The aryl hydrocarbon receptor interacts with nuclear factor erythroid 2-related factor 2 to mediate induction of NAD(P)H:quinoneoxidoreductase 1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Abstract

NAD(P)H:quinoneoxidoreductase 1 (NQO1) belongs to a group of the aryl hydrocarbon receptor (AhR) battery of drug-metabolizing enzymes that are characteristically induced by both AhR agonists and nuclear factor erythroid 2-related factor 2 (Nrf2) activators. We have previously reported that induction of Nqo1 by the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hepa1c1c7 cells involves Nrf2 (Ma et al., Biochem J 377, 205-213, 2004). Here we analyzed the molecular mechanism of induction. Induction required AhR and its DNA-binding partner Arnt because induction was not observed in AhR or Arnt-defective cells, but induction was restored upon reconstitution of the variant cells with functional AhR or Arnt. Induction also required Nrf2, as induction by benzo[a]pyrene was lost in the liver of Nrf2 knockout mice similarly to induction by butyl hydroxyanisol, demonstrating a cross-interaction between the AhR and Nrf2 pathways for induction in vivo. TCDD increased the protein level and induced the nuclear accumulation of Nrf2 with a delayed kinetics compared with activation of AhR. Chromatin immunoprecipitation revealed that TCDD recruited both AhR and Nrf2 to the Nqo1 promoter enhancer region containing a DRE and an ARE in time-dependent manners. Co-immunoprecipitation experiments revealed that, in addition to AhR-Arnt binding, TCDD induced an interaction between AhR and Nrf2 as well as Keap1. The findings reveal that TCDD induces multi protein complexes to mediate cross-interaction between the AhR and Nrf2 pathways, uncovering a novel mechanistic aspect of gene regulation by environmental chemicals through AhR and Nrf2.

Keywords: ARE; Ah receptor; Cross-interaction; NQO1; Nrf2; TCDD.

Fig. 1

Induction of Cyp1a1 and Nqo1 by TCDD in hepa1c1c7 and variant cells. WT (hepa1c1c7), AhR-D, or Arnt-D cells and the variant cells reconstituted with AhR, Arnt, or their mutants were described under Material and Methods. Cells were grown to confluency and were treated with DMSO or TCDD (1 nM) for 4 h. Total RNA was analyzed for mRNA expression of Cyp1a1 (A) and Nqo1 (B) using real time-PCR. Relative amounts of cDNA were calculated from C_T values for each sample as described in Methods for real-time PCR. Data represents means ± standard deviations from triplet samples. The same experiments were repeated two more times with consistent results.

Fig. 2

Induction of Cyp1a1 and Nqo1 in Nrf2 WT and KO mice. Adult Nrf2 WT and KO male mice in a C57BL/6 J background were treated with B[a]P or BHA as described under Material and Methods. Corn oil was used as vehicle control. Total RNA was prepared from the liver. Expression of Cyp1a1 (A) and Nqo1 (B) mRNAs was measured by real-time PCR as described for Fig. 1. Data represents means ± standard deviations from six RNA preparations from each genotype and treatment.

Fig. 3

Stabilization of Nrf2 protein by TCDD. Hepa1c1c7 cells were grown to confluency in 10-cm plates and were treated with tBHQ (30 μM) or TCDD (1 nM) for 4 h. (A) Immunoblotting of Nrf2 in total cell lysate. (B) Immunoprecipitation of Nrf2. Cycloheximide (CHX) was used to inhibit Nrf2 protein synthesis. Antibodies used for IB and IP were described under Material.

Fig. 4

Nuclear localization of Nrf2 and AhR. Hepa1c1c7 cells were treated with TCDD (1 nM, 4 h). Nuclear extracts were prepared at indicated time points using the Nuclei EZ PREP reagents (Sigma). Treatment with tBHQ (30 μM, 4 h) was used a as a positive control for activation of Nrf2. Nrf2 and AhR were blotted using specific antibodies described under Material. Lamin A was used as a marker for nuclear fraction and Actin as a marker for cytoplasmic contamination.

Fig. 5

Binding of AhR to Nqo1 enhancer. Hepa1c1c7 cells were treated with TCDD (1 nM) or tBHQ (30 μM) for 4 h. ChIP assay was performed with anti-AhR antibodies to immunoprecipitate AhR-bound enhancer sequences. Specific DNA sequences were quantified by real-time PCR with primers specific for the ARE enhancer region or the coding region of Nqo1 (as a negative control) (A), or were amplified by real-time PCR and visualized on agarose gel (B). Quantitative data represents means ± standard deviations from three samples. The rabbit serum was used as the negative control for antibody and 10% of the input was included to show normalized input.

Fig. 6

Binding of Nrf2 to Nqo1 enhancer. Hepa1c1c7 cells were treated with tBHQ (30 μM) or TCDD (1 nM) for 4 h. ChIP analysis was performed with anti-Nrf2 antibodies to immunoprecipitate Nrf2-bound enhancer sequences. Specific DNA sequences were quantified by real-time PCR with primers specific for the ARE enhancer region or the coding region of Nqo1 (as a negative control) (A), or were amplified by real-time PCR and visualized on agarose gel (B). Quantitative data represents means ± standard deviations from three samples. The rabbit serum was used as the negative control for antibody and 10% of the input was included to show normalized input.

Fig. 7

Time curves of tBHQ or TCDD-induced binding of Nrf2 and AhR to Nqo1 enhancer. Hepa1c1c7 cells were treated with tBHQ (30 μM) or TCDD (1 nM) for up to 5 h. ChIP was performed at indicated time points with antibodies against Nrf2 or AhR and real-time PCR was performed to amplify co-immunoprecipitated enhancer sequences. ChIP was performed using the protocol described under Material and Methods.

Fig. 8

Effect of TCDD on Ho-1 expression. Hepa1c1c cells were treated with tBHQ (30 μM, 4 h, positive control); CHX (10 μg/ml, 4 h); TCDD (1 nM, 4 h), or combinations of the agents. Total RNA was prepared and expression of Ho-1 mRNA was examined by real-time PCR as described under Material and Methods. Results represent means ± standard deviations from triplicate samples. The same experiment was repeated twice with similar results.

Fig. 9

Interaction between AhR and Nrf2. Cells were treated with tBHQ (30 μM) or TCDD (1 nM) for 4 h. Cell lysate was prepared and immunoprecipitation was performed as described under Material and Methods. (A) AhR-Arnt interaction. (B) AhR-Nrf2 interaction. (C) AhR-Keap1 interaction.

Fig. 10

A working model of Nqo1 induction by AhR agonists. AhR agonists, such as TCDD and B[a]P, binds and activates AhR. Activated AhR translocates into the nucleus and dimerizes with Arnt in order to bind to DRE of the Nqo1 enhancer. Activated AhR also interacts with Nrf2, either directly or through the Nrf2/Keap1 complex, to inhibit Nrf2 degradation leading to Nrf2 nuclear accumulation, dimerization of Nrf2 with a small Maf, and increased binding of Nrf2/Maf to ARE. AhR may physically interact with Nrf2 at the enhancer. The concerted actions of AhR and Nrf2 are responsible for Nqo1 induction by AhR agonists.