Abstract
The most important biliary efflux transporter known so far is the multidrug resistance protein 2 (MRP2). Previously, we isolated and characterized the 5'-flanking region of the rat mrp2 gene. In the present study, we performed site-directed mutagenesis experiments indicating that both a Y-Box and a GC-Box in the rat mrp2 promoter are essential for the full basal expression of the gene, but have no significant relevance for its inducibility by the chemical carcinogen 2-acetylaminofluorene. Gel mobility shift experiments demonstrated the binding of the transcription factor CBF/NF-Y, but not of EFIA/YB-1, to the Y-Box. Site-directed mutations in the Y-Box decreasing reporter gene activity of a promoter construct prevented the binding of NF-Y. Consequently, NF-Y contributes substantially to the basal expression of the gene. A site-directed mutation in the GC-Box also reduced basal expression and resulted in a reduced complex formation with the transcription factor Sp1. The corresponding region of the human MRP2 promoter comprises no Sp1 site, but a Y-Box-like element binding YB-1 but not NF-Y, which, however, does not contribute to basal expression. In conclusion, NF-Y and Sp1 binding sites play a decisive role in the basal expression of the rat mrp2 gene, while the human MRP2 gene is regulated differently.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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2-Acetylaminofluorene / pharmacology
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ATP Binding Cassette Transporter, Subfamily B / biosynthesis*
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ATP Binding Cassette Transporter, Subfamily B / genetics
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ATP Binding Cassette Transporter, Subfamily B / metabolism
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ATP-Binding Cassette Transporters*
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Animals
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Binding Sites
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CCAAT-Binding Factor / metabolism*
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Carcinogens / pharmacology
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Carcinoma, Hepatocellular / genetics
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Carcinoma, Hepatocellular / metabolism
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Carrier Proteins / biosynthesis*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Gene Expression Regulation, Neoplastic* / drug effects
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Humans
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Liver Neoplasms / genetics
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Liver Neoplasms / metabolism
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Liver Neoplasms, Experimental / genetics
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Liver Neoplasms, Experimental / metabolism
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Membrane Transport Proteins*
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins*
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Mutagenesis, Site-Directed / physiology
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Promoter Regions, Genetic
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Rats
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Sp1 Transcription Factor / metabolism*
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Transfection
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Tumor Cells, Cultured
Substances
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ABCC2 protein, human
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ATP Binding Cassette Transporter, Subfamily B
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ATP-Binding Cassette Transporters
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Abcc2 protein, rat
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CCAAT-Binding Factor
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Carcinogens
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Carrier Proteins
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Membrane Transport Proteins
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins
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Sp1 Transcription Factor
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2-Acetylaminofluorene
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multidrug resistance-associated protein 1