The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C 4 (LTC 4 ) (K m ؍ 0.1 and 1 M). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC 4 and methotrexate transport in insideout membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH 2 -proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, K m (LTC 4 ) values of the chimera decreased ϳ4-fold and K m (methotrexate) values increased ϳ5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased ϳ3-fold; (ii) when the NH 2 -proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH 2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC 4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC 4 and anticancer agents. Two representative genes for the ATP binding cassette (ABC)1 transporter superfamily proteins, P-gp/MDR1 and MRP1, mediate acquisition of a multidrug resistance phenotype through altered membrane transport of various anticancer agents in tumor cells (1, 2). MRP1 confers resistance to a number of relatively hydrophobic natural product drugs including certain anthracyclines, epipodophyllotoxins, methotrexate, and vinca alkaloids (3-7). However, unlike P-gp, MRP1 can also transport a wide range of relatively hydrophilic anionic compounds including potential physiological substrates such as LTC 4 and E 2 17G (8 -15). Topology studies of MRP1 have demonstrated that MRP1 and P-gp share a similar core structure consisting of two membrane-spanning domains (MSD2 and MSD3) and two nucleotide-binding domains (NBD1 and NBD2), referred to as the MDR-like core (16). The primary distinguishing characteristic of MRP1 and its related proteins, MRP2, -3, -6, and -7, is an additional NH 2 -terminal region forming a membrane-spanning domain (MSD1) with five
The human multidrug resistance protein 2 (MRP2/ABCC2), expressed on the bile canalicular membrane, mediates the multispecific efflux of several organic anions, including conjugates of glucuronate, sulfate, and glutathione. Expression of MRP2 can be altered in response to environmental stimuli such as cholestasis and jaundice. We previously reported that MRP2 mRNA expression levels are decreased in the nontumorous part of hepatitis C virus-infected human liver tissues, and that inflammatory cytokines inhibit MRP2 expression in human hepatic (HepG2) cells. We investigated the molecular mechanisms by which inflammatory cytokines modulate MRP2 gene expression in hepatic cells. Treatment of human hepatic cells with interleukin-1 (IL-1) or tumor necrosis factor ␣ resulted in a decrease in the protein and mRNA levels of MRP2. IL-1 inhibited the transcriptional activity of MRP2 promoter constructs by 40%, and this inhibition of MRP2 promoter activity was mediated through the interferon stimulatory response element (ISRE). Electrophoretic mobility shift assays with IL-1-treated nuclear extracts showed a decrease in the formation of DNA protein complexes, specifically those including interferon regulatory factor 3 (IRF3). Expression of recombinant human IRF3 increased MRP2 promoter activity. Treatment with a specific extracellular signal-regulated kinase inhibitor relieved IL-1-induced MRP2 mRNA downregulation and abrogated the binding of IRF3 to the ISRE element. In conclusion, IL-1 induces downregulation of the MRP2 gene by inactivating IRF3 binding to ISRE on the MRP2 promoter in human hepatic cells; this inactivation is accomplished via interference with the extracellular signal-regulated kinase pathway.
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