Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway Is Involved in Estradiol 17β-D-Glucuronide-Induced Cholestasis: Complementarity with Classical Protein Kinase C
Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway Is Involved in Estradiol 17β-D-Glucuronide-Induced Cholestasis: Complementarity with Classical Protein Kinase C
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DOI：
10.1002/hep.23846
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发表时间：
2010-10-01
期刊：
影响因子：
13.5
通讯作者：
Roma, Marcelo G.
中科院分区：
文献类型：
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作者：
Boaglio, Andrea C.;Zucchetti, Andres E.;Roma, Marcelo G.
雌二醇 17 β-D-葡萄糖醛酸 (E(2)17G) 是一种内源性胆汁淤积代谢物，可诱导与胆汁分泌相关的小管转运蛋白的内吞内化：胆汁盐输出泵 (Bsep) 和多药耐药相关蛋白 2 (Mrp2)。我们评估了磷酸肌醇 3 激酶 (PI3K) 是否参与 E(2)17G 诱导的胆汁淤积。根据对最终 PI3K 效应子蛋白激酶 B (Akt) 磷酸化的评估，E(2)17G 激活 PI3K。当将 PI3K 抑制剂渥曼青霉素 (WM) 预先给予离体大鼠肝细胞对 (IRHC) 时，它部分阻止了 E(2)17G 诱导的 IRHC 分泌荧光 Bsep 和 Mrp2 底物（分别为胆酰赖氨酰荧光素和谷胱甘肽甲基荧光素）比例的降低。另一种 PI3K 抑制剂 2-Morpholin-4-yl-8-phenychromen-4-one 和 Ala 抑制剂 (Calbiochem 124005) 显示出类似的保护作用。 IRHC 免疫染色和共聚焦显微镜分析表明，E(2)17G 诱导的 Bsep 和 Mrp2 的内吞内化被 WM 广泛阻止；这种效应被微管破坏剂秋水仙碱完全阻断。 WM 的保护作用是经典蛋白激酶 C (cPKC) 抑制剂 5,6,7,13-四氢-13-甲基-5-氧代-12H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-12-丙腈 (Go6976) 提供的保护作用的补充；这表明 PI3K 和 cPKC 信号通路在 E(2)17G 诱导的胆汁淤积中存在差异性和互补性。在离体灌注大鼠肝脏中，门静脉内注射 E(2)17G 引发 Bsep 和 Mrp2 的内吞作用，并且伴随着胆汁流量的持续减少以及 Bsep 和 Mrp2 底物 [H-3]牛磺胆酸盐和谷胱甘肽的胆汁排泄，直到灌注期结束。与Go6976不同，WM并没有阻止最初的衰退，但它极大地加速了这些参数的恢复正常，以及Bsep和Mrp2以微管依赖性方式重新插入微管膜。结论：PI3K/Akt 信号通路通过 cPKC 内吞的小管转运蛋白持续内化，参与 E(2)17G 诱导的胆道分泌衰竭。 （肝病学 2010；52：1465-1476）
Estradiol 17 beta-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Ala inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo [2,3-a] pyrrolo[3,4-c] carbazole-12-propanenitrile (Go6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intra-portal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [H-3]taurocholate and glutathione until the end of the perfusion period. Unlike Go6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. Conclusion: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC. (HEPATOLOGY 2010;52:1465-1476)