription[19]; (5) TCDD induces cytochrome P450s which accelerate the metabolism of estrogens13, 20.

The expression of the ER gene is auto-regulated[21]. It has been shown that the promoter region of the human ER gene contains XREs and also potential Sp1 sites[19], which are known to interact with AhR[22]. Nonetheless, experimental evidence is lacking regarding the effects of TCDD on the transcription of ER gene. In an earlier study, we showed that TCDD decreases ER mRNA levels in several tissues[18]. A reduced pool of the steady-state level of mRNA could result from reduced gene transcription, or alternatively, it could be due to accelerated mRNA turnover or both. In this study, we examined the mechanism of this reduction.

Study of transcriptional regulation of ER gene requires measurement of the steady-state level of mRNA as well as the kinetics of output of hnRNA from pre-assembled cellular transcriptional machinery. The techniques that are often used include Northern blot and nuclear run-on assays. These techniques are neither quantitative nor sensitive enough for studying transcription of ER gene which is expressed at low levels in many tissues. In this study, we extended the competitive RT-PCR procedure to evaluate the changes of the primary, un-spliced ER transcript, the ER hnRNA. Similar PCR-based methods have been used by other investigators23, 24, 25, 26, 27to study transcriptional regulation in place of the conventional run-on assay which is laborious, less accurate and requires handling large amounts of radioisotopes.

In this study, we measured the relative changes of ER mRNA and hnRNA in TCDD-treated C57BL/6J (B6) mice and DBA/2J (D2) mice. The B6 mice express the AhR from the Ah^b allele that has a high binding affinity and capacity for TCDD, while D2 mice express AhR from Ah^d allele with much lower binding affinity and capacity for TCDD[28]. These allelic differences provide the genetic backdrop for testing the involvement of the AhR in the TCDD-induced antiestrogenicity.

Section snippets

Chemicals and reagents

Restriction enzymes, T4 ligase, 100 bp DNA ladder, SuperScript II RNase H⁻ reverse transcriptase, Taq DNA polymerase, Random primer and custom-made oligonucleotide primers were purchased from Gibco (Gaithersburg, MD). GENECLEAN II kit was from Bio101 (La Jolla, CA), spin columns were obtained from Qiagen (Chatsworth, CA), TA cloning vector (pCRII) was from Invitrogen (San Diego, CA). The sequence analysis was performed in the core facility at UMDNJ-Robert Wood Johnson Medical School. TCDD was

Animals and TCDD treatment

Four days post-dosing, there were no overt signs of toxicity and no significant changes in body weight associated with TCDD treatment. However, there was a significant increase in the liver-to-body weight ratios in B6 mice (28%, p<0.01, n=5). TCDD-treated D2 mice exhibited a slight, yet statistically insignificant, increase (8.6%, p>0.1, n=5) in liver to body weight ratio (Fig. 4(D)).

Inverse PCR to amplify the DNA sequences across the intron–exon boundary

To develop a quantitative PCR method to measure ER hnRNA, we first had to clone the intronic sequences adjacent

Discussion

In an earlier study, we reported that TCDD treatment suppressed levels of ER mRNA in multiple tissues of female CD-1 mice with the suppression in ovary and uterus being most pronounced[18]. The decreases in ER mRNA after TCDD treatment raised the possibility that expression of the ER gene is regulated by the AhR at the level of transcription. The current study was designed to investigate this possibility. By using competitive RT-PCR, we demonstrated TCDD treatment caused significant decreases

Acknowledgements

This study was supported in part by NIEHS ES05002 and NCI CA42493. We thank Dr Steven W. Ward of the Division of Urology, UMDNJ-Robert Johnson Medical School for the critical review of the manuscript and helpful suggestions. The research was funded in part by NIEHS ES05022 (M. A. G) and NCI CA 42493 (T. T.).

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Most of the toxic effects of TCDD are mediated through binding to aryl hydrocarbon receptor (AhR) that acts as a multifunctional molecular switch regulating endo- and xenobiotic metabolism, cell growth and proliferation (Bock and Kohle, 2006). The mechanisms underlying TCDD-induced reproductive disorders have been speculated to involve reduced expression of sex steroid and LH receptors (Ohsako et al., 2001; Tian et al., 1998; Fukuzawa et al., 2004) by promoting ubiquitination and degradation of receptor protein (Ohtake et al., 2007), altered steroidogenesis (Mutoh et al., 2006; Myllymaki et al., 2005), modulating of ER signaling by a co-regulatory-like function of activated AhR/aryl hydrocarbon receptor nuclear translocator (ARNT) (Ohtake et al., 2003) and/or induction of cytochrome P450 1 family enzymes resulting in enhanced inactivation of steroid hormones (Badawi et al., 2000). Recent data have shown that mixtures of endocrine disrupting chemicals with diverse modes of action can display cumulative, dose additive or synergistic effects on male reproductive tract development (Metzdorff et al., 2007; Veeramachaneni et al., 2007; Hass et al., 2007; Christiansen et al., 2009; Rider et al., 2008; Jacobsen et al., 2010).
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The resulting heterodimeric complex binds to a specific DNA sequence, the dioxin response element (DRE, also called xenobiotic response element, XRE; or aryl hydrocarbon response element, AhrE) (Denison et al., 1989; Hapgood et al., 1989; Nebert et al., 2000), recruits protein coactivators and induces target gene transcription (Whitlock, 1999; Xu et al., 2000). Importantly, activation of the Ahr pathway also interferes with Esr pathways through a number of mechanisms (Beischlag and Perdew, 2005; DeVito et al., 1992; Kietz et al., 2004; Matthews and Gustafsson, 2006; Matthews et al., 2005; Ohtake et al., 2003, 2007; Safe and Wormke, 2003; Tian et al., 1998a,b). Considering the critical roles of E2 and its receptors in the regulation of the female HPG axis (Green et al., 1986; Greene et al., 1986), the deleterious effects of TCDD on female reproduction may be due to antiestrogenic effects.
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Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Abstract

Introduction

Section snippets

Chemicals and reagents

Animals and TCDD treatment

Inverse PCR to amplify the DNA sequences across the intron–exon boundary

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Toxicol. Lett.

Biochem. Pharmacol.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Anal. Biochem.

Toxicol. Appl. Pharmacol.

Binding of 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin to a common Ah receptor site in mouse and rat hepatic cytosols

Eur. J. Biochem.

Stereospecific high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol

J. Biol. Chem.

Cloning of the Ah-receptor cDNA reveals a distinctive ligand activated transcription factor

Proc. Natl. Acad. Sci. U.S.A.

Cloning of a factor required for activity of the Ah (dioxin) receptor

Science