Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblasts

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Abstract

The nuclear factor-E2 p45-related factor (Nrf) 2 is a transcription factor for the antioxidant responsive element-mediated induction of enzymes responsible for conjugation. Since multidrug resistance-associated protein1 (Mrp1/Abcc1) is an ATP-binding cassette transporter which plays an important role in the cellular extrusion of conjugated metabolites and, therefore, acts synergistically with conjugating enzymes for the cellular detoxification of xenobiotics, we examined the possibility that Nrf2 is also involved in the expression of Mrp1 in mouse embryo fibroblasts. The constitutive expression levels of Mrp1 mRNA and protein were significantly lower in Nrf2 (−/−) cells compared with those in wild type cells. In addition, significant induction by diethyl maleate was observed in wild type, but not in Nrf2 (−/−), cells, suggesting the involvement of Nrf2 in both the constitutive and inducible mRNA and protein expression of Mrp1. In addition, the uptake of [3H]2,4-dinitrophenyl-S-glutathione, a typical substrate of Mrp1, into isolated membrane vesicles also demonstrated that Nrf2 regulates the transport activity of glutathione conjugates in mouse fibroblasts. This is the first demonstration that Nrf2 is required for the constitutive and inducible expression of Mrp family proteins.

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Materials and methods

Materials. Unlabeled and [3H]labeled 2,4-dinitrophenyl-S-glutathione (DNP-SG; 50 μCi/nmol) were synthesized enzymatically with unlabeled and [glycine-2-3H]GSH (NEN Life Sciences), 1-chloro-2,4-dinitrobenzene, and GST (Sigma Chemical, St. Louis, MO) as described previously [19]. Monoclonal anti-Mrp1 antibody (MRPr1) was purchased from Alexis Biochemicals (San Diego, CA). All other chemicals used were commercially available and of reagent grade.

Mouse embryo fibroblasts, prepared either from wild

Involvement of Nrf2 in the expression of Mrp1 in mouse embryo fibroblasts

The amount of Mrp1 mRNA was compared in control and Nrf2 (−/−) fibroblasts by real time PCR under both constitutive and inducible conditions (Fig. 1A). We also examined the expression of GST-Pi (Fig. 1B) and γ-GCS-h (Fig. 1C) as the positive control, since the involvement of Nrf2 in the expression of these enzymes has been reported. Constitutive expression of Mrp1, GST-Pi, and γ-GCS-h was reduced in Nrf2 (−/−) fibroblasts to 38%, 51%, and 32% of that observed in the control fibroblasts,

Discussion

The purpose of the present study was to investigate the involvement of Nrf2 in the expression of Mrp1. Using embryo fibroblasts prepared from control and Nrf 2 (−/−) mice, we compared the mRNA and protein levels of Mrp1 (Fig. 1, Fig. 2). Initially, we quantified the mRNA expression levels of Mrp1 by real time PCR using a Light Cycler. The constitutive mRNA expression of Mrp1 was significantly lower in fibroblasts prepared from Nrf2 (−/−) mice compared with wild type fibroblasts (Fig. 1).

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