Abstract
The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3′,4′-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers.
Highlights
Elucidating the mechanisms underlying transcription is crucial to our understanding of how cells and organisms respond to physiological signals and environmental stimuli
The presence of aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and estrogen receptor-a (ERa) at the dioxin-inducible Cytochrome P4501A1 (CYP1A1) enhancer and the E2-inducible pS2 promoter has already been documented in MCF7 cells and other breast cancer cell lines [11,19,20,30]
Preliminary studies have identified ECC1 human endometrial cells as an ideal system to study dioxin disruption of estrogen signaling [31,32] very little is known concerning the roles of AHR, ARNT and estrogen receptor (ER) and their respective interactions in this cell line
Summary
Elucidating the mechanisms underlying transcription is crucial to our understanding of how cells and organisms respond to physiological signals and environmental stimuli. The AHRC is capable of recruiting regulatory proteins, such as steroid receptor coactivator-1 (SRC-1), CREB binding protein (CBP/p300), NCoA2/GRIP1 [11,12], receptor-interacting-protein 140 (RIP140) [13], CoCoA [14], GAC63 [15], NcoA4 [16] and TRIP230 [17], which play significant roles in determining the activity of TCDD-induced gene transcription. These co-activators and co-repressors incorporate themselves into multimeric complexes that modify chromatin structure, stabilize core transcriptional machinery, and mediate RNA chain elongation [18]. In addition to these classic transcriptional co-activators and co-repressors, AHR is recruited by other transcription factors during transcription, including estrogen receptor-a (ERa) [11,19,20] and NF-kB [21] to modify their intrinsic activities
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