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, 395 (3), 599-609

Role of Nrf2 in the Regulation of the Mrp2 (ABCC2) Gene

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Role of Nrf2 in the Regulation of the Mrp2 (ABCC2) Gene

Valeska Vollrath et al. Biochem J.

Abstract

The Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate-cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE-Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gsta1/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, beta-NF (beta-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5'-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (-1391 to -1381) and ARE-1 (-95 to -85). Deletion analyses demonstrated that the proximal region (-185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2-protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.

Figures

Figure 1
Figure 1. Co-induction of Mrp2, GCLC and Gsta1/Gsta2 genes by BHA in the mouse liver
Total RNA and proteins were isolated from the livers of control and BHA-treated animals. The levels of Mrp2, GCLC and Gsta1/Gsta2 mRNAs and proteins were determined by Northern (A) and Western (B and C) blotting respectively, as described in the Materials and methods section. (D) Nuclear run-on assay. Isolated nuclei from hepatocytes of control and BHA-treated mice were incubated in the presence of [α-32P]UTP. The nuclear-labelled RNA was used as a hybridization probe against the specific DNA probes that were immobilized on a nylon membrane (ø-X174 plasmid DNA was included to assess non-specific hybridization). Sizes of bands are given in either kb or kDa, as indicated. P-gp, P-glycoprotein.
Figure 2
Figure 2. Dose-dependence and time course of the induction of Mrp2 and GCLC mRNAs by xenobiotics in hepatoma cell lines
Mouse Hepa 1-6 cells (A) and human HepG2 cells (B) were exposed to different concentrations of xenobiotics (BHA, 2,4,5-T, 2AAF or β-NF) for 24 h. Total RNA (10 μg) from control (DMSO) and treated cells was subjected to Northern blot analysis using specific probes for mouse or human genes. In order to determinate the time course of the induction of Mrp2 (C) and GCLC (D) mRNAs, Hepa 1-6 cells were incubated in medium containing 0.1% DMSO (control), 25 μM β-NF, 250 μM BHA, 500 μM 2,4,5-T or 200 μM 2AAF. At various intervals up to 24 h, RNA was harvested, and the specific mRNA levels were determined by Northern blotting. The relative contents of Mrp2 and GCLC were determined using the PhosphorImager system. The mRNA levels were normalized to values observed in control cells, and are plotted as the relative amounts of mRNA. Results are means±S.D. for four independent experiments.
Figure 3
Figure 3. Nucleotide sequence of the 5′-flanking region of the Mrp2 gene
The nucleotide sequence of the 5′-flanking region of the mouse Mrp2 gene from nucleotides −1895 to +103 is shown with numbering relative to the transcription start site (+1, arrow). The consensus binding sites for putative regulatory elements are underlined, and the respective transcription factors are given above the sequences. The positions of the putative ARE-1 (−95 to −85) and ARE-2 (−1391 to −1381) sequences are indicated. The cloning primer (GSP2N) is also indicated by an arrow, and the ATG start codon is shown in boldface. The full nucleotide sequence is available at GenBank® under accession number AY905402. The 5′-flanking region of the mouse Mrp2 gene was isolated, cloned and sequenced as described in the Materials and methods section. AP-1, activator protein 1.
Figure 4
Figure 4. Deletion analysis of the 5′-flanking region of the Mrp2 gene
The 5′-flanking region constructs were transiently transfected into Hepa 1-6 cells, and after 24 h the LUC (A) and CAT (B) activities were determined. The results are expressed as the percentage activity of the entire isolated 5′-flanking region (with p−1895/+99-LUC or p−1895/+99-CAT normalized to 100%), after subtraction of the activity of the empty plasmid reporter. Transfections were performed in triplicate, and results are means±S.D. (C) Schematic representation of the 5′-flanking region of the Mrp2 gene. The reporter gene constructs and the putative binding sites for transcription factors, including ARE-1 and ARE-2 sequences, are shown. AP-1, activator protein 1.
Figure 5
Figure 5. Activity of the Mrp2 reporter gene constructs induced by xenobiotics
(A) Hepa 1-6 cells were transfected with constructs containing either both ARE sequences (p−1895/+99-CAT) or only the ARE-1-like sequence (p−185/+99-CAT). CAT activity was measured after treatment with 250 μM BHA, 500 μM 2,4,5-T or 25 μM β-NF for 24 h. (B) Results expressed as the induction relative to the activity of the construct treated with vehicle (0.1% DMSO). Results are means±S.D. for three independent experiments.
Figure 6
Figure 6. Overexpression of Nrf2 enhances the activity of the Mrp2 5′-flanking region
The construct p−1895/+99-CAT or p−185/+99-CAT was co-transfected into Hepa 1-6 cells with either an Nrf2 expression plasmid or one of two Nrf2-mutant expression plasmids: p-Nrf2-TA or p-Nrf2-DN. The activity is shown relative to that obtained with the plasmid pBR322 used as a control. Results are means±S.D. for six independent experiments.
Figure 7
Figure 7. ARE sequences of the 5′-flanking region of the Mrp2 gene bind to the Nrf2–protein complexes
(A) EMSA was performed using nuclear extracts (5 μg) obtained from control Hepa 1-6 cells or cells overexpressing Nrf2-DN, and the radiolabelled probe containing the ARE-1 or ARE-2 sequence. For competition experiments, the unlabelled (cold) probe was included at a 50-fold molar excess. (B) EMSA was performed using nuclear extracts (5 μg) from Hepa 1-6 cells (control) or cells overexpressing Nrf2-DN and the radiolabelled probe containing the ARE-1 or mutant ARE-1 sequence (ARE-1M). For competition experiments, the unlabelled (cold) probe was included at a 10-, 25- or 50-fold molar excess. The positions of the DNA–protein complexes are indicated by asterisks (bands a, b and c).
Figure 8
Figure 8. Xenobiotics increased the binding of Nrf2–protein complexes to the ARE-1 sequence
(A) EMSA was performed using nuclear extracts (5 μg) obtained from control HepG2 cells or those treated with 25 μM β-NF or 250 μM BHA for 3 h and the radiolabelled probe containing the ARE-1 or ARE-2 sequence. For competition experiments, the unlabelled (cold) probe was included at a 50-fold molar excess. (B) A supershift assay was performed using nuclear extracts (15 μg) from HepG2 cells (control) or those treated with BHA and the radiolabelled probe containing the ARE-1 sequence in the presence or absence of anti-Nrf2 antibody (Ab). The shifted bands are denoted by lines, and supershifted bands are denoted by the bracket.
Figure 9
Figure 9. Sequence alignment of the human, mouse and rat MRP2/Mrp2 5′-flanking regions
ClustalW alignment of the proximal 5′-flanking region of each gene is shown. Shaded boxes indicate nucleotides that are identical across all three species. The conserved regions containing transcription-factor-binding sites and mouse ARE-1 and human ARE-like core sequence are boxed. The transcription start sites for mouse and human are indicated by *M and *H respectively. USF, upstream stimulatory factor.

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