Sequence Analysis and Functional Characterization of the 5′-Flanking Region of the Rat Multidrug Resistance Protein 2 (MRP2) Gene

Abstract

Gene expression of the canalicular conjugate transportermrp2is inducible by treatment with the DNA-damaging agents 2-acetylaminofluorene (50 and 100 μM), and cisplatin (20 μM) in primary rat hepatocytes as well as in the rat hepatoma cell line H4IIE. Furthermore, phenobarbital (1 and 2 mM) inducesmrp2gene expression, probably explaining the increase in bile-salt-independent bile flow caused by phenobarbital, while the cholestatic drug ethinyl estradiol (10−6M) leads to an increase inmrp2mRNA but decreases Mrp2 protein level probablyviaa posttranscriptional mechanism. The 5′-flanking region of the ratmrp2gene was sequenced and cloned into a luciferase reporter vector. Transient transfection assays with reporter vectors containing unidirectionally deleted 5′-flanking regions using H4IIE cells indicate that two different sequences of 17 and 37 bases comprising a Y-Box and a GC-Box are required formrp2gene basal expression. Sequences mediating 2-AAF induction are located within a region 250 bases upstream of the translation start site while the inducing effect of phenobarbital seems to be mediated by another domain located further upstream.