Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

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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

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions Masafumi Imamura a,b , Takashi Kojima b,⁎, Mengdong Lan b , Seiichi Son a,b , Masaki Murata b , Makoto Osanai b , Hideki Chiba b , Koichi Hirata a , Norimasa Sawada b a

Department of Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan Department of Pathology, Sapporo Medical University School of Medicine, S1. W17. Sapporo 060-8556, Japan

b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected

Received 20 December 2006

and play crucial roles in the barrier to keep bile in bile canaculi away from the blood

Revised version received

circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral

1 March 2007

hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although

Accepted 15 March 2007

claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells,

Available online 20 March 2007

the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of

Keywords:

hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal

Claudin-2

hepatocytes. In this study, we examined whether OSM could induce expression and

Hepatocyte

function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured

Oncostatin M

proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative

Signal transduction

rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi. © 2007 Elsevier Inc. All rights reserved.

Introduction Hepatic tight junctions (TJs) play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation, which we call the blood–billiary barrier [1–3]. Intrahepatic cholestasis or impairment of bile flow is an important manifestation of inherited and acquired liver disease. In

intrahepatic cholestasis leading to the leakage of bile into blood, it is well-known that a loss or decrease of TJ function is observed as well as downregulation of gap junction function and disruption of the myosin–actin network [4,5]. Although hepatic TJs are influenced by various cytokines during cholestasis, the mechanisms of downregulation of TJ functions are still unclear.

⁎ Corresponding author. Fax: +81 11 613 5665. E-mail address: [email protected] (T. Kojima). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.03.010

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TJs, which are located at the most apical aspect of the lateral membranes of epithelial and endothelial cells, perform two functions, namely the control of paracellular diffusion of ions and small molecules (barrier function) and the restriction of intramembrane diffusion of proteins and lipids between the membrane poles (fence function) [6–9]. They show a particular netlike meshwork of fibrils formed by the integral membrane proteins, occludin, the claudin family and JAM. Several peripheral membrane proteins, ZO-1, -2, -3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6, and ASIP, an atypical protein kinase C-interacting protein, were reported [7,10]. The claudin family, consisting of 24 members, is solely responsible for forming TJ strands and shows tissue- and cellspecific expression of individual members [7]. Several lines of evidence point to claudins as the basis for the selective size, charge, and conductance properties of the paracellular pathway [11]. In murine livers, claudin-1, -2, -3, -5 and -14 are detected and claudin-1, -2, -3 are expressed in the bile canaliculus region of hepatocytes [3,12]. In the rat liver, claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule [13,14]. When cDNAs of claudin-1 and -2 were introduced into mouse L fibroblasts lacking TJs, claudin1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face that were occupied by chains of particles [15]. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells [16]. There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of TJ strands. Claudin-1 and -4 are expressed both in MDCK I and II cells, whereas the expression of claudin-2 is restricted to MDCK II cells. When dog claudin-2 cDNA was introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells, the TER values of MDCK I clones stably expressing claudin-2 fell to the levels of MDCK II cells [17]. Furthermore, with the knockdown of endogenous claudin-2 expression in MDCK cells using siRNA against claudin-2, removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity [18]. Previous studies have indicated the involvement of distinct signal transduction pathways in the regulation of claudin-2 expression [19–23]. In primary cultures of rat hepatocytes, IL1β treatment induces proteins and mRNA of claudin-2 via p38 MAPK and PI3K pathways [22]. In oncogenic Ras- or Raf-1transfectants, claudin-2 is downregulated through MAPK signal pathways [20,24]. In primary cultures and hepatic cell lines derived from occludin-deficient mice, claudin-2 expression is increased by downregulation of activated MAPK and Akt [23]. It has recently been reported that cingulin regulates claudin-2 expression through the small GTPase RhoA [25]. Furthermore, the claudin-2 promoter in the human intestine is activated by transcription factors CDX2, HNF-1alpha, and GATA-4 in a cooperative manner [26]. A snail gene that is closely associated with epithelial mesenchymal transition

(EMT) induces a moderate decrease in claudin-2 [27–29]. Although it is thought that claudin-2 may be a key molecule in hepatic TJs, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a member of the IL-6 family of cytokines and is considered to be a multifunctional cytokine implicated in the activation, proliferation and/or differentiation of several cell types such as hepatocytes, osteoblasts and lung epithelial cells [30–32]. OSM is produced by hematopoietic cells and induces differentiation of fetal hepatic cells, conferring various metabolic activities of the adult liver [34]. It can also induce formation of E-cadherin-based adherens junctions via K-ras [35]. OSM exerts its action via the signal transducers gp130, LIF receptor and OSM receptor, leading to the activation of the JAK/STAT and MAPK cascades [33]. In the cirrhotic human liver, expression of OSM is upregulated [36]. However, the effects of OSM on the expression and function of hepatic TJs are not known. In the present study, we found that OSM could induce expression of claudin-2 in rodent hepatic cells in vitro via distinct signal transduction pathways including PKC. Induction of claudin-2 by OSM in rodent hepatocytes enhanced paracellular barrier function and regulated the size selectivity of paracellular permeability.

Materials and methods Cytokine, inhibitors, and antibodies Mouse oncostatin M (OSM) was purchased from R&D Systems, Inc. (Minneapolis, MN). The protein kinase C inhibitor (GF109203), MAP-kinase inhibitor (PD98059), p38 MAP-kinase inhibitor (SB203580), and PI3-kinase inhibitor (LY294002) were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA). The mouse monoclonal antibody to occludin and rabbit polyclonal antibodies to occludin, claudin-1, claudin-2 and rabbit anti-claudin-3 were obtained from Zymed Laboratories (San Francisco, CA). The rabbit polyclonal anti-JAM-A antibody was a kind gift of Dr. T. Kita. Rabbit polyclonal antiERK1/2 and rabbit polyclonal anti-phospho-p44/42 MAPK (phospho-ERK1/2) antibodies were purchased from Promega Corporation (Madison, WI). TRITC–phalloidin was purchased from Sigma. Secondary antibodies, Alexa 488 (green)-conjugated anti-rabbit IgG, and Alexa 592 (red)-conjugated antimouse IgG, were purchased from Molecular Probes Inc. (Eugene, OR). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from DAKO (A/S, Denmark). The enhanced chemiluminescence (ECL) Western blotting system was obtained from Amersham Corp. (Buckinghamshire, UK). 14C-mannitol (50–62 mCi/mmol) and 14 C-inulin (5–20 mCi/mmol) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Mouse hepatic cell line and culture CHST8 mouse hepatic cells were transfected with human connexin 32 cDNA as described previously [37]. The cells were incubated with DMEM medium (GIBCO BRL, Gaithersburg, MD) with 4% fetal bovine serum (Sigma Co., St. Louis, MO) and

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antibiotics in a humidified 5% CO2–95% air incubator at 37 °C. The cells were treated with 10 ng/ml OSM for 24 h–144 h. Some cells were treated with 10 μM GF109203, 10 μM PD98059, 10 μM SB203580 and 10 μM LY294002 at 1 h before treatment with 10 ng/ml OSM for 24 h.

Isolation and culture of rat hepatocytes Male Sprague–Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) (range, 300–400 g) were used to isolate hepatocytes by the two-step liver perfusion method of Seglen with some modification. Briefly, the liver was perfused in situ through the portal vein with 150 ml of Ca2+, Mg2+-free Hanks' balanced salt solution (HBSS) supplemented with 0.5 mM EGTA (Sigma), 0.5 mg/l insulin from bovine pancreas (Sigma), and antibiotics. After the initial perfusion, the liver was perfused with 200 ml of HBSS containing 40 mg of collagenase (Yakult Co., Tokyo, Japan) for 10 min. The isolated cells were purified by Percoll isodensity centrifugation. Viability of the cells, as judged by the trypan blue exclusion test, was more than 90% in these experiments. The cells were suspended in L-15 medium (Gibco BRL, Gaithersburg, MD) with 0.2% bovine serum albumin (Sigma), 20 mM HEPES (Sigma), 0.5 mg/l insulin (Sigma), 10− 7 M dexamethasone (Sigma), 1 g/l galactose (Sigma), 30 mg/1 proline (Sigma), 20 mM NaHCO3, 10 ng/ml EGF (Collaborative Res., Lexington, MA), and antibiotics (modified L-15 medium). The isolated hepatocytes

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were plated at a density of 5.5 × 105 cells/ml on 35- and 60-mm culture dishes (Corning Glass Works, Coming, NY), which were coated with rat tail collagen (500 μg dried tendon/ml in 0.1% acetic acid), and placed in a humidified 5% CO2–95% air incubator at 37 °C. The medium was replaced with fresh medium every other day. After 96 h of culture, 2% DMSO (Aldrich Chemical Co. Inc., Milwaukee, WI) and 10− 7 M glucagon (Glucagon S, Yamanouchi, Tokyo, Japan) were added to the modified L-15 medium and then the cells were cultured until 9 days after plating. The cells were treated with 10 ng/ml OSM for 24 h at days 3, 5, and 9 after plating. Some cells were treated with 10 μM GF109203, 50 μM PD98059, 20 μM SB203580 and 20 μM LY294002 at 1 h before treatment with 10 ng/ml OSM for 24 h.

Western blot analysis For Western blotting of total cell 1ysates, the dishes were washed with phosphate-buffered saline twice, and 300 μl of sample buffer (1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride [PMSF; Sigma]) was added to 60-mm dishes. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 s. The protein concentration of the samples was determined using a BCA Protein Assay Reagent Kit (Pierce Chemical Co, Rockford, IL, USA). Western blot analysis was performed as described previously. Briefly, aliquots of 15 μg of protein/lane for each sample were separated by electrophor-

Fig. 1 – Immunocytochemistry for claudin-1, -2, occludin and JAM-A in immortalized mouse hepatocytes. Scale bar: 20 μm. Claudin-1, occludin and JAM-A are strongly localized at the cell border, whereas claudin-2 is faintly stained.

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esis in 4/20% gradient sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) (Daiichi Pure Chemicals Co., Tokyo, Japan). After electrophoretic transfer to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), the membranes were saturated with a blocking buffer (25 mM Tris, pH 8.0, 125 mM NaC1, 0.1% Tween 20, and 4% skim milk) for 30 min at room temperature. The quality of the transfer was controlled by Ponceau S staining of the membranes. The membranes were incubated with anti-claudin-1, anti-claudin-2, anti-claudin-3, anti-occludin, anti-JAM-A, anti-pMAPK, anti-ERK, or anti-actin antibodies at room temperature for 1 h. Membranes were incubated with horseradish peroxidase-conjugated antimouse IgG or anti-rabbit IgG at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting system.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted and purified using Trizol (Invitrogen). One microgram of total RNA was reverse transcribed (RT) into cDNA using a mixture of oligo (dT) and Superscript ™ II RTase under the recommended conditions (Invitrogen). Each cDNA synthesis was performed in a total volume of 20 μl for 50 min at 42 °C and terminated by incubation for 15 min at 70 °C. PCR containing 100 pM primer pairs and 1.0 ml of the 20 ml total RT reaction was performed in 20 ml of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.4 mM dNTPs, and 0.5 U of Taq DNA polymerase (Takara), applying 25 or 30 cycles with cycle times of 15 s at 96 °C, 30 s at 55 °C, and 60 s at 72 °C. Final elongation time was 7 min at 72 °C. Ten microliters of the 20 μl total PCR reaction was analyzed in 1% agarose gel after staining with ethidium bromide. Primers used to detect claudin-1, claudin-2, claudin-3, occludin, JAM-A, mouse OSM-receptorβ (OSMRβ) and rat OSMRβ by RT-PCR had the following sequences: claudin-1 (sense 5′-GCTGCTGGGTTTCATCCTG-3′ and antisense 5′-CACATAGTCTTTCCCACTAGAAG-3′, amplicon length: 619 bp), claudin-2 (sense 5′-GCAAACAGGCTCCGAAGATACT-3′ and antisense 5′-GAGATGATGCCCAAGTACAGAG-3′, amplicon length: 546 bp), claudin-3 (sense 5′-TGCTGTTCCTTCTCGCCGCC-3′ and antisense 5′-CTTAGACGAAGTCCATGCGG-3′, amplicon length 247 bp), occludin (sense 5′TCAGGGAATATCCACCTATCACTTCAG-3′ and antisense 5′-CATCAGCAGCAGCCATGTACTCTTCAC-3′, amplicon length: 186 bp), JAM-A (sense 5′-GGTCAAGGTCAAGCTCAT-3′ and antisense 5′-CTGAGTAAGGCAAATGCAG-3′, amplicon length 765 bp), mouse OSMRβ (sense 5′-ATCCAAAGGCTCCGCAGGAC3′ and antisense 5′-GTAAGGTTGCAGGTCAAGGC-3′, amplicon length 419 bp), rat OSMRβ (sense 5′-GGACCTGCATCCCTACAAGA-3′, and antisense 5′-TGGGACCTACGTTCTTCCAC-3′, amplicon length 248 bp). To provide a qualitative control for reaction efficiency, PCR reactions were performed with primers coding for the housekeeping gene G3PDH (sense 5,ACCACAGTCCATGCCATCAC-3, and antisense 5′-TCCACCACCCTGTTGCTGTA-3′, amplicon length: 452 bp).

Immunofluorescence microscopy For immunocytochemistry, cells grown on coated-glass coverslips were fixed with an ethanol and acetone mixture (1:1) at

−20 °C for 10 min. After the coverslips were rinsed with PBS, some were incubated with anti-claudin-1, anti-claudin-2, anti-occludin and anti-JAM-A antibodies, and TRITC–phalloidin (Sigma) at room temperature for 1 h and then were incubated with Alexa 488 (green)-conjugated anti-rabbit IgG or Alexia 592 (red)-conjugated anti-mouse IgG at room temperature for 1 h. The specimens were examined using a laser-scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA).

Freeze fracture analysis Cells were cultured on 60-mm dishes. Dishes were washed with PBS, and the cells were scraped from the dishes and collected in microcentrifuge tubes. They were fixed then with 2.5% glutaraldehyde/0.1 M PBS (pH 7.3). After fixation, they were immersed in 40% glycerin solution. The specimens were

Fig. 2 – Changes of tight junction proteins in immortalized mouse hepatocytes after treatment with 10 ng/ml OSM. (A) In Western blotting, expression of claudin-2 protein is markedly increased at 24 h after treatment with OSM, whereas expression of claudin-1, occludin and JAM-A is unchanged. (B) In RT-PCR, mRNAs of claudin-2 and OSMRβ are increased at 24 h after treatment with 10 ng/ml OSM, whereas expression of claudin-1, occludin and JAM-A is unchanged. In Western blotting (C) and RT-PCR (D), the increase in protein and mRNA of claudin-2 was observed in a time-dependent manner.

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Fig. 3 – Immunocytochemistry for claudin-2 (A) and freeze fracture replicas (B) in immortalized mouse hepatocytes at 24 h after treatment with 10 ng/ml OSM. (A) Claudin-2 appears in most cells and strongly localizes at cell borders after treatment with OSM. Scale bars: 20 μm. (B) Well-developed network of many TJ strands is observed in the hepatocytes after treatment with OSM, whereas parallel lines of TJ strands are observed in control hepatocytes. Scale bar: 50 nm.

mounted on a copper stage, cooled in liquid nitrogen, and fractured at − 150 to −160 °C in a JFD-7000 freeze fracture device (JEOL Ltd., Tokyo, Japan). Platinum–carbon replicas were made without etching. After thawing, the replicas were floated on filtered 10% sodium hypochlorite solution for 30 min in a Teflon dish. Replicas were washed in distilled water, mounted on copper grids, and examined at an acceleration voltage of 100 kV with a JEOL-1200EX transmission electron microscope (JEOL Ltd.).

Measurement of transepithelial electrical resistance (TER) Cells were cultured to confluence on 12-mm Transwell with 0.4-μm pore size filters (Corning Inc., NY) coated with rat tail collagen. TER was measured in culture medium or in P buffer containing 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.3, using an EVOM voltameter with an ENDOHM-12 (World Precision Instruments) on a heating plate (Fine, Tokyo, Japan) adjusted to

Fig. 4 – Barrier function measured as TER in immortalized mouse hepatocytes after treatment with 10 ng/ml OSM (n = 6). The values of TER were significantly increased in the hepatocytes from day 1 after treatment with OSM compared to the control. **p < 0.01 versus control.

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37 °C. To examine the effects of Na+ and Cl− on TER separately, some of the TER measurements were performed in modified P buffer with 140 mM NaCl replaced by 140 mM arginine–HCl, or 140 mM lysine–HCl. At neutral pH, arginine and lysine are positively charged in the solution. HCl dissociates into H+ and Cl− in the solution, and H+ will combine with OH− to form water at pH 7.3. The TER values are expressed in standard units of ohms per square centimeters and presented as the mean ± SD. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells.

Measurement of permeability (fluxes of FITC–dextrans, 14 C-inulin and 14C-mannitol) To determine the paracellular flux, the cells were cultured on 12-mm Transwell, 0.4-μm pore size filters (Corning Inc.), and fluorescein isothiocyanate (FITC)–dextran (MW 500 kDa or 70 kDa)-, 14C-inulin (MW 5 kDa)- or 14C-mannitol (MW 182 Da)containing medium was added to the inner chamber. Samples were collected from the outer chamber at 15, 30, 60 and 90 min. The fluorescent signals were measured at an excitation wavelength 485 nm and emission one of 535 nm (Wallac

Fig. 5 – Barrier function measured as paracellular fluxes using FITC–dextran (MW 500 kDa and 70 kDa), 14C-inulin (MW 5 kDa) and 14C-mannitol (MW 182 Da) in immortalized mouse hepatocytes at day 1 after treatment with 10 ng/ml OSM (n = 3). (A) Paracellular fluxes of FITC–dextran of 70 kDa and 500 kDa and 14C-inulin, but not 14C-mannitol, were decreased after treatment with 10 ng/ml OSM compared to the control. (B) Paracellular flux rates of FITC–dextran of 70 kDa and 500 kDa, 14C-inulin, 14 C-mannitol were 91%, 73%, 65% and 32%, respectively. *p < 0.05 versus control.

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1420 Multilabel Counter ARVO™ MX, Perkin Elmer). FITC– dextran fluxes were calculated as ng/μl. The 14C-inulin and 14 C-mannitol signals were measured with a liquid scintillation counter (Beckman LS-6500). The results were expressed as clearance per hour per square centimeter (inulin: pmol/h/cm2, mannitol: nmol/h/cm2). The paracellular flux rate was calculated as the measured value of the OSM-treated group/that of control group at 90 min.

BrdU labeling index To examine the BrdU labeling index, immunocytochemical staining for 5′-bromo-deoxy-uridine (BrdU) was performed. Twenty micromolar BrdU was added to each 35-mm dish containing coated-glass coverslips at 24 h before fixation. The samples were fixed with an ethanol and acetone mixture (1:1) at − 20 °C for 10 min and then incubated in 2 N–HCl for 15 min. After the samples were rinsed in PBS, they were incubated with the anti-BrdU antibody at room temperature for 1 h and then incubated with Alexa 488 (green)-conjugated anti-mouse IgG at room temperature for 1 h. Samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 2 min. Nuclei from 3 random fields were analyzed in each slide. The percentage of cells of DNA synthesis was calculated as BrdUpositive nuclei/DAPI-positive nuclei, and shown as the BrdU index.

Data analysis Signals were quantified using the Scion Image Beta 4.02 Win (Scion Corporation, Frederick, Ml, USA). Each set of results shown is representative of three separate experiments. Results are given as means ± SEM. Differences between groups were tested by the two-tailed Student's t test for unpaired data.

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treatment with OSM, whereas expression of claudin-1, occludin and JAM-A was unchanged (Fig. 2A). In RT-PCR, mRNAs of claudin-2 and OSMRβ were increased at 24 h after treatment with OSM, whereas those of claudin-1, occludin and JAM-A were unchanged (Fig. 2B). The increases in protein and mRNA of claudin-2 were observed in a time-dependent manner (Figs. 2C and D). We examined the change in the localization of claudin-2 in immortalized mouse hepatocytes at 24 h after treatment with 10 ng/ml OSM. Immunoreactivity to claudin-2 appeared in most cells and was predominantly localized at cell borders (Fig. 3A). However, no changes in localization of claudin-1, occludin or JAM-A were observed (data not shown).

Change of TJ strands in immortalized mouse hepatocytes after treatment with OSM To examine changes of TJ strands in immortalized mouse hepatocytes at 24 h after treatment with 10 ng/ml OSM, we performed freeze fracture analysis. In the control, TJ strands were discontinuous at the P face, which was occupied by chains of particles (Fig. 3B). In the hepatocytes treated with OSM, a well-developed network of TJ strands was observed, whereas parallel lines of TJ strands were observed in the control (Fig. 3B).

Change of barrier function in immortalized mouse hepatocytes after treatment with OSM To examine changes of the barrier function of TJ in immortalized mouse hepatocytes after treatment with OSM, TER and paracellular fluxes were measured using FITC–dextran (MW 500 kDa and 70 kDa), 14C-inulin (MW 5 kDa) and 14C-mannitol (MW 182 Da). The values of TER were significantly increased

Results Localization of integral TJ proteins in immortalized mouse hepatocytes To examine the localization of TJ proteins in immortalized mouse hepatocytes, we performed immunocytochemistry for claudin-1, -2, occludin and JAM-A. In the hepatocytes, claudin1, occludin and JAM-A were exclusively localized at the cell borders, whereas claudin-2 was faintly stained (Fig. 1).

Changes in expression and localization of claudin-2 in immortalized mouse hepatocytes after treatment with OSM In Western blotting of the control hepatocytes, proteins of claudin-1, -2, occludin and JAM-A were detected. Only the expression of claudin-2 was low (Fig. 2A). In RT-PCR of the control hepatocytes, mRNAs of claudin-1, -2, occludin, JAM-A and OSMRβ were detected (Fig. 2B). We examined changes in expression of proteins and mRNAs of claudin-1, -2, occludin and JAM-A in immortalized mouse hepatocytes after treatment with 10 ng/ml OSM for 24– 96 h. Claudin-2 protein was markedly increased at 24 h after

Fig. 6 – Change of paracellular Cl− and Na+ conductance in immortalized mouse hepatocytes at day 1 after treatment with 10 ng/ml OSM. TER was measured in culture medium (n = 6) or in P buffer (n = 6) or in the modified P buffer with 140 mM NaCl replaced by 140 mM arginine–HCl (n = 4), or 140 mM lysine–HCl (n = 4). When NaCl was substituted by arginine–HCl or lysine–HCl, the increase in TER after treatment with OSM was not affected. *p < 0.05 and **p < 0.01 versus control.

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Changes of paracellular Cl− and Na+ conductance in immortalized mouse hepatocytes after treatment with OSM It is known that claudin-2 forms cation-selective channels in MDCK cells [16]. To examine whether claudin-2 expression induced by treatment with OSM affected the paracellular conductance to Na+ or Cl−, some of the TER measurements were performed in modified P buffer with 140 mM NaCl replaced by 140 mM arginine–HCl, or 140 mM lysine–HCl. However, when NaCl was substituted by arginine–HCl or lysine–HCl, the significant increase in TER after treatment with 10 ng/ml OSM was not affected (Fig. 6).

Changes in expression of tight junction proteins in primary cultured rat hepatocytes after treatment with OSM To examine effects of OSM on tight junctions of proliferative rat hepatocytes in primary culture, the cells cultured at days 3, 5 and 9 after plating were treated with 10 ng/ml OSM for 24 h.

Fig. 7 – BrdU labeling indices at days 3, 5 and 9 after plating in primary cultured rat hepatocytes (n = 3). BrdU labeling indices at days 3, 5 and 9 after plating were 54%, 23% and 10%, respectively. Scale bar: 40 μm. **p < 0.01 versus Day3.

from day 1 after treatment with 10 ng/ml OSM and they depended on the time in culture (Fig. 4). Paracellular fluxes of FITC–dextran of 70 kDa and 500 kDa and 14C-inulin, but not 14 C-mannitol, were clearly decreased at day 1 after treatment with 10 ng/ml OSM compared to the control (Fig. 5A). Paracellular flux rates of FITC–dextran of 500 kDa and 70 kDa, 14C-inulin, 14C-mannitol were shown to be 32%, 65%, 73% and 91%, respectively (Fig. 5B). These results indicated that changes of permeability after treatment with OSM depended on molecular size.

Fig. 8 – Changes of tight junction proteins in primary cultured rat hepatocytes 24 h after treatment with 10 ng/ml OSM. In Western blotting (A) and in RT-PCR (B) of the primary rat hepatocytes after treatment with OSM, increases of claudin-2 and decreases of claudin-1 were observed at days 3, 5 and 9 after plating.

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In this culture system, the BrdU labeling indices at days 3, 5 and 9 after plating were 54%, 23% and 10%, respectively (Fig. 7). In Western blotting and RT-PCR of the primary rat hepatocytes after treatment with 10 ng/ml OSM, an increase of claudin-2 and a decrease of claudin-1 were observed at days 3, 5 and 9 after plating (Figs. 8A, B). In RT-PCR, OSMRβ mRNA was increased at days 3 and 5 after plating. At day 3 after plating when the BrdU labeling index was high, expression in protein and mRNA of claudin-2 was much increased by treatment with OSM (Figs. 8A, B). Expression of claudin-3, occludin and JAM-A was unchanged (Fig. 8). In immunocytochemistry for the control hepatocytes at day 3 after plating, claudin-2 was not detected but claudin-1 was strongly expressed at cell borders (Fig. 9). In the hepatocytes after treatment with 10 ng/ml OSM, the appearance of claudin-2 and a decrease of claudin-1 were observed at cell borders compared to the control (Fig. 9).

Effects of PKC, MAP-kinase, p38 MAP-kinase and PI3-kinase inhibitors on expression of claudin-2 induced by treatment with OSM To investigate which signal transduction pathways were involved in the upregulation of claudin-2 in the immortalized mouse hepatocytes and the primary cultured rat hepatocytes after treatment with OSM, we used the PKC, MAP-kinase, p38 MAP-kinase and PI3-kinase inhibitors, GF109203, PD98059,

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SB203580 and LY294002, respectively. In immortalized mouse hepatocytes, upregulation of claudin-2 protein after treatment with 10 ng/ml OSM, was strongly inhibited by GF109203, SB203580 and LY294002, and was slightly inhibited by PD98059 (Fig. 10A). In primary cultured rat hepatocytes at day 3 after plating, upregulation of claudin-2 protein after treatment with 10 ng/ml OSM, was strongly inhibited by GF109203 and was slightly inhibited by LY294002, whereas PD98059 and SB203580 did not affect it (Fig. 10B).

Discussion In the present study, we found that OSM induced expression of the integral tight junction protein claudin-2 in rodent hepatic cells in vitro via distinct signal transduction pathways including PKC. Expression of claudin-2 by OSM in rodent hepatic cells enhanced paracellular barrier function and regulated size in the paracellular permeability. Claudin-2 is a structural component of TJs in the liver, kidney, pancreas, stomach, and small intestine [7,38]. In the rat liver, claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-3 is uniformly expressed [14]. In the present study, we focused on the effects of OSM on TJs of hepatocytes because OSM induces differentiation of fetal hepatic cells and can induce formation of E-cadherin-based adherens junctions of hepatocytes

Fig. 9 – Changes in localization of claudin-1 and -2 in primary cultured rat hepatocytes at 24 h after treatment with 10 ng/ml OSM. Scale bar: 20 μm. In the control hepatocytes at day 3 after plating, claudin-2 is not detected and claudin-1 is strongly expressed at cell borders. In the hepatocytes after treatment with OSM, the appearance of claudin-2 and a decrease of claudin-1 are observed at cell borders.

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Fig. 10 – Effects of a PKC inhibitor (GF109203), a MAP-kinase inhibitor (PD98059), a p38 MAP-kinase inhibitor (SB203580) and a PI3-kinase inhibitor (LY294002) on expression of claudin-2 protein induced by treatment with OSM (n = 3). (A) In immortalized mouse hepatocytes, upregulation of claudin-2 protein after treatment with 10 ng/ml OSM was strongly inhibited by GF109203, SB203580 and LY294002, and was slightly inhibited by PD98059. *p < 0.05 and **p < 0.01 versus NT. (B) In primary cultured rat hepatocytes at day 3 after plating, upregulation of claudin-2 protein after treatment with 10 ng/ml OSM was strongly inhibited by GF109203 and was slightly inhibited by LY294002. *p < 0.05 versus NT.

[34,35]. We used immortalized mouse hepatocytes and primary cultured proliferative rat hepatocytes in which claudin-2 expression was low. In the immortalized mouse hepatocytes and primary cultured proliferative rat hepatocytes after treatment with OSM, an increase in protein and mRNA of claudin-2 was observed. In the immortalized mouse hepatocytes, upregulation of claudin-2 by OSM was controlled through PKC, MAPK, p38 MAPK and PI3K signaling pathways. In primary cultured rat hepatocytes at day 3 after plating, upregulation of claudin-2 by OSM, was controlled through PKC and PI3K signaling pathways. In primary cultures of adult rat hepatocytes, IL-1beta regulated expression of claudin-2 via p38

MAPK and PI3K [22]. IL-17 induced claudin-2 expression via MAPK in T84 cells [19]. The MAPK signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells [39]. In hepatocytes derived from occludin-deficient mice, claudin-2 expression was increased via MAPK and PI3K [23]. These results indicated that the increase of claudin-2 expression induced by OSM in rodent hepatic cells was regulated through distinct signaling pathways including PKC. In the transfection experiments, claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular E face that were occupied by chains of particles in freeze fracture replicas [15]. In the control of the immortalized mouse hepatocytes, the TJ strands were discontinuous at the P face, and were seen as chains of particles. In the hepatocytes after treatment with OSM, well-developed networks of the TJ strands were observed on the wide membranes of the hepatocytes. The results of freeze fracture electron microscopy supported that immunoreactivity of claudin-2 induced by OSM was exclusively localized at cell borders and that the increase of claudin-2 expression enhanced TJ barrier function. Claudins create the barrier and regulate electrical resistance, size, and ionic charge selectivity [40]. Claudin-2 expression induces cation-selective channels in TJs of epithelial cells [16], and high expression of claudin-2 decreases TER in the transfection experiments [17,39]. Furthermore, with the knockdown of endogenous claudin-2 expression in MDCK cells by siRNA, removal of claudin-2 depresses the permeation of Na+ and results in the loss of cation selectivity [18]. In contrast, expression of claudin-2 increases TER by only 20% and does not change the ionic selectivity in low-resistance MDCK II cells [41]. On the other hand, claudin-5 causes a selective increase in paracellular permeability of small molecules in endothelial cells [42,43]. In the present study, in immortalized mouse hepatocytes, the increase of claudin-2 induced by OSM enhanced paracellular barrier function measured as TER and paracellular fluxes. The paracellular permeability depended on molecular size. However, in the immortalized mouse hepatocytes used in this study, expression of claudin-2 induced by OSM did not affect the paracellular conductance to Na+ or Cl−. These finding indicated that claudin-2 in hepatocytes might create the barrier function and regulate size but not the ionic charge selectivity in paracellular permeability. Alternatively, since the TER of the cells was quite low, changes of Na+ permeability, possibly induced by OSM, might not influence the barrier function. OSM plays a role in many biological processes such as inflammation, hematopoiesis, embryonic development and tissue remodeling in various tissues [31]. It is produced by hematopoietic cells and induces differentiation of fetal hepatic cells and formation of E-cadherin-based adherens junctions via K-ras [34,35]. In the adult liver, OSM may play roles in metabolism, inflammation, tissue remodeling and regeneration of hepatocytes [31]. OSMR-mediated signaling is required for the early proliferative response of hepatocytes to hepatectomy as well as toxic injury [44]. In the cirrhotic human liver, expression of OSM is upregulated [36]. In the present study, higher induction of claudin-2 by OSM was observed in highly proliferative adult rat hepatocytes. Thus, induction of claudin-2 by OSM in hepatocytes may play a

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crucial role in hepatocyte remodeling, regeneration and liver tissue. Activation of the signaling transduction pathways mediated by OSM requires the binding of the cytokine to either the type I OSM receptor (LIFR/gp130) or type II OSM receptor (OSMR/ gp130). Whereas human OSM has the capability to evoke signals both via LIFR/gp130 and OSMR/gp130 heterodimers, murine OSM solely utilizes the OSMR/gp130 heterodimer for signal transduction [45,46]. In human fetal hepatocytes, treatment with OSM increases cell size and enhances cell differentiation and the formation of bile canaliculi [32]. These findings may explain the partly inconsistent observations in this study, because both mouse and rat hepatocytes were used. Furthermore, a decrease of claudin-1 was observed in the rat hepatocytes after treatment with murine OSM, whereas in the mouse hepatocytes, murine OSM did not affect expression of claudin-1. In the future, when we perform further experiments using human hepatocytes and human OSM, there may be different responses of claudins and signaling pathways from murine hepatocytes and rodent OSM. In conclusion, induction of claudin-2 by OSM in rodent hepatocytes creates the barrier function and regulates size selectivity in paracellular permeability. These results suggest that, in the cirrhotic human liver, claudin-2 may be induced by the upregulation of OSM and affect bile canalicular seal.

Acknowledgments We thank Ms. E. Suzuki (Sapporo Medical University) for the technical support. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports Science, and Technology, and the Ministry of Health, Labour and Welfare of Japan and the Akiyama Foundation and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.

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