A novel xenobiotic responsive element regulated by aryl hydrocarbon receptor is involved in the induction of BCRP/ABCG2 in LS174T cells

Abstract

Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17β-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR-responsive element (AhRE5) located -2357/-2333bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug-drug interactions.

Figure 1. Induction of ABCG2 in LS174T cells

A) LS174T cells were treated for 24 hr with ligands for PPARα [Wy 14643 (10 µM), CLF (10 µM)], PPARγ [TGZ (10 µM)] and activators for AhR [EGB (100 µg/ml), Resv (100 µM), OMP (50 µM), 3MC (1 µM)]. ABCG2 expression was measured by real-time RT-PCR, normalized by GAPDH expression and presented as fold induction over DMSO (0.1%) control. B) LS174T cells were treated with the same batch of chemicals as described in A for 72 hr. Whole cell lysates (45 µg each) were loaded for Western immunoblot analysis of ABCG2 as described under Materials and Methods. C) LS174T cells were treated with 3MC at the concentrations ranging from 0.05 µM to 5 µM for 24 hr before ABCG2 mRNA expression was detected as outlined in A. The nonlinear regression analysis was carried out to obtain estimate of the EC₅₀ value for 3MC. D) LS174T cells treated with 3MC at concentrations from 0.05 µM to 5 µM for 72 hr. Whole cell lysate (45 µg each) were subjected to immunoblot analysis of ABCG2 as described in Materials and Methods.

Figure 2. AhR is required for 3MC-mediated induction of ABCG2 in LS174T cells

A) LS174T cells were transfected with non-specific siRNA (siRNA-NC) or AhR-specific siRNA (siRNA-AhR). Forty-eight hours after transfection down-regulation of ABCG2 was measured by real-time RT-PCR. In addition to the bar figure, the RT-PCR products of AhR knock down were also visualized on agarose gel. B) In a parallel experiment, LS174T cells transfected with siRNA-NC or siRNA-AhR were treated with DMSO control or 3MC (1 µM) for 24 hr. ABCG2 expression was determined by real-time RT-PCR, and expressed as fold over siRNA-NC DMSO control.

Figure 3. Identification of AhR response elements in the ABCG2 promoter

A) The schematic figure depicts the prototypical AhR response element (AhRE) sequences and five putative elements within the −3.8 kb of the ABCG2 promoter region. The core sequences were bolded and underlined. LS174T cells were transfected with B) the BCRP-1.2kb reporter construct (containing 3 putative AhREs) or C) the BCRP-3.8kb reporter construct (containing 5 putative AhREs), and treated with AhR agonists EGB (100 µg/ml), Resv (100 µM), OMP (50 µM) or 3MC at 1 µM and 5 µM. Luciferase activities were measured and data were presented as fold activation over DMSO control.

Figure 4. Activation of the putative AhREs of ABCG2 in LS174T cells

A) LS174T cells were transfected with promoter deletion constructs and treated with 3MC (1µM) for 24 hr. The number of putative AhREs was illustrated in the promoter schematic to the left. B) LS174T cells were transfected with BCRP-3.8kb reporter constructs with site-directed mutagenesis of AhRE1, 4 and 5 in a number of configurations: BCRP-3.8-Δ1, BCRP-3.8-Δ5, BCRP-3.8-Δ1/4, BCRP-3.8-Δ1/5, and BCRP-3.8-Δ4/5. Transfected cells were then treated with 3MC (1µM) for 24 hr. In both A and B, luciferase activities were measured and presented as fold activation over DMSO control.

Figure 5. Recruitment of AhR to the ABCG2 promoter

A) Chromatin immunoprecipitation assays were carried out in LS174T cells treated with DMSO control or 3MC as described in Materials and Methods. Chromatin was precipitated with antibodies to IgG (non-specific) or AhR. PCR primers were specific to the regions containing AhRE1, 4 or 5. B) Mouse Hepa-1 cells were transfected with BCRP-3.8kb or BCRP-3.8-Δ5 promoter construct, following the treatment of DMSO or 3MC. After precipitation with IgG or AhR antibody, AhRE5 region was amplified by real-time PCR.