Colorado PROFILES, The Colorado Clinical and Translational Sciences Institute (CCTSI)
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The overall objective of the present proposal is to define further the cellular and molecular mechanisms underlying the ontogenic expression of hepatic bile acid transport. It is our hypothesis that the developmental regulation of these proteins which mediate uptake and secretion of bile acids occurs predominantly at the level of transcription in response to ontogenic programming, the hormonal milieu, and an increasing substrate [ bile acid ] pool. The following specific aims are designed to test this hypothesis as well as to determine if alternative or complementary mechanisms (translational and post-translational) are involved in the process. The specific aims are to determine: (1) the factors involved in the developmental expression of the basolateral Na+-dependent and the canalicular potential-driven bile acid transporters; (2) the number of canalicular bile acid transporters, their driving forces [potential vs. ATP dependent] and their expression during perinatal development; and (3) the molecular mechanisms underlying the ontogenic and tissue-specific expression of the basolateral and canalicular bile acid transporters. An integrated experimental approach will employ membrane vesicle transport assays, immunochemical quantitation and immunocytochemical localization of the transport proteins, and Northern blot analysis of transporter mRNA levels. These studies will determine whether the canalicular ATP-dependent bile acid transporter is functional in neonatal liver and whether its activity can be distinguished from the potential- driven system through its pattern of expression and through selective reconstitution of transport activity into synthetic proteoliposomes. The cloning of the basolateral Na+-dependent and the canalicular potential- driven bile acid transporter genes including their promoters will allow the identification of regulatory elements involved in the ontogenesis of transport activity and mediating the effects of hormones and bile acids on gene transcription. The cis-acting elements and trans-acting factors that direct the liver-specific and ontogenic expression of these transporters will be defined through an analysis of DNA and nuclear protein interactions. The cis-regulatory regions of these genes will be characterized further by linking various portions of the promoter regions to a firefly luciferase reporter for transfection assays in cultured primary rat hepatocytes. The ability of bile acids to regulate the promoter activities of the transporters will be investigated in experiments utilizing in vitro transfection assays. Transgenic mice will be used to assess DNA regulatory regions required to direct the correct acinar, developmental, and tissue-specific pattern for expression of the Na+-dependent bile acid transporter. These studies will provide further insights into the factors contributing to the physiologic immaturity of hepatic excretory function and susceptibility to cholestatic liver disease in the newborn.
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