Progressive familial intrahepatic cholestasis type 1 (PFIC1; Byler's disease, OMIM 211600) is a systemic disease, whose principal manifestation is hepatic. Biliary excretion of bile salts is diminished in PFIC1 resulting in progressive cholestatic liver disease. A mouse model of PFIC1 is characterized by inappropriately enhanced intestinal uptake of bile salts. Defects in ATP8B1 underlie PFIC1 by as yet to be determined mechanisms. FIC1, a P-type ATPase membrane protein, is found in liver, intestine and pancreas. Functional studies in liver membranes and CHOK1 cells ascribe an aminophospholipid flippase activity to FIC1. Preliminary studies in our laboratory have shown that activity of the Farnesoid X-Receptor (FXR) is diminished in children with FIC1 disease and in FIC1 anti-sense treated Caco-2 cells. Absence of FIC1 activity is associated with a disruption in the nuclear localization of FXR. Diminished FXR activity may lead to reduced hepatic canalicular bile acid excretion and enhanced ileal bile acid transporter via effects on the promoters for these transporters. FIC1 is an intergral membrane protein that may alter the asymmetry of aminophospholipids in the lipid bilayer. We hypothesize that changes in FIC1 expression alter membrane asymmetry and transduce an unknown intracellular signaling pathway that alters the post-translational modification of FXR. Regulatory response to alterations in FIC1 expression will be studied in cell lines (e.g. Caco-2, CHO) utilizing siRNA and FIC1 expression constructs. Downstream targets will be identified by microarray analysis with confirmation by northern blot or RT/PCR. Relevant signal transduction pathways will be identified in the presence or absence of FIC1 utilizing well characterized inhibitors, antibodies and proteomic approaches. The specific post-translation modifications of FXR will be determined by biochemical and mutational analysis. These innovative studies will delineate a novel set of mechanisms that underlie the pathogenesis of PFIC1 and will develop a new area of cellular regulation of lipid membrane asymmetry.
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