Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, Amsterdam, The Netherlands.
J Hepatol. 2010 Nov;53(5):918-26. doi: 10.1016/j.jhep.2010.05.015. Epub 2010 Jul 17.
BACKGROUND & AIMS: Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110α/β/γ), specific functions of which, in liver, are unclear. In other cell types, p110γ is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110γ mediates bile salt-induced hepatocyte cell death.
Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110γ was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA.
All bile salts tested activated p110β, while p110α was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110γ. Inhibition of p110γ attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110γ markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor α- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation.
PI3K p110γ is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK.
甘氨胆酸（GCDC）和牛磺胆酸（TLC）是具有肝毒性和胆汁淤积性的胆盐，而牛磺熊脱氧胆酸（TUDC）则具有细胞保护和抗胆汁淤积作用。然而，它们都通过磷脂酰肌醇-3-激酶（PI3K）依赖性机制（“PI3K 悖论”）发挥作用。肝细胞表达三种催化 PI3K Ⅰ类同工型（p110α/β/γ），但其在肝脏中的具体功能尚不清楚。在其他细胞类型中，p110γ 与有害作用相关。为了阐明 PI3K 之谜，我们确定了疏水性和亲水性胆盐是否会以不同的方式激活肝细胞中的不同 p110 同工型，以及 p110γ 是否介导胆盐诱导的肝细胞死亡。
建立了同工型特异性 PI3K 活性测定法，以确定胆盐在大鼠肝细胞中对同工型的激活作用。通过免疫印迹法测定 Akt 和 JNK 的激活情况。用疏水性胆盐刺激后，通过 Hoechst 染色后形态学观察和 caspase-3/-7 活性或 caspase-3 切割分析来确定肝细胞凋亡。用特定的 siRNA 进行药理学抑制（AS604850）或敲低抑制 PI3K p110γ 的活性或表达。
所有测试的胆盐均激活了 p110β，而 TUDC 和 GCDC 仅激活了 p110α。有趣的是，只有疏水性胆盐激活了 p110γ。p110γ 的抑制减弱了 GCDC 诱导的 Akt 和 JNK 激活，但不改变 TUDC 或 cAMP 诱导的大鼠肝细胞中的 Akt 信号转导。抑制或敲低 p110γ 可显著减弱大鼠肝细胞和人肝癌细胞系中疏水性胆盐诱导的凋亡，但不改变 Fas、肿瘤坏死因子α和依托泊苷诱导的凋亡。钙（++）耗竭可防止 GCDC 在大鼠肝细胞中的毒性，但不影响 GCDC 诱导的 Akt 和 JNK 激活。
PI3K p110γ 被疏水性胆盐激活，但不被亲水性胆盐激活。胆盐诱导的肝细胞凋亡部分通过 PI3K p110γ 依赖性信号通路介导，可能涉及 JNK。
