Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis.
Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis.
复制标题
人阻塞性胆汁淤积中，小管膜 MRP2/ABCC2 内化由 Ezrin Thr567 磷酸化决定。
DOI：
10.1016/j.jhep.2015.07.016
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发表时间：
2015-12
影响因子：
25.7
通讯作者：
Chen W
中科院分区：
文献类型：
--
作者：
Chai J;Cai SY;Liu X;Lian W;Chen S;Zhang L;Feng X;Cheng Y;He X;He Y;Chen L;Wang R;Wang H;Boyer JL;Chen W
多药耐药相关蛋白2（MRP2）排泄结合的有机阴离子，包括胆红素和胆汁酸。MRP2功能异常导致患者黄疸。啮齿类动物的研究表明，Radixin在决定Mrp2小管膜表达中起关键作用。然而，目前尚不清楚胆汁淤积中人肝MRP2的表达是如何调节的。我们评估了胆管结石阻塞引起的阻塞性胆汁淤积患者肝脏MRP2的表达，并研究了HepG2细胞中的调节机制。Western blot检测到阻塞性胆汁淤积患者（n=30）肝脏MRP2蛋白表达显著降低至非胆汁淤积对照组（n=23）的25%。免疫沉淀鉴定了Ezrin而不是Radixin与人肝脏中的MRP2相关，并且磷酸Ezrin Thr567的增加量与胆汁淤积肝脏中共沉淀的MRP2的量呈正相关，而Ezrin和Radixin总蛋白水平在胆汁淤积中没有变化。进一步的详细研究表明Ezrin Thr567磷酸化在HepG2细胞中的MRP 2内化中起重要作用。由于在这些胆汁淤积的肝脏中检测到PKCα、δ和ε的表达增加，我们进一步证实这些PKC刺激HepG2细胞中Ezrin磷酸化并降低MRP 2膜表达。最后，我们确定GP78为参与MRP 2蛋白酶体降解的关键泛素连接酶E3。胆汁淤积期间肝PKC的活化导致Ezrin Thr567磷酸化，导致MRP2内化和降解，其中涉及泛素连接酶E3 GP78。这一过程为阻塞性胆汁淤积患者的黄疸提供了一种机制性解释。
Multidrug resistance–associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.