Elsevier

Biochemical Pharmacology

Volume 81, Issue 1, 1 January 2011, Pages 178-184
Biochemical Pharmacology

LPS-induced dissociation of multidrug resistance-associated protein 2 (Mrp2) and radixin is associated with Mrp2 selective internalization in rats

https://doi.org/10.1016/j.bcp.2010.09.016Get rights and content

Abstract

Multidrug resistance-associated protein 2 (Mrp2) is an ATP-dependent export pump that mediates the formation of bile-salt-independent bile flow. Disruption of the canalicular localization of Mrp2, without changes in its expression, is observed in chronic liver failure and is accompanied by oxidative stress. We reported previously that Mrp2 is rapidly internalized from the canalicular membrane during acute oxidative stress induced by lipopolysaccharide (LPS) in the rat liver. A disturbance in the colocalization of Mrp2 and radixin (which crosslinks actin with interacting membrane proteins) and endocytic retrieval of Mrp2 are present in chronic liver failure. However, the C-terminal phosphorylation status of radixin (p-radixin; functional form) and its protein–protein interaction with Mrp2 were not examined in the pathological cholestatic situation. In this study, we examined whether the C-terminal phosphorylation status of radixin and its interaction with Mrp2 were affected by LPS-induced experimental liver failure with cholestasis, and whether this condition was accompanied by Mrp2 internalization in the rat liver. At 3 h after LPS treatment, the canalicular expression of Mrp2 was decreased, without variation of the other canalicular transporters. Similarly, the canalicular localization of radixin was decreased after LPS treatment. These results show that LPS treatment decreased the total amount of the active form of p-radixin and the amount of radixin that coimmunoprecipitated with Mrp2, and that LPS treatment impaired the protein–protein interaction between Mrp2 and radixin. In conclusion, LPS-induced cholestasis seems to be caused by posttranscriptional regulation of Mrp2, which is due to the disruption of its interaction with radixin and by its dephosphorylation.

Introduction

The multidrug resistance-associated protein 2 (MRP2) and the bile-salt export pump (BSEP/ABCB11) are involved in the formation of bile-salt-independent and -dependent bile flow, respectively. The canalicular secretion of several amphiphilic organic anions, including bilirubin glucuronides, glutathione (GSH), and its conjugates, is mediated by MRP2, which is a conjugate export pump encoded by the mrp2 gene [1]. In addition to the static expression of MRP2 on the canalicular surface, its dynamic insertion and internalization processes are of great importance, as its steady-state expression level is directly dependent on these turnover rates. Therefore, disruption of these turnover rates is thought to lead to cholestatic jaundice. Oxidative stress markers are closely related with the progression of chronic cholestatic disorder in patients with chronic hepatic failure (primary biliary cirrhosis (PBC) and hepatitis C virus (HCV) infection) [2], [3]. Disruption of the canalicular localization of MRP2, without variation in its expression, is observed in the liver of these patients [4]. Several studies demonstrated that experimental cholestasis induced by bile duct ligation (BDL) [5], taurolithocholic acid [6], estradiol-17β glucuronide [7], and phalloidin [8] is closely associated with impaired excretory function of Mrp2, which may be attributed to its endocytic retrieval from the canalicular membrane. We also demonstrated that Mrp2 internalization is observed under ethacrynic acid (EA)- and tert-butylhydroperoxide-induced acute oxidative stress in experimental perfused rat livers [9], [10], [11]. Moreover, oxidative-stress-triggered signaling pathways that lead to the activation of novel protein kinase C (nPKC) cause the rapid retrieval of Mrp2 from the canalicular surface in the rat liver [10]. In experimental liver failure with cholestasis, treatment with lipopolysaccharide (LPS), which is a bacterial endotoxin, causes the rapid retrieval of Mrp2 from the canalicular membrane [12]. In addition, we showed that oxidative stress is a triggering factor of LPS-induced Mrp2 internalization [13].

The ezrin–radixin–moesin (ERM) family of proteins crosslinks cytoskeletal actin filament (F-actin) with integral membrane proteins. This anchoring function of ERM is regulated by the C-terminal phosphorylation status of these proteins, via the transition between active (C-terminal phosphorylated) and inactive (C-terminal dephosphorylated) forms [14]. Radixin is the dominant ERM protein in the canalicular membrane of the liver. Mice lacking radixin develop conjugated hyperbilirubinemia because of the impaired localization of Mrp2 on the canalicular membrane surface, which suggests that radixin is required for the canalicular localization of Mrp2 [15]. Disturbed colocalization of Mrp2/MRP2 and radixin, as well as endocytic retrieval of MRP2, are observed in the liver of PBC patients and several experimental cholestatic models, which may be associated with a failure in the anchoring of Mrp2/MRP2 to the canalicular membrane [4], [16], [17], [18]. However, the C-terminal phosphorylation status of radixin (p-radixin) and its protein–protein interaction with Mrp2/MRP2 were not examined in these pathological cholestatic situations. Recently, we demonstrated that the activation of conventional PKC (cPKC) impaired the localization of Mrp2 at the apical membrane surface of the rat intestine, but not of the multidrug resistance protein 1 (Mdr1, which is another apical membrane protein). This was accompanied by the downregulation of C-terminally phosphorylated ezrin (which is expressed dominantly in the intestine) and by a decrease in its interaction with Mrp2 [19]. Here, we propose that the LPS-induced alteration in the phosphorylation status of radixin may cause its dissociation from Mrp2 and lead to the internalization of Mrp2 in the rat liver. In addition, we examined whether the effect of LPS on protein localization was specific to Mrp2, rather than a nonspecific event accompanied by the internalization of other biliary transporters, such as Bsep and Mdr1.

Section snippets

Chemicals and antibodies

LPS was obtained from Wako Pure Chemical (Osaka, Japan). A rabbit anti-Mrp2 polyclonal antibody (EAG15) was raised against the C-terminal 12-amino-acid sequence of rat Mrp2. Rabbit anti-phosphorylated ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) (p-ERM) antibody was purchased from Millipore (Temecula, CA). Goat anti-rat/Alexa Fluor 488 and goat anti-mouse/Alexa Fluor 546 antibodies were from Molecular Probes (Eugene, OR). Mouse anti-Mrp2 (M2III5) antibody was from Abcam (Cambridge, MA).

Effect of LPS on the hepatic injury and redox statuses

LPS induces liver injury via the production of inflammatory cytokines and reactive oxygen species (ROS). We evaluated serum ALT level and hepatic GSH content as indexes of liver injury and oxidative stress, respectively. In LPS-treated rats, serum ALT leakage was significantly increased by about 10 times compared with the saline-treated control (Table 1). Hepatic GSH content was decreased by LPS treatment to 45.9 ± 8.2% of the saline-treated control (Table 1).

Effect of LPS on the localization of canalicular membrane proteins

Previous reports indicate that rapid

Discussion

Impaired steady-state canalicular surface expression of Mrp2 and jaundice are observed in radixin knockout mice. Therefore, radixin is now considered as the primary molecule that anchors Mrp2 to F-actin [15]. In parallel with this study, we revealed that the LPS-induced rapid retrieval of Mrp2 from the canalicular surface accompanied by GSH decrease is also triggered by oxidative stress [13]. In the present study, Mrp2 expression at the canalicular membrane was reduced to 71%, without changing

Acknowledgments

We thank Prof. Sachiko Tsukita for kindly providing the rat anti-radixin (R21) antibody. This work was supported by a Grant-in-Aid for Scientific Research (A) (21249003) and a Grant-in-Aid for Young Scientists (B) (21790141) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References (36)

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