Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene.
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.
Vollrath
Valeska
V
Department of Gastroenterology, School of Medicine, Catholic University of Chile, Casilla 114-D, Santiago, Chile. valeska@med.puc.cl
Wielandt
Ana M
AM
Iruretagoyena
Mirentxu
M
Chianale
Jose
J
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001483:Base Sequence; D001646:Bile / Q000187:drug effects / Q000378:metabolism; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D015533:Transcriptional Activation; D019324:beta-Naphthoflavone / Q000494:pharmacology
C000715087:ABCC2 protein, human; D000975:Antioxidants; D007527:Isoenzymes; C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D002083:Butylated Hydroxyanisole; D019324:beta-Naphthoflavone; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005721:Glutamate-Cysteine Ligase