Biochemical and Biophysical Research Communications
Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblasts
Section snippets
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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