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Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):552-559; doi:10.1093/carcin/bgn003
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Activation of RON differentially regulates claudin expression and localization: role of claudin-1 in RON-mediated epithelial cell motility

Kun Zhang1,2, Hang-Ping Yao1 and Ming-Hai Wang1,2,*

1 Laboratory of Cancer Biology and Therapeutics, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, People’s Republic of China and
2 Cancer Biology Center and Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of Pharmacy, Amarillo, TX 79106, USA

* To whom correspondence should be addressed. Tel: +1 806 356 4015, ext. 248; Fax: +1 806 356 4034;Email: minghai.wang{at}ttuhsc.edu


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Claudins are integral membrane proteins essential in tight junction formation and function. Altered expression of claudins has been implicated in epithelial malignant transformation. We report here that activation of recepteur d'origine nantais (RON) differentially regulates tight junction function and claudin expression. In Martin-Darby canine kidney (MDCK) cells, macrophage-stimulating protein-induced RON activation or expression of constitutively active variant RON160 significantly disrupted cellular tight junctions and reduced transepithelial electrical resistance. These changes were featured by diminished claudin-1 expression and redistribution of claudin-3 and -4 into cytoplasmic compartments. The inhibition of claudin-1 was also seen in breast cancer T-47D cells. By analyzing the signaling events, we found that activation of the extracellular signal-regulated kinase 1/2 pathway is required for RON-mediated inhibition of claudin-1 expression and redistribution of claudin-3 and -4. Results from luciferase reporter assays showed that inhibition is acted at the transcriptional levels because RON activation decreases claudin-1 promoter activities and increases transcriptional repressor Snail-1 expression. Functional analysis further revealed that reduced claudin-1 expression is linked to increased motilities of MDCK and T-47D cells as evident in cell migration and wound-healing assays. Forced expression of claudin-1 prevented RON-mediated cell migration and restored cell morphologies to their original epithelial appearance. In conclusion, RON activation differentially regulates claudin expression in epithelial cells. Inhibition of claudin-1 expression may represent a novel mechanism that contributes to RON-mediated invasive activities, leading to increased tumor malignancy.

Abbreviations: EGF, epidermal growth factor; Erk1/2, extracellular signal-regulated kinase 1/2; HGF, hepatocyte growth factor; MAP, mitogen-activated protein; MDCK, Martin-Darby canine kidney; MSP, macrophage-stimulating protein; RON, recepteur d'origine nantais; TER, transepithelial electrical resistance; ZO-1, zonulae occludens protein-1


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Tight junctions are one of the epithelial junction complexes, which play a critical role in establishing and maintaining epithelial cell polarity and integrity (1). Structurally, tight junctions are composed of a belt of anastomosing strands of proteins and lipids surrounding the lateral membrane of epithelial cells (2). The created barrier regulates paracellular movement of water and solutes across epithelia (1,2). There functions can be determined by measuring transepithelial electrical resistance (TER) across the epithelial cell monolayer (3). Decreased TER accompanied with increased permeability has been found in various pathological conditions, which is an indication of loss of epithelial barrier function (1–3).

The backbones of tight junctions are a group of integral membrane proteins known as claudins (4). Currently, 24 claudin proteins including tight junction-related proteins occludin and zonulae occludens protein-1 (ZO-1) have been identified (2,4). According to their in vivo distribution and functional experiments, several claudins including claudin-1 and -4, are considered to increase, whereas other claudins, especially claudin-2, are indicated to decrease TER (2–4). Dynamic regulation of tight junction function is fundamental to many physiological processes (1–4). Disruption of tight junctions is also a hallmark in epithelial cancer development and malignant progression (5). Altered expression of claudins such as loss of claudin-1 expression or overexpression of claudin-3 and -4 is correlated with enhanced invasive and malignant transformation of certain epithelial cancers (6–9). Clinically, changes in claudin expression are strong prognostic indicators for certain invasive cancers (6–8). Thus, determining the roles of claudins in cancer pathogenesis should provide insight into the mechanisms underlying tumor progression toward malignancy.

Recepteur d'origine nantais (RON) is a transmembrane protein belonging to a subfamily of receptor tyrosine kinases, which include MET and C-Sea (10). RON is mainly present in epithelial cells and regulates cell differentiation, growth, migration and survival (11). Immunohistochemistry of tumor tissue microarrays has recently revealed that RON is overexpressed in significant numbers in primary cancer samples derived from pancreas (33%), bladder (36%), skin (37%), thyroid (42%), lung (48%), colon (51%) and breast (56%) (12). In the event of epithelial cell transformation and tumorigenic progression, altered RON expression often displays constitutive phosphorylation and activation of downstream signaling pathways including mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (11). The signaling cascades lead to cell dissociation and morphological changes with increased motile-invasive phenotypes known as epithelial to mesenchymal transition (11,13). Epithelial to mesenchymal transition is featured by the loss of epithelial phenotypes and the gain of mesenchymal markers such as expression of vimentin and smooth muscle actin-{alpha} and increasingly recognized a process essential for cancer invasion and metastasis (14,15). Overexpression of RON is also accompanied with generation of various RON variants (16). Currently, six RON variants, produced mainly by alternative pre-messenger RNA splicing process, have been identified in different types of cancer cells (16). RON160, a constitutively active variant (17), is a typical example. The protein is produced by a RON messenger RNA transcript through alternative splicing that eliminates 109 amino acids in the RON extracellular domain (17). These amino acids are encoded by exons 5 and 6, which constitute the part of the extracellular domains of the RON β-chain (18). The deletion results in conformational changes leading to constitutive tyrosine phosphorylation (17). Studies in vitro and in vivo have shown that RON160 is an active variant capable of regulating various tumorigenic activities (17,19,20).

The present work is to study the roles of tight junction protein claudins in RON-mediated cell motile activities. Martin-Darby canine kidney (MDCK) cells were used as a model because of their typical epithelial phenotypes and intact junction structures. We demonstrated that activation of RON disrupts tight junctions with impaired transcellular resistance. These activities were mediated through differential regulation of claudin expression, which is featured by inhibition of claudin-1 expression and intracellular redistribution of claudin-3 and -4. Additional evidence indicated that inhibition of claudin-1 expression is a critical event involved in RON-mediated cell migration. Thus, differential regulation of claudin expression and distribution is a mechanism underlying RON-mediated cell migration in epithelial cells.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines and reagents
MDCK or MDCK cells expressing human RON (MDCK-RON) or RON160 (MDCK-RON160) were established by plasmid transfection as described previously (19). Expression of RON or RON160 was determined by western blot analysis. Human breast cancer cell lines Du4475, T-47D and BT-483 were from American Type Culture Collection (Manassas, VA). Mouse monoclonal antibodies to claudin-1, -3, -4, -7 and ZO-1, respectively, were from Zymed (San Francisco, CA). PD98059 (PD), SB203580 (SB) and wortmannin (WT) were from Calbiochem (San Diego, CA). Luciferase reporter vector pGL3-CLD1p (containing a human claudin-1 promoter sequences) (21) and pGL3-CLD4p (containing a human claudin-4 promoter fragment) (22) were kindly provided by Drs S.Vilaró (University of Barcelona, Barcelona, Spain) and P.J.Morin (National Institutes of Health, Bethesda, MD), respectively. Generation of a human claudin-1 promoter fragment with double mutations in the conserved two E-boxes sequences was carried out using polymerase chain reaction techniques as reported previously (17,21). The core 5'-CA(G/C)(G/C)TG-3' sequences of the E-boxes (E1 and E2) in the claudin-1 promoter sequences was mutated to 5'-TG(G/C)(G/C)TG-3' (21). The mutated fragment was then inserted into the pGL3 luciferase reporter vector (designated as pGL3-CLD1pM1-2). An expression vector containing human claudin-1 cDNA (pCDNA3.1-CLD1) (23) was provided by Dr Weeraratna (National Institutes of Health).

Measurement of TER
Cells (2 x 105 cells) were seeded on a polycarbonate filter with a pore size of 0.4 µm and allowed to attach for 24 h to form a monolayer. Before stimulation, cells were washed in phosphate-buffered saline and then stimulated at 37°C for 15 min with macrophage-stimulating protein (MSP) (2 nM) with or without inhibitors such as PD98059 (50 µM), SB203580 (20 µM) or wortmannin (100 nM). A Millicell-ERS volt-ohm meter (Millipore, Temecular, CA) was used to determine TER value in each sample (24). Final TER values were calculated by subtracting blank value and normalized for the area of the filter.

Cellular protein preparation and western blot analysis
Preparation of Triton X-100 soluble or insoluble proteins was carried out according to a published protocol (24) with slight modification. Briefly, cells were lysed in lysis buffer (24) and centrifuged at 13 000 r.p.m. for 20 min. Supernatant was collected as the soluble fraction. The insoluble fraction was obtained by dissolving the pellet in solubilization buffer (10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid, pH 7.2, 1% sodium dodecyl sulfate, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid and various proteases inhibitors) and then subjected to repeated sonication pulses. After a short incubation, samples were centrifuged at 13 000 r.p.m. for 20 min. The resulting supernatants were used as the insoluble fraction. Equal amount of proteins were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reduced conditions and transferred to Immobilon-P membrane (Millipore, MA). Individual proteins were detected by western blot analysis using specific antibodies as described previously (17). The same membrane was also stripped and reprobed with anti-actin antibodies to ensure the equal sample loading (17).

Immunofluorescent staining
The method was performed as described previously (13). Cells cultured in coverslip were fixed with 3.7% formaldehyde in phosphate-buffered saline and then treated with 0.2% Triton X-100 in phosphate-buffered saline for 10 min. After blocking non-specific binding, cells were incubated with antibodies specific to claudin-1, -3, -4, -7 or ZO-1 followed by secondary antibodies conjugated with fluorescein isothiocyanate. Fluorescent staining was observed under an Olympus microscope equipped with fluorescent apparatus and photographed with a digital camera.

Luciferase report assays for claudin promoter activities
MDCK, MDCK-RON or MDCK-RON160 cells in a six-well plate were transfected with 3 µg of pGL3-CLD1p, pGl3-CLD1pM1-2 or pGL3-CLD4p as described previously (25). The control vector pRL-SV40 (Promega, Madison, WI) was used for normalization of transfection efficiency. After a 48 h incubation, cells were lysed and assayed for luciferase activities using a Turner Designs Luminometer TD20/20 (Promega).

Cell migration assays
Two methods were used. One is the use of a 48-well migration chamber to determine cell migration in response to MSP stimulation (19). Polycarbonate membranes (Neuro Probe, Gaithersburg, MD) were coated with 50 µg/ml type IV collagen. Cells transfected with pCDNA3.1-CLD1 or control vector were used. Migrated cells were determined after cells were incubated for 24 h. The other is the wound-healing assay, in which a wound area was created in a cell monolayer by a plastic tip (26). After MSP stimulation for 12 or 24 h, the percentages of open spaces covered by migrated cells were determined.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Activation of RON or RON160 decreases E-cadherin and claudin-1 expression in MDCK cells
RON or RON160 expression and activation in MDCK cells (Figure 1A) results in diminished E-cadherin expression and increased vimentin accumulation (Figure 1B and C). As expected, these activities were also seen in MDCK cells expressing active variant RON160 with constitutive tyrosine phosphorylation (Figure 1A and B). Morphological changes accompanied with reorganization of actin were also seen in RON- or RON160-expressing cells (Figure 1C). These results suggest that RON or RON160 expression regulates adherens junctions in MDCK cells.


Figure 1
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  		Fig. 1. Expression of RON or RON160 results in diminished E-cadherin expression, increased vimentin expression, actin reorganization and reduced claudin-1 expression. (A) Expression and activation of RON or RON160 in MDCK cells. Cells were stimulated with MSP (2 nM) for 10 min. Cellular proteins (200 µg per sample) were immunoprecipitated with anti-RON IgG and followed by western blot analysis using monoclonal antibodies to phosphor-tyrosine (top panel). The membrane was also reprobed with rabbit anti-RON IgG (bottom panel). (B) Regulation of E-cadherin and vimentin expression in MDCK cells. Proteins (50 µg per sample) were subjected to western blot analysis using antibodies to E-cadherin or vimentin. The membrane was then reprobed for β-actin for the loading control. (C) Fluorescent analysis of E-cadherin and vimentin expression in MDCK cells. Cells cultured for 24 h were subjected to immunofluorescent analysis using antibodies to E-cadherin, vimentin or β-actin as detailed in Materials and Methods. (D) Diminished claudin-1 expression in MDCK cells. Cells were stimulated with or without MSP (2 nM) for 24 h. Proteins (50 µg per sample) were separated in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reduced conditions. Claudins were detected in western blot analysis using individual antibodies. Levels of individual claudins were quantitatively measured. Membranes were also reprobed with actin antibodies for the loading controls. One of three experiments with similar results.

 
To determine if tight junctions are regulated, we studied claudin expression by western blot analysis. Among five proteins analyzed, a significant reduction of claudin-1 expression was observed in RON160-expressing MDCK cells but not in MDCK-RON cells (Figure 1D). However, when MDCK-RON cells were stimulated with MSP for a prolonged period (up to 24 h), the levels of claudin-1 were reduced. MSP stimulation of MDCK-RON cells also caused a moderate but statistically significant increase of claudin-3 and -4 expression. The moderate increase was also seen and statistically significant in MDCK-RON160 cells. No effects on claudin-7 and ZO-1 expression were observed (data not shown). These results suggest that MSP-induced RON activation differentially regulates claudin expression in MDCK cells.

Activation of RON or expression of RON160 reduces the overall TER
TER is a functional feature reflecting the structural and biological integrities of tight junctions (1). We measured TER to see if reduced claudin-1 expression impairs tight junctions. As shown in Figure 2A, levels of TER in control MDCK cells were not affected following treatment of MSP or individual inhibitors. However, a decrease of TER was observed in MSP-stimulated MDCK-RON cells. Decreased TER was also seen in MDCK-RON160 cells (Figure 2B), in which RON160 is constitutive active (Figure 1A). These results suggest that RON activation is required to decrease TER. Next, the effect of small chemical inhibitors on RON-mediated TER reduction was assessed. Among three inhibitors used, only PD98059, a specific MAP kinase inhibitor, prevented the MSP-induced TER reduction in MDCK-RON cells. No effect of SB203580 or wortmannin was observed.


Figure 2
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  		Fig. 2. MSP-dependent or -independent MAP kinase activation is involved in regulation of TER and claudin expression. (A) Effect of RON or RON160 activation on TER. MDCK or MDCK-RON cells were cultured on a polycarbonate filter as detailed in Materials and Methods. Before TER measurement, cells were stimulated with or without 2 nM of MSP in the presence of PD98059 (50 µM), SB203580 (20 µM) or wortmannin (100 nM). TER was measured using a Millicell-ERS volt-ohm meter (Millipore, Temecular, CA) as described previously (23). (B) Effect of claudin-1 expression on TER. MDCK-RON160 cells in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were transiently transfected with 3 µg of pcDNA3.1-CLD-1 for 72 h. MDCK cells were used as the control. TER was measured as described in (A). (C) Kinetic effect of MSP-induced RON activation on claudin expression. MDCK-RON cells cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were stimulated with MSP (2 nM) for various times. Proteins (50 µg per sample) from cell lysates subjected to western blot analysis using antibodies to claudin-1, -3, -4, -7 and ZO-1, respectively. Proteins from MDCK-RON160 cells served as the control. (D) Effect of Erk1/2 activation on RON- or RON160-mediated inhibition of claudin expression. MDCK-RON cells were stimulated with 2 nM of MSP for 24 h in the presence of small chemical inhibitors PD98059 (50 µM), wortmannin (100 nM) or SB203580 (20 µM), respectively. (E) MDCK-RON160 cells were treated with individual inhibitors for 12 and 24 h. Cellular proteins were subjected to western blot analysis using antibodies specific to phospho-Erk1/2, Erk1/2, claudin-1, -3 and -4. The membranes were also probed for β-actin as the loading control. One of three experiments with similar results.

 
To determine if diminished claudin-1 expression is responsible for reduced TER, MDCK-RON160 cells were transiently transfected with pcDNA3.1 vector containing the full length of claudin-1 cDNA. The forced expression of claudin-1 (Figure 2B) partially recovered the TER activities, suggesting that reduced claudin-1 expression is responsible for the reduced TER. Thus, MSP-dependent or -independent RON activation reduces TER in MDCK cells. The impaired tight junction functions are associated with activation of MAP kinase and reduction of claudin-1 expression.

MSP-induced RON activation differentially regulates claudin expression in a time-dependent manner
To study the inhibitory effect of RON on claudin expression in more detail, claudin expression at different time points after MSP stimulation was determined in MDCK-RON cells. MDCK-RON160 cells with diminished claudin-1 expression served as the positive control. Results in Figure 2C show that the inhibitory effect of MSP was seen as early as 12 h after cells were stimulated by MSP. More than 90% of inhibition was achieved 24 h after MSP stimulation, which lasted up to 72 h. Moreover, increased claudin-3 or -4 expression (up to 2-fold of increase) was also observed. In contrast, no changes were seen at levels of claudin-7 and ZO-1. These results suggest that RON-mediated inhibition of claudin-1 is significant and long lasting. The effects of MSP on upregulation of claudin-3 and -4 expression were relatively moderate.

Activation of extracellular signal-regulated kinase 1/2 MAP kinase is required for RON-mediated inhibition of claudin-1 expression
To determine signaling proteins involved in the inhibitory activities, MDCK-RON cells were stimulated with MSP for 24 h in the presence or absence of small chemical inhibitors such as PD98059, SB203580 and wortmannin. Results in Figure 2D show that MSP-induced extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation is associated with diminished claudin-1 expression. Inhibition of Erk1/2 phosphorylation by PD98059 resulted in reexpression of claudin-1. PD98059 also inhibited the moderately increased claudin-3 and -4 in MSP-stimulated cells. Phosphorylation of AKT and p38 MAP kinase was observed in MSP-stimulated cells; however, their activation was not involved in the inhibitory effect because wortmannin or SB203580 did not show any modulating effect (data not shown).

The involvement of Erk1/2 was also evaluated in MDCK-RON160 cells, in which Erk1/2 is constitutively active (Figure 2E). Again, treatment of cells with PD98059 resulted in reappearance of claudin-1. PD98059 also showed regulatory effects on claudin-3 and -4 expression. Levels of claudin-3 and -4 were significantly reduced after Erk1/2 was inhibited by PD98059. No regulatory effects were observed in cells treated with SB203580 or wortmannin. Thus, activation of Erk1/2 is correlated with the inhibitory effect of RON or RON160 on claudin-1 expression.

Ligand-dependent or -independent RON activation differentially regulates the promoter activities of claudin-1 and -4
To determine if diminished claudin-1 expression is manifested at the transcriptional level, MDCK, MDCK-RON or RON160 cells were transiently transfected with luciferase reporter vectors containing the promoter fragments of claudin-1. Cells transfected with vectors containing human claudin-4 promoter sequences were also analyzed. Results in Figure 3A showed that luciferase activities driven by the claudin-1 promoter was significantly reduced when MDCK-RON cells were stimulated with MSP. The reduced luciferase activities were also seen in MDCK-RON160 cells. However, when cells were transfected with the claudin-1 promoter containing mutations in the two E-boxes (claudin-1 and M1-2), the inhibitory effect of activated RON or RON160 on luciferase activities was diminished. Luciferase activities driven by the claudin-4 promoter was slightly upregulated in unstimulated MDCK-RON cells but increased when MSP was used. The increased claudin-4 promoter activities were also observed in MDCK-RON160 cells. Thus, activation of RON or RON160 inhibits the claudin-1 promoter transcription but enhances claudin-4 promoter activities.


Figure 3
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  		Fig. 3. RON activation differentially regulates promoter activities of claudin-1 and -4, increases transcription repressor Snail-1 expression and causes cellular redistribution of claudins. (A) Effect of RON activation on claudin promoter activity. MDCK, MDCK-RON or RON160 cells at ~60–70% of confluence in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were transiently transfected with pGL3-CLD1p, pGL3-CLD1pM1-2, or pGL3-CLD4p vectors (3 µg per well). Cells were also transfected with pRL-SV40 control vector to normalize the transfection efficiency. Transfected MDCK-RON cells were stimulated with 2 nM of MSP for 24 h. Luciferase report activities were measured 48 h after transfection. (B) Association of MSP-induced Snail-1 expression with diminished claudin-1 expression: MDCK-RON cells in Dulbecco's modified Eagle's medium with 5% fetal bovine serum were stimulated with 2 nM of MSP for various times. Cellular proteins were subjected to western blot analysis using antibodies to claudin-1 or Snail-1. Membranes were also probed for actin as the loading control. (C) Effect of RON activation on claudin cellular distribution: Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. MDCK-RON cells were stimulated with 2 nM of MSP for 24 h in the presence or absence of PD98059 (50 µM) before lysis. Cellular proteins from MDCK and MDCK-RON cells were fractioned into Triton X-100 soluble and insoluble portions. Samples (50 µg per lane) were subjected to western blot analysis to detect claudin expression using individual antibodies. β-actin was used as the loading control. (D) Fluorescent analysis of claudin expression and localization after RON activation. MDCK, MDCK-RON or -RON160 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a six-well plate for 24 h. To activate RON, MDCK-RON cells were stimulated with MSP (2 nM) for additional 24 h. Cells were fixed and permeabilized followed by immunofluorescent analysis using antibodies to individual claudins. Representative results shown here are from one of three experiments.

 
Increased Snail expression correlates with RON-mediated inhibition of claudin-1 expression
Transcription factor Snail-1 has been shown to inhibit claudin-1 transcription in MDCK cells (21). To see if RON activation increases Snail-1 expression and if such increase correlates with claudin-1 inhibition, MSP-induced Snail-1 expression was studied. MSP-induced RON activation results in increased Snail-1 expression in a time-dependent manner (Figure 3B). The induction was peaked at ~4–6 h after MSP stimulation and lasted up to 24 h. This time course is ~6 h ahead of RON-mediated inhibitory activities, suggesting that increased Snail-1 expression is associated with diminished claudin-1 expression.

Activation of RON or RON160 differentially regulates cellular localization of claudins
The intracellular redistribution of claudins is a mechanism that determines tight junction functions (3,4). Analysis of claudins in Triton X-100 soluble or insoluble fractions is a way to determine if RON activation affects claudin distribution and localization (24). In MDCK or MDCK-RON cells, claudin-1, -3, -4 and ZO-1 are mainly in the insoluble protein fraction (Figure 3C), indicating their membrane localization. Claudin-7 is mainly in the soluble fraction or cytoplasmic distribution. When MDCK-RON cells were stimulated with MSP, the amount of claudin-1 in the insoluble fraction was reduced. Accompanied with this change were the increased amounts of claudin-3 and -4 in the soluble fraction, indicating the redistribution of these two claudins. No changes were seen in claudin-7 and ZO-1. By inhibiting Erk1/2 activities, PD98059 was able to prevent MSP-induced reduction of claudin-1 in the insoluble fraction. PD98059 also prevented the increased accumulation of claudin-3 and -4 in the soluble fraction. No effect of SB203580 or wortmannin was seen (data not shown). Thus, RON activation not only inhibits claudin-1 expression but also regulates claudin-3 and -4 distributions with increased cytoplasmic localization.

To validate these results, immunofluorescent analysis was performed. Results in Figure 3D confirmed the membrane localization of claudin-1, -3, -4 and ZO-1 and the mixed expression patterns of claudin-7 in MDCK and in unstimulated MDCK-RON cells. When MDCK-RON cells were stimulated with MSP, the diminished claudin-1 expression was observed. Moreover, increased cytoplasmic/prenuclear localization of claudin-3 and -4 was observed. These changes were also seen in MDCK-RON160 cells. No changes in distributions of claudin-7 and ZO-1 in MSP-stimulated MDCK-RON or MDCK-RON160 cells were observed. These results were consistent with those from western blot analysis. Thus, MSP-induced RON activation results in diminished claudin-1 expression, which is accompanied with increased cytoplasmic localization of claudin-3 and -4.

Diminished claudin-1 expression is involved in RON160-mediated migration activities
MDCK or MDCK-RON160 cells were transfected to express human claudin-1. As shown in Figure 4A, moderate increase of claudin-1 (almost 1-fold) was observed in transfected MDCK cells. Claudin-1 expression, comparable with parental MDCK cells, was also seen in MDCK-RON160 cells (Figure 4A). Using these cells in wound-healing assays, it was shown that RON160 expression resulted in increased migration of MDCK cells. Almost all open spaces were covered by migrated cells within a 24 h period (Figure 4B). In contrast, cell migration was slowed down when claudin-1 was reexpressed in MDCK-RON160 cells. In this case, only half of the open space was filled. To validate these activities further, the chamber-based cell migration assay was performed (Figure 4C). Consistent with the wound-healing assay, RON160 expression increased cell migration, which was further enhanced by MSP. Reexpression of claudin-1 not only reduced spontaneous migration of MDCK-RON160 cells but also prevented MSP-induced enhancement of cell migration. The quantitative results are shown in Figure 4D, suggesting that reduced claudin-1 expression is involved in RON-mediated migration of MDCK cells.


Figure 4
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  		Fig. 4. Reduced claudin-1 expression results in increased migration of MDCK cells expressing RON160. (A) Expression of human claudin-1 in MDCK or MDCK-RON160 cells. Cells were transfected with pCDNA3.1-CLD1. Proteins from stable cell lines were subjected to western blot analysis using antibodies to claudin-1. (B) MDCK or MDCK-RON160 cells in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were transiently transfected with pcDNA3.1-CLD1 (3 µg DNA per dish). After incubation for 48 h, a wound area was created. Cells transfected with the control pCDNA3.1 vector were used as the control. Cell migration was then monitored. (C) A 48-well migration chamber was used to determine the effect of forced claudin-1 expression on MDCK cell migration. After transfection of the pcDNA3.1-CLD1 vector for 48 h, MDCK-RON160 cells were used in the migration chamber as detailed in Materials and Methods. MSP (2 nM) was used as the attractant. Migration was determined after a 12 h incubation. (D) Quantitative analysis of cell migration in MDCK or MDCK-RON160 cells expressing human claudin-1. Migrated cells from (C) were counted in randomly selected three areas. Representative results shown here are from one of three experiments.

 
Effect of RON activation on claudin-1 expression and cell migration in breast cancer T-47D cells
Results from above studies prompted us to study if the effect of RON on claudins occurs in cancer cells. As shown in Figure 5A, among three RON-positive breast tumor lines tested, only T47-D cells expressed claudin-1 (Figure 5A). After MSP stimulation, claudin-1 expression was progressively reduced in T-47D cells (Figure 5B), which is accompanied with scatter-like morphological changes (Figure 5C). MSP stimulation also resulted in increased cell migration as indicated in wound-healing assays (Figure 5D). Thus, inhibition of claudin-1 expression is not limited in MDCK cells. RON activation also inhibits claudin-1 expression in breast cancer such as T-47D cells.


Figure 5
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  		Fig. 5. Association of RON-mediated reduction of claudin-1 and increased cell migration in breast cancer T47-D cells. (A) Proteins (50 µg per sample) from lysates of T-47D, Du4475 and BT-483 were analyzed in western blotting for RON and claudin-1 expression by specific antibodies, respectively. β-actin was used as the loading control. (B) T-47D cells (5 x 105 cells per well) were stimulated with MSP for various times. Cellular proteins (50 µg per sample) were analyzed for claudin-1 expression in western blotting. (C) T-47D cells in RPMI + 5% fetal bovine serum were stimulated with 2 nM of MSP for 24 h. Morphological changes were observed under a microscope and photographed (magnification x200). (D) The monolayer of T-47D cells was wounded by a plastic tip. Cells were then stimulated with 2 nM of MSP for 24 h. The percentages of wounded area covered by migrated cells were determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The purpose of this study is to study cellular proteins involved in epithelial cell migration mediated by RON or its constitutive active variant RON160. MDCK cells were chosen as the model because of their well-characterized epithelial phenotypes and junction structures. Overexpression of RON and its oncogenic variants is known to increase cell migration and invasiveness (11,27). These activities have been demonstrated in various cancers including those from colon, breast and pancreas (19,28,29). Several structural and signaling proteins are considered as critical players in regulating these activities (11,27). For example, inhibition of E-cadherin expression, accompanied with disruption of adherens junctions, contributes significantly to RON-mediated invasive activities (13,30). The role of E-cadherin in promoting cancer motility is well documented (31,32). Thus, it is not surprising that regulation of E-cadherin expression is utilized as a mechanism in RON-mediated tumorigenic activities. Results from this study provided new evidence that regulation of tight junction protein claudin expression is a critical event in RON-mediated cellular motility. We showed that ligand-dependent RON activation disrupts tight junctions and impairs their functions, which is characterized by diminished claudin-1 expression, redistribution of claudin-3 and -4 and reduction in TER. These activities can be reproduced in MDCK cells expressing the constitutively active variant RON160 and in breast cancer T-47D cells. Thus, activation of RON or expression of RON160 has a fundamental impact on integrity and function of epithelial cell–cell junctions. Alterations of these cellular structures are critical components in RON-mediated tumorigenic activities that facilitate malignant progression.

As structural components of tight junctions, the overall ratio among claudins expressed in a particular cell type and their homo- and heterophilic interactions are critically important in determining the characteristics of its tight junctions (2). These structural features are subjected to regulation under various conditions, especially during tumorigenic transformation of epithelial cells. Differential effects of growth factors such as epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on regulation of claudin expression have been reported (24,33,34). In MDCK cells, EGF-induced EGF receptor activation caused the inhibition of claudin-2 expression (24). EGF stimulation also induces cellular redistribution and increased expression of claudin-1, -3 and -4 (24). These changes were accompanied with increased transepithelial resistance across the MDCK cell monolayer (20). In contrast, stimulation of MDCK cells with HGF resulted in an overall increase in claudin-1 and -4 expressions (24). No major differences in claudin-2 and -3 expressions were observed (24,33). The patterns of claudin expression regulated by activated RON or RON160 in MDCK cells were different from those induced by EGF or HGF. MSP inhibited claudin-1 expression and caused redistribution of claudin-3 and -4. These differences may reflect the differences in signaling events mediated by their corresponding receptors. For example, both MSP and HGF induce cell dissociation, scattering and epithelial to mesenchymal transition in MDCK cells with diminished E-cadherin expression (8,35). MSP or HGF stimulation also caused a marked reduction in TER (33) with increased permeability, indicating the impairment of tight junctions. However, their effects on claudin-1 expression were opposite. HGF increases whereas MSP inhibits claudin-1 expression in MDCK cells. Clearly, different growth factors induce different patterns of claudin expression and distribution, which may have different biological significance.

Various mechanisms have been implicated in regulation of claudin expression, particularly claudin-1 (21,36). In colon cancers, Smad4, the signal component of transforming growth factor-β family, represses claudin-1 transcription through modulation of T-cell factor/lymphocyte enhancer factor activities (36). The Snail family of transcription factors including Snail and Slug has also been implicated as transcriptional repressors of claudin-1 in MDCK cells (21). Results from this study indicate that activation of Erk1/2 signaling cascades is the first step required for RON to exert the inhibitory effect on claudin-1 expression. Inhibition of Erk1/2 phosphorylation by specific inhibitor PD98059 prevented the RON-mediated effect and restored claudin-1 expression. Activated Erk1/2 signaling is also involved in redistribution of claudin-3 and -4 into cytoplasmic/prenuclear compartments. Measurement of promoter activities of the claudin-1 gene has revealed that the inhibitory effect of RON or RON160 acts at transcription levels. Luciferase activities driven by the promoter of the claudin-1 gene were significantly reduced in MDCK cells expressing constitutively active RON160. By correlating expression of transcription repressors, it was reasoned that Snail is a potential factor responsible for inhibition of claudin-1 gene transcription. As shown in Figure 3B, increased Snail expression in RON- or RON160-expressing MDCK cells is inversely correlated with diminished claudin-1 expression. In contrast, Slug expression was not changed during the process of inhibiting claudin-1 expression. Previous studies have uncovered a direct link between the activation of Erk1/2 pathways with increased Snail expression (21). As shown in MDCK cells, activation of the Erk1/2 pathway is required for transcriptional induction of Snail expression (21). Moreover, overexpression of Snail downregulates claudin-1 expression at both messenger RNA and protein levels in MDCK cells (21). In addition, Snail is able to repress effectively claudin-1-driven reporter gene constructs containing the promoter sequences. Additional evidence further demonstrated that Snail binds to the E-box motifs present in the claudin-1 promoter (21). Results from our study showed that Erk1/2 phosphorylation and increased Snail-1 expression are associated with reduction of claudin-1 expression. However, we do not have direct evidence to show if this is the main pathway connecting RON activation and reduction of claudin-1 expression. Nevertheless, our results support a hypothesis that RON-mediated inhibition of claudin-1 expression in MDCK cells is channeled through the functions of transcription repressor Snail. Definitely, other mechanisms play a role in regulation of claudin-1 expression. Thus, it will be interest in the future to dissect the detailed cellular events underlying RON-mediated inhibition of claudin expression.

The discovery that decreased claudin-1 expression is involved in RON-mediated cell migration, not only in MDCK but also in breast cancer cells, is interesting. It reaffirms our notion that regulation of junction protein expression such as claudin-1 and E-cadherin is a general mechanism responsible for RON-mediated cell migration and invasiveness. More importantly, it demonstrated that altered claudin-1 expression is not merely a sign for dysfunction of tight junctions, but a pathogenic factor contributing to RON-mediated cancer migration. Diminished claudin-1 expression has been documented in several epithelial cancers including hepatocellular carcinomas (6) and breast cancers (8,37). In breast cancer samples, loss of claudin-1 expression occurs in the majority of invasive breast carcinoma cells with lymph node involvement (8,37). It also correlates with several clinicopathological parameters such as the short disease-free interval (8,37). Currently, the mechanism underlying the loss of claudin-1 expression is unknown. No genetic alterations in the promoter and coding sequences have been found in breast cancers cells (38). It is probably that transcription repression of claudin-1 gene expression or increased protein degradation is involved in diminished claudin-1 expression. Results from our studies provide a potential explanation that RON-mediated inhibition of claudin-1 expression might be one mechanism for the observed loss of claudin-1 in breast cancers cells. Since RON is highly expressed and activated in the majority of breast cancer samples and cell lines (12,39), it will be interest in the future to verify if indeed this is a mechanism responsible for diminished claudin-1 expression in the majority of invasive breast cancers.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Key grant (30430700) from Natural Sciences Foundation of China to M.H.W.; R01 grant (CA91980) from USA National Institute of Health to M.H.W.


   Acknowledgments
 
We thank Drs Edward J.Leonard for providing MSP, S.Vilaro for pGL3-CLD1p vector, P.J.Morin for pGl3-CLD4p construct and A.T.Weeratna for pcDNA3.1-CLD1 expression vector. The assistance from Ms K.Bohn (Texas Tech University Health sciences Center School of Pharmacy in Amarillo) in editing the manuscript was greatly appreciated.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received September 3, 2007; revised December 13, 2007; accepted December 25, 2007.


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