Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis

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• 作者：Jin Chai¹ ; Shi-Ying Cai² ; Xiaocong Liu¹ ; Wei Lian¹ ; Sheng Chen³ ; Liangjun Zhang¹ ; Xinchan Feng¹ ; Ying Cheng¹ ; Xiaochong He⁴ ; Yu He⁵ ; Lei Chen¹ ; Rongquan Wang¹ ; Huaizhi Wang⁵ ; ^{whuaizhi@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; James L. Boyer² ; Wensheng Chen¹ ; ^{wenshengchen@hotmail.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：ERAD ; endoplasmic reticulum (ER)-associated degradation ; ERM ; Ezrin/Radixin/Moesin ; GAPDH ; glyceroldehyde 3-phosphate dehydrogenase ; ALP ; alkaline phosphatase ; IF ; immunofluorescence ; IHC ; immunohistochemistry ; MRP2 ; multidrug resistance-associated protein 2 ; PKCα ; protein kinase C alpha ; PKCδ ; protein kinase delta ; PKCε ; protein kinase epsilon
• 刊名：Journal of Hepatology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：63
• 期：6
• 页码：1440-1448
• 全文大小：2276 K

文摘

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.

Methods

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.

Results

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.

Conclusions

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.