Ezrin and radixin both regulate the apical membrane localization of ABCC2 (MRP2) in human intestinal epithelial Caco-2 cells

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Ezrin and radixin both regulate the apical membrane localization of ABCC2 (MRP2) in human intestinal epithelial Caco-2 cells Qing Yang a,b,1 , Reiko Onuki b,c,1 , Chikako Nakai b , Yuichi Sugiyama b,⁎ a

School of Pharmacy, Fudan University, Yixueyuan Road 138, Shanghai, China Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Science, the University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan c Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, 135-0064, Japan b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Multidrug resistance-associated protein ABCC2 (MRP2) is widely expressed in mammalian

Received 22 February 2007

tissues including intestine, liver and kidney, and it has been shown to be located

Revised version received

exclusively on the apical membrane of polarized cells. Recently, several reports suggest

13 June 2007

that apical membrane localization of ABCC2 (Mrp2) was regulated by radixin in rodent

Accepted 24 July 2007

liver. To investigate the mechanism underlying this apical membrane targeting of MRP2 in

Available online 7 August 2007

human intestine, we chose Caco-2 cells as a model to examine the unique roles of ezrin and radixin. Following immunostaining, radixin and ezrin were found to be concentrated

Keywords:

at the apical membrane of Caco-2 cells. Using the RNAi method, radixin and ezrin stable

Apical membrane localization

knockdown Caco-2 cells were constructed. A cell surface biotinylation experiment with

of ABCC2 (MRP2)

radixin or ezrin stable knockdown Caco-2 cells showed that radixin or ezrin deficiency

Radixin

caused the loss of ABCC2 (MRP2) from the cell surface. An immunoprecipitation assay

Ezrin

showed that radixin and ezrin were associated with ABCC2 (MRP2). These findings

RNA interference

indicate that both ezrin and radixin are independently required for the apical membrane

Caco-2 cell

localization of ABCC2 (MRP2) in Caco-2 cells. Radixin and ezrin play similar roles in the apical membrane localization of ABCC2 (MRP2) and their expression level and subcellular distribution are important factors in the regulation of ABCC2 (MRP2) at the posttranscriptional level. © 2007 Elsevier Inc. All rights reserved.

Introduction The multidrug resistance protein ABCC2 (MRP2) is an ATPbinding cassette (ABC) transporter and it is localized to the brush border membrane of proximal tubule cells, the canalicular membrane of hepatocytes and the mucosal epithelium

of the intestine. Substrates of ABCC2 (MRP2) include conjugated and unconjugated organic anions, such as glutathione conjugates, glucuronide conjugates, leukotrienes, methotrexate, ochratoxin A and PAH [1]. The apical membrane localization of ABCC2 (MRP2) in enterocytes allows it to limit xenobiotic absorption and also mediate intestinal xenobiotic

⁎ Corresponding author. Fax: +81 3 5841 4766. E-mail address: [email protected] (Y. Sugiyama). 1 First and second authors contributed equally to this work. 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.07.033

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secretion and in hepatocytes it plays an important role in biliary excretion. ERM proteins (ezrin, radixin and moesin) are a closely related family of membrane-cytoskeleton crosslinkers. ERM proteins share two conserved domains: the NH2-terminal FERM (four point one, ezrin, radixin and moesin) domain, which directly binds to the cytoplasmic region of integral membrane proteins, such as CD44, CD43, ICAM-1 and ICAM-2 [2–6], and the Cterminal, which directly interacts with actin filaments [7–10]. ERM proteins also crosslink actin filaments indirectly to membrane proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the Na+/H+ exchanger-3 (NHE3) and the β2-adrenergic receptor through a PDZ-containing protein, EBP50, a relative of NHE-RF [11,12]. The crosslinking activity of ERM is regulated by the small GTP-binding protein Rho [5]. Based on these interactions, ERM proteins are thought to play key roles in morphology, motility, signal transduction and apoptosis [13–15]. Regulation of the localization of ABCC2 (Mrp2) by radixin was first reported by Tsukita group. They constructed the radixin deficient mouse and found that such mouse exhibits a loss of ABCC2 (Mrp2) from bile canalicular membranes without changing its expression level. This leads to conjugated hyperbilirubinemia, a disorder that is also induced by mutations in the gene encoding ABCC2 (MRP2) itself as seen in the human Dubin-Johnson syndrome [16]. Recently, the original radixin knockout mice were backcrossed to confirm the importance of radixin for the localization of ABCC2 (Mrp2) [17]. Sandwich cultured rat hepatocytes treated with siRNA targeting for radixin exhibited a marked reduction in the canalicular membrane structure and dissociation of ABCC2 (Mrp2) from apical membrane [18]. These observations suggest that radixin is a key molecule for the regulation of the apical localization of ABCC2 (MRP2) in rodents. In the case of humans, the samples from a patient with non-icteric primary biliary cirrhosis (PBC) stage III exhibited irregular ABCC2 (MRP2) immunostaining suggesting the redistribution of ABCC2 (MRP2) into intracellular structures and areas of irregular ABCC2 (MRP2) immunostaining also exhibit significantly reduced radixin immunostaining [19] although this evidence does not directly support the relationship between ABCC2 (MRP2) and radixin. It is known that radixin is the only ERM protein in hepatocytes [16] but, in most established cell lines, more than two species of ERM proteins are co-expressed in different ratios. Although the responses of ERM proteins to kinase are different, there is a functional overlap rather than tissue-specific requirements among vertebrate ERM proteins [20,21,16]. Besides radixin, other ERM proteins might also be involved in the apical membrane localization of ABCC2 (MRP2). Moesin exhibits quite a different cellular distribution from radixin and ezrin that concentrate at the apical membrane of epithelial cells, and it is found primarily in endothelial cells [22,23]. Thus, we initiated a study focusing on the role of radixin and ezrin. Polarized Caco-2 cells endogenously expressing ABCC2 (MRP2), radixin and ezrin are suitable for such a study. In this study, we established radixin and ezrin knockdown Caco-2 cells by the RNA interference method and the loss of ABCC2 (MRP2) from the cell surface was observed unless these cell lines still exhibited cell polarity. This shows that both radixin and ezrin contribute to the apical membrane localization of ABCC2 (MRP2) in human intestinal epithelial Caco-2 cells.

Materials and methods Materials and antibodies Monoclonal antibody to human ABCC2 (MRP2) (M2III-6) was purchased from Alexis (San Diego, CA). Anti-actin monoclonal antibody was purchased from Chemicon (Temecula, CA). Polyclonal antibody Ezrin (C-19) against all ERM proteins was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Ezrin-specific monoclonal antibody M11 was kind gift from Prof. S. Tsukita [16]. Monoclonal anti-moesin clone 38/87 that recognized moesin and radixin was purchased from Sigma. Monoclonal ezrin/p81/80K/Cytovillin Ab-1 (3C12) against ezrin was purchased from Lab Vision (Fremont, CA). A mouse monoclonal antibody C219 against P-gp and a rabbit polyclonal antibody against ZO-1 were purchased from Signet (Dedham, MA) and Zymed Laboratories (South San Francisco, CA), respectively. Caco-2 cells at passage 40–60 were maintained in a culture medium (CM) consisting of Dulbecco's Modified Eagle's Medium with 4500 mg/l glucose (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), nonessential amino acids (NEAA; Invitrogen), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Invitrogen) at 37 °C with 5% CO2 and 95% humidity. To obtain differentiated Caco-2 cells in a short period, cells were cultured in differentiation medium consisting of Entero-Stim Differentiation Medium (BD Bioscience, Bedford, MA) supplemented with MITO+ Serum Extender (BD Bioscience, Bedford, MA) as described previously [24].

Western blotting Cells were collected and suspended in 500 μl PBS containing 0.5 mg PMSF and disrupted by a 10 s burst of sonication. After centrifugation for 10 min at 3500 rpm, the supernatant was collected as a total lysate. Total lysate separated by SDS–PAGE was transferred to a polyvinylidene fluoride (PVDF) membrane (Pall, East Hills, NY) and probed with various antibodies. HRP conjugated secondary antibodies (Amersham Bioscience) were detected by chemiluminescence with ECL and a Western Blotting Detection Kit (Amersham Bioscience).

Immunocytochemistry The method for short-term Caco-2 cell culture established by Yamashita et al. [24] was modified for immunocytochemistry using glass coverslips. Caco-2 cells were seeded on glass coverslips at a density of approximately 1.0 × 105 cells/cm2 with culture medium. Culture medium was replaced with differentiation medium 2 days after the start of cultivation. Differentiation medium was changed again on day 4, and cells were used on day 5. Cells grown on glass coverslips were washed with PBS then fixed at 4 °C with cold MeOH for 10 min to preserve their three-dimensional structure. Cells were then permeabilized for 5 min with 0.1% Triton X-100 and blocked for 1 h with 5% bovine serum albumin. Cells were incubated for 1 h with monoclonal antibodies to ezrin (M11 rat anti-mouse ezrin mAb, no dilution) and radixin (anti-moesin clone 38/87, diluted 2000-fold). After

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rinsing with PBS, cells were incubated for 1 h with goat anti-rat IgG Alexa 568 for ezrin (diluted 250-fold) and goat anti-mouse IgG Alexa 568 for radixin (diluted 250-fold). Cells were further incubated for 1 h with ZO-1 (diluted 100-fold) and rinsed. Antirabbit IgG Alexa 488 (diluted-250) were used for detection of ZO-1 signal. To-PRO-3 iodide (diluted 200-fold) was used for counterstaining of nuclei. Confocal laser scanning was performed using an LSM 510 (Carl Zeiss Oberkochen, Germany) and FV1000 (Olympus Tokyo, Japan).

(reverse); ezrin, 5′-CTATGAGGAGAAGACAAAGAAG-3′ (forward) and 5′-TTCTTCTCTGCCTCAGTGAT-3′ (reverse); reduced glyceraldehydes 3-phosphate dehydrogenase (GAPDH) 5′-ATGGTGAAGGTCGG-3' (forward) and 5′-TTACTCCTTGGAGGCCATGT3′) (reverse). The level of GAPDH mRNA was used as an internal control. Aliquots of PCR product were electrophoresed on 2% agarose gels.

siRNA preparation and transfection

Caco-2 cells were seeded in 12 well plates at a density of 1.5 × 105 cells/well and grown for 2 days. Cells were then washed twice with ice-cold PBS-Ca/Mg (phosphate-buffered saline (PBS) containing 0.1 mM/l CaCl2 and 1 mM/l MgCl2) and incubated twice with NHS-SS-biotin (Pierce Biotechnology, Rockford, IL) for 30 min on ice. Cells were washed with quenching buffer (100 mM glycine in PBS-Ca/Mg) and incubated at 4 °C for 15 min to remove unreacted biotin. Subsequently, cells were washed twice with PBS-Ca/Mg then disrupted with 100 μl lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.1% Triton X-100, pH 7.5) containing 1% SDS and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 15 min. The lysate was supplemented with 900 μl lysis buffer to reduce the SDS concentration. Streptavidin–agarose beads (Pierce Biotechnology) were added to the above diluted lysate followed by incubation at 4 °C overnight with end-over-end rotation. The beads were washed three times with lysis buffer, twice with high-salt lysis buffer (50 mM Tris, 500 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.1% Triton X-100, pH 7.5) and once with low-salt lysis buffer (50 mM Tris, pH 7.5). The biotinylated proteins were eluted from the beads with 30 μl 1× SDS loading buffer (obtained by dilution of 3× SDS loading buffer (Biolabs, Hertfordshire, UK) with PBS) at 60 °C for 5 min. The eluent was subjected to western blotting.

Two sets of siRNA sequences targeted for human radixin and three sets of siRNA sequences targeted for ezrin were chosen: for radixin, R3, 1457–1479 (5′-ATGATGAACACGATGAGAATAAT-3′) and R4, 1463–1485 (5′-AACACGATGAGAATAATGCTGAA-3′); for ezrin, E1, 1214–1236 (5′-GACAGGCGGTGGATCAGATAAAG-3′); E5, 1212–1234 (5′-GAGACAGGCGGTGGATCAGATAA-3′) and E6, 963–984 (5′-AACAGCTGGAAACAGAGAAGAAA-3′). The sequence derived from Euglena gracilis, which has no homology with the human genome, was used as a control sequence (5′-TTGCGCGCTTTGTAGGATTCGTT-3′). The siRNAs were synthesized with a CUGA ® 7 in vitro siRNA Synthesis Kit (NIPPON GENE, Tokyo, Japan). The integrity of the siRNA duplexes was confirmed by electrophoresis on 8% nondenaturing polyacrylamide gel. Caco-2 cells were seeded in 10 cm culture dishes at 2.4 × 106 cells/dish, grown for 12 h then transfected with synthetic siRNA (10 μg/well) using a GeneSilencer™ siRNA Transfection Reagent Kit (Gene Therapy Systems) according to the manufacturer's protocol. Total RNA was isolated to examine siRNA efficiency after a 48 h transfection. Control cells were treated with Gene Silencer Reagent and OPTI-MEM medium.

siRNA expression vector construction and transfection Chemically synthesized oligonucleotide encoding an siRNA sense strand, loop (5′-ttcaagaga-3′) and an siRNA antisense strand were annealed with corresponding complementary single-stranded DNA oligonucleotide. The resulting doublestranded DNA (dsDNA) was inserted into SacI and KpnI sites of the piGENE™ tRNA Pur (iGENE, Ibaraki, Japan) to generate radixin, ezrin or control siRNA expression vector. Caco-2 cells were seeded in 10 cm dishes at 1.0 × 106 cells/ dish, grown for 12 h then transfected with the constructed R3, R4 (for radixin) and E6 (for ezrin) siRNA expression vector (5 μg/well) using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. At 8 h after the initiation of transfection, the plasmid–lipofectamine solution was removed, and the cell culture medium was added. The transfected Caco-2 cells were selected in cell culture medium containing puromycin (5.0 μg/ml) to obtain the stable transfectant.

Biotinylation

Immunoprecipitation Caco-2 cells from 10 culture dishes, 10 cm in diameter, were lysed with 1 ml RIPA buffer (0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 1 mM pamidino phenylmethylsulfonyl fluoride (PMSF) and 10 μg/ml leupeptin) for 20 min at 4 °C. Cell lysates were pre-cleared with protein A (or G)–sepharose 4B Fast Flow (Sigma) for 1 h at 4 °C. Radixin and ezrin were immunoprecipitated from the precleared lysate with monoclonal anti-moesin clone 38/87 (or control mouse IgG) and monoclonal antibody ezrin/p81/80K (or control mouse IgG), respectively, for 3 h at 4 °C, in the presence of protein A or G–sepharose (4B Fast Flow, Sigma). Immunoprecipitated beads were washed then boiled in SDS sample buffer, separated by SDS–PAGE and immunoblotted with the M2III-6 antibody against ABCC2 (MRP2) protein, ezrin (C-19) against radixin and ezrin and anti-actin antibody against actin.

RT-PCR analysis Total RNA was isolated from cells with Isogen™ (Nippon Gene) according to the manufacturer's instructions and quantified by UV absorbance spectroscopy. RT-PCR was performed using an RT-PCR Kit (AMV) Ver. 3.0 (Takara, Shiga, Japan). The sequences of the primers used were: radixin, 5′-CTCGAAAAGCTCTAGAACTGG-3′(forward) and 5′-GGTTCATTACCCCTTCATTTG-3′

Results Expression of ERM proteins in Caco-2 cells It has been reported that all three ERM proteins are expressed in several cell lines, such as KB cells and A431 cells [25,26]. Western

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Fig. 1 – Immunoblotting analysis of the expression of ERM proteins in HepG2 and Caco-2 cells. Cell lysate (40 μg) was separated by SDS–PAGE (8.5%). The bands of ezrin (E), radixin (R) and moesin (M) were detected by polyclonal antibody for ezrin (C-19).

blotting analysis performed with pAb C-19 recognizes all ERM proteins in ABCC2 (MRP2) endogenously expressing cells, HepG2 and Caco-2 cells (Fig. 1). In HepG2 cells, C-19 recognized three bands around 80 kDa, which corresponded to ezrin, radixin and

Fig. 3 – ABCC2 (MRP2) associates with radixin and ezrin. Radixin and ezrin were immunoprecipitated from Caco-2 cells lysates with mouse monoclonal anti-moesin clone 38/87 and mouse monoclonal antibody ezrin/p81/80K, respectively, and mouse IgG was used as a negative control for immunoprecipitation (Con.). Upper panels: Western blotting for ABCC2 (MRP2) in radixin or ezrin immunoprecipitates (IP) with monoclonal antibody (M2III-6). Middle panels: western blotting for radixin and ezrin in radixin and ezrin immunoprecipitates, respectively, with polyclonal antibody ezrin (C-19). Lower panels: western blotting for actin in radixin or ezrin immunoprecipitates with anti-actin antibody.

moesin from the top, whereas Caco-2 cells lacked moesin. Polarized LLC-PK1 cells were also found to express all ERM proteins (data not shown). Because of the different subcellular distributions, radixin and ezrin rather than moesin are supposed to be involved in the special localization of ABCC2 (MRP2). Caco-2 cells are suitable for investigating the role of ezrin and radixin in the membrane stability of ABCC2 (MRP2).

Fig. 2 – Immunofluorescence localization of radixin or ezrin in Caco-2 cell monolayers. (A) Monolayer of Caco-2 cells was double stained for radixin (red; anti-moesin clone 38/87) and ZO-1 (green). (B) Monolayer was double stained for ezrin (red; M11) and ZO-1 (green). Nuclei were stained with To-PRO-3 iodide (blue). Scale bar: 20 μm.

Fig. 4 – Effect of siRNAs on radixin or ezrin expression in Caco-2 cells. Ten micrograms of each siRNA as indicated (mock, control siRNA; E1, E5 and E6, ezrin specific siRNA; R3 and R4, radixin specific siRNA) or water (Con.) was transiently transfected into Caco-2 cells. Forty eight hours later, total RNA was isolated and the level of radixin or ezrin was measured by RT-PCR.

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Apical localization of radixin and ezrin in polarized Caco-2 cells Next, differentiated monolayer Caco-2 cells were obtained by a short-term method as described previously [24]. Immunostaining was performed to identify the subcellular localization of radixin and ezrin in the Caco-2 cells. As shown in Fig. 2, both radixin and ezrin were localized at the apical membrane bordered by ZO-1.

ABCC2 (MRP2) associated with radixin and ezrin in Caco-2 cells We performed an immunoprecipitation experiment to assess the level of ABCC2 (MRP2) which is associated with ezrin, radixin and actin in Caco-2 cells. ABCC2 (MRP2) and actin were detected in immunoprecipitates from Caco-2 cell lysates using antiradixin or anti-ezrin antibodies (Fig. 3). This result suggested

Fig. 6 – Immunofluorescent localizations of ZO-1 and actin in ezrin E6-4 (A) or radixin R4-5 (B) knockdown Caco-2 cell monolayers. ZO-1 (green) and actin (red) were stained with their specific antibody, respectively. Nuclei were stained with To-PRO-3 iodide (blue). Scale bar: 40 μm.

that ABCC2 (MRP2) could be associated with ezrin, radixin and actin and this association could play an important role in the unique apical membrane localization of ABCC2 (MRP2).

Transient suppression of radixin and ezrin by synthesized siRNA duplex

Fig. 5 – Western blot analysis of radixin or ezrin expression in siRNA stably expressed Caco-2 cells. Lysate proteins were isolated from each clone and separated with SDS–PAGE (10%). The expression of radixin was examined by monoclonal anti-moesin clone 38/87 against moesin and radixin. Ezrin was probed with specific monoclonal antibody M11. Each lane was loaded with 20 μg lysate protein.

Two siRNAs (R3 and R4) designed to target radixin, three siRNAs specific for ezrin (E1, E5 and E6), together with control siRNA were introduced into Caco-2 cells. The expression level of radixin or ezrin mRNA was examined by RT-PCR after a 48h incubation. No significant change in target mRNAs was observed in cells that were untreated (con.) and treated with control siRNA (mock). However, treatment with either R4 siRNA or E6 siRNA produced a significant reduction in the level of radixin or ezrin mRNA (Fig. 4). Similarly, R3 siRNA reduced the level of radixin mRNA, but E1 siRNA and E5 siRNA had no

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Caco-2 cells were transfected with radixin (R3 and R4) or ezrin (E6) siRNA expression vectors, and clones of each type of siRNAexpressing cells were established after puromycin selection. To evaluate the inhibitory effects against radixin or ezrin gene expression in these cells, western blotting was performed. R3-9 and R3-12 expressing R3 siRNA as well as clones R4-5 and R4-7 expressing R4 siRNA showed RNAi efficacy on the radixin gene to different extents (Fig. 5A). We also observed the inhibition of ezrin expression in two clones, E6-4 and E6-9, expressing E6 siRNA (Fig. 5B). Subcellular localization of ZO-1, a common tight junction marker, and actin were immunostained normally in E6-4 and R4-5 which suggests that these knockdown cell lines still exhibited cell polarity. Although the growth of knockdown cell lines is slow in comparison with control or mock cells, these cells retain their polarity (Fig. 6). Interestingly, in the ezrindeficient clone E6-9, the expression level of radixin was also reduced. Neither the radixin nor the ezrin knockdown clone showed compensatory up-regulation of other ERM family members, similar to the results obtained in ezrin, radixin or moesin gene deficient mice [27,16,21].

cells. Western blotting with proteins from the cell surface showed that the amount of ABCC2 (MRP2) in radixin or ezrin knockdown clones was significantly reduced, in comparison with that of wild-type Caco-2 cells or Caco-2 cells containing control siRNA vector (Fig. 7A). Interestingly, the level of ABCC2 (MRP2) on the cell surface was dramatically reduced in the E6-9 cells. The reason for this might be due to the fact that not only ezrin but also radixin was reduced in E6-9 cells. Several independent biotinylation analyses showed that the respective radixin KD/wild ratios of ABCC2 (MRP2) expression were: 41.98 ± 13.09 (R3-9), 41.09± 16.94 (R3-12), 33.79± 23.10 (R4-5), 31.05± 22.83 (R4-7) and the respective ezrin KD/wild ratios of ABCC2 (MRP2) expression were: 27.23± 16.61 (E6-4), 24.48± 14.39 (E6-9) (Fig. 7B). To determine whether radixin or ezrin deficiency also reduced the total amount of ABCC2 (MRP2) in Caco-2 cells, we performed western blotting of the cell lysate. In contrast to the result of the biotinylation assay, the total amount of ABCC2 (MRP2) in radixin or ezrin knockdown clones was less likely to be reduced (Fig. 8). These results indicated that there was a reduction of radixin or ezrin in Caco-2 cells caused by the loss of ABCC2 (MRP2) from the cell surface without reducing its total amount. We investigated the expression of P-gp in whole cells and the cell surface using clone R3-12, E6-4 and E6-9 (Fig. 9) to determine whether the reduction of radixin or ezrin also affects the localization of other apical transporters. Unlike the result of ABCC2 (MRP2), radixin or ezrin deficiency reduced the amount of P-gp at the cell surface as well as in whole cells.

Loss of ABCC2 (MRP2) from the cell surface in radixin or ezrin knockdown Caco-2 cells

Discussion

We investigated the cell surface expression of ABCC2 (MRP2) by biotinylation with wild-type and siRNA stably expressing Caco-2

Several factors influence the trafficking and localization of ABCC2 (MRP2), such as glycosylation of ABCC2 (MRP2) [28] and

marked effect on ezrin mRNA. Therefore, the efficacies of these synthesized siRNAs (R3, R4 and E6) in suppressing the target gene were confirmed.

Suppression of radixin or ezrin in siRNA stably expressed Caco-2 cells

Fig. 7 – ABCC2 (MRP2) at the apical membrane was reduced in ezrin or radixin knockdown Caco-2 cells. The lysate of cell surface protein in wild-type and siRNA stably expressed Caco-2 cells was isolated using biotinylation. (A) Cell surface proteins were separated by SDS–PAGE and analyzed with an antibody (M2III-6) specific for ABCC2 (MRP2). (B) Expression ratio of ABCC2 (MRP2) in ezrin or radixin knockdown cells relative to wild-type cells. Data are means ± SD (n = 3 or 4). *P b 0.01 versus wild-type cell.

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Fig. 8 – Western blot analysis of total ABCC2 (MRP2) expression in siRNA stably expressed Caco-2 cells. Total lysate was isolated from each clone and separated with SDS–PAGE (7% for E6-4, E6-9, R4-5 and R4-7; 8.5% for R3-9 and R3-12). The expression of ABCC2 (MRP2) was examined using a monoclonal antibody (M2III-6) against ABCC2 (MRP2). Each lane was loaded with 20 μg.

orientation of the microtubules [29,30] or actin cytoskeleton [31,32]. Notably, the actin filament organization as well as the indirect association of the actin filament with ion pumps through ERM proteins may alter the localization of ion pumps [33,34]. Several in vivo and in vitro rodent studies have been published describing radixin as a critical determinant of the apical membrane localization of ABCC2 (Mrp2) in the liver [16– 18]. However, ezrin is also known to regulate the localization of some apical membrane proteins in the mouse intestine [35]. In the case of humans, abnormal localization of ABCC2 (MRP2) as well as localization of radixin were reported in the autopsies on PBC (stage III) patients. However, this phenomenon is not a direct evidence of a relationship between ABCC2 (MRP2) and radixin and cell line studies are needed. Therefore, we employed human intestinal epithelial Caco-2 cells to investigate this issue. Radixin and ezrin are the only ERM proteins endogenously expressed in Caco-2 cell, and they have been found to localize at the apical membrane and interact with ABCC2 (MRP2) and actin (Figs. 1–3). In order to examine the role of radixin and ezrin in the localization of ABCC2 (MRP2), effective siRNAs targeting radixin or ezrin were selected and siRNA stably expressing cells were constructed (Figs. 4 and 5). Although the growth of knockdown cell lines is slow in comparison with control or mock cells, immunostaining with anti-ZO-1 antibody revealed the junctional localization of ZO-1 in every

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knockdown cell line, suggesting that these cells retain their polarity (Fig. 6). The siRNA treatments targeting radixin or ezrin both caused the loss of ABCC2 (MRP2) from the cell surface (Fig. 7), while the level of total ABCC2 (MRP2) in these cell lines showed no significant change (Fig. 8). Our study suggests that radixin and ezrin may mediate the apical membrane localization of ABCC2 (MRP2) via their interaction with ABCC2 (MRP2) and actin in human cells. It has been found that the C-terminus of ABCC2 (MRP2) binds the N-terminus of radixin directly [16]. ABCC2 (MRP2) has a PDZbinding motif at its C-terminus and interacts with a PDZ domain containing the PDZK1 protein [36]. ERM proteins can interact with several PDZ domain containing proteins such as EBP50/NHE-RF and E3KARP [11,37]. Thus, ABCC2 (MRP2) might bind radixin and ezrin directly via its C-terminal cytoplasmic domain or indirectly through the PDZ domain containing proteins. Similarly, P-gp association with actin through ERM plays a key role in the localizing of P-gp in well-defined membrane sites in human lymphoid cells [34]. It has been reported that radixin is required not only for the localization of ABCC2 (Mrp2) to the apical membrane, but also for other canalicular membrane transporters such as Bsep and Mdr1 in rat hepatocytes [18]. However, in radixin knockout mice, radixin deficiency selectively affected the canalicular membrane localization of ABCC2 (Mrp2) compared with P-gp at the early development [16]. However, a later report suggests that disruption of ABCC2 (Mrp2) on the canalicular membrane leads to a variable compensatory increase in basolateral transporter Mrp3, depending on the genetic background and no significant difference was observed in the case of the amount of P-gp even in adult KO mice [17]. In the ezrin-deficient clone E6-9 and the radixin-deficient clone R3-12, we showed that the expression of P-gp in whole cells and the cell surface was reduced in both cases (Fig. 9). These discrepancies may be attributed to species and cell type differences. Because there is a high degree of homology among ERM members, they have been presumed to be functionally redundant. A recent study of ERM proteins has led to a dispute as to whether they are redundant proteins. In moesin-deficient mice and isolated moesin-deficient cells, targeted disruption of the moesin gene did not induce compensatory up-regulation of other ERM members. Also, in isolated moesin-deficient cells, the ERM-dependent functions were not affected [21]. However, the tissue distribution and primary structures of ERM proteins

Fig. 9 – Western blot analysis of P-gp expression in the whole cell and cell surface in ezrin or radixin knockdown cells. Whole cell lysate protein was isolated from each clone and separated with 7% SDS–PAGE, each lane was loaded with 20 μg (upper panels). The lysate of cell surface protein was isolated by biotinylation and separated by 7% SDS–PAGE (lower panels). The expression of P-gp was examined using a monoclonal antibody (C219) against P-gp.

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indicate that these are not simply redundant proteins [38]. It is interesting to note that moesin-deficient mice are viable with no gross abnormalities, and radixin-deficient mice are viable but develop hyperbilirubinemia with defective localization of ABCC2 (Mrp2) to the bile canalicular membrane [16]. Furthermore, radixin or moesin knockout mice can grow normally but ezrin knockout mice do not survive past weaning [35,27]. Our results showed that all the ERM proteins, radixin and ezrin in Caco-2 cells contributed to the apical localization of ABCC2 (MRP2). Also, the suppression of both radixin and ezrin caused a more marked disappearance of ABCC2 (MRP2) from the cell surface. This appears to support the hypothesis that ERM proteins are functionally redundant. Caco-2 cells have been used to predict intestinal paracellular and transcellular transport. The expression and localization of ERM proteins in small intestine tissue are different from that in Caco-2 cells. In the small intestine, ezrin is highly enriched on the apical aspect of epithelial cells while radixin and moesin are found in the lamina propria of endothelial cells [39,35]. Due to its restricted pattern of localization in polarized epithelia, ezrin has been reported to be crucial in the epithelial organization in the intestine [35,27]. The effect of ezrin on the apical membrane localization of ABCC2 (MRP2) in Caco-2 cells may also be seen in the intestine. Radixin and ezrin are expressed at the apical surface of kidney proximal tubules and, therefore, they may play a role in the ABCC2 (MRP2) localization in the kidney. Confirmation using knockdown and knockout mice is now in progress. Finally, our ERM knockdown Caco-2 cells, together with ERM-deficient animals, are good systems for studying the role of ERM protein in the localization of some apical membrane proteins.

[6]

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Acknowledgments We thank Prof. Sachiko Tsukita at Kyoto University for helpful comments on the manuscript. This work was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

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