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Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan

	Abstract

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Nectins, Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules and cadherins, Ca²⁺-dependent cell–cell adhesion molecules, are associated through their respective cytoplasmic tail-binding proteins, afadin and catenins and play roles in formation of adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin-like molecule-5 (Necl-5) is a Ca²⁺-independent Ig-like molecule which does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, one member of the nectin family. Necl-5 does not directly bind afadin and therefore is not associated with cadherins. Necl-5 regulates cell motility and proliferation in cooperation with integrins and growth factor receptors, when it does not interact with nectin-3. We studied here a role of the heterophilic trans-interaction of Necl-5 with nectin-3 in cell–cell adhesion using L cells stably expressing Necl-5, nectin-3 and E-cadherin (Necl-5-nectin-3-EL cells). Afadin, E-cadherin and catenins were recruited to the nectin-3 side, but not to the Necl-5 side, of the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3. The anti-Necl-5 monoclonal antibody, which specifically inhibited the heterophilic trans-interaction of Necl-5 with nectin-3, inhibited the formation of the E-cadherin-based AJs in Necl-5-nectin-3-EL cells. These results indicate that Necl-5 plays roles not only in cell motility and proliferation but also in cell–cell adhesion in cooperation with nectin-3.

	Introduction

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Necl-5/Tage4/poliovirus receptor (PVR)/CD155 is an immunoglobulin (Ig)-like molecule having a domain structure consisting of one extracellular region with three Ig-like loops, one transmembrane region and one cytoplasmic region (Mendelsohn et al. 1989; Koike et al. 1990; Chadeneau et al. 1994, 1996). Human PVR/CD155 was originally identified as the human PVR (Mendelsohn et al. 1989; Koike et al. 1990), whereas Tage4 was originally identified as the product of a gene over-expressed in rat and mouse colon carcinoma (Chadeneau et al. 1994, 1996). PVR/CD155 has also been shown to be over-expressed in human colorectal carcinoma and malignant glioma (Masson et al. 2001; Gromeier et al. 2000). The PVR/CD155 gene and the Tage4 gene are likely to be derived from the common ancestor gene (Baury et al. 2001; Ravens et al. 2003) and tentatively renamed nectin-like molecule-5, Necl-5 (Takai et al. 2003). Nectin-like molecule (Necl) is the name given to a group of Ig-like molecules of which domain structures are similar to, but slightly different from, those of nectins, Ca²⁺-independent Ig-like cell–cell adhesion molecules (Takai et al. 2003). Although Necl-5 had been shown to be the PVR (Mendelsohn et al. 1989; Koike et al. 1990), its physiological role remained unknown for a long time. We (Ikeda et al. 2003) and Mueller & Wimmer (2003) have recently shown, independently, that Necl-5 heterophilically trans-interacts selectively with nectin-3, a member of the nectin family (Ikeda et al. 2003), although Necl-5 does not homophilically trans-interact (Aoki et al. 1997).

The nectin family consists of four members, nectin-1, -2, -3 and -4 (Takai & Nakanishi 2003; Takai et al. 2003). Each nectin forms homo-cis-dimers, followed by formation of trans-dimers (trans-interaction), eventually causing cell–cell adhesion. Nectin-3, furthermore, forms heterophilic trans-dimers with either nectin-1 or -2. Nectin-4 forms heterophilic trans-dimers with nectin-1, but nectin-2 does not form heterophilic trans-dimers with nectin-1. Of these heterophilic trans-interactions, the trans-interaction between nectin-1 and -3 is the strongest and that between nectin-2 and -3 is the next strongest. Nectins bind afadin, an actin filament-binding protein that connects nectins to the actin cytoskeleton (Takai & Nakanishi 2003; Takai et al. 2003), as cadherins bind the - and ß-catenin complex which connects cadherins to the actin cytoskeleton (Takeichi 1995; Nagafuchi 2001; Perez-Moreno et al. 2003). Although Necl-5 has a domain structure similar to those of nectins, it does not bind afadin. Nectins first form cell–cell adhesion where E-cadherin is recruited, resulting in formation of adherens junctions (AJs) in epithelial cells and fibroblasts (Takai & Nakanishi 2003; Takai et al. 2003). In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through the reorganization of the actin cytoskeleton (Shimizu & Takai 2003; Takai et al. 2003). AJs are a major junctional structure in both epithelial and nonepithelial cells. AJs play key roles in formation and maintenance of tight junctions and desmosomes in epithelial cells (Takeichi 1995; Nagafuchi 2001; Perez-Moreno et al. 2003).

We have recently found that Necl-5 enhances serum- and growth factor-induced cell motility in an integrin-dependent manner, when Necl-5 does not interact with nectin-3 (Ikeda et al. 2004). More recently, we have revealed that Necl-5 furthermore enhances the serum- and growth factor-induced cell proliferation, when Necl-5 does not interact with nectin-3 (Kakunaga et al. 2004). Thus, Necl-5 regulates both cell motility and proliferation, when Necl-5 does not interact with nectin-3. However, the role of the heterophilic trans-interaction of Necl-5 with nectin-3 remains unknown. We examined here whether the heterophilic trans-interaction of Necl-5 with nectin-3 is involved in cell–cell adhesion at the initial stage, because Necl-5 localizes at the leading edges of migrating cells (Ikeda et al. 2004) and nectins are involved in cell–cell adhesion (Takai & Nakanishi 2003; Takai et al. 2003). Our results indicate that Necl-5 plays roles not only in cell motility and proliferation but also in cell–cell adhesion in cooperation with nectin-3 and suggest that Necl-5 and nectins play a role in crosstalk of cell adhesion, motility and proliferation signals.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Recruitment of afadin to the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3

Necl-5 does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3 (Ikeda et al. 2003; Mueller & Wimmer 2003). While nectin-3 directly binds afadin, Necl-5 does not (Ikeda et al. 2003). We first examined whether afadin was concentrated at the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3. For this purpose, we utilized microbeads coated with the extracellular region of Necl-5 fused with the Fc portion of IgG (Lef-5), microbeads coated with the extracellular region of nectin-3 fused with the Fc portion of IgG (Nef-3), L cells stably expressing Necl-5 (Necl-5-L cells) and L cells stably expressing nectin-3 (nectin-3-L cells). When nectin-3-L cells were incubated with the Lef-5-coated beads for 60 min, the immunofluorescence signal for nectin-3 was concentrated at the contact sites between the Lef-5-coated beads and the nectin-3-L cells (Fig. 1A). The signal for afadin was concentrated at the contact sites between the Lef-5-coated beads and the nectin-3-L cells (Fig. 1A). When Necl-5-L cells were incubated with the Nef-3-coated beads for 60 min, the signal for Necl-5 was concentrated at the contact sites between the Nef-3-coated beads and the Necl-5-L cells (Fig. 1B). However, the signal for afadin was not concentrated at the contact sites between the Nef-3-coated beads and the Necl-5-L cells (Fig. 1B). These results indicate that afadin is concentrated at the nectin-3 side of the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3.

View larger version (67K): [in this window] [in a new window]   Figure 1  Recruitment of afadin to the contact sites between the Lef-5-coated beads and nectin-3-L cells, but not to those between the Nef-3-coated beads and Necl-5-L cells. (A) The Lef-5-coated beads were added to nectin-3-L cells, followed by incubation at 37 °C for 60 min. After the incubation, the cells were fixed, followed by immunostaining for nectin-3 and afadin using the anti-nectin-3 and anti-afadin Abs, respectively. (B) The Nef-3-coated beads were added to Necl-5-L cells, followed by incubation at 37 °C for 60 min. After the incubation, the cells were fixed, followed by immunostaining for Necl-5 and afadin using the anti-Necl-5 and anti-afadin Abs, respectively. DIC, a differential interference contrast image. Bars, 10 µm. The results are representative of three independent experiments.

Recruitment of E-cadherin, -catenin and ß-catenin to the nectin-3 side of the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3

We have previously shown that nectins recruit E-cadherin, -catenin and ß-catenin to the nectin-based cell–cell adhesion sites through afadin (Takai & Nakanishi 2003; Takai et al. 2003). Therefore, we next examined whether E-cadherin, -catenin and ß-catenin were concentrated at the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3. For this purpose, we utilized the Lef-5-coated beads, the Nef-3-coated beads and L cells stably expressing Necl-5, nectin-3 and E-cadherin (Necl-5-nectin-3-EL cells). When Necl-5-nectin-3-EL cells were incubated with the Nef-3-coated beads for 60 min, the immunofluorescence signal for Necl-5 was concentrated at the contact sites between the Nef-3-coated beads and the Necl-5-nectin-3-EL cells (Fig. 2). However, the signal for afadin, E-cadherin, -catenin or ß-catenin was not concentrated at the contact sites between the Nef-3-coated beads and the Necl-5-nectin-3-EL cells (Fig. 2 and data not shown). When Necl-5-nectin-3-EL cells were incubated with the Lef-5-coated beads for 60 min, the signal for nectin-3 and afadin were concentrated at the contact sites between the Lef-5-coated beads and the Necl-5-nectin-3-EL cells (Fig. 3). The signals for E-cadherin, -catenin and ß-catenin were also concentrated at the contact sites between the Lef-5-coated beads and the Necl-5-nectin-3-EL cells (Fig. 3 and data not shown). These results indicate that E-cadherin, -catenin and ß-catenin are concentrated at the nectin-3 side of the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3.

View larger version (78K): [in this window] [in a new window]   Figure 2  No recruitment of E-cadherin and ß-catenin to the contact sites between the Nef-3-coated beads and Necl-5-nectin-3-EL cells. The Nef-3-coated beads were added to Necl-5-nectin-3-EL cells, followed by incubation at 37 °C for 60 min. After the incubation, the cells were fixed, followed by immunostaining for Necl-5, afadin, E-cadherin and ß-catenin using the anti-Necl-5, anti-afadin, anti-E-cadherin and anti-ß-catenin Abs, respectively. DIC, a differential interference contrast image. Bars, 10 µm. The results are representative of three independent experiments.

View larger version (60K): [in this window] [in a new window]   Figure 3  Recruitment of E-cadherin and ß-catenin to the contact sites between the Lef-5-coated beads and Necl-5-nectin-3-EL cells. The Lef-5 coated beads were added to Necl-5-nectin-3-EL cells, followed by incubation at 37 °C for 60 min. After the incubation, the cells were fixed, followed by immunostaining for nectin-3, afadin, E-cadherin and ß-catenin using the anti-FLAG, anti-afadin, anti-E-cadherin and anti-ß-catenin Abs, respectively. DIC, a differential interference contrast image. Bars, 10 µm. The results are representative of three independent experiments.

We then examined whether the heterophilic trans-interaction of Necl-5 with nectin-3 is involved in the formation of the E-cadherin-based cell–cell adhesion in Necl-5-nectin-3-EL cells. For this purpose, we utilized the anti-Necl-5 monoclonal antibody (mAb) which inhibits the trans-interaction of Necl-5 with nectin-3 (Ikeda et al. 2003). When Necl-5-nectin-3-EL cells were cultured at 2 mM Ca²⁺, the immunofluorescence signals for nectin-3 and ß-catenin were concentrated at the cell–cell contact sites (Fig. 4A, Normal Ca²⁺). The signals for E-cadherin, -catenin and afadin were also concentrated at the cell–cell contact sites (data not shown). The signal for Necl-5 was diffusely observed along the plasma membrane including both the cell–cell contact sites and the free surface (Fig. 4A, Normal Ca²⁺). When the cells were cultured at 2 µM Ca²⁺ in the absence of the anti-Necl-5 mAb for 4 h, the cells rounded up, and the signal for nectin-3 remained at the cell–cell contact sites, whereas that for ß-catenin at the cell–cell contact sites was reduced and diffusely observed along the plasma membrane including both the cell–cell contact sites and the free surface (Fig. 4A, Low Ca²⁺). The signal for Necl-5 was diffusely observed along the plasma membrane including both the cell–cell contact sites and the free surface (Fig. 4A, Low Ca²⁺). The signals for E-cadherin and -catenin were also diffusely observed along the plasma membrane including both the cell–cell contact sites and the free surface (data not shown). When the cells, precultured at 2 µM Ca²⁺ for 4 h, were re-cultured at 2 mM Ca²⁺ in the absence of the anti-Necl-5 mAb, the cells re-flattened, the signal for nectin-3 was concentrated only at the cell–cell contact sites, and that for ß-catenin was re-concentrated at the cell–cell contact sites (Fig. 4A, Normal Ca²⁺ 15 min, 30 min, 60 min and 120 min). The signals for E-cadherin and -catenin were also re-concentrated at the cell–cell contact sites (data not shown). In contrast, when the cells, precultured at 2 µM Ca²⁺ for 4 h, were re-cultured at 2 mM Ca²⁺ in the presence of the anti-Necl-5 mAb, the cells similarly re-flattened, the signal for nectin-3 was diffusely observed along the plasma membrane, and that for ß-catenin was not re-concentrated at the cell–cell contact sites (Fig. 4B, Normal Ca²⁺ 15 min, 30 min, 60 min and 120 min). The signal for E-cadherin or -catenin was not re-concentrated at the cell–cell contact sites (data not shown). These results indicate that the anti-Necl-5 mAb inhibits the heterophilic trans-interaction of Necl-5 with nectin-3, resulting in reduction of the formation of the E-cadherin-based cell–cell adhesion in Necl-5-nectin-3-EL cells.

View larger version (90K): [in this window] [in a new window]   Figure 4  Reduction by the anti-Necl-5 mAb of the formation of E-cadherin-based AJs in Necl-5-nectin-3-EL cells. (A) Necl-5-nectin-3-EL cells were incubated in the absence of the anti-Necl-5 mAb at 2 µM Ca2+ for 4 h. After the incubation, the cells were further incubated in the absence of the anti-Necl-5 mAb at 2 mM Ca2+ for indicated periods of time. The cells were fixed, followed by immunostaining for nectin-3, ß-catenin and Necl-5 using the anti-FLAG, anti-ß-catenin and anti-Necl-5 Abs, respectively. (B) Necl-5-nectin-3-EL cells were incubated in the presence of the anti-Necl-5 mAb at 2 µM Ca2+ for 4 h. After the incubation, the cells were further incubated in the presence of the anti-Necl-5 mAb at 2 mM Ca2+ for indicated periods of time. The cells were fixed, followed by immunostaining for nectin-3, ß-catenin and Necl-5 using the anti-FLAG, anti-ß-catenin and anti-Necl-5 Abs, respectively. Bars, 10 µm. The results are representative of three independent experiments.

	Discussion

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We have previously shown by the intercellular motility assay that the heterophilic trans-interaction of Necl-5 of Necl-5-L and V12Ras-NIH3T3 cells with nectin-3 of L cells stably expressing nectin-3 (nectin-3-L cells) enhances motility of Necl-5-L and V12Ras-NIH3T3 cells on a confluent culture of nectin-3-L cells (Ikeda et al. 2003). These observations are apparently inconsistent with the present observations. However, it has been suggested by use of the intercellular motility assay that the dynamic formation of cadherin-based AJs enhances motility of L cells stably expressing E-cadherin (EL cells) on a confluent culture of EL cells (Nagafuchi et al. 1994). Taken together, it is likely that the hetrophilic trans-interaction of Necl-5 with nectin-3 plays a role in cell–cell adhesion at the initial stage but that this trans-interaction is involved in intercellular motility even after AJs are formed.

Necl-5 has been shown to be functionally associated with integrin _Vß₃ (Mueller & Wimmer 2003; Ikeda et al. 2004). Here we have shown that the functional association of Necl-5 with the formation of cadherin-based AJs through nectin-3. The crosstalk between the cell–cell and cell-matrix adhesions have been known for a long time to play important roles for regulation of cell motility, proliferation and adhesion. Cell motility, proliferation and adhesion of non-transformed normal cells are dynamic, well regulated and critical for many events, including tissue patterning, morphogenesis and maintenance of normal tissues (Takeichi 1995; Gumbiner 1996; Lauffenburger & Horwitz 1996). Cells disrupt cell–cell adhesion and start to migrate in response to extracellular cues, such as growth factors, cytokines and extracellular matrix molecules (Gumbiner 2000; Thiery 2002). When migrating cells contact other cells, they apparently stop movement and proliferation and adhere to each other to become confluent (Abercrombie 1979; Martz & Steinberg 1972). This phenomenon is known for a long time as contact inhibition of cell movement and proliferation. Transformation of cells causes disruption of cell–cell adhesion, increase of cell motility and loss of contact inhibition of cell movement and proliferation, eventually leading transformed cells to uncontrolled cell proliferation, invasion into surrounding tissues and finally metastasis to other organs (Thiery 2002). The direct interaction of Necl-5 with nectin-3 may serve as a connector between integrins and cadherins and may be involved in the regulation of contact inhibition of cell movement and proliferation. Since Necl-5 has been shown to be up-regulated in various transformed cells, the up-regulation of Necl-5 may be involved in loss of contact inhibition of cell movement and proliferation of the transformed cells.

	Experimental procedures

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Cells and establishment of transfectants

Necl-5-L or nectin-3-L cells (L cells stably expressing exogenous Necl-5 or nectin-3, respectively) were prepared as described (Satoh-Horikawa et al. 2000; Ikeda et al. 2003). Nectin-3-EL cells (L cells stably expressing exogenous FLAG-tagged-nectin-3 and E-cadherin) were prepared as previously described (Hoshino et al. 2004). Necl-5-nectin-3-EL cells (L cells stably expressing exogenous Necl-5, FLAG-tagged-nectin-3 and E-cadherin) were obtained by transfection with pCAGIPuro-Necl-5 using LipofectAMINE PLUS reagent (Invitrogen) in nectin-3-EL cells.

Antibodies

A rat anti-nectin-3 mAb, a rat anti-Necl-5 mAb (1A8-8) and a rabbit anti-Necl-5 polyclonal Ab (pAb) were prepared as described (Satoh-Horikawa et al. 2000; Ikeda et al. 2003). A mouse anti-afadin mAb was prepared as described (Sakisaka et al. 1999). A rat anti-E-cadherin mAb (ECCD-2) was supplied by Dr M Takeichi (Centre for Developmental Biology, RIKEN, Kobe, Japan). A rabbit anti-ß-catenin pAb and a mouse anti-FLAG mAb were obtained from SIGMA. Secondary Abs for immunofluorescence microscopy were obtained from Chemicon International, Inc. Nef-3 (the extracellular region of nectin-3 fused with the Fc portion of IgG) or Lef-5 (the extracellular region of Necl-5 fused with the Fc portion of IgG) were prepared as described (Satoh-Horikawa et al. 2000; Ikeda et al. 2003).

Assay for bead-cell contact

The bead-cell contact was assayed as described (Honda et al. 2003). Briefly, Necl-5-L, nectin-3-L or Necl-5-nectin-3-EL cells were seeded on each well of a 24-well plate and cultured in DMEM containing 10% FCS for 12 h. Latex-sulphate microbeads coated with Nef-3 Lef-5, or IgG were added to each well. After 60 min incubation, the cells were fixed and immunostained. The samples were analysed by the immuofluorescence microscopy as described (Mandai et al. 1997; Takahashi et al. 1999).

Ca²⁺ switch assay

Ca²⁺ switch experiments were assayed as described (Honda et al. 2003). Briefly, Necl-5-nectin-3-EL cells were washed with PBS and cultured at 2 mM Ca²⁺ in DMEM for 60 min. The medium was replaced with DMEM containing 5 mM EGTA (2 µM Ca²⁺) and then the cells were cultured for 4 h in the presence or absence of 50 µg/mL the anti-Necl-5 mAb. After the incubation, the cells were washed with DMEM and further cultured at 2 mM Ca²⁺ in DMEM for 30 min in the presence or absence of 50 µg/mL the anti-Necl-5 mAb. The cells were then fixed with PBS containing 1% formaldehyde for 15 min and permealized with PBS containing 0.2% Triton X-100 for 15 min. After being blocked in TBS-C (50 mM Tris/HCl, pH 7.5, 200 mM NaCl and 2 mM CaCl₂) containing 1% BSA for 1 h, the samples were incubated with indicated Abs in the same buffer for 1 h. The samples were washed with TBS-C for 5 min 3 times and incubated with the secondary Abs in TBS-C containing 1% BSA for 30 min. The samples were washed and analysed by the immuofluorescence microscopy as described (Mandai et al. 1997; Takahashi et al. 1999).

	Acknowledgements

 
The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (2002, 2003).

	Footnotes

 
Communicated by: Shoichiro Tsukita

^* Correspondence: E-mail: ytakai{at}molbio.med.osaka-u.ac.jp

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Received: 18 May 2004
Accepted: 4 June 2004

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