Bile secretion is a major function of the liver which is frequently impaired in diseases of the liver resulting in the syndrome of cholestasis. The long term objectives of this grant (since 1973) have been to characterize the basic transport mechanisms in hepatocytes and bile duct epithelial cells (cholangiocytes) that determine the secretion of bile and to define, at the cellular and molecular level, alternations in these mechanisms that result in cholestatic liver disease. Thus the specific aims of this proposal are: 1) to understand the molecular mechanisms for transcriptional regulation of membrane transporters in liver, kidney and intestine that are important determinants of bile formation and that undergo adaptive regulation in cholestatic liver injury. In particular we will examine the role of nuclear transcription factors in adaptive responses of several ABC transporters, Mrp2, Mrp3 and Bsep and the influence of cytokines on this process. Mechanisms of transcriptional regulation of the human MRP3 promoter will be sought as well as the effects of cholestasis on the expression of organic cation transporters (Octs) and Mrp4 in liver, kidney and intestine and the ameliorating effects of ursodeoxycholic acid. By understanding the mechanisms of these adaptive changes, ways may be found to diminish cholestatic liver injury; (2) to characterize post-transcriptional mechanisms of regulation of the expression of canalicular ABC transporters. Utilizing a series of Bsep-GFP mutants that cause PFIC-II in children, we will determine if chemical chaperones can correct defects in targeting of these mutants to apical domains when expressed in MDCK cells using confocal microscopy. The effects of agonists and inhibitors on post-transcriptional Bsep-GFP targeting will also be examined. The role of """"""""tethering"""""""" proteins (ezrin, radixin, EKARP, EBP50) in the targeting of Mrp2, Bsep and Mdr1 to the canalicular domain in hepatocytes will also be examined; (3) caracterization of transport mechanisms in cholangiocytes. The effects of gender and estrogens on the role of these tethering proteins will also be compared in cyclic AMP mediated secretion in cholangiocytes.
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