The overall objective of this proposal continues to be to define the critical cellular mechanisms culminating in lethal liver cell injury. Because of the importance of cholestatic liver injury in human disease, our program is working on the cellular mechanisms causing hepatocyte injury during cholestasis. Specifically, our long-term goal is to understand the cellular mechanisms by which bile salts, which accumulate in the liver during cholestasis, modulate hepatocyte apoptosis. Based on our preliminary data, we propose the novel CENTRAL HYPOTHESIS that hvdrophobic hepatotoxic bile salts induce hepatocvte apoptosis by a Fas-dependent mechanism while more hydrophilic bile salts inhibit apoptosis by actively stimulating a phosphoinositide 3-kinase (PI3K)-dependent survival pathway. We will now employ current and complementary molecular, biochemical, and cell biological approaches to ascertain how bile salts modulate apoptotic effector processes. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that toxic bile salts induce hepatocyte apoptosis by a Fas-dependent process resulting in: a) Fas oligomerization independent of Fas ligand; and b) formation of a death-inducing signaling complex (DISC) with caspase 8 activation. SECOND, we will test the hypothesis that toxic bile salts increase plasma membrane Fas receptors: a) by a mechanism dependent upon a redistribution of pre-existing cytoplasmic Fas to the plasma membrane via a microtubule-dependent transport pathway; and b) resulting in a mechanism of apoptosis dependent upon intracellular Fas translocation to the cell surface. FINALLY, non toxic, hydrophilic bile salts directly signal cell survival pathways by a phosphoinositide 3-kinase (PI3K)-dependent mechanism resulting in: a) activation of the antiapoptotic atypical protein kinase c isoforms (PKC? and PKCX.); and b) inhibition of apoptosis by atypical PKC-dependent activation of nuclear factor kappa B (NF-KB) and/or caspase phosphorylation. The proposal is innovative conceptually and technically as it tests new concepts for both bile salt-mediated cytotoxicity and apoptosis in general using sophisticated methodologies. The significance of the information generated is that it will help provide a framework for the potential development of novel therapeutic strategies effective in attenuating cholestatic liver injury.
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