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Originally published In Press as doi:10.1074/jbc.M605666200 on November 12, 2006

J. Biol. Chem., Vol. 282, Issue 3, 1830-1837, January 19, 2007
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JAM-C Regulates Tight Junctions and Integrin-mediated Cell Adhesion and Migration*Formula

Guillaume Mandicourt{ddagger}, Sandra Iden§, Klaus Ebnet§, Michel Aurrand-Lions{ddagger}, and Beat A. Imhof{ddagger}1

From the {ddagger}Department of Pathology and Immunology, the University Medical Center, CH 1211 Geneva 4, Switzerland and the §Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Munster, D-48149 Münster, Germany

Received for publication, June 13, 2006 , and in revised form, September 29, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tight junctions play a fundamental role in the generation of cell polarity in epithelial and endothelial cells. They delimitate the apical and the basolateral plasma membrane domains and restrict the free diffusion of integral membrane proteins and lipids between them (1). The Junctional Adhesion Molecules (JAMs)2 belong to the immunoglobulin superfamily and have been shown to be associated to tight junctions of epithelial and endothelial cells (2–4). The protein JAM-A is involved in the formation of tight junctions, acting as an anchor recruiting polarity complex proteins to intercellular junctions via its PDZ binding motif (5–7). Similarly to JAM-A, previous studies have shown that the PDZ binding site of JAM-C interacts with Par-3 and ZO-1, both acting as scaf-folds for larger polarity complexes (8).

Maintenance of cell polarity is crucial for tissue integrity. The development of cancers is frequently associated with loss of cellular apico-basal polarity, enabling tumor epithelial cells to escape the constraints imposed by intercellular junctions and tissue organization. Epithelial plasticity ranges from subtle phenotypic changes to complete epithelial-to-mesenchymal transition (9). Mesenchymal transformation is characterized by disruption of intercellular contacts, loss of epithelium-specific proteins, switch to a mesenchymal gene expression pattern, and gain of invasive properties (10). In contrast, during morphogenesis, epithelial cells transiently down-modulate or relocalize junctional proteins without losing cell-cell contacts (11, 12). This occurs during development but has a fundamental role in tumor progression from adenoma to carcinoma. Disorganization of tight junctions occurs concomitantly with activation of the cytoskeleton machinery into a pro-migratory state of the cell, due in part to activation of integrin complexes that provide the physical link between the actin cytoskeleton and the extracellular environment (13). Indeed, by interacting with their ligands, including the members of the immunoglobulin superfamily, integrins initiate intracellular responses that lead to an increase in cell migration, adhesion, or integrin activation (14, 15). The latter is also controlled by "inside-out" signals that tune the affinity of integrins for their ligands (16).

JAM-C expression has been found in carcinoma cell lines (17). However, the role of JAM-C during tumor formation remains to be determined. In the present study, we investigated the role of JAM-C in tight junction plasticity and integrin activation associated to the tumor process. We took advantage of the tumor cell line KLN205, which is devoid of JAM-C expression and displays an intermediate transformed phenotype, representing an ideal model to test the function of JAM-C during epithelial-to mesenchymal transformation (18). We show that JAM-C expression in KLN205 cells restores an epithelial phenotype in respect to tight junction formation and trans-epithelial electrical resistance. In addition, mutation of the serine 281 amino acid of JAM-C into alanine (J-C S281A) abolishes this effect and induces a pro-adhesive and pro-migratory phenotype of the cells. In agreement, we show that the S281A mutation of JAM-C activates beta3 integrins. Hence, we demonstrate that the JAM-C phosphorylation status is involved in the switch from a static, polarized state to a pro-migratory phenotype occurring during neoplastic progression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors—JAM-C and JAM-CEGFP constructs have been previously described (19). The point mutation in J-C S281A was generated by a PCR-based approach using Pfu-Turbo® DNA polymerase (Stratagene). Briefly, mutagenic primers were used to amplify the plasmidic matrix DNA encoding the full-length sequence of JAM-C. After 15 cycles of amplification, the PCR mix was digested using DpnI to degrade the matrix plasmidic DNA and recover amplification products that were transformed in bacteria. Mutated plasmids were then sequenced on both strands using the Thermo Sequence Fluorescent-labeled Primer Cycle Sequencing kit (Amersham Biosciences) and the LI-COR DNA Analysis System (MWG-Biotech GmbH, Ebersberg, Germany). From this product, the J-C S281AEGFP construct was generated with the same strategy as the one used for the generation of JAM-CEGFP.

Antibodies—The rat monoclonal antibody against mouse JAM-C (F26) was previously described (18). The KM16 anti-beta1 integrin and the HMbeta3 anti-beta3 integrin are from BD Biosciences. The polyclonal anti-ZO-1 is from Zymed Laboratories (Invitrogen). F-actin was visualized with Texas Red®-X phalloidin (Molecular Probes Inc., Eugene, OR). The 9EG7 anti-beta1 integrin was kindly provided by D. Vestweber, Münster, Germany (20).

Cell Lines and Transfections—KLN205 cells (21) were cultured in Dulbecco's modified Eagle's medium and CHO cells in F12 medium (Invitrogen), both supplemented with antibiotics and 10% fetal calf serum. TransIT®-LT1 Transfection Reagent (Mirus Bio Corp.) was used for stable transfection according to the manufacturer's recommendation. Cells were cultured in the presence of 1 mg/ml geneticin (Invitrogen) to select for stable expressing cells. JAM-C-expressing cells were selected using fluorescence-activated cell sorting (FacStar; BD Biosciences) after immunostaining with appropriate antibodies. Transfected cells were selected for comparable amounts of JAM-C expression and used without clonal selection to avoid artifacts due to clonal variations (supplemental Fig. S1).

Western Blot—Cells were incubated in lysis buffer (50 mM Tris·HCl, pH 7.4, and 150 mM NaCl with 0.5% Triton X-100) containing protease inhibitors (chymostatin, leupeptin, anti-pain A, pepstatin A, each at 1 µg/ml, Sigma) for 30 min on ice. Afterward, the lysed cells were transferred to an Eppendorf tube and centrifuged. The supernatant was collected, and protein content was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce). Equal amounts of total extracts (50 µg) were separated by 10% SDS-PAGE before transfer onto a nitrocellulose membrane (Hybond ECL; Amersham Biosciences). Uniform loading of lanes was verified both by immunoblotting of total actin and Ponceau red staining. The membrane was blocked for 60 min at room temperature in PBS containing 0.05% (v/v) Tween 20 (PBS-Tween) and 5% (w/v) nonfat milk powder and then incubated either overnight at 4 °C or for 2 h at room temperature with primary antibodies anti-JAM-C rat monoclonal (F26) or anti-total actin rabbit serum (1:5000) (gift from G. Gabbiani, University of Geneva). After extensive washing in PBS-Tween, the membrane was incubated for 60 min at room temperature with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). The membrane was then washed extensively in PBS-Tween, and antigen-antibody complexes were detected by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham Biosciences).


Figure 1
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  		FIGURE 1.
JAM-C is not expressed in KLN205 cells and colocalizes with tight junctional marker ZO-1 upon transfection. A, total extracts of confluent non-transfected or JAM-C-transfected KLN205 were immunoblotted with antibody against JAM-C. Uniform loading of lanes was verified by immunoblotting of total actin. B, micrographs showing the localization of JAM-C (green) and ZO-1 (red) in wild-type KLN205 (upper panel) and JAM-C-transfected KLN205 (lower panel) cells. Bar,10 µm.

 
In Vivo Labeling and Phosphoamino Acid Analysis—Confluent KLN205 cells stably expressing enhanced green fluorescent protein-tagged JAM-C wild-type or JAM-C S281A were washed in phosphate-free Dulbecco's modified Eagle's medium and subsequently metabolically labeled for 14 h in phosphate-free Dulbecco's modified Eagle's medium containing [32P]orthophosphate (0.5 mCi/ml) supplemented with 0.5% fetal calf serum. Cells were lysed in lysis buffer (50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1% (v/v) Nonidet P-40, 10% glycerol, 10 µg of aprotinin/ml, 1 mM Pefabloc, 10 µg of leupeptin/ml, 1 µg of pepstatin A/ml, 1 mM dithiothreitol), and JAM-C was immunoprecipitated using affinity-purified polyclonal rabbit antibodies. Phosphorylated proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After excision of the bands corresponding to JAM-C, amino acids were released by acid hydrolysis with constant boiling hydrochloric acid for 1 h at 100°C and separated by two-dimensional electrophoresis on thin layer cellulose plates using a Hunter HTLE 7000 apparatus (C. B. S. Scientific Company, Inc.) according to the manufacturer's protocol.

Epithelial Cultures Using the Air/Liquid System—For JAM-C junctional distribution study and trans-epithelial electrical resistance (TER) measurement, KLN205 cells were cultured in an air/liquid system to allow optimal organization of monolayers (22). Briefly, cells were grown on 0.4-µm pore size polycarbonate filters (Nunc, Roskilde, Denmark). When confluence was reached, the medium in the upper chamber was removed, allowing air contact with the cells and the complete polarization of the monolayer. The setting up of the polarity of the monolayer was followed by the measurement of TER using a WPI EVOM Electrod port (World Precision Instruments). It is important to note that the TER of a non-polarized KLN205 monolayer is usually ~500 Ohms x cm2 and increases up to >2000 Ohms x cm2 when tightness of cell-cell contacts is complete.


Figure 2
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  		FIGURE 2.
Serine 281 of JAM-C is phosphorylated and regulates intercellular localization of the protein. A, alignment of human and murine amino acid sequences of JAM-A, JAM-B, and JAM-C cytoplasmic tails. Identical and homologous amino acids are shaded in dark and light gray, respectively. The star indicates the conserved serine 281; the PDZ binding motif is underlined. B, part a, confluent KLN205 cells stably expressing enhanced green fluorescent protein-tagged JAM-C wild-type (WT) or JAM-C mutant S281A (JAM-C S281A) were metabolically labeled with [32P]orthophosphate and subjected to immunoprecipitation with JAM-C antibodies. Note that equal amounts of JAM-C immunoprecipitates were used in the following phosphopeptide analysis. Part b, phosphoamino acid analysis of JAM-C. Immunoprecipitates shown in part a were hydrolyzed, the resulting amino acids were subjected to two-dimensional electrophoresis, and the remaining phosphorylation signals were displayed by 32P autoradiography. The circles indicate the positions of comigrating cold phosphoamino acids. Sample origins are indicated by black crosses. Arrows indicate electrophoresis directions at respective pH conditions. Note that JAM-C is phosphorylated exclusively on serine residues (JAM-C WT, left panel) and that a major portion of the phosphorylation signal gets lost by substitution of serine 281 to alanine (JAM-C S281A, right panel), indicating that Ser-281 reflects the main phosphorylation site in JAM-C expressed by KLN205 cells. Pi, free phosphate residues; pSer, phosphoserine; pThr, phosphothreonine; pTyr, phosphotyrosine. C, micrographs showing the localization of JAM-C in CHO cells transfected with the indicated mutant forms of the protein. Bar,5 µm. J-C S281A displays enrichment at cell-cell contacts. D, micrographs showing the localization of JAM-C in KLN205 cells transfected with the indicated mutant forms of the protein. Bar,10 µm. Similarly to CHO cells, J-C S281A displays enrichment at cell-cell contacts.

 
Immunofluorescent Staining—For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with paraformaldehyde containing 0.01% Triton for 5 min prior to washing with PBS containing 0.2% bovine serum albumin (Sigma). Cells were then incubated with the corresponding primary antibody for 1 h, washed, and further incubated with secondary antibody coupled to fluorescein isothiocyanate or Texas Red (Jackson Immunoresearch Laboratories, Inc.). Images were acquired using a confocal microscope Zeiss LSM510 (Zeiss, Oberkochen, Germany).

Fluorescence-activated Cell Sorting Analysis—Trypsinized adherent cells were collected, washed once with 10% fetal calf serum Dulbecco's modified Eagle's medium, and then resuspended in 0.2% bovine serum albumin/PBS. Cells were incubated with corresponding primary antibody on ice for 30–60 min. After two washes with PBS, cells were incubated with the appropriate isotype-matched phycoerythrin-conjugated antibody (Jackson Immunoresearch Laboratories, Inc.) on ice for 30–60 min. Cells were washed twice with PBS followed by flow cytometry using a FACScan (BD Biosciences). The flow cytometer was calibrated using single phycoerythrin-stained cells. Results of individual cell lines are expressed as a plot of frequency versus log fluorescence.

Adhesion Assay—KLN205 cells were labeled using PKH26 Red Fluorescent Cell Linker kit (Sigma) 1 day before use to avoid any problem with toxicity. Adhesion was performed in 96-well plates (Costar 9017; Corning Inc., Corning, NY) coated with either 10 µg/ml of fibronectin or different concentrations of vitronectin diluted in Hanks' Balanced Salt Solution (Invitrogen) supplemented with 0.2% bovine serum albumin, 1.25 mM CaCl2, and 0.8 mM MgCl2, for 60 min at 37 °C in 10% CO2. Cells were fixed with 1% paraformaldehyde and washed four times. The fluorescence of adherent cells was quantified in a CytoFluor plate reader with excitation at 544 nm and emission at 590 nm (FlexStation; Molecular Devices). For blocking studies, cells were incubated with the 9EG7 anti-beta1 integrin and the HMbeta3 anti-beta3 integrin-blocking antibodies, the KM16 anti-beta1 integrin non-blocking antibody, or the integrin-blocking RGD peptide (Bachem, Bubendorf, Switzerland) during the adhesion period. For adhesion on vitronectin, the time of incubation was reduced to 30 min.

Spreading Assay—KLN205 cells were plated on glass coverslips coated with 10 µg/ml of fibronectin. After 90 min, the majority of cells adhered to the fibronectin. Cells were fixed and then washed several times, and photographs were taken with phase contrast optics using an epifluorescent microscope (Zeiss-Axiovert 100, Zurich, Switzerland), with CP Achromat x 10/0.25 Ph1 objective (Zeiss), equipped with a C4742-95-10 digital CCD camera (Hamamatsu Photonics) controlled by the Open-lab software (Improvision, Coventry, UK). Cells were considered as unspread when they appeared round and bright and spread when they appeared darker. The ratio was calculated by the number of spread cells divided by the total number of adherent cells.

Migration Assay—Cell migration was studied by wounding assays. Cells were plated at high density (106 cells/ml) on glass coverslips and grown for 2 days in order to reach a dense, contact-inhibited monolayer. A wound was then created by scratching the monolayer with a yellow pipette tip, and the closure of the wound by migrating cells was followed over 8 h by time lapse microscopy using the same equipment as for the spreading assay completed with an incubation chamber with the temperature and CO2 set at 37 °C and 10%, respectively.

Statistical Analysis—Bar graphs represent the mean and ± S.E. All experiments were evaluated with the Student's t test, using GraphPad Prism (www.graphpad.com/). p < 0.05 was considered statistically significant. The symbol * indicates a value of p < 0.05. **, p < 0.01. ***, p < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Junctional Localization of JAM-C in Epithelial Cells Is Controlled by Phosphorylation of Serine 281—To explore the function of JAM-C in polarized cells, we took advantage of the mouse tumor cell line of epithelial origin, KLN205, which lacks endogenous expression of JAM-C (Fig. 1). When expressed in KLN205 cells JAM-C colocalized with ZO-1 (Fig. 1B). Having previously shown that the localization of JAM-C at cell-cell borders of non-polarized cells is negatively regulated by serine phosphorylation (8), we looked to see whether it is the case in the polarized KLN205 cells. The cytoplasmic tail of JAM-C contains a single phosphorylatable serine residue conserved across species (Fig. 2A). To analyze whether this serine residue is involved in cell-cell contact localization of JAM-C, we generated stable KLN205 cell lines expressing either JAM-C or JAM-C S281A. By phosphoamino acid analysis, we found that JAM-C is phosphorylated on serine residues in these cells (Fig. 2B). Importantly, the serine phosphorylation signal was markedly reduced in cells expressing JAM-C S281A despite the same amounts of JAM-C immunoprecipitated from the two cell lines (Fig. 2B and supplemental Fig. S1), indicating that JAM-C is phosphorylated on serine 281 in KLN205 cells. Interestingly, this mutation affected the localization of the protein at cell borders. The J-C S281A mutated protein appeared highly enriched to cell-cell borders of CHO cells (Fig. 2C). In KLN205 cells, the distribution of the mutated protein to cell borders was more diffuse as compared with the wild-type protein, which was localized to the apical region of the lateral membrane domain (Fig. 2D). To further compare the localization of JAM-C and the J-C S281A mutated protein along the lateral membrane, KLN205 cells were grown in an air/liquid culture system that allows a full apico-basolateral polarization of the cells. Under these conditions, the wild-type protein was localized to the tight junctional region of KLN205 cell contacts (Fig. 3A). By contrast, the serine 281 unphosphorylatable mutant of JAM-C (J-C S281A) was massively enriched at cell-cell borders and displayed a lateral localization, overflowing the tight junction area (Fig. 3A), showing that serine 281 phosphorylation regulates JAM-C localization not only in the poorly polarized CHO cells but also in the more polarized KLN205 cells. In addition, expression of JAM-C in KLN205 cells resulted in a better organization of the cell monolayer as depicted by the regular square-shaped morphology of the cells, suggesting that JAM-C may have improved apico-basal polarity of KLN205 cells.


Figure 3
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  		FIGURE 3.
JAM-C promotes full polarization of KLN205 monolayers. A, micrographs showing the localization of JAM-C (green) and actin (red) in non-transfected (NT) and transfected KLN205 cells. Pictures were obtained with cells grown on filters in an air/liquid system and are presented as z-axis reconstruction. The organization of the monolayer is improved in JAM-C-expressing cells as compared with cells expressing the S281A mutant form of JAM-C or non-transfected cells. Bars,10 µm. B, transepithelial electrical resistance (TER) measured on monolayers of non-transfected and transfected KLN205 cells. In agreement with the apparent improved organization of the monolayer, TER is increased by 400% in JAM-C-expressing cells as compared with cells expressing the S281A mutant form of JAM-C or non-transfected cells. Similar results were obtained in three independent experiments, and one representative experiment is shown. ***, p < 0.001.

 
Because the TER of cell monolayers reflects the polarity of epithelial cells (22), we compared the TER of non-transfected, JAM-C and J-C S281A-transfected KLN205 cells. We found that the expression of JAM-C increases the TER up to 2200 Ohms x cm2, whereas the TER of non-transfected or J-C S281A-transfected cells remains at ~500 Ohms x cm2 (Fig. 3B). This is in agreement with the observation that the morphology of intercellular contacts was improved upon JAM-C transfection as visualized by electron microscopy (supplemental Fig. S2). These results indicate that JAM-C improves the junctional barrier properties of KLN205 monolayers and that this effect is dependent on JAM-C phosphorylation at serine 281.


Figure 4
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  		FIGURE 4.
JAM-C S281A mutation regulates cell adhesion to fibronectin. KLN205 cells expressing J-C S281A show an increased adhesion to fibronectin compared with non-transfected (NT) or JAM-C-transfected cells. Results are expressed as a percentage of total cells (input) after 60 min at 37 °C. Similar results were obtained in three independent experiments, and one representative experiment is shown. *, p < 0.05.

 


Figure 5
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  		FIGURE 5.
JAM-C S281A mutation decreases cell spreading on fibronectin. KLN205 cells expressing J-C S281A present a decreased spreading on fibronectin as compared with non-transfected (NT) or JAM-C-transfected cells. Results are expressed as a percentage of spread cells among adhering cells after 90 min at 37 °C. Similar results were obtained in three independent experiments, and one representative experiment is shown. **, p < 0.01.

 
JAM-C S281A Increases Cell Adhesion and Migration—Having shown that JAM-C improves tightness of cellular contacts in carcinoma cells and restores a more epithelial phenotype, we addressed the question whether JAM-C affects other cardinal features of tumor cells, such as adhesion and migration. Surprisingly, we found no significant difference in adhesion of untransfected and JAM-C-transfected KLN205 cells on different substrates. In contrast, expression of the J-C S281A mutant dramatically increased cell adhesion to the extracellular matrix protein fibronectin (Fig. 4). Similar results were obtained using CHO- and Madin-Darby canine kidney-transfected cells (supplemental Fig. S3), suggesting that serine 281 of JAM-C is critically involved in the control of cell interaction with the extracellular matrix.


Figure 6
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  		FIGURE 6.
JAM-C regulates cell migration and wound closure. A, representative micrographs of wound healing performed on non-transfected KLN205 cells or cells expressing JAM-C or the S281A mutated form of the protein are shown. Pictures obtained at time 0 and 6 h after wounding are shown. The closure of the wound is accelerated in cells expressing the wild-type form and the S281A mutated form of JAM-C as compared with untransfected cells. Bar, 100 µm. B, quantification of wound closure by monolayers of KLN205 cells shown in panel A. The area of wound opening after 6 h is expressed as a percentage of opening at time 0 (filled columns). Measurements were performed in triplicate (three independent wound measures for each type of cells), and results are expressed as mean ± S.E. Similar results were obtained in three independent experiments, and one representative experiment is shown. ***, p < 0.001. C, quantification of wound closure at different time points after wounding monolayers expressing the wild-type form or the S281A mutated form of JAM-C. Results are expressed as in panel B except that the normalization is done at 2 h after wounding. The speed of wound closure is significantly increased in cells expressing the mutated form of JAM-C (dashed columns) as compared with cells expressing the wild-type form of the molecule (filled columns). *, p < 0.05; **, p < 0.01.

 
Fibronectin is a versatile substrate allowing cells to adhere, spread, and migrate. We then investigated whether JAM-C expression influences cell spreading and found that the J-C S281A unphosphorylatable mutant significantly reduced cell spreading when compared with cells expressing no or wild-type JAM-C (Fig. 5). Because the JAM-C phosphorylation state modulated both adhesion and spreading of cells to fibronectin, it was likely that migration was also affected. Wounding assays using cell monolayers showed that mutant J-C S281A substantially increased and wild-type JAM-C slightly increased cell migration when compared with non-transfected cells (Fig. 6). This differential effect occurred immediately after cell wounding and was maximal after 6 h. At this time, J-C S281A-transfected KLN205 cells completely closed the wound whereas untransfected cells still kept it open. The differences observed in the wounding assay were not due to other mechanisms such as cell proliferation because growth rates of the different cell types were identical (supplemental Fig. S4).


Figure 7
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  		FIGURE 7.
JAM-C S281A mutation impairs beta1 integrin activation. A, adhesion to fibronectin of non-transfected KLN205 cells (NT) or cells expressing wild-type or the S281A mutated form of JAM-C was tested in the presence of the anti-beta1 integrin-blocking antibody 9EG7 (dashed columns), the anti-beta1 integrin-non-blocking antibody KM16 (white columns), or a control isotype-matched antibody (filled columns). Although beta1 integrin contributes >50% to the adhesion of non-transfected or JAM-C wild-type-expressing cells, no contribution of this integrin is found for the adhesion of J-C S281A-expressing cells. Because of variation in the number of adherent cells between cell types (see Fig. 3), results are expressed as a percentage of control adhesion observed for each cell type (filled columns). Similar results were obtained in three independent experiments, and one representative experiment is shown. *, p < 0.05. B, spreading of non-transfected KLN205 cells (NT)or cells expressing wild-type or the S281A mutated form of JAM-C on fibronectin in the presence of the anti-beta1 integrin-blocking antibody 9EG7 (dashed columns), the anti-beta1 integrin-non-blocking antibody KM16 (white columns), or control isotype-matched antibody (filled columns). Results are expressed as a percentage of control as in panel A. Similar results were obtained in three independent experiments, and one representative experiment is shown. *, p < 0.05; **, p < 0.01. C, flow cytometry histogram profiles showing beta1 integrin expression on the cell lines used in panels A and B. Comparable amounts of beta1 integrin surface expression are found on the indicated three cell lines.

 
JAM-C Controls Activation of beta1 and beta3 Integrins—Our results suggest that serine 281 of the cytoplasmic tail of JAM-C regulates the interaction of KLN205 cells to the extracellular matrix. Hence we investigated whether this occurs via regulation of integrin avidity for their ligands. KLN205 cells express both beta1 and beta3 integrin subunits, which are receptors for fibronectin (Figs. 7C and 8C). We first focused on beta1 integrins, which are the main receptors for fibronectin, and performed adhesion and spreading assay in the presence of the anti-beta1-blocking antibody 9EG7. Blocking antibody against beta1 integrin reduced the adhesion and spreading of non-transfected and JAM-C-transfected KLN205 cells on fibronectin, but not of J-C S281A cells (Fig. 7, A and B). We could exclude that this was due to signal delivery through beta1 integrin by the 9EG7 antibody, because the non-blocking antibody KM16 had no effect on adhesion or spreading. In addition, levels of beta1 integrin surface expression were not affected by expression of JAM-C or J-C S281A (Fig. 7C). These observations indicate that signals delivered through S281A mutation of JAM-C down-regulate beta1 integrin-dependent interaction of the cells with the extracellular matrix, whereas adhesion is increased.

In the absence of beta1 integrin function, cells use beta3 integrins as alternative receptors for fibronectin (23). To test whether beta3 integrins are also involved in our system we used the anti-beta3 antibody HMbeta3 to block adhesion of transfected cells to fibronectin. As shown in Fig. 8A, the blocking effect of HMbeta3 was substantially increased using J-C S281A-transfected cells compared with cells transfected with the wild-type protein. This result was confirmed using a RGD peptide as antagonist of fibronectin binding integrins. Thus, it is likely that the J-C S281A mutant increases the activation state of beta3 integrin, although this cannot be tested directly because activation-dependent antibodies against mouse beta3 integrin do not exist. We therefore tested the activation state of beta3 integrin using adhesion assays on graded concentrations of vitronectin, a more specific ligand for this integrin (23, 24). The J-C S281A-transfected cells adhered better to vitronectin than non-transfected or JAM-C-expressing cells at all tested concentrations (Fig. 8B). This was not due to changes in beta3 integrin expression (Fig. 8C), suggesting a higher activation state of the beta3 integrin in cells transfected with J-C S281A.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The function of JAM proteins has been previously linked to their intercellular localization (25–27). The protein JAM-C has been found either in desmosomes or tight junctions of epithelial cells (18, 28). In the present study, we report that JAM-C expressed in the KLN205 carcinoma cells localizes to tight junctions. This leads to a better organization of the cells in monolayer and to increased tightness of cell-cell contacts. Interestingly, mutation of serine 281 of JAM-C into alanine abolishes this effect and mislocalizes the protein.

Tight junctions regulate cell polarity by restricting the free diffusion of integral membrane proteins and lipids between the apical and basolateral plasma membranes (1). Among several molecules involved in this process, the polarity complex molecules PAR-3 and PAR-6, by forming a ternary complex with atypical protein kinase C, play a central role in the generation of tight junctions and cell polarity (29, 30). Indeed, overexpressing a mutated form of PAR-6 lacking the atypical protein kinase C binding domain or a dominant-negative form of atypical protein kinase C disrupts the integrity of tight junctions measured by TER or para-cellular permeability (31, 32). Interestingly, JAM-C directly associates to PAR-3 (8). In addition, PATJ, which is part of a second polarity complex made of PALS-1-PATJ-CRB1, has been shown to coprecipitate with JAM-C in spermatids (33, 34). Based on our observations that JAM-C localizes to tight junctions in the KLN205 carcinoma cells, we postulate that JAM-C interacting with PAR-3 via its PDZ binding motif acts as an anchor to recruit polarity complexes. This consolidates pre-existing tight junctions and reinforces polarization of the cells. In contrast, the S281A mutation, which does not affect the PDZ binding motif (8), leads to lateral accumulation of JAM-C out of tight junctions. In this environment, mutated JAM-C may not be able to further interact with polarity complexes and affect tight junctions. This would be in agreement with the unique properties of J-C S281A, which does not decrease the TER of KLN205 cells but increases adhesion and migration of the cells.


Figure 8
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  		FIGURE 8.
JAM-C S281A mutation activates beta3 integrin. A, adhesion to fibronectin of nontransfected KLN205 cells (white columns) or cells expressing wild-type (dashed columns) or the S281A mutated (filled columns) form of JAM-C was tested in the presence of the anti-beta3 integrin-blocking antibody HMbeta3 or the integrin-blocking RGD peptide. Appropriate controls are shown. Because of variation in the number of adherent cells between cell types (see Fig. 3), results are normalized to 100% for the adhesion of each cell type observed in the absence of treatment. The contribution ofbeta3 integrin to the adhesion of the cells is substantially increased in KLN205 cells expressing the S281A mutated form of JAM-C. Similar results were obtained in three independent experiments, and one representative experiment is shown. *, p < 0.05; **, p < 0.01. B, although dose-dependent adhesion to vitronectin is observed for non-transfected KLN205 cells (NT) or cells expressing wild-type or the S281A mutated form of JAM-C, the adhesion of J-C S281A-expressing cells was substantially increased at all concentrations of vitronectin. Results are expressed as a percentage of adherent cells after 30 min at 37 °C. Similar results were obtained in three independent experiments, and one representative experiment is shown. *, p < 0.05; **, p < 0.01. C, flow cytometry histogram profiles showing beta3 integrin expression on the cell lines used in panels A and B. Comparable amounts of beta3 integrin surface expression were found on the indicated three cell lines.

 
Migration is a complex cellular behavior that involves protrusion and adhesion at the cell front and contraction and detachment at the rear (35). During these processes, different integrins provide the physical link between the actin cytoskeleton and the extracellular environment (36). The conversion from a static state to a pro-migratory state observed in JAM-C mutant cells correlates with differential modulation of specific integrins. The S281A mutant form of JAM-C reduces beta1 integrin activity and enhances beta3 integrin-mediated interaction with the extracellular matrix, suggesting that phosphorylation and dephosphorylation of JAM-C on serine 281 modulate the activation state of beta1 and beta3 integrins. Because JAM-C is located at the lateral cell border and integrins at basal focal adhesions, the question remains how JAM-C and integrin function are linked. Despite several studies showing direct transinteractions between JAMs and integrins (37–41), we did not detect overlapping staining between JAM-C and beta1 and beta3 integrins in our mouse model cells (data non shown). Alternatively, constitutive dephosphorylation of JAM-C on serine 281 may activate a signal transduction pathway in order to regulate the interaction of the cell with the extracellular matrix. We tested several pathways, including phosphoinositide-3'OH-kinase, Rho-associated kinase, and myosin light chain kinase by adding specific inhibitors during adhesion assays. Surprisingly, J-C S281A-expressing cells are resistant to the inhibitors tested, contrary to non-transfected and JAM-C-transfected cells (data not shown). This unexpected result indicates that other mechanisms are involved in connecting JAM-C with integrin activity.

During development, epithelial cells can transiently lose cell-cell adhesion and exhibit a migratory behavior (42, 43). A similar phenotypic switch occurs during carcinogenesis. Indeed, breakdown of intercellular contacts and gain of invasive properties are hallmarks of malignancy (10, 44, 45). KLN205 cells are carcinoma cells that have lost some of the epithelial phenotype. The wild-type KLN205 monolayers exhibit low electrical resistance and poor epithelial organization as compared with KLN205 cells expressing JAM-C. The J-C S281A mutant does not affect monolayer organization but converts the cell into a pro-migratory phenotype via activation of beta3 integrins and deactivation of beta1 integrins. JAM-C is expressed by human intestinal epithelial cells (28) and by the non-small lung carcinoma cell line NCI-H522 (17). Hence, it is likely that JAM-C is present in different phosphoisoforms in epithelial and carcinoma cells. However, whether JAM-C is up-regulated and/or phospho-modulated in tumors and whether JAM-C dephosphorylation in tumors correlates with their metastatic potential need further investigation.

In summary, our findings underline the functional dichotomy of JAM proteins as adhesion molecules, intercellular junctional organizers, and outside-in signaling entities. Mutation of a unique serine residue in JAM-C cytoplasmic tail controls JAM-C junctional localization and downstream events related to cell migration, epithelial differentiation, and carcinogenesis.


   FOOTNOTES
 
* This work was supported by Le Fonds National Suisse Grants 3100AO-100697/1 (to B. A. I.) and 310000-112551/1 (to M. A. L.), La Ligue Contre Le Cancer Grant OCS-01653-02-2005, and Deutsche Forschungsgemeinschaft Grant EB16012-3, SPP1111* (to K. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4. Back

1 To whom correspondence should be addressed: Dept. of Pathology and Immunology, University Medical Center, 1 Rue Michel-Servet, CH 1211 Geneva 4, Switzerland. Tel.: 41-22-379-57-47; Fax: 41-22-379-57-46; E-mail: beat.imhof{at}medecine.unige.ch.

2 The abbreviations used are: JAM, junctional adhesion molecule; CHO, Chinese hamster ovary; TER, transepithelial electrical resistance; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Bernard Wehrle-Haller for technical advice, Dominique Ducrest-Gay, Marie-Claude Jacquier, and Dr. Laurence Zulianello for technical expertise, and Dr. Paul Frederick Bradfield and Dr. Chrystelle Lamagna for critical reading of the manuscript. We thank Dr. Friedemann Kiefer for scientific support and advice in the two-dimensional separation method.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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