Abstract：
OBJECTIVE: Estradiol 17β-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation.
METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence.
RESULTS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis.
CONCLUSIONS: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence.
RESULTS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis.
CONCLUSIONS: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
摘要：
目的：雌二醇17β-D-葡萄糖醛酸苷（E217G）通过引发胞吞作用并进一步在细胞内保留小管转运蛋白Bsep和Mrp2，以cPKC和PI3K依赖性方式诱导胆汁淤积，分别。妊娠引起的胆汁淤积与E217G胆汁淤积作用有关，常规用熊去氧胆酸(UDCA)治疗。由于UDCA在E217G诱导的胆汁淤积中的保护机制尚不清楚，我们在这里确定了它的主要代谢物，牛磺熊去氧胆酸盐（TUDC），可以通过抵消cPKC和PI3K/Akt激活来防止小管转运蛋白的内吞作用。
方法：通过免疫印迹在分离的大鼠肝细胞中评估cPKC和PI3K/Akt的激活（评估膜结合和磷酸化形式，分别)。通过评估其荧光底物的顶端积累，在分离的大鼠肝细胞偶联（IRHCs）中定量了Bsep/Mrp2功能，CLF和GS-MF，分别。我们还研究过,在孤立的,灌流大鼠肝脏（IPRL），Bsep和Mrp2传输功能的状态，通过TC和DNP-SG的胆汁排泄来评估，分别,和Bsep/Mrp2免疫荧光定位。
结果：E217G激活了cPKC和PI3K/Akt依赖性信号，用TUDC预处理显著减弱了这些激活。在IRHC中，TUDC阻止了E217G诱导的CLF和GS-MF顶端积累的减少，和蛋白质磷酸酶的抑制剂未能抵消这种保护。在IPRL中，E217G诱导胆汁流量和胆汁排泄TC和DNP-SG的急性减少，这是由TUDC阻止的。免疫荧光研究表明，TUDC可预防E217G诱导的Bsep/Mrp2内吞作用。
结论：TUDC恢复了E217G受损的Bsep/Mrp2的功能和定位，通过以不依赖蛋白磷酸酶的方式防止cPKC和PI3K/Akt激活。
方法：通过免疫印迹在分离的大鼠肝细胞中评估cPKC和PI3K/Akt的激活（评估膜结合和磷酸化形式，分别)。通过评估其荧光底物的顶端积累，在分离的大鼠肝细胞偶联（IRHCs）中定量了Bsep/Mrp2功能，CLF和GS-MF，分别。我们还研究过,在孤立的,灌流大鼠肝脏（IPRL），Bsep和Mrp2传输功能的状态，通过TC和DNP-SG的胆汁排泄来评估，分别,和Bsep/Mrp2免疫荧光定位。
结果：E217G激活了cPKC和PI3K/Akt依赖性信号，用TUDC预处理显著减弱了这些激活。在IRHC中，TUDC阻止了E217G诱导的CLF和GS-MF顶端积累的减少，和蛋白质磷酸酶的抑制剂未能抵消这种保护。在IPRL中，E217G诱导胆汁流量和胆汁排泄TC和DNP-SG的急性减少，这是由TUDC阻止的。免疫荧光研究表明，TUDC可预防E217G诱导的Bsep/Mrp2内吞作用。
结论：TUDC恢复了E217G受损的Bsep/Mrp2的功能和定位，通过以不依赖蛋白磷酸酶的方式防止cPKC和PI3K/Akt激活。
