This is a renewal application to further define the ontogeny of hepatic ion transport as well as define the mechanisms by which transporters are modified under conditions of cholestasis. The overall objective of this competing renewal application is further defined by the mechanisms underlying the expression of basolateral and canalicular transporters for bile acids and other organic anions during development and cholestatic liver disease. Since transcriptional activation of the liver Na+ /bile acid cotransporter gene (Ntcp) is largely responsible for the abrupt expression of transport activity in the perinatal period, the investigators propose that a change in chromating structure, reflected in an increase in DNASE I hypersensitivity and a decrease in gene methylation precedes the onset of transcription during development. Transgenic mice expressing various portions of the 5' untranslated region linked to the luciferase reporter gene will be used to define sequences that direct tissue- and developmental stage-specific expression of Ntcp. Transgenic animals will be used to show that the rapid postnatal increase in Ntcp transcription could in part be mediated by prolactin. To demonstrate the importance of the Na+/bile acid cotransporter to hepatobiliary function, the Ntcp gene will be rendered nonfunctional in mice by targeted disruption. The phenotypes of the mice with null mutations will be defined including whether liver disease (cholestasis) is induced and how the mutation alters hepatobiliary function and enterohepatic cycling of bile acids. The signaling mediating the targeting of the liver and ileal Na+/bile acid cotransporters to the plasma membrane will be determined through transfection and analysis of native transporters, truncated mutants lacking cytoplasmic tails, and chimeric transporters comprised of portions of the liver and ileal transporters. Finally, the development of the canalicular multi specific organic anion transporter (cMOAT/Mrp1) will be defined including a correlation of transport activity in canalicular membrane vesicles with levels of Mrp2 mRNA and protein. The investigators will determine the mechanisms underlying the effects of bile acids and the inflammatory cytokines TNF-alpha and IL-beta on expression of Ntcp and Mrp2 through transfection of chimeric promoter/reporter gene constructs into primary hepatocytes. DNA foot printing and gel mobility shift assays will determine the importance of known or novel cis-acting elements and trans-activating factors in regulating gene expression. These studies should provide insights into the biology and pathobiology of hepatic anion transporters and help explain the susceptibility of the infant to develop cholestatic liver disease.

R37HD020632

1985-09-01

2009-03-31