The overall objective of this proposal is to understand the cellular mechanisms by which bile salts, which accumulate in the liver during cholestasis, modulate hepatocyte apoptosis. The central hypothesis proposed is that hydrophobic hepatotoxic bile salts induce hepatocyte apoptosis by a Fas-dependent mechanism while more hydrophilic bile salts inhibit apoptosis by actively stimulating a phosphoinositide 3-kinase (PI3K)-dependent survival pathway. The proposal will use complementary molecular, biochemical, and cell biological approaches to ascertain how bile salts modulate apoptotic effector processes in three specific aims.
Specific aim one will 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.
Specific aim two 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.
Specific aim three will test the hypothesis that non toxic, hydrophilic bile salts directly signal cell survival pathways by a PI3K-dependent mechanism resulting in: a) activation of anti-apoptotic atypical protein kinase c isoforms; and b) inhibition of apoptosis by atypical PKC-dependent activation of nuclear factor kappa B (NF-kappa B) and/or caspase phosphorylation.
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