Instituto de Fisiología Experimental (IFISE) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas - Universidad Nacional de Rosario, Suipacha 570, S2002LRL Rosario, Argentina.
Instituto de Fisiología Experimental (IFISE) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas - Universidad Nacional de Rosario, Suipacha 570, S2002LRL Rosario, Argentina.
Biochim Biophys Acta Mol Basis Dis. 2018 Apr;1864(4 Pt A):1072-1085. doi: 10.1016/j.bbadis.2018.01.015. Epub 2018 Jan 31.
Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.
由于胆汁酸转运体（如 BSEP 和 MRP2）的内化和细胞内滞留增加导致的胆汁酸分泌受损是胆汁淤积的主要共同发病机制。然而，控制这一过程的机制尚不清楚。我们对雌二醇 17β-D-葡糖苷酸（E17G）诱导的胆汁淤积进行了特征描述，这是一种部分模拟妊娠诱导性胆汁淤积的实验模型。网格蛋白介导的内吞作用（CME）抑制剂，如单丹磺酰尸胺（MDC）或钾耗竭，但不是胞饮介导的内吞作用抑制剂 Filipin 和 Genistein，可阻止 E17G 诱导的 BSEP 和 MRP2 内化，并阻止这些转运体在分离的大鼠肝细胞对（IRHC）中的活性受损。免疫荧光和共聚焦显微镜研究表明，在 E17G 处理的 IRHC 中，MRP2 与网格蛋白、AP2 和 Rab5 的共定位显著增加，这三种都是 CME 机制的重要组成部分。在夹心培养的大鼠肝细胞中用 siRNA 敲低 AP2 可完全阻止 E17G 诱导的 BSEP 和 MRP2 内化。MDC 可显著阻止这种内化，以及在分离和灌注的肝脏中 E17G 诱导的胆汁流量和 BSEP 和 MRP2 底物的胆汁分泌受损。在对照大鼠中，BSEP 和 MRP2 主要存在于筏（富含小窝蛋白）微域中，而在 E17G 处理的动物肝脏中则主要存在于非筏（富含网格蛋白）微域中，从那里它们可以很容易地被招募到 CME 中。总之，我们的研究结果表明，CME 是 E17G 诱导的胆汁淤积中 BSEP 和 MRP2 内化的机制。这些转运体从小窝蛋白到非小窝蛋白微域的转移可能是在胆汁淤积条件下这些转运体被内化的先决条件。
