Research Article

4-Phenylbutyrate modulates ubiquitination of hepatocanalicularMRP2 and reduces serum total bilirubin concentration

Hisamitsu Hayashi1,⇑, Tadahaya Mizuno1, Reiko Horikawa2, Hironori Nagasaka3,Takashi Yabuki1, Hajime Takikawa4, Yuichi Sugiyama1,⇑

1Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo 113-0033, Japan; 2Division of Endocrinology and Metabolism, National Center for Child Health and Development, 2-10-1 Okura,

Setagaya-ku, Tokyo 157-8535, Japan; 3Division of Metabolism, Chiba Children’s Hospital, 579-1 Heta Cho, Midori-ku, Chiba 266-0007, Japan;4Department of Medicine, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo173-8605, Japan

Background & Aims: Multidrug resistance-associated protein 2 MRP2/Mrp2 is degraded via ubiquitination-mediated targeting

(in humans, MRP2; in rodents, Mrp2) mediates biliary excretionof bilirubin glucuronides. Therefore, upregulation of MRP2/Mrp2 expression may improve hyperbilirubinemia. We investi-gated the effects of 4-phenylbutyrate (4PBA), a drug used to treatornithine transcarbamylase deficiency (OTCD), on the cell surfaceexpression and transport function of MRP2/Mrp2 and serum T-Bilconcentration.Methods: MRP2-expressing MDCKII (MRP2-MDCKII) cells andrats were studied to explore the change induced by 4PBA treat-ment in the cell surface expression and transport function ofMRP2/Mrp2 and its underlying mechanism. Serum and liverspecimens from OTCD patients were analyzed to examine theeffect of 4PBA on hepatic MRP2 expression and serum T-Bil con-centration in humans.Results: In MRP2-MDCKII cells and the rat liver, 4PBA increasedthe cell surface expression and transport function of MRP2/Mrp2. In patients with OTCD, hepatic MRP2 expression increasedand serum T-Bil concentration decreased significantly after 4PBAtreatment. In vitro studies designed to explore the mechanismunderlying this drug action suggested that cell surface-resident

Journal of Hepatology 20

Keywords: ABC transporter; Jaundice; Protein degradation.Received 17 August 2011; received in revised form 25 November 2011; accepted 28November 2011; available online 13 January 2012⇑ Corresponding authors. Address: Laboratory of Molecular Pharmacokinetics,Department of Medical Pharmaceutics, Graduate School of PharmaceuticalSciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,Japan. Tel.: +81 3 5841 4770/4773; fax: +81 3 5841 4766.E-mail addresses: hayapi@mol.f.u-tokyo.ac.jp (H. Hayashi), sugiyama@mol.f.u-tokyo.ac.jp (Y. Sugiyama).Abbreviations: MRP2/Mrp2, human/rat isoforms of multidrug resistance-associ-ated protein 2; BSEP/Bsep, human/rat isoforms of the bile salt export pump; DJS,Dubin–Johnson Syndrome; TR�, transport deficient rat; EHBR, Eisai hyperbiliru-binemic rat; 4PBA, 4-phenylbutyrate; OTCD, ornithine transcarbamylase defi-ciency; P-gp, P-glycoprotein; SD, Sprague–Dawley; T-Bil, total bilirubin; CMVs,canalicular membrane vesicles; LTx, liver transplantation; qPCR, quantitativePCR; E217bG, 17b estradiol 17b-D-glucuronide; Css, plasma, plasma concentrations atsteady-state; Vss, bile, biliary excretion rate at steady-state; Css, liver, average hepaticconcentration at steady-state; CLtotal, total plasma clearance; CLbile, plasma, biliaryclearance normalized by circulating plasma; CLbile, liver, biliary clearance normalizedby the liver concentration; DPPIV, dipeptidyl peptidase IV; Ub, ubiquitin; UbDGG, Ubin which the two glycines in C-terminal end were deleted; UbDGG/I44A, incorporatingUb I44A in addition to deletion of the last two glycines; E.V., empty vector; TUDCA,tauroursodeoxycholic acid; SUMO, small Ub-like modifier.

to the endosomal/lysosomal degradation pathway and that4PBA inhibits the degradation of cell surface-resident MRP2/Mrp2 by reducing its susceptibility to ubiquitination.Conclusions: 4PBA activates MRP2/Mrp2 function throughincreased expression of MRP2/Mrp2 at the hepatocanalicularmembrane by modulating its ubiquitination, and therebydecreases serum T-Bil concentration. 4PBA has thus therapeuticpotential for improving hyperbilirubinemia.� 2012 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Multidrug resistance-associated protein 2 (in humans, MRP2; inrodents, Mrp2) belongs to the ABC transporter family and islocated at the bile canalicular membrane. It plays an indispens-able role in the biliary excretion of a wide variety of organicanions, including glutathione, glutathione conjugates, and sul-fated and glucuronidated bile acids [1]. Together with the func-tion of the bile salt export pump (in humans, BSEP; in rodents,Bsep), an ABC transporter involved in extrusion of monovalentbile salts into bile [2], the MRP2/Mrp2-dependent secretion ofthese solutes provides the osmotic driving force for the formationof bile [3].

MRP2/Mrp2 is also important for the biliary excretion of bili-rubin glucuronides. Bilirubin, an end product of heme catabolismin mammals, is taken up by hepatocytes and excreted into thebile after conjugation mainly with glucuronic acid. Therefore,MRP2/Mrp2 function plays a pivotal role in homeostasis of biliru-bin, as shown in Dubin–Johnson Syndrome (DJS) and transportdeficient (TR�) and Eisai hyperbilirubinemic rats (EHBR), all ofwhich are caused by a congenital absence of MRP2/Mrp2 andshow hyperbilirubinemia [4–6]. These results suggest that upreg-ulation of MRP2 expression will improve hyperbilirubinemia inpatients with DJS.

We reported previously that treatment with 4-phenylbutyrate(4PBA), a drug used to treat ornithine transcarbamylasedeficiency (OTCD), increased the cell surface expression and thetransport capacity of BSEP/Bsep at a clinically achievable

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JOURNAL OF HEPATOLOGY

concentration in OTCD patients by prolonging the half-life ofBSEP/Bsep on the plasma membrane [7]. Little is known aboutthe post-translational mechanism that regulates cell surfaceexpression of MRP2/Mrp2 and BSEP/Bsep except that radixinand Na+/H+ exchanger regulatory factor 1 tether and recycleMrp2 to the canalicular membrane via interaction with the PDZdomain [8–11] and that HAX-1 and myosin II regulatory lightchain mediate the internalization of Bsep from the apical mem-brane and its trafficking to the apical membrane, respectively[12,13]. However, both transporters are considered to share thesame machinery in part, because in patients with intrahepaticand obstructive cholestasis, canalicular immunolabeling of bothtransporters is disrupted, whereas the canalicular expression ofP-glycoprotein (in humans, P-gp; in rodents, p-gp), an ABC trans-porter belonging to the same subfamily as BSEP/Bsep, is pre-served [14,15]. Disrupted canalicular localization of bothtransporters was also observed in experimental cholestatic ani-mal models [16,17]. These previous reports have generated inter-est in the effects of 4PBA on the expression level and transportfunction of MRP2/Mrp2.

Our current study explored the influence of 4PBA on theexpression level and transport function of MRP2/Mrp2 inMRP2-expressing MDCKII (MRP2-MDCKII) cells, McA-RH7777cells, and Sprague–Dawley (SD) rats and on the hepatic MRP2expression and the serum total bilirubin (T-Bil) concentrationin OTCD patients. The mechanism underlying this drug actionwas also investigated using MRP2-MDCKII cells, McA-RH7777cells, and SD rats.

Materials and methods

A detailed description of Materials and methods is presented in the Supplemen-tary material. All materials and methods used standard techniques and commer-cially available reagents.

Animals

Male SD rats (6–7 weeks old) were purchased from Nippon SLC (Shizuoka, Japan).All animals were maintained under standard conditions with a reverse dark–lightcycle and were treated humanely. Food and water were available ad libitum. SDrats given 0.6 g/kg/day 4PBA or vehicle by gavage in three divided doses for10 days were subjected to the preparation of canalicular membrane vesicles(CMVs) and the in vivo infusion study. The studies reported in this manuscriptwere carried out in accordance with the guidelines provided by the InstitutionalAnimal Care Committee (Graduate School of Pharmaceutical Sciences, The Uni-versity of Tokyo, Tokyo, Japan).

Sampling of liver tissue from and measurement of laboratory parameters in OTCDpatients

Small liver samples were collected from seven OTCD patients who received 4PBAtreatment (OTCD-4PBA(+)) when they underwent liver transplantation (LTx). Thefirst control group consisted of organ donors whose liver specimens wereobtained before subsequent transplantation. Because the amount of donor livertissue was matched with the body size of the recipient before transplantation,the liver segment that remained after size matching was available for this study.For the second control group (OTCD-4PBA(�)), samples were obtained by liverneedle biopsy from three OTCD patients (5-R, 6-R, and 7-R in Table 2) from thetest group mentioned above before 4PBA treatment. This second control groupalso included a liver specimen taken from an OTCD patient (8-R in Table 2)who underwent LTx without 4PBA treatment. Liver specimens were immediatelysnap-frozen in liquid nitrogen and preserved at �70 �C after sampling. Serum wascollected on two occasions: firstly, on the day before beginning 4PBA treatment,and secondly, on the day before LTx after daily administration of 4PBA to OTCDpatients or from organ donors who did not receive 4PBA treatment. The results

Journal of Hepatology 2012

of laboratory analyses of these samples are summarized in Table 2. The studywas approved by the institutional ethics review boards and informed consentwas obtained from all patients’ parents before assessment and from organ donorsbefore liver transplantation.

Statistical analysis

Experiments were repeated at least three times, and the data in the figures arepresented as the mean ± SE. The significance of differences between two variablesand multiple variables was calculated at the 95% confidence level by Student’s ttest and by one-way ANOVA with Tukey’s test, respectively, using Prism software(GraphPad Software, Inc., La Jolla, CA).

Results

4PBA-mediated upregulation of MRP2/Mrp2 expression at the cellsurface

We used MRP2-MDCKII cells and McA-RH7777 cells, a rat hepa-toma cell line, to investigate the effect of 4PBA on MRP2/Mrp2, asthe immunocytochemical analysis in this study showed thatexogenously overexpressed MRP2 in MRP2-MDCKII cells andendogenously expressed Mrp2 in McA-RH7777 cells are predom-inantly localized at the apical membrane (Supplementary Fig. 1),consistent with their cellular localization in hepatocytes. InMRP2-MDCKII cells, 4PBA treatment altered the expression levelof MRP2 in a concentration- and time-dependent manner; theoptimal condition was 1 mM for 24 h (Fig. 1A and B), which is aclinically achievable concentration [7]. A cell surface biotinyla-tion study demonstrated that, under optimal conditions (1 mM,24 h), 4PBA increased MRP2 expression at the plasma membrane2.5-fold (Fig. 1C). The expression level of endogenously expressedP-gp, another apical transporter, was not affected in the wholecell lysate and cell surface fraction (Fig. 1A–C). In contrast tothe increase in MRP2 protein, quantitative PCR (qPCR) analysisshowed that MRP2 mRNA expression was not significantlyincreased by 4PBA treatment (p = 0.17) (Fig. 1D). The expressionof MRP2 mRNA was evaluated after normalizing the values rela-tive to the expression of b-actin, because b-actin mRNA expres-sion was not affected by 4PBA treatment (data not shown). InMcA-RH7777 cells as well as in MRP2-MDCKII cells, 4PBA treat-ment increased endogenous Mrp2, without significantly changingMrp2 mRNA level, but did not affect endogenous P-gp expression(Fig. 1A, B, and D).

To examine this 4PBA effect in vivo, SD rats were subjected toan infusion study with [3H]-17b estradiol 17b-D-glucuronide([3H]-E217bG), a typical MRP2/Mrp2 substrate [18], and to apreparation of CMVs after 10-day administration of 0.6 g/kg/day4PBA, a dose approved in children [7]. In this infusion experi-ment, the plasma concentrations at steady state (Css, plasma) andbiliary excretion rate at steady state (Vss, bile) of [3H]-E217bGdid not differ significantly between vehicle- and 4PBA-treatedSD rats, whereas the average hepatic concentration at steadystate (Css, liver) of [3H]-E217bG was 1.6-fold lower in 4PBA-treatedrats than in vehicle-treated rats (Table 1). Consequently, totalplasma clearance (CLtotal) and biliary clearance normalized bythe circulating plasma concentrations (CLbile, plasma) for [3H]-E217bG did not differ significantly between vehicle- and 4PBA-treated SD rats. By contrast, biliary clearance normalized by theliver concentration (CLbile, liver) for [3H]-E217bG was 1.8-foldhigher in 4PBA-treated rats than in vehicle-treated rats (Table 1).These results suggest that 4PBA increases the biliary excretion of

vol. 56 j 1136–1144 1137

A

C D E F

G H

B

0 6 12 24Incubation time (h)

MRP2

β-actin

0 6 12 24Incubation time (h)

MRP2-MDCKII McA-RH7777

MRP2-

MDCKII McA-

RH7777

Concentration (mM)

McA-RH7777

P-gp

MRP2

P-gp

Mrp2

β-actin

P-gp

Mrp2

β-actin

P-gp

Concentration (mM)

MRP2

β-actin

MRP2-MDCKII

P-gp

5037

250150

kDa

250150

250150

kDa

250150

5037

250150

kDa

250150

5037

250150

kDa

250150

5037

250150

kDa

250150

0 0.1 1 2.5 0 0.1 1 2.5

Vehicle

4PBA

Mrp2

DPPIV

175kDa

83

Incubation time (h)0 6 12 24

Incubation time (h)0 6 12 24

Incubation time (min)

MR

P2/β

-act

inex

pres

sion

(a.u

.)

43210 M

rp2/

β-ac

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sion

(a.u

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mR

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on (a

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expr

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.u.)

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0.0

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-gp

expr

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on (a

.u.)

43210

0.2

0.1

0.0

DMSO4P

BA

**

**

*

***

**

***

**

Concentration (mM)0 0.1 1 2.5

MR

P2/β

-act

inex

pres

sion

(a.u

.)

*

Concentration (mM)0 0.1 1 2.5

43210 M

rp2/

β-ac

tinex

pres

sion

(a.u

.)

43210

*

**

DMSO4PBA

Vehicle4PBA

DMSO4PBA

Vehicle4PBA

n.s.

n.s. Upt

ake

(pm

ol/m

g pr

otei

n) 10075502500.0 0.5 1.0 1.5 2.0

V (pmol/min/mg protein)

V/S

(µl/m

in/m

g pr

otei

n) 504030201000 250 500 750 1000

Vehicle4PBA

Fig. 1. Effect of 4PBA treatment on MRP2 expression in MDCKII cells and on endogenous Mrp2 expression in McA-RH7777 cells and the rat’s liver. (A and B) Effect of4PBA treatment on MRP2/Mrp2 expression. MRP2-MDCKII and McA-RH7777 cells were treated with 1 mM 4PBA for the indicated times (A) or with the indicated 4PBAconcentrations for 24 h (B) before preparation of the whole cell lysate. (C) Determination of MRP2 expression on plasma membrane in MRP2-MDCKII cells. Cell surfacefractions were isolated after 1 mM 4PBA treatment for 24 h, by cell surface biotinylation. (D) Measurement of MRP2/Mrp2 mRNA expression. RNA was isolated from MRP2-MDCKII and McA-RH7777 cells after 1 mM 4PBA treatment for 24 h. Each bar represents the mean ± SE of three independent experiments. n.s., not significant. (E–H) CMVsprepared from SD rats given 0.6 g/kg/day 4PBA or vehicle for 10 days were used for these studies. (E and F) Time profile and Eadie–Hofstee plot of uptake of [3H]-E217bG byCMVs prepared from 4PBA- and vehicle-treated SD rats. The uptake of 1 lM [3H]-E217bG for the times indicated (E) or for 1 min in the presence of various concentrations(0.1–100 lM) of E217bG (F) was examined at 37 �C in the medium containing 5 mmol/L ATP or AMP. The uptake of [3H]-E217bG was obtained by subtracting the value inthe presence of AMP from that in the presence of ATP. Data are presented as mean ± SE of three independent experiments. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001. (G) Determinationof Mrp2 expression in CMVs. (H) Measurement of Mrp2 mRNA expression. Each bar represents the mean ± SE of three independent experiments. In (A–C and G), arepresentative result of three independent experiments is shown.

Research Article

[3H]-E217bG via the canalicular membrane. Although Vss, bile wasnot increased by treatment with 4PBA, the absence of an effectcan be explained by considering that Vss, bile is determined notonly by the biliary excretion process across the canalicular mem-brane but also by other parameters, and that there is a rate-lim-iting step for the biliary excretion process. Vss, bile is calculatedusing the equation Vss, bile = CLbile, plasma � Css, plasma [7], where,based on the well-stirred model, CLbile, plasma is calculated fromhepatic blood flow rate (QH), the unbound fraction in blood (fb),and intrinsic hepatic clearance (CLint, h) as follows: CLbile, plasma =QH � fb � CLint, h/(QH + fb � CLint, h) [19]. Therefore, if fb � CLint, h,for which Mrp2 function is a component, is much higher thanQH (fb � CLint, h� QH), CLbile, plasma is approximated to QH, indicat-ing that both CLbile, plasma and Vss, bile are determined in a bloodflow-limited manner and are independent of Mrp2 function. This

1138 Journal of Hepatology 2012

is true for [3H]-E217bG because CLbile, plasma for this compound(33.5 ± 1.1 and 40.2 ± 2.6 ml/min/kg for vehicle- and 4PBA-trea-ted rats, respectively,) is close to QH. Even if such a postulate(fb � CLint, h� QH) was not the case for [3H]-E217bG, it isassumed that CLbile, plasma will not be affected by Mrp2 functionfor the following reason: CLint, h, a component of CLbile, plasma,includes the intrinsic clearance of uptake (PSinf) and efflux (PSeff)across the sinusoidal membrane and intrinsic biliary clearance(PSbile), in which Mrp2 is involved, and is expressed as CLint, h =PSinf � PSbile/(PSbile + PSeff) [20]. In the case of [3H]-E217bG, PSbile

is considered to be much higher than PSeff (PSbile� PSeff) becauseMRP2 facilitates the transport of [3H]-E217bG across the canalic-ular membrane [18], whereas there is no evidence for the pres-ence of such transporters on the sinusoidal membrane underphysiological conditions. Therefore, the CLint, h for [3H]-E217bG

vol. 56 j 1136–1144

Table 1. Pharmacokinetic parameters of [3H]-E217bG during constant infusion at 0.6 g/kg/day for 10 days into 4BPA- or vehicle-treated SD rats.

Css, plasma(pg/ml)

Css, liver(pg/ml)

Vss, bile(pg/min/kg)

CLtotal(ml/min/kg)

CLbile, plasma(ml/min/kg)

CLbile, liver(ml/min/kg)

Vehicle (n = 4) 642 ± 32 325 ± 24 2.14 ± 0.07 29.4 ± 1.5 33.5 ± 1.1 6.70 ± 0.474PBA (n = 5) 549 ± 32 202 ± 14** 2.20 ± 0.11 34.5 ± 2.0 40.2 ± 2.6 11.8 ± 0.4***

Data represents the mean ± SE. Significantly different from vehicle-treated SD rats by Student’s t test (⁄⁄p <0.01; ⁄⁄⁄p <0.001).

Table 2. Clinical and laboratory parameters of OTCD patients and donors before LTx.

Clinical parameters Laboratory parameters

Patients Age at LTx Sex Administration period of 4PBA

AST (IU/L) ALT (IU/L) GGT (IU/L) T-Bil (mg/dl)

OTCD patients 1-R 3 mo M 3 mo 26 (38) 21 (26) 216 (595) 0.34 (2.03)2-R 5 mo M 3 mo 66 (113) 61 (58) 174 (616) 0.10 (0.36)3-R 5 mo M 5 mo 23 (22) 26 (14) 281 (40) 0.70 (4.06)4-R 7 mo M 1 mo 49 (38) 41 (31) 211 (19) 0.08 (0.47)5-R* 3 yr 4 mo F 2 mo 35 (32) 35 (39) 42 (61) 0.20 (0.70)6-R* 4 yr 6 mo F 2 mo 35 (35) 34 (31) 44 (50) 0.30 (0.80)7-R* 5 yr 8 mo F 3 mo 54 (71) 61 (95) 35 (78) 0.20 (0.80)8-R 2 yr 9 mo F Not treated 37 26 14 0.32

Donors 1-D 29 yr F Not treated 29 17 13 0.922-D 37 yr M Not treated 16 25 32 0.523-D 35 yr M Not treated 20 34 37 0.524-D 26 yr F Not treated 14 11 11 1.095-D 33 yr F Not treated 12 11 6 0.386-D 35 yr F Not treated 17 9 7 0.917-D 32 yr F Not treated 20 24 16 0.568-D 37 yr M Not treated 16 17 15 0.559-D 37 yr F Not treated 16 15 7 0.9210-D 38 yr F Not treated 14 11 9 0.56

Note. The parenthetic values are recorded before 4BPA treatment.Normal values: AST, <35 IU/L; ALT, <35 IU/L ; c-GTP, <60 IU/L; T-Bil, <1 mg/dl.⁄Patients undergoing a liver needle biopsy for diagnostic reasons before 4BPA treatment.LTx, liver transplantation; AST, aspartate transaminase; ALT, alanine aminotransferase; GGT, c-glutamyl transpeptidase; T-Bil, total bilirubin; R, recipient; D, donor; yr,years; mo, months; M, male; F, female.

JOURNAL OF HEPATOLOGY

is nearly equal to PSinf, indicating that Mrp2 makes no contribu-tion to CLint, h, CLbile, plasma, and Vss, bile.

The ATP-dependent uptake of [3H]-E217bG by CMVs was lin-ear up to 2 min; within the linear range of uptake, the ATP-dependent uptake was 2.5-fold higher at 2 min in 4PBA-treatedrats than in vesicles from vehicle-treated rats (Fig. 1E). Kineticanalysis revealed that the initial ATP-dependent uptake of [3H]-E217bG into CMVs from 4PBA- and vehicle-treated rats could bedescribed by a saturable component with apparent Km values of7.6 ± 2.4 and 5.5 ± 1.0 lM, and a non-saturable component witha clearance of 3.2 ± 0.8 and 2.5 ± 0.2 ll/min/mg protein, respec-tively (Fig. 1F). These Km values for [3H]-E217bG are consistentwith previously reported values [18]. Given that the CMVs fromthe EHBR almost completely eliminated the ATP-dependentuptake of [3H]-E217bG [18] and that the uptake study usingmembrane vesicles from rat Mrp2-expressing Sf9 cells demon-strated that the Mrp2-dependent transport of [3H]-E217bG

Journal of Hepatology 2012

involved both a high-affinity site and a lower-affinity non-satura-ble site [21], it is likely that the presence of the two componentsin Fig. 1F is due to Mrp2-mediated [3H]-E217bG transport com-prised of its high-affinity site and its lower-affinity non-saturablesite, and is not due to the involvement of multiple transporters.4PBA treatment increased the Vmax value of [3H]-E217bG from79.1 ± 21.7 to 274 ± 52 pmol/min/mg protein (Fig. 1F). Westernblot analysis of CMVs demonstrated that 4PBA treatmentincreased the Mrp2 expression level by 3.2-fold, whereas dipep-tidyl peptidase-4 (DPPIV) expression level was not affected(Fig. 1G). In this study, DPPIV was selected as a loading controlbecause we previously found that its expression on the plasmamembrane was not affected by 4PBA treatment [7]. The changein the Mrp2 expression level was close to the change in its Vmax

values for [3H]-E217bG. These results suggest that 4PBA treat-ment at a clinically relevant dosage used in humans can increasefunctional Mrp2 expression at the canalicular membrane, and

vol. 56 j 1136–1144 1139

A

C D

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MRP2/GAPDH

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NA

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8

710

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2.53.54.04.5

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l (m

g/dl

)OTCD-4PBA(-)OTCD-4PBA(+)

*

Fig. 2. Hepatic MRP2 expression and serum T-Bil concentration in patientswith OTCD. (A) MRP2 expression in membrane fractions prepared from livers ofOTCD-4PBA(+), OTCD-4PBA(�), and donors. (B) MRP2 mRNA expression in liversof OTCD-4PBA(+), OTCD-4PBA(�), and donors. (C and D) Serum T-Bil concentra-tion in OTCD patients. Serum collected from OTCD-4PBA(+), OTCD-4PBA(�), anddonors was analyzed. (C) The mean values of individual specimens in each groupare shown. (D) The changes from before to after 4PBA treatment in the samepatients are shown. (A–D) The parenthetic values are the number of specimens.Each bar represents the mean ± SE of individual specimens. ⁄p <0.05.

DMSO

4PBA

IB: MRP2

0 12 24250150

kDa

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Incubation time (h)

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Incubation time (h)

**

DMSO4PBA

Rem

aini

ng b

iotin

-la

bele

d M

RP2

(% o

f 0 h

)

125100

5075

250

0 12 24

Fig. 3. Effect of 4PBA treatment on the degradation rate of cell surface-resident MRP2 in MDCKII cells. (Upper panel) Degradation rate of cell surface-resident MRP2. MRP2-MDCKII cells were treated for 24 h, with or without 1 mM4PBA, before the cell surface biotinylation. The biotinylated cells were incubatedfor the indicated times at 37 �C, with or without 1 mM 4PBA, and then analyzed tomeasure the remaining biotinylated proteins as described in the Supplementarymaterial. (Lower panel) Quantification of band intensity representing MRP2. Theintensity of the band representing MRP2 is expressed as a percentage of MRP2present at 0 h. All data are presented as mean ± SE of six independent experi-ments. ⁄p <0.05.

Research Article

consequently, increase [3H]-E217bG transport via the canalicularmembrane in SD rats.

qPCR analysis demonstrated no change in Mrp2 mRNA expres-sion by 4PBA treatment in the SD rat liver (Fig. 1H).

Increase in hepatic MRP2 expression and decrease in serum T-Bilconcentration in OTCD patients after 4PBA treatment

4PBA was approved originally for the treatment of OTCD patients.We investigated the effect of 4PBA on MRP2 in human liver spec-imens and serum from OTCD patients before and after the begin-ning of 4PBA treatment. These OTCD patients had serumconcentrations of AST, ALT, and GGT, indices of liver function,that were mostly within normal ranges before 4PBA treatment(Table 2). 4PBA treatment did not change these values signifi-cantly and did not induce any adverse events.

The mRNA and protein expression levels of MRP2 were com-pared using liver specimens obtained from OTCD patients beforethey were given 4PBA treatment (OTCD-4PBA(�)), samples fromthe same patients after they were given 4PBA treatment (OTCD-4PBA(+)), and samples from healthy organ donors. The samples ofOTCD-4PBA(�) were obtained by liver needle biopsy of threeOTCD patients (5-R, 6-R, and 7-R) before 4PBA treatment andthe liver specimen from an OTCD patient (8-R) who underwentLTx without 4PBA treatment. Western blot analysis of membranefractions showed that MRP2 expression level in OTCD-4PBA(+)was 1.7- and 2.4-fold higher than in donors and OTCD-4PBA(�),respectively, after normalizing the values by that of b-actin(Fig. 2A). By contrast, qPCR analysis showed similar expressionof MRP2 mRNA in donors, OTCD-4PBA(�), and OTCD-4PBA(+)(Fig. 2B).

1140 Journal of Hepatology 2012

The effect of 4PBA on serum T-Bil concentration was evalu-ated using serum collected from patients before 4PBA treatmentand before LTx after the beginning of 4PBA treatment. Serum T-Bil concentration in OTCD patients and donors was within thenormal range except for two patients (1-R and 3-R), whose valuesbefore 4PBA treatment were above the normal range because ofphysiological neonatal jaundice (Table 2). The mean serum T-Bilconcentration was 3.5- and 6.8-fold lower in OTCD-4PBA(+) thanin donors and OTCD-4PBA(�), respectively (Fig. 2C). The highermean serum T-Bil concentration in OTCD-4PBA(�) than in donorscan be attributed to the inclusion of data for two OTCD-4PBA(�)patients with physiological neonatal jaundice (1-R and 3-R) in theanalysis. When the data for these two OTCD patients wereexcluded from the analysis, the mean serum T-Bil concentrationfor the OTCD-4PBA(�) group was similar to that of the donors(0.63 mg/dl and 0.69 mg/dl for OTCD-4PBA(�) and donors,respectively). Serum T-Bil concentration was compared withinthe same patients at different times; serum T-Bil concentrationdecreased significantly in all patients after 4PBA treatment(p = 0.0498) (Fig. 2D).

4PBA-mediated prolongation of the half-life of cell surface-residentMRP2

Because 4PBA increased the cell surface expression and transportfunction of MRP2/Mrp2 without changing MRP2/Mrp2 mRNAexpression (Figs. 1 and 2) and had no effect maturation ofMRP2/Mrp2 from an ER-resident immature form to a cell sur-face-resident mature form (Supplementary Fig. 2), we investi-gated the effect of 4PBA on the half-life of cell surface-residentMRP2/Mrp2. The study using cell surface biotinylation found that4PBA treatment inhibited the degradation of cell surface-resident

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MRP2 in MRP2-MDCKII cells (Fig. 3). The amounts of biotin-labeled MRP2 remaining after the 12- and 24-h incubations were2.1- and 2.6-fold higher in 4PBA-treated cells than in DMSO-trea-ted cells. 4PBA treatment did not change the degradation rate ofcell surface-resident P-gp (data not shown).

Ubiquitination-mediated lysosomal degradation of MRP2/Mrp2 onthe plasma membrane

Ubiquitin (Ub) modifications at one, two, or several differentlysine residues in cell surface-resident membrane proteins actas a degradation signal by their accelerated internalization and/or their sorting to be incorporated into multivesicular bodiesrather than recycling to the plasma membrane in the early endo-some, which leads to lysosomal degradation [22].

Incubation with typical lysosomal inhibitors (chloroquine,NH4Cl, and bafilomycin) at commonly used concentrationsresulted in about a 2.5-fold increase of MRP2 expression inMRP2-MDCKII cells and of endogenous Mrp2 expression inMcA-RH7777 cells (Fig. 4A). The cell surface biotinylation studyshowed that chloroquine treatment prolonged the half-life of cellsurface-resident MRP2 in MRP2-MDCKII cells (Fig. 4B). Chloro-quine increased the amount of biotin-labeled MRP2 remainingby 2.1- and 2.4-fold after the 12- and 24-h chases, respectively.

The overexpression of UbDGG, which lacks the glycine–glycinesequence at the C-terminus, inhibits the ubiquitination-mediatedsorting of cargoes [23,24]. By contrast, the overexpression ofUbDGG/I44A, which incorporates Ub I44A into UbDGG, is utilizedas a negative control of UbDGG [23,24]. The study with cell surfacebiotinylation showed that overexpression of N-terminally His-tagged (His)-UbDGG significantly prolonged the half-life of cellsurface-resident MRP2 in MRP2-MDCKII cells, whereas His-UbDGG/I44A overexpression had no effect (Fig. 4C and D). Theamounts of biotin-labeled MRP2 remaining after the 12- and24-h chases were 1.7- and 3.3-fold higher, respectively, in theHis-UbDGG-overexpressing cells than in those with the emptyvector (E.V.). Unlike the effect on MRP2, overexpression of His-UbDGG had no significant effect on the degradation of the trans-ferrin receptor on the plasma membrane (data not shown), whichis independent of ubiquitination [25]. Together, these results sug-gest that MRP2 is sorted from the plasma membrane to the deg-radation pathway through its ubiquitination and is finallydegraded in lysosomes.

Involvement of ubiquitination of MRP2/Mrp2 in the 4PBA-mediatedinhibition of degradation of cell surface-resident MRP2/Mrp2

In the immunoprecipitates with anti-MRP2 antibody from acrude membrane fraction of MRP2-MDCKII cells and McA-RH7777 cells and from CMVs of SD rats, ubiquitinated MRP2/Mrp2 (MRP2/Mrp2-Ub) and MRP2/Mrp2 were detected as asmear band around the 225 kDa and 190 kDa bands, respectively(Fig. 4E). Given that the molecular weight of Ub is 8 kDa, thisimplies the presence of cell surface-resident MRP2/Mrp2 carryingnot a polyubiquitin chain, but one, two, or several Ub modifica-tions at several different sites, which can function as the lyso-somal degradation signal for membrane proteins [25,26].Quantitative densitometric analysis showed that 4PBA treatmentdecreased the ratio of MRP2/Mrp2-Ub to MRP2/Mrp2 to 38% inMRP2-MDCKII cells, 17% in McA-RH7777 cells, and 33% in therat liver (Fig. 4E).

Journal of Hepatology 2012

Whereas 4PBA treatment significantly decreased the degrada-tion rate of cell surface-resident MRP2 in E.V.-transfectedMRP2-MDCKII cells, 4PBA treatment had no effect inHis-UbDGG-overexpressing MRP2-MDCKII cells, on which theubiquitination reaction and ubiquitination-mediated processesare inhibited (Fig. 4F). Taken together, these results suggest that4PBA reduces the susceptibility of cell surface-resident MRP2/Mrp2 to ubiquitination, which is needed for the degradation ofcell surface-resident MRP2/Mrp2, and thereby prolongs itshalf-life.

Discussion

The main findings in this study are that 4PBA increased the cellsurface expression and transport function of MRP2/Mrp2 withoutsignificantly changing the MRP2/Mrp2 mRNA level and that 4PBAdecreased serum T-Bil concentration. Treatment with 4PBA at aclinically relevant concentration increased the cell surfaceexpression of MRP2/Mrp2 in MRP2-MDCKII cells and in the SDrat liver (Fig. 1C and G). In SD rats, the increased Mrp2 expressionat the canalicular membrane was accompanied by increased bil-iary excretion of Mrp2 substrate (Fig. 1E, F, and Table 1). In OTCDpatients, 4PBA treatment increased hepatic MRP2 expression anddecreased serum T-Bil concentration (Fig. 2A, C, and D). Theseresults suggest that this drug increases the biliary excretion ofbilirubin glucuronides through the activation of MRP2/Mrp2function, which reduces serum T-Bil concentration.

We also identified the degradation pathway of MRP2/Mrp2 onthe plasma membrane and that this process is implicated in themechanism responsible for the effects of 4PBA. The half-life ofcell surface-resident MRP2 in MRP2-MDCKII cells was extendedin cells overexpressing His-UbDGG, a dominant negative form ofUb, and by treatment with chloroquine, a typical lysosomal inhib-itor (Fig. 4B and D). The immunoprecipitation studies showedthat MRP2/Mrp2-Ub on the plasma membrane was modifiedwith only a small number of Ub molecules, which act as a lyso-somal degradation signal for membrane proteins (Fig. 4E). Takentogether, these results suggest that the cell surface-residentMRP2/Mrp2 is degraded through ubiquitination-mediated target-ing to the endosomal–lysosomal degradation pathway, as hasbeen demonstrated for other membrane proteins [22–24]. Aftertrafficking to the canalicular membrane, MRP2/Mrp2 is distrib-uted between the bile canalicular membrane and intracellularpools, and is finally removed from this cycle to the degradationpathway [27]. However, little is known about the molecularmechanism of MRP2/Mrp2 degradation. Our finding is the firstto show the regulatory mechanism responsible for the degrada-tion of cell surface-resident MRP2/Mrp2. We also showed that4PBA treatment decreased the amount of MRP2/Mrp2-Ub in themembrane-enriched fraction of MRP2-MDCKII cells, McA-RH7777 cells, and in CMVs from SD rats (Fig. 4E) and that theinhibitory effect of 4PBA on the degradation of cell surface-resi-dent MRP2 was not observed in His-UbDGG expressing MRP2-MDCKII cells (Fig. 4F). Thus, we identified a new pharmacologicalaction of 4PBA whereby 4PBA reduces the susceptibility to ubiq-uitination of cell surface-resident MRP2/Mrp2 and consequentlyinhibits its degradation. To our knowledge, no drugs that regulateintracellular sorting by modulating the ubiquitination status ofproteins have been approved.

Mutagenesis studies on a mutation of MRP2-encoding gene,which is present in DJS patients, suggested that some DJS patients

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Fig. 4. Involvement of ubiquitination in the degradation pathway of cell surface-resident MRP2/Mrp2. (A) Effect of lysosomal inhibitors on MRP2/Mrp2 expression.Before the preparation of whole cell lysates, MRP2-MDCKII and McA-RH7777 cells were treated with 30 lM chloroquine for 12 h, 2.5 mM NH4Cl for 24 h, or 1 lMbafilomycin for 24 h. (B) Effect of chloroquine treatment on the degradation rate of cell surface-resident MRP2. MRP2-MDCKII cells were treated for 12 h, with or without30 lM chloroquine and analyzed as described in Fig. 3. Western blot analysis and quantification of band intensity representing MRP2 are shown. All data are presented asmean ± SE of three independent experiments. ⁄p <0.05. (C) Expression of the mutated form of Ub in MRP2-MDCKII cells expressing His-UbDGG, His-UbDGG/I44A, or emptyvector. (D) The effect of the mutated form of Ub on the degradation rate of cell surface-resident MRP2 in MRP2-MDCKII cells. MRP2-MDCKII cells transfected with theindicated plasmid were analyzed as described in Fig. 3. Western blot analysis and quantification of band intensity representing MRP2 are shown. All data are presented asmean ± SE of six independent experiments. ⁄p <0.05, ⁄⁄⁄p <0.001 relative to empty vector.-expressing cells. (E) Susceptibility to ubiquitination of MRP2 in 4PBA-treatedMRP2-MDCKII cells and of Mrp2 in 4PBA-treated McA-RH7777 cells and SD rats. (Upper panel) MRP2/Mrp2 was immunoprecipitated from solubilized crude membranefractions of MRP2-MDCKII and McA-RH7777 cells 24 h after 4PBA or DMSO treatment with anti-MRP2 antibody or from solubilized CMVs prepared from SD rats given 4PBAor vehicle at 0.6 g/kg/day by gavage for 10 days with anti-MRP2 antibody. Immunoprecipitates were analyzed by Western blotting. (Lower panel) Quantification of MRP2/Mrp2-Ub normalized to MPR2/Mrp2. Data were derived from the band intensity representing MRP2/Mrp2-Ub and MRP2/Mrp2 in immunoprecipitates with anti-MRP2antibody by Image Gauge software. (F) Effect of 4PBA treatment on the degradation rate of cell surface-resident MRP2 in MRP2-MDCKII cells expressing His-UbDGG or emptyvector. MRP2-MDCKII cells transfected with the indicated plasmid were treated for 24 h, with or without 1 mM 4PBA, and analyzed as described in Fig. 3. Western blotanalysis and quantification of band intensity representing MRP2 are shown. All data are presented as mean ± SE of three independent experiments. ⁄p <0.05, ⁄⁄p <0.01. In (A,C, and E), a representative result of three independent experiments is shown.

Research Article

express MRP2 on the canalicular membranes of hepatocytes withreduced, but not completely deficient, transport function [28].Because 4PBA raises the hepatocanalicular expression of MRP2

1142 Journal of Hepatology 2012

and thereby activates the biliary excretion of bilirubin glucuron-ides, this drug could have a beneficial effect on DJS patients withthis kind of mutation, which does not induce loss of function of

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MRP2. Ursodeoxycholic acid and its taurine-conjugated deriva-tive, tauroursodeoxycholic acid (TUDCA), have a wide variety ofcholeretic effects including increasing the canalicular expressionof MRP2/Mrp2 and BSEP/Bsep [29]. Although both 4PBA andTUDCA increase MRP2/Mrp2 and BSEP/Bsep expression at thecanalicular membrane, the mechanism responsible for the effectsof 4PBA differs completely from that of TUDCA, which stimulatesthe insertion of both transporters into the canalicular membrane[30]. Therefore, co-administration of 4PBA with drugs such asTUDCA may confer an additional therapeutic effect in patientswith DJS who have low but not a complete loss of MRP2 function.Moreover, based on our previous study demonstrating the inhib-itory effect of 4PBA on the degradation of BSEP on the plasmamembrane [7], 4PBA may be a novel mechanism-based drug forseveral forms of intrahepatic cholestasis such as sepsis- anddrug-induced cholestasis and cholestasis in pregnancy becausestudies using liver specimens from patients and animal modelshave suggested that the intracellular accumulation and reducedexpression of BSEP can be attributed to increased endocytosis fol-lowed by degradation [15,31,32].

At present, we do not know the reason why this drug reducesthe ubiquitination susceptibility of MRP2/Mrp2 on the plasmamembrane. One possible mechanism is that 4PBA inhibits theactivity of the enzymes involved in the ubiquitination of MRP2/Mrp2. The ubiquitination of substrate proteins is mediated bythe covalent attachment of Ub by the sequential action of threeenzymes: a Ub-activating enzyme E1, a Ub-conjugating enzymeE2, and a Ub ligase E3 [26]. Among these three enzymes, E3sare thought to ensure the correct timing, localization, and speci-ficity of the ubiquitination reaction [26]. Because 4PBA reducesthe amount of both BSEP/Bsep-Ub and MRP2/Mrp2-Ub on theplasma membrane (Fig. 4E) [23] but has no effect on the degrada-tion rate of P-gp and DPP IV [7], it is possible that 4PBA acts onthe ability of E3 to ubiquitinate specifically both BSEP/Bsep andMRP2/Mrp2. Previously reported E3s that can mediate the ubiq-uitination of Bsep [33] may be the potential targeted moleculesfor 4PBA. The promotion of small Ub-like modifier (SUMO)ylationof MRP2/Mrp2 is another possible mechanism because a recentstudy reported that Mrp2 is modified by SUMO, which competesfor lysine residues with Ub and protects from Ub-dependent deg-radation [34]. Further in vitro studies will unravel the detailedmechanism underlying that 4PBA treatment decreases theamount of MRP2/Mrp2-Ub and BSEP/Bsep-Ub on plasma mem-brane and help develop a more potent compound that increasesMRP2/Mrp2 and BSEP/Bsep expression at the canalicular mem-brane and improves hepatobiliary diseases associated with thedecreased both transporters.

In conclusion, we have shown that 4PBA treatment at a clini-cally relevant concentration activates MRP2/Mrp2 functionthrough its induction on canalicular membrane and reducesserum T-Bil concentration. The mechanism underlying this4PBA action involves the inhibition of the degradation of cell sur-face-resident MRP2/Mrp2 by decreasing its susceptibility to ubiq-uitination. Together with our findings that 4PBA increases the cellsurface expression and transport activity of Bsep in rats [7] andthe hepatic expression of BSEP in OTCD patients after 4PBA treat-ment [35], these results suggest that 4PBA could be a new phar-macological agent for patients with intrahepatic cholestasis, DJSpatients with low, but not completely deficient, MRP2 transportactivity. Future clinical research is needed to show the utilityand safety of 4PBA as a therapy for these liver diseases.

Journal of Hepatology 2012

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Financial support

This study was supported by the Program for Promotion of Fun-damental Studies in Health Sciences of the National Institute ofBiomedical Innovation (NIBIO) to Y.S. and a Grant-in-Aid forYoung Scientists (B) (23790175) to H.H.

Acknowledgements

The authors thank Dr. Piet Borst (The Netherlands Cancer Insti-tute, Amsterdam, The Netherlands) for providing MRP2-MDCKIIcells and Dr. Larissa Kogleck (The University of Tokyo, Tokyo, Ja-pan) for advice on the manuscript.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jhep.2011.11.021.

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