This proposal focuses on the function and expression of MRP2 (ABCC2), the ABC transporter that mediates the biliary excretion of glucuronide and glutathione conjugates from the hepatocyte into bile. The proposal builds on our findings that 1) MRP2 transports tauroursodeoxycholate (TUDC), a bile acid used for the treatment of cholestatic liver disease, including intrahepatic cholestasis of pregnancy (ICP), 2) estradiol-17[3- D-glucuronide (E217G) causes increased permeability of the hepatocellular tight junction that coincides with endocytic retrieval of MRP2 and the bile salt export pump (BSEP) from the canalicular membrane and decreased bile flow, and 3) identification of upstream open reading frames (uORF) in the 5' leader sequences of rat,human and mouse MRP2.
Aim 1 will characterize MRP2-mediated transport of hydrophilic bile acids, including TUDC, (3-muricholicacid and tetrahydroxy bile acids, and the importance of heterotropic and homotropic activation in their transport. We will also examine the ability of MRP2 variants to transport these bile acids. We will use an MRP2 baculovirus expression system in Sf9 cells to characterize transport.
Aim 2 will determine the role of protein kinase C (PKC) and tight junction permeability in E217G-induced cholestasis. We will test the hypothesis that E217G disrupts hepatocyte tight junctions in a PKC-dependent manner that coincides with endocytic retrieval of key transporters, MRP2 and BSEP, from the canalicular membrane, leading to cholestasis. We will use 4 model systems to test this hypothesis: in vivo studies in the rat, the isolated perfused rat liver, isolated rat hepatocyte couplets, and HepG2 cells coupled with confocal fluorescence and electron microscopy, to monitor the localization of transporters and tight junction proteins after treatment with E217G and in the presence and absence of PKC inhibitors.
Aim 3 will characterize the importance of the uORF in rat,human and mouse MRP2 mRNA on the translational regulation of MRP2 protein. We will characterize the differential tissue expression of MRP2 mRNA transcripts expressing the uORF and use site-directed mutagenesis to disrupt the uORF coupled with in vitro translation assays to investigate the importance of leaky scanning and the sequence of the peptide encoded by the uORF in regulating translation of Mrp2/MRP2. We will also determine whether the peptide encoded by the rat uORF is expressed in vivo. Significance: MRP2 is critical for elimination of polar metabolites from liver and kidney, as well decreasing the intestinal absorption of dietary carcinogensand its expression in liver is decreasedin pregnancy. Characterization of mechanisms of regulation of the activity, localization and expression of MRP2 can lead to improved drug therapy for women in pregnancy and development of therapeutic measures to increase MRP2 function in cholestatic liver disease, particularly in ICP.
