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Am J Physiol Gastrointest Liver Physiol 295: G1035-G1041, 2008. First published September 11, 2008; doi:10.1152/ajpgi.90404.2008
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LIVER AND BILIARY TRACT

Canalicular Mrp2 localization is reversibly regulated by the intracellular redox status

Shuichi Sekine,1 Kousei Ito,2 and Toshiharu Horie1

1Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba; and 2Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Submitted 30 June 2008 ; accepted in final form 9 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is known to be a common feature of cholestatic syndrome. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile salt-independent bile flow, under acute oxidative stress, and a series of signaling pathways finally leading to the activation of novel protein kinase C were involved in this mechanism; however, it has been unclear whether the internalized Mrp2 localization was relocalized to the canalicular membrane when the intracellular redox status was recovered from oxidative stress. In this study, we demonstrated that decreased canalicular expression of Mrp2 induced by tertiary-butyl hydroperoxide (t-BHP) was recovered to the canalicular membrane by the replenishment of GSH by GSH-ethyl ester, a cell-permeable form of GSH. Moreover, pretreatment of isolated rat hepatocytes with colchicine and PKA inhibitor did not affect the t-BHP-induced Mrp2 internalization process but did prevent the Mrp2 recycling process induced by GSH replenishment. Moreover, intracellular cAMP concentration similarly changed with the change of intracellular GSH content. Taken together, our data clearly indicate that the redox-sensitive balance of PKA/PKC activation regulates the reversible Mrp2 localization in two different pathways, the microtubule-independent internalization pathway and -dependent recycling pathway of Mrp2.

cholestasis; biliary transporter; internalization; intracellular redox; protein kinase

THE CANALICULAR MEMBRANE OF rat hepatocytes contains multidrug resistance-associated protein 2/ATP-binding cassette (ABC) transporter family 2 (Mrp2/Abcc2), which is responsible for the biliary excretion of several amphiphilic organic anions, including bilirubin glucuronides, glutathione (GSH), and its conjugates (9). The defective expression of MRP2 in the canalicular membrane seen in patients with Dubin-Johnson syndrome causes conjugated hyperbilirubinemia; therefore, the importance of the canalicular expression of MRP2 has been widely recognized in the clinical setting. In addition to the inherited deficiency of MRP2 expression, the transporter expression is regulated on a short- and long-term basis in normal subjects, with important implications for the pathogenesis of cholestatic syndromes (17, 20). Short-term regulation includes rapid retrieval of the transporter from the canalicular membrane of hepatocytes and its translocation into cytosol in putative vesicles under the influence of hyperosmolarity (18, 28), endotoxin (19), phalloidin (27), bile acid (2), and oxidative stress (11, 29). On the other hand, long-term regulation includes mrp2 gene expression, which is affected by lipopolysaccharide (20), bile duct ligation, or a variety of drugs (14). In addition to the static expression of Mrp2 on the canalicular surface, its dynamic insertion and internalization processes are of great importance because the steady-state expression level directly depends on these turnover rates. Therefore, collapse of these turnover rates leads to changes of Mrp2 localization.

In patients with chronic hepatic failure (primary biliary cirrhosis and hepatitis C virus infection) with chronic cholestatic disorder, the disrupted canalicular localization of Mrp2 was observed (16). Moreover, oxidative stress markers were closely related in these chronic liver failure patients (1). These observations strongly suggested that not only the mRNA expression of canalicular transporters but also their localization were equally important in human liver failure. We have previously revealed that Mrp2 was rapidly internalized under ethacrynic acid (EA)-induced acute oxidative stress in the perfused rat liver. Because both GSH itself and the resulting glutathione conjugates are substrates of Mrp2, rapid downregulation of Mrp2 under GSH-depleting conditions seems a favorable feedback mechanism for hepatocytes to retain their intracellular GSH. Although it is not known whether the downregulation of Mrp2 actually contributes to cytoprotection, constitutive overexpression of human MPP2 in MDCKII cells accelerates 4-hydroxynonenal-induced GSH depletion and necrosis compared with control MDCKII cells (10).

Here we propose the hypothesis that internalized Mrp2 might return to the canalicular membrane surface when the intracellular GSH level recovers. In this study, we examined this hypothesis using a rat liver perfusion study and isolated rat hepatocyte couplets (IRHCs). Our results showed that Mrp2 localization was recovered to the canalicular membrane by the replenishment of intracellular GSH. Furthermore, microtubule polymerization and cAMP-dependent PKA are essential factors for the recycling process, but not for the internalization process of Mrp2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. Tertiary-butyl hydroperoxide (t-BHP) and glutathione-reduced ethyl ester (GSH-EE) were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). PKA inhibitor 14-22 amide, cell-permeable myristoylated, was from Calbiochem (Darmstadt, Germany). The cAMP enzyme immunoassay (EIA) system was from GE Healthcare (St. Giles, UK). Rabbit anti-Mrp2 antiserum was raised against the 12-amino acid sequence at the carboxy terminus of rat Mrp2 (6). All other chemicals and solvents were of analytical grade.

Animals. Male Sprague-Dawley rats (SDR) weighing 170 to 220 g (6–7 wk) were used throughout the experiments (Japan SLC, Shizuoka, Japan). The animals were treated humanely in accordance with the guidelines issued by the National Institutes of Health. Animal protocols were approved by the Animal Research Committee of Chiba University.

Rat liver perfusion and crude membrane preparation. Rat livers were perfused in situ in a nonrecirculating system with Krebs-Henseleit bicarbonate (KHB) buffer (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 5 mM glucose, pH 7.4 at 37°C). Rat livers were perfused with or without 500 µM t-BHP for 30 min and subsequently 3 mM GSH-EE was perfused for 1 h. Liver specimens (2 g) were then snap frozen in liquid nitrogen. The crude membrane (CrM) was prepared as previously described (34). CrM fractions (5 µg protein) and homogenates (35 µg protein) were subjected to immunoblot analysis as described previously (12, 31). Enrichment of the crude membrane fraction was confirmed by immunoblot experiment with antibodies for dipeptidyl peptidase IV (DPPIV), a canalicular marker protein, or rab4, an endosomal marker protein. As a result, expression of DPPIV was ~18- to 20-fold higher whereas that of rab4 was ~8–10 times lower in crude membrane fraction compared with homogenate (data not shown). Relative enrichment of these marker proteins was not affected by the perfusion with t-BHP and/or GSH-EE.

Semiquantitative real-time RT-PCR. Total mRNA was prepared from perfused rat liver using RNA-Solv reagent (OMEGA Bio-tek, Doraville, GA). Reverse transcription was performed with 1 µg of total RNA using Takara RNA PCR kit ver. 3.0 (Takarabio, Shiga, Japan). Real-time PCR was performed to quantify the mRNA expression relative to β-actin using the qPCR Mastermix for SYBR Green 1 (Eurogentee SA, Seraing, Belgium). Real-time RT-PCR amplification was determined utilizing an ABI Prism 7000 machine (Applied Biosystems, Foster City, CA).

IRHCs preparation. Hepatocytes were isolated from SDR liver by limited collagenase digestion as previously described (7). After isolation, rat hepatocytes were maintained (5x105 cells/ml) at 4°C in Williams' medium E supplemented with 10% fetal bovine serum, penicillin (100,000 units/ml), and streptomycin (100 µg/ml) until seeding. Cell viability, assessed by Trypan blue exclusion, remained greater than 95%.

Processing of cells. The processing of the cell was performed as described previously with some modification (3). Briefly, hepatocytes were plated at a density of 2.5x106 cells per 60-mm culture dish and kept for 3 h at 37°C in a CO2 incubator. Cells were added to 100 µM t-BHP and incubated for 10 min, and subsequently the medium was replaced with 3 mM GSH-EE for 30 min. After the treatments, cells were scraped into ice-cold buffer A (20 mM Tris, 250 mM sucrose, 5 mM EGTA, 1 mM MgCl2) supplemented with 1 mM phenylmethylsulfonyl fluoride and 0.1% protease inhibitor cocktail and then immediately sonicated (VP-5S, Taitec, Saitama, Japan) and centrifuged at 150,000 g for 40 min at 4°C, and resulting pellet was used as the membrane fraction. Membrane fractions were subjected to immunoblot analysis. The blotted membranes were probed with PKC{alpha}-, PKC{delta}-, and PKC{varepsilon}-specific antibodies followed by a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were detected with an enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ) and analyzed with an LAS1000 chemiluminescence image detector (Fuji Photo Film, Tokyo, Japan).

Measurement of intracellular GSH content. Samples were mixed with 3-fluorotyrosine as an internal standard followed by filtration through a 0.45-µm syringe filter (Millex-LH; Millipore, Bedford, MA). HPLC was performed as described previously (15). Briefly, an Inertsil ODS column (4.6-mm inner diameter x 250 mm; GL Sciences, Tokyo, Japan) was used with a mobile phase (0.1% trifluoroacetic acid-methanol = 20:1) at a flow rate of 1.0 ml/min. The eluate from the column was mixed with solution containing 18.6 mM o-phthalaldehyde and 17.1 mM 2-mercaptoethanol in 100 mM carbonate buffer (pH 10.5), which was delivered at a rate of 0.2 ml/min. The mixture was then passed through a stainless steel coil at 70°C to facilitate derivatization. A fluorescence detector was used and operated at an excitation wavelength of 355 nm and an emission wavelength of 425 nm. The concentration of GSH was calculated with reference to the height of a standard GSH sample.

Immunofluorescence analysis. Hepatocytes were plated on glass coverslips at a density of 2.0x105 cells/ml per 12 wells and incubated in a CO2 incubator for 3 h to form IRHCs, which were then further incubated for 10 min with or without 100 µM t-BHP. In some experiments, cells were pretreated with PKA inhibitor (200 nM), colchicine (100 nM), and its inactive analog lumicolchicine (100 nM) for 2 h prior to exposure to t-BHP. IRHCs were fixed with 4% formaldehyde/PBS for 15 min and permeabilized in 0.1% Triton X-100/PBS for 10 min. Mrp2 and {alpha}-tublin were probed with anti-Mrp2 antiserum (1:50) and anti-{alpha}-tublin antibody (Abcam, Cambridge, UK) (1:200) for 1 h, respectively. After being washed with PBS, samples were incubated for 1 h with FITC-conjugated secondary antibody (1:250). All antibodies were diluted in PBS containing 1% bovine serum albumin. In some experiment, samples were treated with 0.2 mg/ml RNase A in PBS for 20 min at room temperature just before being mounted on a Vectashield containing propidium iodide (Vector Laboratories, Burlingame, CA). Cells were analyzed in a blinded fashion with a confocal laser scanning microscope, LSM510 type (Carl Zeiss, Jena, Germany). Ten fields per coverslip were chosen randomly, and all the cells (30–50 cells per field) and canalicular Mrp2 appeared in the field were counted with a x40 objective lens. Mrp2-positive canaliculus was defined as the Mrp2 staining between two or more hepatocytes couplets irrespective of the lumen size.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced form of GSH level in livers treated with or without t-BHP and GSH-EE. t-BHP is known to be highly extracted by the liver in a single perfusion (30) and catalyzed by glutathione peroxidase (32), whereas GSH-EE is known as a cell-permeable form of GSH that undergoes hydrolysis by intracellular esterases, thereby increasing the intracellular GSH level rapidly.

As previously reported, 500 µM t-BHP perfusion led to a decrease of the hepatic reduced form of GSH by more than half of control within 30 min after perfusion (29). In addition, hepatic GSH content remained low after subsequent KHB buffer perfusion for 60 min. On the other hand, subsequent 3 mM GSH-EE perfusion for 60 min replenished the hepatic GSH content almost completely (91.1 ± 4.6% of control) (Fig. 1B).


Figure 1
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  		Fig. 1. Effect of glutathione-reduced ethyl ester (GSH-EE) liver perfusion on decrease of intrahepatic GSH content and bile flow induced by tertiary-butyl hydroperoxide (t-BHP) perfusion. The Sprague-Dawley rat (SDR) liver was preperfused with or without t-BHP (500 µM) for 30 min and then subsequently perfused with or without GSH-EE (3 mM) for 60 min (A). GSH content (B) and bile flow (C) were measured at indicated time points after the start of t-BHP perfusion. KHB, Krebs-Henseleit buffer. Results are given as means ± SE of 3 independent liver perfusions. **P < 0.01 compared with control. #P < 0.05 and ##P < 0.01 compared with t-BHP perfusion.

 
Effect of t-BHP and GSH-EE on bile flow in rat liver perfusion. Bile flow mainly consists of two canalicular transporters. The bile salt export pump (Bsep/Abcb11) and Mrp2 are involved in bile salt-dependent and -independent bile flow formation, respectively; therefore, in the liver perfusion study, the bile flow rate is often estimated as index of transporter activity. As reported previously, perfusion of 500 µM t-BHP for 30 min rapidly decreases bile flow (57.2 ± 11.2% of control at 30 min) (Fig. 1C) (29). Bile flow did not recover but gradually decreased even after switching the perfusate to KHB buffer without t-BHP (41.1 ± 7.0% of control at 90 min). On the other hand, bile flow increased immediately after switching the perfusate from t-BHP to GSH-EE and finally recovered to the control value (101.2 ± 5.7% of control) within 30 min after the perfusate was changed (Fig. 1C). GSH-EE perfusion per se did not affect basal bile flow or hepatic GSH (Fig. 1B). These data imply that replenishment of intracellular GSH restores the function of canalicular transporters to the control level.

Effect of GSH-EE on the localization of canalicular transporters in perfused rat liver. We have previously demonstrated that Mrp2 was internalized in the liver perfused with EA and this process was triggered by the decrease of intracellular GSH content (11). Therefore, we first tried to confirm whether Mrp2 was also internalized in the liver perfused with t-BHP when GSH content dropped to less than half the control. As shown in Fig. 2, Mrp2 expression in the crude membrane was significantly decreased to ~50% of the control at 30 min after t-BHP perfusion. This indicated that t-BHP, a hydrogen peroxide producer, indirectly consumes GSH to scavenge hydrogen oxide radical, similarly decreasing Mrp2 membrane expression, as was observed under the EA-induced GSH-depleted condition, in which GSH is directly conjugated with EA. Mrp2 membrane expression was restored to the control level by subsequent 60-min perfusion with GSH-EE, but not by control KHB buffer (Fig. 2). GSH-EE perfusion alone for 60 min did not increase Mrp2 expression in the membrane over the control. On the other hand, Mrp2 mRNA and the protein level in total cell lysate were not affected during the experimental period (Fig. 3, A and B). These results indicate that Mrp2 is internalized from and recovered to the membrane surface by GSH depletion and replenishment, respectively, and, most importantly, these are reversible processes regulated by GSH content. On the other hand, Mdr, another canalicular ABC transporter, was not affected by these treatments, as reported previously (data not shown, Ref. 31). Differential effects on canalicular transporters exclude nonspecific effects by GSH depletion, including disruption of the canalicular structure itself or global interference of transporters sorting to the membrane surface.


Figure 2
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  		Fig. 2. Protective effect of GSH-EE liver perfusion on the decrease of membrane-associated Mrp2 induced by t-BHP perfusion. Immunoblot analysis of Mrp2 was performed in 5 µg protein, and crude membrane fraction was prepared from perfused rat livers. Densitometry analysis of the immunoreactive bands was shown below. Values are expressed as the percentage of control at 30 min. Results are given as means ± SE of 3 independent liver perfusions. **P < 0.01 compared with control. ##P < 0.01 and ###P < 0.005 compared with t-BHP perfusion.

 

Figure 3
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  		Fig. 3. Effect of t-BHP and GSH-EE on the Mrp2 protein (A) and mRNA (B) expression in the perfused liver. A: 35 µg protein of perfused liver homogenate was applied and detected with anti-Mrp2 antibody. Band densities of Mrp2 are shown below. Values are expressed as the percentage of control at 30 min. B: Mrp2 mRNA level of perfused liver was measured by the real-time RT-RCR. Mrp2 mRNA level was normalized by the β-actin mRNA level. Results are given as means ± SE of 3 independent liver perfusions.

 
Effect of t-BHP and GSH-EE on GSH content and the localization of Mrp2 in IRHCs. IRHCs is an established experimental system maintaining apical-basal polarity and endogenous liver-specific protein expression on the canalicular membrane. Mrp2 was typically localized to the canalicular space between adjacent hepatocytes couplets, which are reorganized around 3 h after isolation. We calculated the ratio of Mrp2-positive pseudocanaliculi per cell nucleus to define the Mrp2 index as already established (31). By using this system, we previously demonstrated that EA reduces GSH, Ca2+ elevation, NO production, and PKC{delta} and PKC{varepsilon} activation and finally leads to Mrp2 internalization (31). To investigate the molecular mechanism of Mrp2 reinsertion to the canalicular membrane, we confirmed the relationship between intracellular GSH level and the Mrp2 index according to the time schedule shown in Fig. 4A. Exposure to 100 µM t-BHP for 10 min decreased intracellular GSH to about half of the control and recovered completely by subsequent GSH-EE (3 mM) treatment for 30 min treatment (Fig. 4B). Concomitantly, the Mrp2 index was decreased and restored by t-BHP and subsequent GSH-EE treatment, respectively (Fig. 4C). It is worthwhile to note that our Mrp2 index possibly includes Mrp2 in subapical vesicle as well as that on the canalicular membrane surface because of the limited optical resolution. And yet, we did find significant reversible effect on Mrp2-index by t-BHP treatment as observed in our in vivo experiment. These results guarantee that our in vitro method is sensitive enough to evaluate change in the localization of Mrp2 by GSH-depletion/replenishment and that basic machinery to regulate Mrp2 localization under such is retained in IRHCs.


Figure 4
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  		Fig. 4. Effect of t-BHP and GSH-EE treatment on the intracellular GSH content and Mrp2 index in isolated rat hepatocyte couplets (IRHCs). IRHCs were treated without or with t-BHP (100 µM) for 10 min and then treated with or without GSH-EE (3 mM) for 30 min as shown in A. Mrp2 index was assessed as the ratio of Mrp2-positive canaliculi per cell (B). Intracellular GSH content was measured by the postlabeled o-phthalaldehyde HPLC analysis (C). Results are given as means ± SE of 3 independent cell preparations. **P < 0.01, ***P < 0.005 compared with control. ##P < 0.01, ###P < 0.005 compared with t-BHP.

 
Distribution of PKC isoforms in IRHCs. Activation of PKC isoforms sometimes results in rapid alternation of their subcellular localization, especially from the soluble cytosolic fraction to the membranous fraction. We reported that EA-induced oxidative stress resulted in selective translocation of novel protein kinase C (nPKC) isoforms (PKC{delta}, PKC{varepsilon}) to the membranous compartment and was correlated with Mrp2 internalization, but it remains to be determined whether activated PKC isoforms returned to the basal state when intracellular GSH was restored; therefore, the localization of PKC isoforms was investigated. As a result, 100 µM t-BHP exposure for 10 min translocated all the PKC isoforms tested, including PKC{alpha} (isoforms of conventional PKC: Ca2+-dependent PKC family), PKC{delta}, and PKC{varepsilon} to the membrane fraction. Subsequent GSH replenishment significantly suppressed the membrane association of PKC{alpha} to the control level (Fig. 5, A and B). It also had a decreasing effect on PKC{varepsilon} and PKC{delta}, although they still had a significantly higher value than the control. These results indicated that PKCs returned to near the basal level by GSH replenishment, whereas they continued to be high without GSH replenishment. Mrp2 reinsertion to the canalicular membrane seems to correlate with inactivation of PKCs.


Figure 5
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  		Fig. 5. Effect of t-BHP and GSH-EE treatment on the distribution of PKC isoforms in IRHCs. IRHCs were treated as shown in Fig. 4A. Representative immunoblot analyses using PKC{alpha}-, PKC{delta}-, and PKC{varepsilon}-specific antibodies are shown (A). Band densities shown in A were quantified (B). Values are expressed as the percentage of respective controls. Results are given as means ± SE of 3 independent cell preparations. *P < 0.05, **P < 0.01 compared with control. #P < 0.05, ##P < 0.01 compared with t-BHP exposure.

 
Effect of PKA inhibitor on changes to the Mrp2 index. cAMP stimulates bile flow in the perfused rat liver (8) and the sorting of Mrp2 from cytosol to the apical membrane in IRHCs (24). Actually, cAMP content apparently correlated with the Mrp2 index in our IRHC system; that is, it decreased to half of the control by t-BHP treatment and was increased to higher than the control by subsequent GSH replenishment (Fig. 6A). Supporting the importance of the cAMP-PKA signaling pathway, recovery of the Mrp2 index after GSH replenishment was significantly inhibited by the presence of PKA inhibitor (Fig. 6B). Cell-permeable PKA inhibitor at 200 nM, which is much higher than reported Ki value (=36 nM), affected neither the basal expression status nor the internalization process of Mrp2. This indicates that PKA is involved in the exocytic reinsertion of Mrp2 to the canalicular membrane but not in the internalization process or basal maintenance of Mrp2.


Figure 6
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  		Fig. 6. Role of PKA in the recycling process of Mrp2. Effect of t-BHP and GSH-EE treatment on the intracellular cAMP concentration (conc.) in IRHCs (A). IRHCs were treated as shown in Fig. 4A. Intracellular cAMP concentration was measured by the EIA system after the soluble cytosolic fractions were obtained from the cells treated with indicated time points at 10 and 40 min. Effect of PKA inhibitor on the decrease and recovery process of Mrp2 index induced by the change of intracellular redox status in IRHCs (B). Cells were treated with or without PKA inhibitor (200 nM) for 30 min, subsequently treated as shown in Fig. 4A. Finally, Mrp2 index was assessed as the ratio of Mrp2-positive canaliculi per cell. Results are given as means ± SE of 3 independent cell preparations. **P < 0.01, ***P < 0.005 compared with control. #P < 0.05, ##P < 0.01, ###P < 0.005 compared with t-BHP exposure.

 
Effect of microtubule disruptor on changes to the Mrp2 index. Microtubules are known to be an important factor for intracellular vesicular trafficking. In particular, insertion of newly synthesized Mrp2 into the canalicular membrane requires integrity of the microtubule structure (24), but it has not yet been clarified whether the integrity of microtubules also contributes to the recycling process of Mrp2. Therefore, we investigated the effect of colchicine (100 nM), a microtubule disruptor, on the Mrp2 index in our IRHC system. As a result, colchicine had no effect on the t-BHP-induced decrease of the Mrp2 index, whereas it significantly inhibited restoration of the Mrp2 index after GSH replenishment (Fig. 7A). This inhibitory effect on the Mrp2 reinsertion process was not observed with use of lumicolchicine, a structural isomer of colchicine without any disrupting activity for microtubules (Fig. 7A). The microtubule was indeed disrupted by colchicine but not by lumicolchicine as shown in Fig. 7B. This indicates that microtubules are involved in the exocytic reinsertion of Mrp2 to the canalicular membrane, but not in the internalization process.


Figure 7
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  		Fig. 7. Effect of colchicine on the decrease and recovery process of Mrp2 index induced by the change of intracellular redox status in IRHCs. Cells were treated without (control) or with colchicine (100 nM) and its inactive analog, lumicolchicine (100 nM), for 2 h, then treated as shown in Fig. 4A. Finally, cells were fixed and stained for Mrp2. Mrp2 index was assessed as the ratio of Mrp2-positive canaliculi per cell (A). Results are given as means ± SE of 3 independent cell preparations. **P < 0.01 ***P < 0.005 compared with control. ##P < 0.01, ###P < 0.005 compared with t-BHP exposure. Cells were treated with colchicine (100 nM), lumicolchicine (100 nM), or vehicle for 2 h, then added with t-BHP for 10 min. Cells were fixed and stained with anti-{alpha}-tubulin antibody as described in MATERIALS AND METHODS (B). Bars = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Oxidative stress is involved in the pathogenesis and progression of liver diseases, such as alcoholic liver disease and biliary cirrhosis (5). During this process, the expression of biliary efflux transporters is affected (21, 26, 36), and, in this mechanism, the importance of the intracellular GSH content in regulating Mrp2 internalization was suggested from in situ rat liver perfusion studies using EA (12), t-BHP, and chlorodinitrobenzene (30). In addition, we have previously reported that nPKC activation triggered by intracellular GSH reduction leads to Mrp2 internalization as early as 2 min after the addition of EA (31); however, it has not been elucidated whether the internalized Mrp2 localization is recovered by the replenishment of GSH content.

Our series of studies demonstrated that internalized Mrp2 induced by the GSH decrease returned to the canalicular membrane when intracellular GSH was replenished in both the rat liver perfusion system and IRHCs. Long-term exposure (for more than 8 h) to oxidative stress results in the induction of antioxidative stress proteins, including Mrp2, which are regulated by Nrf2 (nuclear factor-E2-related factor 2) in mouse liver (22, 33). On the other hand, it was reported that protein degradation was accelerated through the lysosomal pathway under oxidative stress conditions in rat hepatocyte (4, 23); however, neither mRNA nor the protein amount of Mrp2 in total cell lysates was affected by t-BHP induced oxidative stress, at least during our experimental period. This suggests that the decrease of Mrp2 in the membrane fraction is not caused by changes in the synthesis or degradation rate, but by changes in the localization between the membrane surface and intracellular pool. From this point of view, complete recovery within 1 h after GSH replenishment is likely due to stimulated recycling of the internalized molecules rather than by de novo synthesis.

Recycling of Mrp2 seems to require PKA activity and microtubule integrity because PKA inhibitor and colchicine inhibited this process. It was reported that cAMP stimulated the sorting of Mrp2 and Bsep to the canalicular membrane of primary rat hepatocyte couplets in a microtubule-dependent manner (24). Moreover, it was also demonstrated that a cell-permeable cAMP analog, dibutyryl cAMP (DB-cAMP), which induced stimulation of canalicular formation itself in isolated rat hepatocyte couplets, was inhibited by colchicine treatment, whereas basal canalicular formation in the absence of DB-cAMP was little affected (25). Although PKA is apparently not involved in cAMP-stimulated canalicular formation (25), cAMP and microtubule are at least common factors in hepatocyte canalicular formation and the biliary transporter reinsertion process by GSH replenishment. Collectively, recovery of Mrp2 to the canalicular membrane requires not only the suppression of PKCs to the basal status, but also subsequent activation of PKA. The order of these events can be schematically represented in Fig. 8. The activity balance of PKC and PKA, which depends on GSH content, somehow regulates Mrp2 localization in a reversible manner; however, it remains to be elucidated whether PKA is actually activated when intracellular GSH is replenished.


Figure 8
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  		Fig. 8. Proposed mechanism of the reversible localization of Mrp2 accompanied by the intracellular redox status.

 
Cytoskeleton structures are differentially involved in the internalization and reinsertion process of Mrp2. Microtubules are a key mediator of the relocalization process of Mrp2 to the canalicular membrane surface, whereas they are independent of the internalization process. It is known that kinesin, a motor molecule involved in vesicular membrane trafficking, moving along microtubules toward the plus end in the direction of the cell surface. Moreover, it was reported that soluble PKA (R2{alpha}) forms a complex with the motor proteins (kinesin2, myosin5, dynein) to regulate their binding to vesicular components (13, 35). It is possible that PKA activity might regulate the loading or unloading of Mrp2-containing vesicles on microtubules.

In conclusion, we have demonstrated that Mrp2 and Bsep localizations were reversibly regulated, accompanied by the intracellular redox status. Moreover, in this sorting process of Mrp2, microtubule polymerization and PKA are essential factors, suggesting that the balance of PKC and PKA activations is a key regulator of the reversible trafficking of Mrp2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Horie, Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba Univ., Inohana 1-8-1, Chuo-ku, Chiba, 260-8675, Japan (e-mail: horieto{at}p.chiba-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aboutwerat A, Pemberton PW, Smith A, Burrows PC, McMahon RF, Jain SK, Warnes TW. Oxidant stress is a significant feature of primary biliary cirrhosis. Biochim Biophys Acta 1637: 142–150, 2003.[Medline]
  2. Beuers U, Bilzer M, Chittattu A, Kullak-Ublick GA, Keppler D, Paumgartner G, Dombrowski F. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 33: 1206–1216, 2001.[CrossRef][Web of Science][Medline]
  3. Beuers U, Probst I, Soroka C, Boyer JL, Kullak-Ublick GA, Paumgartner G. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology 29: 477–482, 1999.[CrossRef][Web of Science][Medline]
  4. Bito R, Hino S, Baba A, Tanaka M, Watabe H, Kawabata H. Degradation of oxidative stress-induced denatured albumin in rat liver endothelial cells. Am J Physiol Cell Physiol 289: C531–C542, 2005.[Abstract/Free Full Text]
  5. Bjorneboe A, Bjorneboe GE. Antioxidant status and alcohol-related diseases. Alcohol Alcohol 28: 111–116, 1993.[Abstract/Free Full Text]
  6. Buchler M, Konig J, Brom M, Kartenbeck J, Spring H, Horie T, Keppler D. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 271: 15091–15098, 1996.[Abstract/Free Full Text]
  7. Combettes L, Tran D, Tordjmann T, Laurent M, Berthon B, Claret M. Ca2+-mobilizing hormones induce sequentially ordered Ca2+ signals in multicellular systems of rat hepatocytes. Biochem J 304: 585–594, 1994.[Web of Science][Medline]
  8. Hayakawa T, Bruck R, Ng OC, Boyer JL. DBcAMP stimulates vesicle transport and HRP excretion in isolated perfused rat liver. Am J Physiol Gastrointest Liver Physiol 259: G727–G735, 1990.[Abstract/Free Full Text]
  9. Jedlitschky G, Leier I, Buchholz U, Hummel-Eisenbeiss J, Burchell B, Keppler D. ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2. Biochem J 327: 305–310, 1997.[Web of Science][Medline]
  10. Ji B, Ito K, Horie T. Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II Cells. Chem Res Toxicol 17: 158–164, 2004.[CrossRef][Web of Science][Medline]
  11. Ji B, Ito K, Sekine S, Tajima A, Horie T. Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver. Free Radic Biol Med 37: 1718–1729, 2004.[CrossRef][Web of Science][Medline]
  12. Ji B, Ito K, Sekine S, Tajima A, Horie T. Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver. Free Radic Biol Med 37: 1718–1729, 2004.[CrossRef][Web of Science][Medline]
  13. Kashina AS, Semenova IV, Ivanov PA, Potekhina ES, Zaliapin I, Rodionov VI. Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles. Curr Biol 14: 1877–1881, 2004.[CrossRef][Web of Science][Medline]
  14. Kauffmann HM, Keppler D, Kartenbeck J, Schrenk D. Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes. Hepatology 26: 980–985, 1997.[Web of Science][Medline]
  15. Keller DA, Menzel DB. Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography. Anal Biochem 151: 418–423, 1985.[CrossRef][Web of Science][Medline]
  16. Kojima H, Nies AT, Konig J, Hagmann W, Spring H, Uemura M, Fukui H, Keppler D. Changes in the expression and localization of hepatocellular transporters and radixin in primary biliary cirrhosis. J Hepatol 39: 693–702, 2003.[CrossRef][Web of Science][Medline]
  17. Konig J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1461: 377–394.
  18. Kubitz R, D'Urso D, Keppler D, Haussinger D. Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane. Gastroenterology 113: 1438–1442, 1997.[CrossRef][Web of Science][Medline]
  19. Kubitz R, Warskulat U, Schmitt M, Haussinger D. Dexamethasone- and osmolarity-dependent expression of the multidrug-resistance protein 2 in cultured rat hepatocytes. Biochem J 340: 585–591, 1999.[CrossRef][Web of Science][Medline]
  20. Kubitz R, Wettstein M, Warskulat U, Haussinger D. Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone. Gastroenterology 116: 401–410, 1999.[CrossRef][Web of Science][Medline]
  21. Kullak-Ublick GA, Baretton GB, Oswald M, Renner EL, Paumgartner G, Beuers U. Expression of the hepatocyte canalicular multidrug resistance protein (MRP2) in primary biliary cirrhosis. Hepatol Res 23: 78–82, 2002.[CrossRef][Web of Science][Medline]
  22. Maher JM, Cheng X, Slitt AL, Dieter MZ, Klaassen CD. Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver. Drug Metab Dispos 33: 956–962, 2005.[Abstract/Free Full Text]
  23. Marotta F, Lecroix P, Harada M, Masulair K, Safran P, Lorenzetti A, Ono-Nita SK, Marandola P. Liver exposure to xenobiotics: the aging factor and potentials for functional foods. Rejuvenation Res 9: 338–341, 2006.[CrossRef][Web of Science][Medline]
  24. Roelofsen H, Soroka CJ, Keppler D, Boyer JL. Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets. J Cell Sci 111: 1137–1145, 1998.[Abstract]
  25. Roma MG, Milkiewicz P, Elias E, Coleman R. Control by signaling modulators of the sorting of canalicular transporters in rat hepatocyte couplets: role of the cytoskeleton. Hepatology 32: 1342–1356, 2000.[CrossRef][Web of Science][Medline]
  26. Ros JE, Libbrecht L, Geuken M, Jansen PL, Roskams TA. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease. J Pathol 200: 553–560, 2003.[CrossRef][Web of Science][Medline]
  27. Rost D, Kartenbeck J, Keppler D. Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis. Hepatology 29: 814–821, 1999.[CrossRef][Web of Science][Medline]
  28. Schmitt M, Kubitz R, Lizun S, Wettstein M, Haussinger D. Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity. Hepatology 33: 509–518, 2001.[CrossRef][Web of Science][Medline]
  29. Schmitt M, Kubitz R, Wettstein M, vom Dahl S, Haussinger D. Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene. Biol Chem 381: 487–495, 2000.[CrossRef][Web of Science][Medline]
  30. Schmitt M, Kubitz R, Wettstein M, vom Dahl S, Haussinger D. Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene. Biol Chem 381: 487–495, 2000.[CrossRef][Web of Science][Medline]
  31. Sekine S, Ito K, Horie T. Oxidative stress and Mrp2 internalization. Free Radic Biol Med 40: 2166–2174, 2006.[CrossRef][Web of Science][Medline]
  32. Sitozhevsky AV, Havalkin IV, Ivanov VV, Kondakova IV. Kinetics of tert-butyl hydroperoxide decomposition in erythrocyte suspension. Membr Cell Biol 11: 487–495, 1997.[Medline]
  33. Vollrath V, Wielandt AM, Iruretagoyena M, Chianale J. Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. Biochem J 395: 599–609, 2006.[CrossRef][Web of Science][Medline]
  34. Vos TA, Hooiveld GJ, Koning H, Childs S, Meijer DK, Moshage H, Jansen PL, Muller M. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver. Hepatology 28: 1637–1644, 1998.[CrossRef][Web of Science][Medline]
  35. Zhang W, Zhao Y, Tong C, Wang G, Wang B, Jia J, Jiang J. Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus. Dev Cell 8: 267–278, 2005.[CrossRef][Web of Science][Medline]
  36. Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, Ferenci P, Stauber RE, Krejs GJ, Denk H, Zatloukal K, Trauner M. Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology 33: 633–646, 2001.[CrossRef][Web of Science][Medline]



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E. J. Sanchez Pozzi and M. G. Roma
Putative role for actin organization status in the dynamic localization of canalicular carriers under oxidative stress conditions
Am J Physiol Gastrointest Liver Physiol, April 1, 2009; 296(4): G969 - G969.
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