Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line
Mengdong Lan,
Takashi Kojima1,
Makoto Osanai,
Hideki Chiba and
Norimasa Sawada
Department of Pathology, Sapporo Medical University School of Medicine, S1 W17, Sapporo 060-8556, Japan
1 To whom correspondence should be addressed. Tel: +81 11 611 2111; Fax: +81 11 613 5665; Email: ktakashi{at}sapmed.ac.jp
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Abstract
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The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RTPCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [14C]mannitol and [14C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.
Abbreviations: Cx32, connexin32; DMEM, Dulbecco's modified Eagle's medium; ECL, enhanced chemiluminescence; EMT, epithelial to mesenchymal transition; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, human hepatocellular carcinoma; HRP, horseradish peroxidase
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Introduction
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Raf-1 is the cellular proto-oncogene homologue of v-Raf, a retroviral oncogene, and Raf-1 serine/threonine kinase is a central component of the MAP kinase cascade (1,2). The Raf/MEK/ERK signaling pathway controls fundamental cellular processes, including proliferation, differentiation and survival (3). Many carcinomas with activated Ras/Raf/ERK signaling have undergone epithelial to mesenchymal transition (EMT) in which the epithelial phenotype and polarity, characterized by adherens and tight junctions, and actin organization are lost and a mesenchymal phenotype is aquired (46). Although Ras induces EMT through various cascades of MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase)/Akt and Jun kinase, including the G proteins Rac and Cdc42 (7), the detailed signal transduction pathways involved in Raf-1-induced EMT are as yet unclear.
Cadherin-based adherens junctions play key roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling (8). These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells (914). During EMT, adherens junctions and the actin cytoskeleton are partially dissociated through signaling pathways involving Ras, Src and Wnt (15). Tight junctions, the apical-most component of intercellular junctional complexes, serve as a barrier that prevents solutes and water from passing through the paracellular pathway and as a fence between the apical and basolateral plasma membranes in epithelial cells (16,17). They show a particular net-like meshwork of fibrils formed by the integral membrane proteins, occludin, the claudin family and JAM (18,19). Several peripheral membrane proteins, ZO-1, ZO-2, ZO-3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6 and ASIP, an atypical protein kinase C-interacting protein, have been reported (18,19). Tight junction assembly and function can be modulated by a number of signaling molecules, including cAMP, Ca2+, protein kinase C, Src tyrosine kinases, G proteins, phospholipase C, and diacylglycerol (2023). More recently, Ras-related small GTP-binding proteins, such as RhoA and Rac1, have been reported to regulate tight junction structure and function (2428). In addition, down-regulation of the MAP kinase signaling pathway causes restoration of epithelial cell morphology and the assembly of tight junctions in Ras- or Raf-transfected epithelial cells (29,30).
In the present study we have examined the signal transduction pathways in oncogenic Raf-1-induced EMT, which is characterized by down-regulation of adherens and tight junctions and the reorganization of actin, using an immortalized mouse hepatic cell line. The mouse hepatic cell line was stably transfected with an active Raf-1 gene and then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI 3-kinase inhibitor LY294002 and the c-Src tyrosine kinase inhibitor PP2. We show that oncogenic Raf-1 regulates EMT via distinct signaling pathways, MAP kinase, p38 MAP kinase and c-Src tyrosine kinase, in the mouse hepatic cell line.
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Materials and methods
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Antibodies and reagents
Rabbit polyclonal anti-occludin, anti-claudin-1, anti-claudin-2 and anti-ZO-1 antibodies were purchased from Zymed Laboratories Inc. (San Francisco, CA). Mouse monoclonal anti-E-cadherin was purchased from BD Biosciences Pharmingen (San Diego, CA). Mouse monoclonal anti-pancadherin was purchased from Sigma Chemical Co. (St Louis, MO). Rabbit polyclonal anti-ERK1/2 and rabbit polyclonal anti-pMAPK (phospho-ERK1/2) antibodies were purchased from Promega Corp. (Madison, WI). A rabbit polyclonal anti-c-Src[pY418] antibody was from BioSource (Camarillo, CA). A mouse monoclonal anti-c-Src antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Alexa 488 (green)-conjugated anti-rabbit and mouse IgG antibodies and rhodaminephalloidin were purchased from Molecular Probes Inc. (Eugene, OR). Horseradish peroxidase (HRP)-conjugated anti-rabbit and mouse IgG were obtained from DAKO (A/S, Denmark). The MAP kinase inhibitor (PD98059), p38 MAP kinase inhibitor (SB203580), PI 3-kinase inhibitor (LY294002) and Src kinase family inhibitor (PP2) were purchased from Calbiochem-Novabiochem Corp (San Diego, CA). The enhanced chemiluminescence (ECL) western blotting system was obtained from Amersham Corp. (Buckinghamshire, UK). D-[1-14C]mannitol (5062 mCi/mmol) and inulin-[14C]carboxylic acid (520 mCi/mmol) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).
Cell culture, transfections and treatments
Parental cells were from an immortalized mouse hepatic cell line derived from connexin32 (Cx32)-deficient mice and transfected with the human Cx32 gene as described previously (31,32). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4% fetal bovine serum, 20 mM HEPES, 1 g/l galactose, 30 mg/l proline, 25 mM NaHCO3 and antibiotics and were maintained in a 5% CO2/95% air incubator at 37°C.
For stable transfection, parental cells were transfected with an active Raf-1 gene (33), using LIPOfectamine 2000 (Gibco BRL). After 48 h, the cells were transferred to selection medium containing 2 µg/ml puromycin. When surviving colonies were extensive enough to detect visually they were individually picked and separately propagated. The Raf-1-transfected cells were treated with 50 µM PD98059, 20 µM SB203580, 20 µM LY294002 or 10 µM PP2.
Western blotting
Cultures in 60 mm dishes were washed with phosphate-buffered saline twice and 300 µl of buffer (1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride) was added. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 15 s. Protein concentrations were measured using a BCA kit (Pierce Chemical Co., Rockford, IL). Aliquots of 15 µg protein/lane for each sample were separated by electrophoresis in 12.5% or 4/20 SDSpolyacrylamide gels (Daiichi Pure Chemicals Co., Tokyo, Japan). After electrophoretic transfer to a nitrocellulose membrane (Immobilon; Millipore), the membrane was saturated for 30 min at room temperature with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20 and 4% skimmed milk) and incubated with anti-occludin (1:1000), anti-claudin-1 (1:1000), anti-claudin-2 (1:2000), anti-ZO-1 (1:1000), anti-E-cadherin (1:1000), anti-actin (1:1000), anti-pMAPK (1:1000), anti-ERK1/2 (1:1000), anti-c-Src[pY418] (1:1000) or anti-c-Src (1:1000) antibodies at room temperature for 1 h. The membrane was incubated with HRP-conjugated anti-rabbit or mouse IgG at room temperature for 1 h. The immunoreactive bands were detected using an ECL western blotting system.
RNA isolation and reverse transcription (RT)PCR analysis
Total RNA was extracted and purified using Trizol reagent (Life Technologies). One microgram of total RNA was reverse transcribed into cDNA using a mixture of oligo(dT) and MuLV RTase under the recommended conditions (GeneAmp PCR kit; Perkin Elmer, Branchburg, NJ). Each cDNA synthesis was performed in a total volume of 20 µl for 30 min at 42°C and terminated by incubation for 5 min at 99°C. PCR containing 100 pM primer pairs and 1 µl RT reaction was performed in 20 µl of 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.4 mM dNTPs and 0.5 U Taq DNA polymerase (Takara) for 2530 cycles with cycle times of 15 s at 96°C, 30 s at 55°C and 60 s at 72°C, using a Perkin Elmer/GeneAmp® PCR system 2400. The final elongation time was 7 min at 72°C. Aliquots of 10 µl of the PCR reaction were analyzed in 1% agarose gels after staining with ethidium bromide. Primers used to detect occludin, claudin-1, claudin-2, E-cadherin and ZO-1 by RTPCR had the following sequences: occludin, sense 5'-TCAGGGAATATCCACCTATCACTTCAG-3' and antisense 5'-CATCAGCAGCAGCCATGTACTCTTCAC-3', amplification length 136 bp; claudin-1, sense 5'-GCTGCTGGGTTTCATCCTG-3' and antisense 5'-CACATAGTCTTTCCCACTAGAAG-3', amplification length 619 bp; claudin-2, sense 5'-GCAAACAGGCTCCGAAGATACT-3' and antisense 5'-CTCTGTACTTGGGCATCATCTC-3', amplification length 546 bp; E-cadherin, sense 5'-CGTGATGAAGGTCTCAGCC-3' and antisense 5'-ATGGGGGCTTCATTCAC-3', amplification length 650 bp; ZO-1, sense 5'-CATAGAATAGACTCCCCTGG-3' and antisense 5'-GCTTGAGACCTCATACCTGT-3', amplification length 435 bp. To provide a qualitative control for reaction efficiency, PCR reactions were performed with primers coding for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3', amplification length 452 bp).
Immunocytochemistry
Cells grown on glass coverslips were fixed with cold absolute acetone or an ethanol and acetone 1:1 mixture at 20°C for 10 min. The cells were stained with anti-occludin (1:100), anti-claudin-1 (1:100), anti-claudin-2 (1:100), anti-ZO-1 (1:100) or anti-pancadherin (1:100) antibodies or rhodaminephalloidin (1:200) for 1 h at room temperature. Alexa 488 (green)-conjugated anti-rabbit or mouse IgG was used as the second antibody. The specimens were examined and photographed with an Axioskop 2 plus microscope (Carl Zeiss, Germany) and confocal laser scanning microscope (MRC 1024; Bio-Rad, Hercules, CA). Phase contrast photomicrographs were taken with a Zeiss Axiovert 200 inverted microscope.
Measurement of transepithelial electrical resistance (TER)
Cells grown on semipermeable supports were plated at a density of 2 x 104 cells/filter onto collagen-coated clear polyester membranes of Costar Transwell® filters (0.4 µm pore size, 1 cm2 surface area) and grown to confluence. TER was measured in the wells. Current pulses (4 mA) were passed across the monolayer using a pair of calomel electrodes via KCl salt bridges and the voltage was measured with a conventional voltmeter across the same cell monolayer using a pair of Ag/AgCl electrodes via KCl salt bridges (EVON, World Precision Instruments).
Measurement of permeability (fluxes of [14C]mannitol and [14C]inulin)
Paracellular permeability was assessed by [14C]mannitol and [14C]inulin fluxes through a confluent monolayer. Cells were seeded on Costar Transwell® filters and cultured for 6 days. After cells reached confluence, they were treated or not with 50 µM PD98059 and 20 µM SB203580. To measure paracellular flux, DMEM containing [14C]mannitol at 1 x 105 d.p.m./well and [14C]inulin at 5 x 105 d.p.m./well were added to the apical compartment and the cells were incubated at 37°C after stabilization of transepithelial electrical parameters. After 15, 30, 60, 90 and 120 min incubation, aliquots from the basolateral compartments were collected and the radioactivity was counted in a Beckman LS6500 scintillation counter (Beckman Coulter Inc., Fullerton, CA). Fluxes were expressed as d.p.m./cm2.
Data and statistical analysis
Signals were quantified using the Scion Image 4.02 for Windows (Scion Corp., 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. P < 0.05 was considered significant.
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Results
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Activated Raf-1-modulated morphologic changes in an immortalized mouse hepatic cell line
In an immortalized mouse hepatic cell line transfected with the activated Raf-1 gene, pMAP kinase was increased 3-fold compared with endogenous pMAP kinase of parental cells (Figure 1A). When grown to confluence on plastic dishes, parental cells formed an epithelial monolayer and the cell borders were unclear. Raf-1-transfected cells also formed an epithelial monolayer, but the spaces between cells increased and the cell borders were clear (Figure 1B).

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Fig. 1. (A) Western blot analysis for pMAP kinase and ERK1/2 in parental cells and in Raf-1 transfectant cells. The density of p44MAP kinase was normalized to the expression of ERK1/2. The signals are shown as a bar graph. Expression of p44MAP kinase in Raf-1-transfected cells was increased 3-fold compared with parental cells. **P < 0.01 versus parental strain. (B) Morphological changes in Raf-1-transfectant cells. In Raf-1-transfected cells the spaces between cells are increased and the cell borders are clear. Bar, 20 µm.
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Changes in expression of adherens and tight junctions caused by transfection with Raf-1
To investigate changes in expression of adherens and tight junctions caused by transfection with Raf-1, we examine expression of the adherens junction protein E-cadherin and tight junction proteins occludin, claudin-1, claudin-2 and ZO-1 using western blotting and RTPCR. In Raf-1-transfected cells, the proteins occludin, claudin-1 and claudin-2 were significantly decreased compared with parental cells, whereas the proteins E-cadherin and ZO-1 were slightly reduced (Figure 2A). The mRNAs of occludin and claudin-2, but not claudin-1, E-cadherin or ZO-1, were significantly decreased in Raf-1-transfected cells compared with parental cells (Figure 2B) as determined by RTPCR.

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Fig. 2. Western blot (A) and RTPCR (B) analyses for occludin, claudin-1, claudin-2, ZO-1 and E-cadherin in parental cells and in Raf-1 transfectant cells. The signals were normalized to expression of actin or G3PDH. They are shown as bar graphs. **P < 0.01, *P < 0.05 versus parental strain. (A) Expression of the proteins occludin, claudin-1, claudin-2, ZO-1 and E-cadherin in Raf-1 transfectant cells was significantly decreased compared with parental cells. (B) Expression of the proteins occludin, claudin-2 and E-cadherin in Raf-1 transfectant cells were significantly decreased compared with parental cells.
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Changes in localization of adherens and tight junctions and F-actin caused by transfection with Raf-1
In the immortalized mouse hepatic cell line, occludin, claudin-1, claudin-2, ZO-1, pancadherin and F-actin were localized at cell borders (Figure 3A). In Raf-1-transfected cells, occludin and claudin-2 disappeared from cell borders and ZO-1, pancadherin and F-actin changed from linear to zipper-like patterns (Figure 3A). Confocal laser microscopy demonstrated that ZO-1 and pancadherin were co-localized with F-actin, showing zipper-like structures at cell borders (Figure 3B).

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Fig. 3. (A) Single immunocytochemistry for occludin, caudin-1, claudin-2, ZO-1, pancadherin and F-actin in parental and Raf-1 transfectant cells. In Raf-1-transfected cells, occludin and claudin-2 disappeared from cell borders and ZO-1, pancadherin and F-actin changed from a line to a zipper-like pattern. Bar, 10 µm. (B) Double immunocytochemistry for ZO-1 and F-actin and pancadherin and F-actin in Raf-1-transfected cells. ZO-1 and pancadherin are co-localized with F-actin at cell borders. Bar, 5 µm.
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Changes of barrier functions of tight junctions caused by transfection with Raf-1
To evaluate the effects on the barrier functions of tight junctions of transfection with Raf-1, TER and paracellular fluxes using [14C]mannitol and [14C]inulin were measured. In parental cells, TER gradually increased with time of culture from day 4 after plating. In Raf-1-transfected cells, the level of TER was significantly lower from day 6 after plating than that of parental cells (Figure 4A). When we examined paracellular permeability in the cells at day 6 after plating, paracellular fluxes of mannitol and inulin were significantly increased in Raf-1transfected compared with parental cells (Figure 4B).

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Fig. 4. Barrier function of tight junctions in parental and Raf-1-transfected cells. (A) TER assay in parental and Raf-1 transfectant cells. In Raf-1-transfected cells, the TER level was at a significantly lower level from day 6 after plating than in parental cells. **P < 0.01 versus parental strain. (B) Paracellular permeability assay in parental and Raf-1 transfectant cells. In Raf-1-transfected cells, the paracellular fluxes of mannitol and inulin were significantly increased compared with that of parental cells. *P < 0.05 versus parental strain.
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MAP kinase and p38 MAP kinase, but not PI 3-kinase, inhibitors block changes in expression of adherens and tight junctions caused by transfection with Raf-1
To determine whether the MAP kinase, p38 MAP kinase and PI 3-kinase pathways were involved in changes to the adherens and tight junctions caused by transfection with Raf-1, Raf-1-transfected cells were treated with the specific inhibitors PD98059, SB203580 and LY294002 for 2, 4 and 8 h. Treatment with PD98059 completely inhibited the increase in pMAP kinase expression in Raf-1-transfected cells beginning 2 h after treatment, and expression level was lower than that of parental cells (Figure 5A). Treatment with SB203580 also inhibited the increase in pMAP kinase expression in Raf-1-transfected cells beginning 2 h after treatment, and expression was at the same level as that of parental cells (Figure 5A). Treatment with LY294002 did not affect expression of pMAP kinase in Raf-1-transfected cells (data not shown).

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Fig. 5. (A) Western blot analysis for pMAP kinase and ERK1/2 in Raf-1-transfected cells treated with PD98059 and SB203580 for 2, 4 and 8 h. Treatment with PD98059 and SB203580 inhibited the increase in pMAPK expression in Raf-1-transfected cells. (B) Western blot analysis for occludin, claudin-1, claudin-2, ZO-1 and E-cadherin in Raf-1 transfectant cells treated with PD98059 and SB203580 for 2, 4 and 8 h. In Raf-1-transfected cells treated with PD98059 and SB203580, the proteins claudin-2, claudin-1, ZO-1 and E-cadherin were increased from 2 h. (C) RTPCR analysis for occludin, claudin-1, claudin-2, ZO-1 and E-cadherin in Raf-1 cells treated with PD98059 and SB203580 for 2, 4 and 8 h. In Raf-1-transfected cells treated with PD98059 and SB203580, the mRNAs of claudin-2 and E-cadherin were increased from 2 h. The signals were normalized to the expression of ERK1/2, actin or G3PDH. They are shown as bar graphs. **P < 0.01, *P < 0.05 versus untreated Raf-1-transfected cells.
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We examined the effects of treatment with PD98059 and SB203580 on the down-regulation of adherens and tight junctions caused by transfection with Raf-1, using western blotting and RTPCR. In Raf-1-transfected cells treated with PD98059 and SB203580, claudin-2 protein and mRNA returned to half the level of parental cells beginning 2 h after treatment, whereas expression of occludin never recovered (Figure 5B and C). Expression of the proteins claudin-1, E-cadherin and ZO-1 returned to the levels of parental cells (Figure 5B and C). Treatment with LY294002 did not affect the down-regulation of adherens and tight junctions in Raf-1-transfected cells (data not shown).
MAP kinase and p38 MAP kinase inhibitors block changes in localization of adherens and tight junctions caused by transfection with Raf-1
To determine whether the MAP kinase and p38 MAP kinase pathways were involved in the localization of adherens and tight junction proteins and actin filaments, confluent Raf-1-transfected cells were treated with PD98059 and SB203580 for 4 h. In Raf-1-transfected cells treated with PD98059 and SB203580, claudin-2, but not occludin, reappeared at cell borders and the distribution of ZO-1, pancadherin and F-actin changed from zipper-like structures to linear at cell borders (Figure 6).

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Fig. 6. Single immunocytochemistry for occludin, claudin-1, claudin-2, ZO-1 and pancadherin in Raf-1-transfectant cells treated with PD98059 and SB203580 for 4 h. In Raf-1-tranfected cells treated with PD98059 and SB203580 for 4 h, claudin-2 reappeared at cell borders and the distribution of ZO-1, pancadherin and F-actin changed from zipper-like structures to linear at cell borders. Bar, 10 µm.
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MAP kinase and p38 MAP kinase inhibitors block changes in barrier function of tight junctions caused by transfection with Raf-1
We examined the effects of treatment with PD98059 and SB203580 on changes in barrier function of tight junctions induced by transfection with Raf-1. TER was measured in Raf-1-transfected cells treated with PD98059 and SB203580 for 4, 8 and 24 h. Treatment with PD98059 and SB203580 enhanced TER values at 4 and 8 h, but these returned to the untreated level at 24 h (Figure 7A). Although the paracellular fluxes of mannitol and inulin were measured in Raf-1-transfected cells treated with PD98059 and SB203580 for 6 h, no changes in paracellular permeability were observed after treatment (Figure 7B).

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Fig. 7. Barrier function of tight junctions in Raf-1 transfectant cells treated with PD98059 and SB203580. (A) TER assay in Raf-1-transfectant cells treated with PD98059 and SB203580 for 4, 8 and 24 h. Treatment with PD98059 and SB203580 enhanced TER values at 4 and 8 h. **P < 0.01 versus untreated Raf-1-transfected cells. (B) Paracellular permeability assay in Raf-1 transfectant cells treated with PD98059 and SB203580 for 8 h. No change in paracellular permeability was observed after treatment with PD98059 and SB203580.
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c-Src tyrosine kinase inhibitor blocks down-regulation of occludin caused by transfection with Raf-1
Despite recovery of the tight junction protein claudin-2 in Raf-1-transfected cells treated with PD98059 and SB203580, down-regulation of the tight junction protein occludin caused by transfection with Raf-1 was maintained. It was reported that c-Src binds to occludin and phosphorylates it on tyrosine residues (34). In the present study, to investigate whether the c-Src kinase pathway is involved in down-regulation of occludin caused by transfection with Raf-1, Raf-1-transfected cells were treated wth the specific Src kinase inhibitor PP2 for 2, 4, 8 and 24 h. In Raf-1-transfected cells, the activation of tyrosine-phosphorylated c-Src (p-c-Src) was slightly increased compared with parental cells, as demonstrated by western blotting with an anti-c-Src [pY418] antibody (Figure 8A). In Raf-1-transfected cells treated with PP2, p-c-Src decreased beginning from 2 h (Figure 8A). We examined expression of occludin and claudin-2 in Raf-1-transfected cells treated with PP2. Expression of occludin, but not claudin-2, mRNA and protein increased beginning from 4 and 8 h, respectively (Figure 8A and B). Immunofluorescence staining showed that occludin, but not claudin-2, in Raf-1-transfected cells treated with PP2 for 4 h reappeared at cell borders (Figure 8C). However, treatment with PP2 did not affect TER values in Raf-1-transfected cells (Figure 8D).

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Fig. 8. (A) Western blot analysis for p-c-Src, c-Src, occludin and claudin-2 in Raf-1-transfected cells treated with PP2 for 2, 4, 8 and 24 h. In Raf-1-transfected cells treated with PP2, p-c-Src was decreased from 2 h and expression of occludin protein was increased from 8 h. The signals were normalized to the expression of c-Src or actin. They are shown as a bar graph. (B) RTPCR analysis for occludin and claudin-2 in Raf-1 cells treated with PP2 for 2, 4, 8 and 24 h. Expression of occludin mRNA was increased from 4 h. The signals were normalized to expression of G3PDH. They are shown as a bar graph. (C) Single immunocytochemistry for occludin and claudin-2 in Raf-1 transfectant cells treated with PP2 for 8 h. Occludin immunoreactivity reappeared at cell borders. Bar, 10 µm. (D) TER assay in Raf-1 transfectant cells treated with PP2 for 2, 4, 6, 8, 10 and 12 h. Treatment with PP2 did not affect TER values in Raf-1-transfected cells.
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Discussion
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In the present study we have examined the signal transduction pathways in oncogenic Raf-1-induced EMT, which is characterized by down-regulation of adherens and tight junctions and reorganization of actin, using an immortalized mouse hepatic cell line. Our results suggest that Raf-1 regulates EMT via distinct signal transduction pathways in hepatocytes.
During EMT cells lose epithelial polarity, resulting in loss or redistribution of adherens and tight junctions, and acquire a spindle-shaped, highly motile fibroblastoid phenotype (46). Many carcinomas with activated Ras/Raf/MEK signaling undergo EMT through various cascades of MAP kinase, PI 3-kinase/Akt and Jun kinase, including the G proteins Rac and Cdc42 (7). Transfection of Raf-1 into salivary gland epithelial cells (Pa-4 cells) resulted in EMT changes, the down-regulation of occludin and claudin-1 and redistribution of the patterns of ZO-1 and E-cadherin (30). In the present study, transfection of Raf-1 into a mouse hepatic cell line caused the down-regulation of occludin and claudin-2 and the redistribution of ZO-1 and pancadherin, even though cell morphology was of the epithelioid phenotype. The down-regulation of occludin and claudin-2 was observed at the mRNA level, together with a decrease in tight junctional barrier function as measured by TER and permeability. More interestingly, ZO-1 and pancadherin formed zipper-like patterns at cell borders and were co-localized with actin filaments when the cells were maintained at confluence in dishes. The changes in distribution of ZO-1, pancadherin and F-actin at cell borders in Raf-1-transfected cells may have caused the increase in the spaces between cells observed by phase contrast microscopy (Figure 1B). These results suggest that Raf-1 regulates expression of occludin and claudin-2 at the transcriptional level and may control the distribution of adherens junctions and actin filaments at cell borders via small GTPases, including Rac or Rho.
Recently it was suggested that there are cross-talk pathways between MAP kinase, p38 MAP kinase and PI 3-kinase (35,36). To investigate the signaling pathways for Raf-1-induced EMT in a mouse hepatic cell line in detail, we treated Raf-1-transfected cells with inhibitors of MAP kinase, p38 MAP kinase and PI 3-kinase. The MAP kinase inhibitor and the p38 MAP kinase inhibitor, but not the PI 3-kinase inhibitor, blocked the down-regulation of claudin-2 but not occludin and the reorganization of ZO-1, pancadherin and actin induced by Raf-1. With regard to barrier function, the value of TER, but not permeability, recovered after treatment with inhibitors of MAP kinase and p38 MAP kinase. Overexpression of claudin-2 in MDCK I cells markedly decreased the tightness of the epithelial barrier, which could be explained by the induction of paracellular cation channels (37). In the present study it is possible that barrier function might have been dependent on the organization of the adherens junction and actin.
Raf-1 is frequently up-regulated in cancer cells, including lung tumor cell lines (38). It was recently reported that increased activity of Raf/MAP kinase was observed in primary human hepatocellular carcinoma (HCC) (39) and in rat HCC induced by a carcinogen (40). In the present study a p38 MAP kinase inhibitor blocked the increase in pMAP kinase induced by Raf-1, whereas a MAP kinase inhibitor strongly blocked expression of all pMAP kinases, including endogenous expression. These results suggest that the p38 MAP kinase inhibitor functions as a specific target reagent for cancer therapy, including for HCC, compared with the MAP kinase inhibitor (41), because the Raf/MEK/ERK signaling pathway controls fundamental cellular processes, including proliferation, differentiation and survival in normal cells (3).
On the other hand, c-Src is localized in the tight junction region in epithelial cells and Src kinase activity may play an important role in the assembly and function of tight junctions (42). It has been reported that c-Src binds to occludin and phosphorylates it on tyrosine residues (34). To investigate whether the Src kinase pathway is involved in the down-regulation of occludin induced by Raf-1, the specific Src kinase inhibitor PP2 was used. In Raf-1-transfected cells, phospho-Src activity was slightly increased and then inhibited by treatment with PP2. Treatment with PP2 induced expression of occludin, but not claudin-2, at the protein and mRNA levels without an increase in the TER value. These results indicate that Raf-1 in part regulates the expression of occludin through a c-Src tyrosine kinase pathway.
In summary, oncogenic Raf-1 regulates EMT, which is characterized by down-regulation of adherens and tight junctions and reorganization of actin, through distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signaling pathways in a mouse hepatic cell line (Figure 9). In addition, both the MAP kinase pathway and the p38 MAP kinase pathway were closely related to Raf-1-induced EMT. p38 MAP kinase is a stress-responsive MAP kinase activated by proinflammatory cytokines and environmental stress (43,44). Furthermore, the p38 MAP kinase cascade is associated with the resistance to apoptosis of human HCC (45). These facts suggest that Raf-1-induced EMT is partly controlled by stress stimuli. More recently it was reported that inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor, which localized in the tight junction regions in cancer cells, leading to increased uptake of adenoviruses and cell killing (46). Although further investigation of the signal transduction pathways involved in Raf-1-induced EMT, is needed, modification of the Raf/MEK/ERK pathway may be important for differentiation-inducing therapy for malignant disease.

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Fig. 9. Potential signaling pathways for EMT induced by oncogenic Raf-1 in the mouse hepatic cell line. AJ, adherens junction; TJ, tight junction.
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Acknowledgments
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We are grateful to Dr H.Takekawa (University of Tokyo) for the activated Raf-1 cDNA. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science and the Ministry of Health, Labour and Welfare of Japan and by the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Suhara Memorial Foundation, the Smoking Research Foundation and the Long-Range Research Initiative Project of Japan Chemical Industry Association.
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Received March 31, 2004;
revised July 17, 2004;
accepted July 21, 2004.