? DESCRIPTION (provided by applicant): Cholangiocytes, which form the bile duct system in the liver, are the major target cells in a number of human cholestatic liver diseases. Although there has been significant improvement in the understanding of the pathophysiology of disease progression over the past two decades, the underlying cellular/molecular mechanisms remain largely unknown. Cholangiocytes are continuously exposed to high concentrations of bile salts at their apical membranes. Bile acids taken up by an apical sodium bile acid transporter (ASBT) on cholangiocytes are unidirectionally transported across the cell and secreted via specific transporters (MDR3 and Ostα/Ostβ) on the basolateral membrane. ASBT has been reported to be regulated by changes in bile acid concentration and inflammatory cytokines. Bile acids taken up by cholangiocytes have been reported to activate a number of intracellular signaling pathways including: PKC, PI3K, MAP Kinase, and ERK 1/2 allowing for normal physiological homeostasis. Loss of ASBT allows only conjugated bile acids (CBAs) to activate plasma membrane receptors as hydrophilic bile acids cannot easily enter cells by simple diffusion. We have recently reported that CBAs activate the AKT and ERK1/2 signaling pathways via the G protein coupled receptor (GPCR) sphingosine-1-phosphate receptor 2 (S1PR2) in hepatocytes and cholangiocytes. The levels of CBAs in serum and liver are significantly elevated in chronic cholestasis, which is correlated with bile duct obstruction. Our preliminary data indicates that: 1 S1PR2 is the predominant S1P receptor expressed in cholangiocytes; 2) taurocholate (TCA)-induced cell proliferation and migration are inhibited by a specific shRNA and an antagonist of S1PR2 in cholangiocytes; 3) bile duct ligation (BDL) induces the up-regulation of S1PR2 gene expression and down-regulation of ASBT expression in mouse primary cholangiocytes; 4) BDL- induced cholangiocyte proliferation and liver fibrosis are significantly reduced in S1PR2-/- mice; 5) both S1PR2 and SphK2 are up-regulated in the liver of mdr2-/- mice (a PSC mouse model). In addition, it has been reported that TCA concentration was dramatically elevated in the liver and serum after BDL in mice. Based on these studies and our preliminary results, we HYPOTHESIZE that CBA-mediated activation of the S1PR2/SphK2 signaling cascades plays a critical role in promoting chronic cholangiopathy in cholestatic liver diseases. Three specific aims are proposed to test our central hypothesis. 1) To define the role of S1PR2 and SphK2 in CBA-mediated cholestatic liver injury using the BDL mouse model; 2) To identify the molecular/cellular mechanisms by which CBA-mediated S1PR2/SphK2 activation promotes cholestatic liver injury; 3) To test the therapeutic strategy for cholestatic liver injury by targeing S1PR2/SphK2 using chemical inhibitors and genetic tools in mdr2-/- mice, a cholestasis model of PSC. Completion of these specific aims wi...