title: Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Chai
authors.affiliation: Liver Center, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Shi-Ying
authors.initials: SY
authors.lastname: Cai
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Xiaocong
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Lian
authors.affiliation: Department of Pediatrics, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Liangjun
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Xinchan
authors.initials: X
authors.lastname: Feng
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: School of Nursing, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Xiaochong
authors.initials: X
authors.lastname: He
authors.affiliation: Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Rongquan
authors.initials: R
authors.lastname: Wang
authors.affiliation: Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China. Electronic address: whuaizhi@gmail.com.
authors.forename: Huaizhi
authors.initials: H
authors.lastname: Wang
authors.affiliation: Liver Center, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
authors.affiliation: Department of Gastroenterology, Third Military Medical University, Chongqing 400038, PR China. Electronic address: wenshengchen@hotmail.com.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Chen
mesh_terms: D000328:Adult; D001648:Bile Canaliculi / Q000378:metabolism; D016022:Case-Control Studies; D002779:Cholestasis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D042882:Gallstones / Q000150:complications; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D008954:Models, Biological; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060285:Receptors, Autocrine Motility Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011493:Protein Kinase C; D012333:RNA, Messenger; D060285:Receptors, Autocrine Motility Factor; D013912:Threonine; D000099149:Ezrin; C000715087:ABCC2 protein, human; C071776:radixin; C555000:AMFR protein, human
keywords: Ezrin phosphorylation; Multidrug resistance-associated protein 2; Protease degradation; Protein kinase C; Ubiquitin ligase E3 GP78