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, 285 (9), 5993-6002

Interaction Between Oxidative Stress Sensor Nrf2 and Xenobiotic-Activated Aryl Hydrocarbon Receptor in the Regulation of the Human Phase II Detoxifying UDP-glucuronosyltransferase 1A10

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Interaction Between Oxidative Stress Sensor Nrf2 and Xenobiotic-Activated Aryl Hydrocarbon Receptor in the Regulation of the Human Phase II Detoxifying UDP-glucuronosyltransferase 1A10

Sandra Kalthoff et al. J Biol Chem.

Abstract

The defense against oxidative stress is a critical feature that prevents cellular and DNA damage. UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of xenobiotics, mutagens, and reactive metabolites and thus act as indirect antioxidants. Aim of this study was to elucidate the regulation of UGTs expressed in the mucosa of the gastrointestinal tract by xenobiotics and the main mediator of antioxidant defense, Nrf2 (nuclear factor erythroid 2-related factor 2). Xenobiotic (XRE) and antioxidant (ARE) response elements were detected in the promoters of UGT1A8, UGT1A9, and UGT1A10. Reporter gene experiments demonstrated XRE-mediated induction by dioxin in addition to tert-butylhydroquinone (ARE)-mediated induction of UGT1A8 and UGT1A10, which are expressed in extrahepatic tissue in humans in vivo. The responsible XRE and ARE motifs were identified by mutagenesis. Small interfering RNA knockdown, electrophoretic mobility shifts, and supershifts identified a functional interaction of Nrf2 and the aryl hydrocarbon receptor (AhR). Induction of UGT1A8 and UGT1A10 requires Nrf2 and AhR. It proceeds by utilizing XRE- as well as ARE-binding motifs. In summary, we demonstrate the coordinated AhR- and Nrf2-dependent transcriptional regulation of human UGT1As. Cellular protection by glucuronidation is thus inducible by xenobiotics via AhR and by oxidative metabolites via Nrf2 linking glucuronidation to cellular protection and defense against oxidative stress.

Figures

FIGURE 1.
FIGURE 1.
A, time-dependent regulation of UGT1A10 mRNA by TCDD (5 nm) and tBHQ (100 μm) in comparison to solvent (dimethyl sulfoxide, DMSO) in KYSE70 cells. The highest induction of UGT1A10 mRNA by TCDD was detectable after 48 h. Maximal tBHQ inducibility was observed after 24 h. B, time-dependent regulation of the UGT1A10 500-bp 5′-upstream region by TCDD and tBHQ in luciferase assay in KYSE70 cells. A maximal up-regulation of luciferase activity was observed after 48 h both by TCDD and tBHQ. WT, wild type.
FIGURE 2.
FIGURE 2.
A, luciferase reporter gene assay with a UGT1A10 wild type promoter construct and mutagenesis of different potential XRE- and ARE-binding sites. A significant reduction of both TCDD- and tBHQ-induced luciferase activity was observed for the constructs with mutagenized XRE-101, XRE-136, and the ARE-149 binding elements compared with wild type. B, comparison of UGT1A8, UGT1A9, and UGT1A10 induction by TCDD and tBHQ. There was a comparable TCDD and tBHQ inducibility for UGT1A8 and UGT1A10, whereas UGT1A9 showed a lower TCDD and no tBHQ inducibility. Significance is determined in relation to control vector. C, mutagenesis of the XRE-101 and ARE-149 binding sites of UGT1A8. Similar to UGT1A10, mutation of these binding sites led to a significant and simultaneous reduction of both TCDD- and tBHQ-induced luciferase activity in comparison to wild type construct. D, mutagenesis of the XRE-101 and ARE-143 binding sites of UGT1A9. In contrast to UGT1A8 and UGT1A10, mutagenesis of the ARE binding site of UGT1A9 did not affect TCDD inducibility. Significance is determined in relation to wild type construct. The XRE sequences of all mutant constructs are replaced with the sequence ‘AAATT,‘ and the ARE sequences are mutagenized to ‘AAATTTAAA‘. KYSE70 cells were treated with TCDD (5 nm) and tBHQ (100 μm) for 48 h (A–D). DMSO, dimethyl sulfoxide; WT, wild type.
FIGURE 3.
FIGURE 3.
A, effect of mutagenesis of the ARE of UGT1A10 corresponding to that of UGT1A9 on the inducibility by TCDD and tBHQ. (The sequences of the constructs are shown in Table 2.) In luciferase assays, UGT1A10 promoter inducibility by TCDD and tBHQ was significantly reduced with an ARE-containing Thr instead of Gly, in comparison to wild type construct. B, chimeric mutagenesis of the UGT1A9 ARE to correspond to the sequence of the UGT1A10 ARE led to tBHQ inducibility previously not observed with the wild type UGT1A9 sequence. (The sequences of the constructs are shown in Table 2.) Significance is determined in comparison to wild type construct. KYSE70 cells were treated with TCDD (5 nm) and tBHQ (100 μm) for 48 h (A and B). DMSO, dimethyl sulfoxide; WT, wild type.
FIGURE 4.
FIGURE 4.
A, Western blot confirmed the knockdown of AhR and Nrf2 by specific siRNA at different time points in KYSE70 cells. Cells were treated with siRNA and incubated 6 h later with either TCDD (5 nm) or tBHQ (100 μm) for an additional 24/48 h. B and C, siRNA-mediated knockdown of AhR and Nrf2 abolished both TCDD and tBHQ inducibility of UGT1A10 and UGT1A8 in luciferase assays. Significance was determined in relation to the construct treated with control siRNA. D, use of AhR siRNA led to a significant reduction of TCDD inducibility of UGT1A9 compared with treatment with control siRNA. Nrf2 siRNA did not affect TCDD inducibility of UGT1A9, which contrasts the findings with UGT1A10 and UGT1A8. In B and C, KYSE70 cells were treated with TCDD (5 nm) and tBHQ (100 μm) for 48 h. DMSO, dimethyl sulfoxide.
FIGURE 5.
FIGURE 5.
A, Western blot of Nrf2 and AhR in comparison to β-actin. KYSE70 cells were incubated with 5 nm TCDD, 100 μm tBHQ, solvent (all incubations for 24 h), and 100 nm AhR/Nr2 siRNA. Analyses of protein quantity showed that there was no induction of Nrf2 by TCDD and no induction of AhR by tBHQ. Furthermore, AhR siRNA does not affect the protein amount of Nrf2, and AhR protein amount was not diminished by Nrf2 siRNA. B, semiquantitative reverse transcription-PCR of Nrf2 and AhR mRNA was performed to show that both Nrf2- and AhR-mRNA amounts are not affected by TCDD or tBHQ in KYSE70 cells. DMSO, dimethyl sulfoxide.
FIGURE 6.
FIGURE 6.
Nuclear extracts for all EMSA experiments were prepared from KYSE70 cells treated with either 5 nm TCDD (for all experiments using XRE-probes) or 100 μmtBHQ (for all experiments using ARE-probes) for 48 h. A, electrophoretic mobility shift assay for AhR (XRE-101) and Nrf2 (ARE-149) binding elements. Shifts are shown in comparison to published consensus sequences and can be competed by excess unlabeled consensus sequence but not by peroxisome proliferator-activated receptor-γ (PPARγ; control) sequence. The ARE-binding site can be competed with excess of unlabeled XRE sequence and the other way around. B, supershift of labeled UGT1A10 oligonucleotides with the addition of specific antibody for AhR and Nrf2. A supershift of UGT1A10 XRE- and ARE-binding site with AhR as well as Nrf2 antibody (Ab) suggests binding of both transcription factors to both sites. C, electrophoretic mobility shift assay for UGT1A10 XRE-136 binding element. A supershifted band is observed with both AhR and Nrf2 antibody, and XRE-136 site can be competed with an excess of unlabeled ARE-149 sequence.

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