Figure 1.
AHR is required for TCDD-mediated repression of E2-induced gene transcription.
Chromatin immunoprecipitation assays of the CYP1A1 enhancer (A) and pS2 promoter (B) regions in ECC-1 cells using antibodies targeting AHR, ARNT or ERα. Cells were treated for 45 min with either DMSO, E2 (10 nM), TCDD (2 nM) or a combination of E2 and TCDD. (C and D) The functional role of AHR in TCDD-mediated transcription. ECC-1 cells were transfected with siRNAs to either GFP (siGFP) as a negative control or AHR (siAHR#3 or siAHR#4) 24 h prior to ligand treatment, then cells were treated with DMSO, E2 (10 nM), TCDD (2 nM) or a combination of E2 and TCDD. The mRNA levels for pS2 (C) and CYP1A1 (D) were determined through real-time RT-PCR and normalized to constitutively active GAPDH expression. (E) ECC-1 cells were transfected with siRNA's to AHR and harvested for whole cell lysates for Western Blot analysis of AHR, ERα and XAP2 protein levels.
Figure 2.
Loss of ARNT shows a cell specific co-activator or co-repressor transcriptional function.
ECC-1 cells (A and B) and MCF7 cells (C and D) were transfected with either scrambled siRNA (siSCX) or siRNA directed to ARNT (siARNT#1 or siARNT#3). Twenty-four hours after transfection, cells were treated with vehicle (DMSO), E2 (10 nM), TCDD (2 nM) or a combination of E2 and TCDD. Gene expression was determined by real-time RT-PCR after isolation and reverse transcription of total RNA. CAT-D and pS2 expression were normalized to constitutively active 36B4 gene expression. (E) Western blot analysis of CAT-D protein levels in MCF7 and ECC-1 cells. Cells were treated with DMSO or E2 (10 nM) and varying concentrations of TCDD (10 pM, 50 pM, 100 pM, 500 pM, 1 nM or 5 nM). After 24 hours of treatment, whole cell lysates were harvested, and Western Blot assays were performed using antibodies directed against CAT-D and α-tubulin. Error bars represent ± S.D. * p<0.05.
Figure 3.
Effect of ARNT knockdown on ARNT, CAT-D and ERα protein levels.
ECC-1 (A and B) and MCF7 (C and D) cells were transfected with either siSCX or siARNT and ligand treated as described in Figure 2. (A and C) Representative Western blots of ARNT, CAT-D, ERα and α-tubulin protein levels. Bar graphs of CAT-D protein levels in ECC-1 (B) and MCF7 (D) cells after normalizing luminescence values to α-tubulin. Open bars represent ligand treatments after siRNA to scrambled negative control (siSCX) and closed (black) bars represent ligand treatments after siRNA to ARNT (siARNT). Experiments were performed three times with essentially identical outcomes.
Figure 4.
Effect of selective aryl hydrocarbon receptor modulators on estrogen-inducible transcription.
The effect of 1 µM DiMNF on E2-inducible CAT-D expression in MCF7 and ECC-1 cells. Cells were treated with the indicated ligands for 24 h prior to RNA isolation. Gene expression was determined as described above. Error bars represent ± S.D. * p<0.05.
Figure 5.
Loss of ARNT promotes cell proliferation in ECC-1 cells and attenuates growth in MCF7 cells.
ECC-1 (A) and MCF7 (B) cells were transfected with either siSCX or siARNT for 6 hours, then trypsinized and reseeded at 10,000 cells/well in 12-well dishes. Twenty-four hours after seeding, cells were treated with either DMSO or E2 (10 nM) and cell counts were conducted at 0, 48 and 96 hours after treatments with a Fuchs-Rosenthal Counting Chamber. At the 48 h time point, cells were treated a second time with 10 nM E2. Experiments were done in triplicate and each trial was counted three times. (C) Western blot analysis of ARNT after siRNA knock-down reveals that significant knock-down was achieved and persists over a 72 h period. Error bars represent ± S.D. * p<0.05.
Figure 6.
A schematic representation of estrogen signaling, describing the transcriptional functions of ER, AHR and ARNT in ECC-1 and MCF7 cells.
The presence of E2 facilitates the assembly of transcriptional modifiers that induce transcription of E2-responsive genes. Ligand activated AHR represses ER-signaling independent of ARNT. The loss of ARNT in ECC-1 cells, where it acts as a co-repressor, leads to increased sensitivity to E2 and increased transcriptional activity at ER regulated genes. The loss of ARNT in MCF7 cells, where it acts as a co-activator, leads to decreased sensitivity to E2 and decreases transcriptional activity at ER regulated genes.