Transcription Dependent − Modulation of Aryl Hydrocarbon Receptor and Promoter-Specific − Estrogen Receptor Subtype

In this study, we examined the role of estrogen receptors (ER) in aryl hydrocarbon receptor (AHR)–dependent transactivation. Chromatin immunoprecipitation assays showed that AHR agonists differentially induced recruitment of ERα to the AHR target genes CYP1A1 and CYP1B1. Cotreatment with 17β-estradiol significantly increased β-naphthoflavone (BNF)– and 2,3,7,8-tetrachlorodibenzop-dioxin–induced recruitment of ERα to CYP1A1, whereas 3,3′-diindolylmethane induced promoter occupancy of ERα at CYP1A1 that was unaffected by cotreatment with 17β-estradiol. Cyclical recruitment of AHR and ERα to CYP1A1 was only observed in cells treated with BNF. Stable and subtype-specific knockdown of ERα or ERβ using shRNA showed that suppression of ERα significantly reduced, whereas knockdown of ERβ significantly enhanced, AHR agonist–induced Cyp1a1 expression in HC11 mouse mammary epithelial cells. AHR agonist–induced Cyp1b1 expression was reduced by ERβ knockdown but unaffected by ERα knockdown. The siRNA-mediated knockdown of ERα in MCF-7 human breast cancer cells did not affect 2,3,7,8-tetrachlorodibenzo-p-dioxin–dependent regulation of CYP1A1 and CYP1B1 mRNA expression. In agreement with our in vitro findings in the HC11 cells, ERα knockout mice exhibit reduced BNF-dependent induction of Cyp1a1 mRNA. These results establish ligandand promoter-specific influences on the cyclical recruitment patterns for AHR and show ER species-, subtype-, and promoter-specific modulation of AHR-dependent transcription. (Mol Cancer Res 2009;7(6):977–86) Introduction The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic-helix-loophelix PER/ARNT/SIM family (1). AHR binds a wide range of endogenous and xenobiotic compounds, including 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD; refs. 2, 3). In the absence of ligand, the AHR is located in the cytoplasm bound to a multichaperone protein complex (4). On ligand binding, the AHR translocates to the nucleus, heterodimerizes with aryl hydrocarbon nuclear translocator (ARNT), and the heterodimer binds to their cognate DNA sequences, termed AHR response elements. The activated AHR/ARNT heterodimer recruits coregulators leading to changes in target gene expression, including the phase I detoxifying monooxygenases cytochrome P4501A1 (CYP1A1) and CYP1B1 (5, 6). Studies of animal models reveal that AHR plays a key role in development, immune function, differentiation, and reproduction (7-11). Estrogens are an important class of hormones that are involved in many physiologic processes (12). Estrogen action is mediated by estrogen receptor (ER)-α and ERβ, which are members of the nuclear receptor superfamily of transcription factors (13). Both ER subtypes regulate gene expression through two different mechanisms: via direct DNA-binding to estrogen response elements or by protein-protein interactions with other transcription factors (14, 15). ERα and ERβ form heterodimers and these heterodimers can modulate the activities of their respective homodimers (16). ERβ exhibits an antagonistic action on ERα-mediated signaling (17, 18), highlighting the importance of balance between the two ER subtypes in estrogen action (19). Activation of AHR by TCDD and related compounds modulates a number of endocrine systems (20), perhaps most notably by interfering with estrogen signaling. The molecular basis for this inhibitory crosstalk is unclear, and may be due to a combination of several proposed mechanisms (21-23). The antiestrogenic effects of TCDD have led to the proposition that AHR agonists may have therapeutic potential in the treatment of estrogen-dependent cancers. One of these chemicals is indole-3-carbinol, which is found in cruciferous vegetables (24). In the gastrointestinal tract, indole-3-carbinol conjugates are hydrolyzed to many products including 3,3′-diindolylmethane (DIM; refs. 24, 25), which is a partial agonist for AHR. DIM has been reported not only to inhibit estrogenic responses (26) but also to activate ERα in the absence of 17β-estradiol (E2) via protein kinase A–mediated phosphorylation (27, 28). Although AHR agonists such as 3-methylcholanthrene have been reported to enhance ER-dependent responses Received 8/22/08; revised 2/10/09; accepted 2/17/09; published OnlineFirst 5/26/09. Grant support: Canadian Institutes of Health Research grant MOP-82715 (J. Matthews), the European Commission–funded CASCADE Network of Excellence (FOOD-CT-2004-506319), and the Swedish Cancer Fund. J. Matthews is a recipient of a fellowship from the David and Astrid Hageléns Foundation and a New Investigator Award from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jason Matthews, Department of Pharmacology and Toxicology, University of Toronto, Medical Sciences Building, Room 4336, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Phone: 416-9460851; Fax: 416-978-6395. E-mail: jason.matthews@utoronto.ca Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-08-0396 Mol Cancer Res 2009;7(6). June 2009 977 Research. on August 20, 2014. © 2009 American Association for Cancer mcr.aacrjournals. org Downloaded from Published OnlineFirst May 26, 2009; DOI: 10.1158/1541-7786.MCR-08-0396