A Molecular Mechanism To Switch the Aryl Hydrocarbon Receptor from a Transcription Factor to an E3 Ubiquitin Ligase

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.

Keywords: E3 ubiquitin ligase; aryl hydrocarbon receptor; aryl hydrocarbon receptor nuclear translocator; aryl hydrocarbon receptor repressor; molecular switch; transcription factor.

FIG 1

The E3 ubiquitin ligase function of the AhR is favored in cells in which AhR transcriptional activity is impaired by ARNT knockdown. (A) Western blotting (top) and relative AhR protein levels (bottom). MCF7 cells were transfected with negative-control siRNA (siCtrl) or siRNA targeting the AhR (siAhR) and incubated for 48 h. The cells were then treated with TCDD (5 nM) for the indicated times. Cycloheximide at 10 μM was included in all treatment regimes. Whole-cell extracts were prepared, separated on SDS-PAGE gels, and analyzed by immunoblotting using ERα, AhR, and β-actin antibodies. The band intensities of ERα immunoblots were quantified by using ImageJ software, and the relative ERα protein levels were normalized to β-actin levels and are presented as mean values from three independent experiments. (B) Cells were treated with 10 nM TCDD in the presence of 10 μM the proteasomal inhibitor MG132 for up to 6 h, and whole-cell extracts were prepared. Extracts were analyzed following immunoprecipitation (IP) with an ERα antibody by immunoblotting using antiubiquitin (anti-Ub). (C) Following transfection with siRNA, MCF7 cells were treated with 10 nM TCDD for 0 to 6 h. Total RNA was prepared, and the expression of the AhR target gene CYP1A1 was analyzed by RT-qPCR. HPRT was used as an endogenous control for ΔΔC_T analysis. Data are presented as means ± SD of results from three independent experiments performed in duplicate. (D) Western blotting (left) and relative AhR protein levels (right). Cells were treated with 10 nM TCDD for 0 to 6 h, and whole-cell extracts were collected. Cell extracts were separated on an SDS-PAGE gel and transferred onto a membrane for immunoblotting. The blots were analyzed by using ERα, ARNT, and β-actin antibodies. The band intensities of ERα immunoblots were quantified by using ImageJ software, and the relative ERα protein levels were normalized to β-actin levels and are presented as mean values from three independent experiments. (E) MCF7 cells were transiently transfected with negative-control siRNA or siRNA against ARNT. Forty-eight hours after transfection, cells were treated with 10 μM the proteasomal inhibitor MG132 and 10 nM TCDD. Cells were harvested, an immunoprecipitation assay was preformed with an ERα antibody, and cells were immunoblotted as indicated. (F) MCF7 cells were treated with the proteasomal inhibitor MG132 (10 μM) and TCDD (10 nM) for 6 h. (Left) Cell extracts were coimmunoprecipitated with anticullin, and immunoblotting was performed as indicated. (Right) MCF7 cells were transfected with Flag-tagged Arnt, and a Flag antibody was used in the coimmunoprecipitation assay. The immunoprecipitation experiments were repeated several times, and the data show the results of a representative experiment.

FIG 2

Degradation of the AhR is impaired in cells where ARNT protein levels are reduced. (A) Western blotting (top) and relative AhR protein levels (bottom). Whole-cell extracts were prepared from TCDD-treated Hepa-1c1c7 (wild-type [WT]) and Hepa C4 (C4) cells at the indicated time points and separated by SDS-PAGE. Immunoblots were analyzed by using AhR and GAPDH antibodies. The band intensities of AhR immunoblots were quantified by using ImageJ software, and the relative AhR protein levels were normalized to GAPDH levels and are presented as mean values from three independent experiments. (B) Western blot analysis of ARNT protein levels in Hepa-1c1c7 cells (WT) and Hepa C4 cells (C4). (C) Western blotting (top) and relative AhR protein levels (bottom). MCF7 cells were transiently transfected with negative-control siRNA and siRNA targeted to ARNT and treated with TCDD. At the indicated time points, cells were harvested, and whole-cell extracts were subjected to immunoblotting. Blots were analyzed for anti-AhR, anti-ARNT, and anti-β-actin. The data show results from a representative experiment. The band intensities of AhR immunoblots were quantified by using ImageJ software, and the relative AhR protein levels were normalized to β-actin levels and are presented as mean values from three independent experiments. (D) ARNT mRNA expression in MCF7 cells transfected with control siRNA or specific siRNA against ARNT. The data represent the means ± SD of results from three independent experiments performed in duplicate.

FIG 3

Overexpression of the AhRR affects AhR signaling by diminishing the transcription factor function of the AhR. (A) AhRR expression levels in MCF7 cells stably overexpressing the AhRR. Clones stably overexpressing the Myc-tagged AhRR were generated by transfection with pEFIRESpuro or pEFIRESpuromycAhRR constructs and subsequent selection with puromycin. Shown are data from analyses of AhRR mRNA expression levels (left) and protein levels (right) in MCF7 control cells and two selected clones. (B) Effect of AhRR overexpression on AhR-regulated gene expression. MCF7 cells were treated with 10 nM TCDD for 6 h. Total RNA was prepared, and the expression of CYP1A1 was analyzed by RT-qPCR. HPRT was used as an endogenous control for ΔΔC_T analysis. Data are presented as means ± SD of results from three independent experiments performed in duplicate. (C) Recruitment of the AhR to the CYP1A1 promoter is impaired by overexpressed AhRR. ChIP was performed on MCF7 cells treated with DMSO or 10 nM TCDD for 2 h, using antibodies (Ab) against AhRR, AhR, and IgG (as a negative control). The immunoprecipitated DNAs were analyzed by quantitative PCR using primers spanning an XRE-containing region of CYP1A1. Data are presented as means ± SD of results from three independent experiments performed in duplicate.

FIG 4

Estrogen receptor alpha signaling is impaired in MCF7 breast cancer cells overexpressing the repressor of AhR function. (A) MCF7 cells were treated with 10 nM E₂ for 24 h, total RNA was prepared, and the expression levels of the estrogen receptor target genes CTSD (left) and PR (right) were analyzed by RT-qPCR. Beta-actin was used as an endogenous control for ΔΔC_T analysis. Data are presented as means ± SD of results from three independent experiments performed in duplicate. (B) TCDD-induced ERα degradation is increased in MCF7 cells stably overexpressing the Myc-tagged AhRR. Shown are data for Western blotting (top) and relative AhR protein levels (bottom). Cells were treated with 10 nM TCDD for the indicated times, and whole-cell extracts were prepared and subjected to immunoblotting with anti-ERα, anti-Myc tag, and anti-β-actin. The band intensities of ERα immunoblots were quantified by using ImageJ software, and the relative ERα protein levels were normalized to β-actin levels and are presented as mean values from three independent experiments. (C) TCDD-induced ERα ubiquitinylation in MCF7 cells stably overexpressing the AhRR. Cells were treated with 10 μM the proteasomal inhibitor MG132 and 10 nM TCDD. Whole-cell extracts were immunoprecipitated with anti-ERα and immunoblotted with antiubiquitin.

FIG 5

Estrogen-dependent tumor growth and ERα levels are reduced in SCID mice injected with MCF7 breast cancer cells stably overexpressing the AhRR. (A) MCF7 control cells and MCF7 cells stably overexpressing the AhRR (AhRR#1 and AhRR#2) were cultured for up to 96 h. At the indicated time points, cells were harvested, and the optical density at 600 nm (OD 600) was measured. Data are presented as means ± SD of results from three independent experiments in quadruplicate (P value of 0.0126). (B) AhRR overexpression does not affect hypoxia-inducible gene factors in MCF7 cells. MCF7 control and AhRR-overexpressing (AhRR#1 and AhRR#2) cells were cultured for 18 h in 21% or 1% oxygen. Cells were subsequently harvested, and expression levels of the hypoxia target genes PGK1, Dec1, and PHD3 were analyzed by RT-qPCR. HPRT was used as an endogenous control for ΔΔC_T analysis. Data are presented as means ± SD from three independent experiments. (C) SCID mice (n = 10) were injected with MCF7 cancer cells with or without overexpressed AhRR (AhRR#2) in the presence of estrogen-releasing pellets. Tumors were measured by palpation. After 22 days, the mice were sacrificed, tumors were removed, and the tumor volume was determined. Data are presented as means ± standard errors of the means (n = 10) (*, P ≤ 0.05; **, P ≤ 0.01 [by two-way analysis of variance]). (D) AhRR mRNA levels in xenograft tumors from SCID mice injected with control cells (Ctrl) or AhRR-overexpressing cells (AhRR#2). Expression levels were analyzed by RT-qPCR. TBP was used as an endogenous control for ΔΔC_T analysis. Data are presented as means ± SD of results from four different tumors. (E) ERα protein levels in tumors from SCID mice injected with control cells (Ctrl) and cells overexpressing the AhRR (AhRR#2). The data show results from a representative experiment with 6 different tumors. (F) Immunohistochemical analysis of ERα levels in tumors from mice injected with (right) or without (left) AhRR-overexpressing cells. The tumors were stained with an antibody against ERα and counterstained with hematoxylin. Data show results from a representative experiment.

FIG 6

Arnt availability regulates the AhR switch from a transcription factor to an E3 ubiquitin ligase. (A and B) If ARNT is available, the AhR functions as a ligand-induced transcription factor (A) and is subsequently degraded via the proteasome complex (B). (C and D) If ARNT is occupied by other partner factors, e.g., the AhRR (C), the AhR functions as an E3 ubiquitin ligase targeting substrate proteins for proteasomal degradation, for instance, ERα. L, AhR ligand; HDACs, histone deacetylases.