MECHANISMS OF LIVER CELL INJURY

这项建议的总体目标是了解细胞 在肝脏中蓄积的胆盐的机制 胆汁淤积，调节肝细胞凋亡。提出的中心假设是 疏水性肝毒性胆盐通过诱导肝细胞凋亡 Fas依赖的机制，而更亲水的胆盐通过 主动刺激磷脂酰肌醇3-激酶(PI3K)依赖性存活 路径。该提案将使用互补的分子、生化和细胞 确定胆盐如何调节细胞凋亡效应的生物学方法 三个具体目标的过程。具体目标之一将检验该假设 毒性胆盐通过Fas依赖性途径诱导肝细胞凋亡 导致a)不依赖于Fas配体的Fas寡聚；以及b)形成 具有caspase 8激活的死亡诱导信号复合体(DISC)。 具体目标二将检验有毒胆盐增加血浆的假设 膜Fas受体：a)通过依赖于再分布的机制 预先存在的细胞质Fas通过微管依赖到质膜 运输途径；以及b)导致一种依赖于 细胞内Fas转位到细胞表面。具体目标三将 检验无毒、亲水性胆盐直接向细胞发出信号的假设 PI3K依赖机制导致的生存途径：a)激活 抗凋亡的非典型蛋白激酶c亚型；以及b)抑制 非典型PKC依赖的核因子-kappaB激活引起的细胞凋亡 (核因子-kappa B)和/或caspase磷酸化。

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.