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

Background & Aims

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.

Introduction

Multidrug resistance-associated protein 2 (MRP2; ABCC2) is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily [1]. It is widely expressed in many human and rodent tissues, such as liver, kidney, intestine, brain, and breast [2], [3], [4]. MRP2/Mrp2 is localized on the apical membrane of epithelial cells in these tissues [1], [2], [3]. Specifically, it is expressed at the canalicular membrane of hepatocytes, the proximal tubule epithelial cells in kidney, and the brush-border membrane in small intestine for excreting conjugated organic anions, including bilirubin, bile acid divalent conjugates and drugs [1], [2]. In addition, MRP2/Mrp2 secretes glutathione into bile and is the major determinant of bile acid-independent bile flow [2]. Dysfunction of MRP2/Mrp2 leads to jaundice and hyperbilirubinemia in patients with Dubin-Johnson syndrome and in GY/TR⁻ EHBR rats [3], [4]. Jaundice is also seen in patients with obstructive cholestasis. Hepatic MRP2 mRNA expression was not changed in these patients [5], [6], but how its protein expression is regulated remains uncertain.

Previous studies in cholestatic rodent models have found reduced hepatic Mrp2 protein expression without changes in mRNA expression [7], [8], [9], indicating that post-transcriptional regulation most likely plays an important role in regulating Mrp2 function. Indeed, loss of Mrp2 from bile canalicular membranes without altering its mRNA expression in Radixin knockout mice and Radixin knockdown rat hepatocytes emphasized the importance of Radixin in the expression and function of rodent Mrp2 at post-transcriptional levels [10], [11]. Radixin is a member of the Ezrin/Radixin/Moesin (ERM) protein family that consists of two more closely related proteins, i.e. Ezrin and Moesin [12]. ERM proteins are tether proteins, they connect certain plasma membrane proteins with actin filaments in cells [12]. The expression of individual ERM proteins varies in different tissues. Specifically, Radixin was abundantly expressed in rat hepatocytes, where Ezrin was undetectable by immunofluorescent labeling [13]. Instead, Ezrin was detected in biliary epithelia in the rat. Claperon et al. confirmed that Ezrin was mainly detected in bile duct epithelial cells in young children [14]. Interestingly, they also detected Ezrin at the canalicular membrane of hepatocytes in the livers of patients with biliary atresia and neonatal sclerosing cholangitis. Furthermore, both Ezrin and Radixin were detected in human hepatoma HepG2 and HuH7 cells [15], [16]. However, it is not known if Ezrin plays any role in MRP2 protein expression in human hepatocytes.

Studies from Caco2 cells and rat intestine tissue indicate that Ezrin regulates MRP2/Mrp2 membrane expression and function [17], [18], [19]. When the intestine was treated with the protein kinase C (PKC)-α specific activator Thymeleatoxin, Ezrin Thr567 phosphorylation, Mrp2 brush-border membrane expression and efflux activity were reduced although the mechanism remained unexplained [18]. In contrast, activation of PKCα stimulated Ezrin phosphorylation in human breast carcinoma cells (MCF-7 cells) [20]. Elevated PKC activity, including PKCα, δ, and ε, was also reported in cholestatic rodent liver [7], [21]. However, it remains to be determined if PKCs are involved in Ezrin phosphorylation in human cholestatic livers, and whether this process alters MRP2 expression and function.

In this report, we investigated the molecular mechanism of MRP2 expression regulation in the livers of patients with gallstone obstructive cholestasis. We found that hepatic MRP2 protein expression was significantly reduced in these patients. Immunoprecipitation identified Ezrin but not Radixin as the ERM protein that interacted with MRP2 in human livers. Phosphorylation of Ezrin positively correlated with its ability to associate with MRP2 protein but negatively correlated with the total amount of MRP2 protein expression. Further studies in HepG2 cells indicated that phosphorylation of Thr567 in Ezrin by PKCs reduced MRP2 membrane expression, whereas ubiquitin E3 GP78 controlled the degradation of internalized MRP2 in human hepatic cells. Our findings provide a novel mechanistic explanation for the pathogenesis of jaundice seen in patients with obstructive cholestasis and possibly other forms of cholestasis. Understanding the regulation of human MRP2 protein expression regulation may lead to new strategies for treating patients with various forms of hyperbilirubinemia.