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Originally published In Press as doi:10.1074/jbc.M312969200 on February 9, 2004

J. Biol. Chem., Vol. 279, Issue 17, 18015-18025, April 23, 2004
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Nectin-like Molecule-5/Tage4 Enhances Cell Migration in an Integrin-dependent, Nectin-3-independent Manner*

Wataru Ikeda{ddagger}, Shigeki Kakunaga{ddagger}, Kyoji Takekuni{ddagger}, Tatsushi Shingai{ddagger}, Keiko Satoh§, Koji Morimoto§, Masakazu Takeuchi§, Toshio Imai§, and Yoshimi Takai{ddagger}

From the {ddagger}Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka and the §KAN Research Institute Inc., 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815, Japan

Received for publication, November 30, 2003 , and in revised form, February 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca2+-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin {alpha}V{beta}3 at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin {alpha}V{beta}3 or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin {alpha}V{beta}3 in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In multicellular organisms, cell migration is essential for normal development and responses to tissue damages and infection throughout life (1, 2). Cell migration is also observed in many diseases, such as cancer and atherosclerosis (3, 4). Cells migrate as individuals or as groups; leukocytes, lymphocytes, and fibroblasts migrate as individuals, whereas epithelial and endothelial cells migrate as groups. Cell migration is divided into at least four mechanistically separate steps: extension of protrusions, formation of new cell-matrix adhesions, contraction of cell body, and tail detachment (1, 5). Cell migration is normally directed and controlled by extracellular cues, such as growth factors, cytokines, and extracellular matrix molecules. These cues stimulate cell surface receptors to initiate intracellular signaling through second messengers, protein kinases, protein phosphatases, and heterotrimeric large and monomeric small G proteins to regulate the multiple steps. When migrating cells contact other cells, they stop migration and proliferation (6, 7). 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 invasion into surrounding tissues and metastasis to other organs (4, 8).

Cell-cell adhesion is mainly mediated by cell-cell adherens junctions (AJs),1 where cadherins are key Ca2+-dependent cell-cell adhesion molecules (5, 9). Cadherins are associated with the actin cytoskeleton through many peripheral membrane proteins, including {alpha}- and {beta}-catenins, {alpha}-actinin, and vinculin, which strengthen cell-cell adhesion activity of cadherins. Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules that also localize at cell-cell AJs and regulate organization of AJs in cooperation with cadherins (10, 11). Nectins are similarly associated with the actin cytoskeleton through afadin. Nectins comprise a family of four members, nectin-1, -2, -3, and -4. Nectins have one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region. All nectins except nectin-4 have a C-terminal conserved motif of four amino acid (aa) residues that interacts with the PDZ domain of afadin. Nectin-4 does not have this motif but binds afadin. Each nectin forms homo-cis-dimers, followed by formation of homo-trans-dimers (homo-trans-interaction), causing cell-cell adhesion. Nectin-3 furthermore forms hetero-trans-dimers (hetero-trans-interaction) with nectin-1 or -2, and the adhesion activity of the hetero-trans-dimers is stronger than that of the homo-trans-dimers. Nectin-4 also forms hetero-trans-dimers with nectin-1. In addition to the cell-cell adhesion activity, nectins have an activity to induce activation of Cdc42 and Rac small G proteins, which regulate cell-cell adhesion through reorganization of the actin cytoskeleton and gene expression through activation of c-Jun N-terminal kinase (12-14). Nectins furthermore directly bind PAR-3, a cell polarity protein, and regulate cell polarization (15).

Five other molecules with one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region have thus far been identified. These include Necl-1/TSLL1/SynCAM3 (16, 17), Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 (17-21), Necl-3/similar to NECL3/SynCAM2 (17), Necl-4/TSLL2/SynCAM4 (16, 17), and Necl-5/Tage4/human poliovirus receptor (PVR)/CD155 (22-25). The domain structures of this group of molecules are similar to those of nectins, but they do not bind afadin (11, 26). We have proposed that this group of molecules is tentatively called nectin-like molecules (Necls). Of these Necls, we focus here on Necl-5/Tage4/PVR/CD155.

Tage4 was originally identified to be the product of a gene overexpressed in rat and mouse colon carcinoma (24, 25). The Tage4 gene has been mapped to rat chromosome 1q22 (27) and mouse 7A2-B1 (28). Northern blot analysis has revealed that the Tage4 mRNA is expressed in normal adult rat and mouse tissues to small extents (24, 25). Tage4 has been shown to mediate entry of porcine pseudorabies virus and bovine herpesvirus 1 (29). PVR/CD155 was originally identified as a human poliovirus receptor (22, 23). The PVR/CD155 gene has been mapped to the long arm of human chromosome 19, and this region is homologous to the regions of rat and mouse Tage4 genes (23, 29). PVR/CD155 has not been thought to be a cell-cell adhesion molecule because it does not homophilically trans-interact (30). PVR/CD155 has been shown to serve as an entry receptor not only for human poliovirus but also for porcine pseudorabies virus and bovine herpesvirus 1 (29, 31). PVR/CD155 is overexpressed in human colorectal carcinoma and malignant glioma (32, 33). PVR/CD155 has been shown to be physically associated with CD44 on human monocyte cell surfaces (34). CD44 is known to be a transmembrane protein that is involved in cell migration and metastasis of cancer cells (35). The extracellular region of PVR/CD155 has been reported to bind to the extracellular matrix molecule vitronectin (36). The cytoplasmic region of PVR/CD155 binds to Tctex-1, a subunit of the dynein motor complex (37). Thus, the roles of Tage4 and PVR/CD155 as viral receptors have been established, but their physiological function(s) remained unknown. We have recently shown that Tage4 does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, that its expression is very low in normal adult tissues but up-regulated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as estimated by Western blotting, and that the heterophilic trans-interaction of Tage4 with nectin-3 enhances intercellular motility of V12Ras-NIH3T3 cells (26). Consistently, Mueller and Wimmer (38) have recently shown that PVR/CD155 heterophilically trans-interacts with nectin-3. The phylogenetic analysis of nectins and Necls cannot clearly conclude that the genes of Tage4 and PVR/CD155 are derived from the common or different ancestor gene (26), and their exact relationship is currently unknown. However, on the basis of similar properties of Tage4 and PVR/CD155 thus far elucidated (29, 39), we tentatively propose here that they are derived from the common ancestor gene and called Necl-5.

Extending these earlier observations, we have first examined here whether Necl-5 regulates motility of cells that do not contact other cells expressing nectin-3 and have found that Necl-5 enhances serum- and platelet-derived growth factor (PDGF)-induced cell motility without its trans-interaction with nectin-3. We have then examined whether the serum-induced, Necl-5-enhanced cell motility requires integrins, because integrins have been shown to play a key role in cell motility (40), and have found that Necl-5 is functionally associated with integrin {alpha}V{beta}3 in cell motility. Analysis on the mode of action of Necl-5 has revealed that it enhances cell motility through activation of Cdc42 and Rac. We show here that Necl-5 regulates serum- and PDGF-induced cell motility in an integrin-dependent, nectin-3-independent manner. We furthermore show that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction—Expression vectors were constructed in pCAGIZeo (41), pFLAG-CMV1 (Sigma), and pFastBac1-Msp-Fc (42). Constructs of Necl-5 contained the following aa: pCAGIZeo-Necl-5-{Delta}CP, aa 1-374 (deletion of the cytoplasmic region); pFLAG-CMV1-Necl-5-{Delta}CP, aa 30-374 (deletion of the signal peptide and the cytoplasmic region); and pFastBac1-Msp-Fc-Necl-5-EC (the extracellular fragment of Necl-5 fused to the human IgG Fc), aa 30-347 (26). To express the extracellular fragment of nectin-3 fused to the human IgG Fc (Nef-3), pFast-Bac1-Msp-Fc-nectin-3-EC (aa 56-400) was prepared as described (43). The fusion protein with IgG Fc was produced as a secreted protein by the baculovirus transfer system (Invitrogen, Carlsbad, CA) and purified by use of protein A-Sepharose beads (Amersham Biosciences) as described (42). pEF-BOS-Myc-NWASP-CRIB and pEF-BOS-Myc-N17Rac1 were prepared as described (44, 45). pRaichu-Rac1 and pRaichu-Cdc42 were kind gifts from Drs. M. Matsuda and T. Nakamura (Osaka University, Osaka, Japan).

Cell Culture, DNA Transfection, and Establishment of Transformants—L cells were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan). L cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. L cell lines stably expressing mouse Necl-5 (full-length, aa 1-409), FLAG-Necl-5 (aa 30-409), or Necl-5, of which the extracellular region was deleted (aa 335-409) (non-tagged Necl-5-L, Necl-5-L, or Necl-5-{Delta}EC-L cells, respectively) were prepared as described (26). NIH3T3 and V12Ras-NIH3T3 cells were maintained in DMEM supplemented with 10% calf serum. V12Ras-NIH3T3 cells were prepared as described (46). An L cell line, an NIH3T3 cell line, or a V12Ras-NIH3T3 cell line stably expressing Necl-5, from which the cytoplasmic region was deleted (Necl-5-{Delta}CP-L, Necl-5-{Delta}CP-NIH3T3, or Necl-5-{Delta}CP-V12Ras-NIH3T3 cells, respectively), was obtained by transfection with pCAGIZeo-Necl-5-{Delta}CP using LipofectAMINE PLUS reagent (Invitrogen). For transient expression of Myc-NWASP, Myc-N17Rac1, FLAG-Necl-5-{Delta}CP, or Necl-5-{Delta}CP, L or NIH3T3 cell lines were transfected with pEF-BOS-Myc-NWASP-CRIB, pEF-BOS-Myc-N17Rac1, or pFLAG-CMV1-Necl-5-{Delta}CP, respectively, as described above.

Antibodies and Reagents—A rat anti-Necl-5 monoclonal antibody (mAb) (1A8-8; mAb-i) was prepared as described (26). Another rat anti-Necl-5 mAb (3A4-2; mAb-s) was raised against the fusion protein of the extracellular region of Necl-5 (aa 30-347) with IgG Fc. A mouse anti-integrin {alpha}V mAb (clone 21), a rat anti-integrin {alpha}V mAb (RMV-7), and an Armenian hamster anti-integrin {beta}3 mAb (2C9.G2) were purchased from BD Biosciences Pharmingen (San Diego, CA). A rabbit anti-integrin {alpha}V polyclonal Ab (pAb) and {beta}3 pAb were purchased from Chemicon (Temecula, CA). Hybridoma cells expressing the mouse anti-Myc mAb (9E10) were obtained from American Type Culture Collection (Manassas, VA). A rabbit anti-FLAG pAb, a mouse anti-FLAG mAb, fatty acid-free bovine serum albumin (BSA), and echistatin were purchased from Sigma. A horseradish peroxidase-conjugated goat anti-mouse IgG was purchased from American Qualex (San Clemente, CA). A horseradish peroxidase-conjugated donkey anti-rabbit IgG and a horseradish peroxidase-conjugated goat anti-Armenian hamster IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Vitronectin was kindly supplied by Dr. K. Sekiguchi (Osaka University, Osaka, Japan). Human recombinant PDGF-BB was purchased from PEPROTECH (Rocky Hill, NJ).

Immunofluorescence Microscopy—Immunofluorescence microscopy of various cell lines was done as described (47). For the experiments using V12Ras-NIH3T3 cells, the samples were fixed with acetone/methanol (1:1) at -20 °C for 1 min. For the experiments using Necl-5-L cells with various anti-integrin-{alpha}v or -{beta}3 Ab, the signal was enhanced using a Tyramide Signal Amplification kit (Molecular Probes, Eugene, OR) according to the protocol from the manufacturer. The samples were analyzed by Radiance 2000 or 2100 confocal laser scanning microscope (Bio-Rad).

Phagokinetic Track Motility Assay—The uniform carpet of gold particles was prepared on glass coverslips as described (48). The colloidal gold-coated coverslips were placed in 35-mm nontreated dishes, and the coverslips were coated with or without various reagents (50 µg/ml of the anti-Necl-5 mAb-s, mAb-i, or Nef-3, or 10 µg/ml of vitronectin) by incubation at room temperature for 1 h. Cells were seeded at a density of 2 x 103 cells/35-mm dish. When the anti-Necl-5 mAb-i or vitronectin was added into the medium, the final concentration of the anti-Necl-5 mAb-i or vitronectin was 50 or 20 µg/ml, respectively. When the cells were incubated in the absence of serum, DMEM supplemented with 0.5% fatty acid-free BSA was used. After incubation for 16 h, the samples were fixed with PBS containing 3.7% formaldehyde and cell motility was analyzed by measuring the areas free of gold particles around a single cell. Five independent experiments were performed, and at least 24 independent samples in each experiment were picked up to determine the areas. The statistical significance was determined by paired t test.

Boyden Chamber Assay—The Boyden chamber assay was performed as described (49) with some modifications. FalconTM cell culture inserts (8.0-µm pores, Becton Dickinson Labware, Franklin Lakes, NJ) were coated with 3 µg/ml vitronectin or 1% BSA at 37 °C for 1 h. The inserts were then blocked with 1% BSA at 37 °C for 30 min. NIH3T3 cell lines, which had been serum starved with DMEM supplemented with 0.5% fatty acid-free BSA for 24 h, were detached with 0.05% trypsin and 0.53 mM EDTA and then treated with a trypsin inhibitor (Sigma). The cells were then resuspended in DMEM supplemented with 0.5% fatty acid-free BSA and seeded at a density of 4 x 104 cells/insert. The cells were incubated at 37 °C for 14 h in the presence or absence of 30 ng/ml of PDGF-BB. PDGF-BB was added only to the bottom well to generate a concentration gradient. After incubation, the inserts were washed with PBS, the cells were fixed using 3.7% formaldehyde and subsequently stained with 0.5% Crystal Violet (Sigma). The cells, which had not migrated, were removed by wiping the top of the membrane with a cotton swap. The number of stained cells in five randomly chosen fields per filter were counted by microscopic examination.

Fluorescent Resonance Energy Transfer (FRET) Imaging—The FRET imaging was performed as described (13) with some modifications. Necl-5-L, Necl-5-{Delta}EC-L, or Necl-5-{Delta}CP-L cells were transfected with pRaichu-Rac1 or pRaichu-Cdc42 using LipofectAMINE PLUS reagent. The FRET probes for wild-type Rac1 and Cdc42 consisted of a CRIB domain of PAK, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras (50). Twenty-four h after the transfection, the cells were replated on the glass base dishes (Iwaki, Tokyo, Japan). Sixteen h after replating, the cells were then imaged with an Olympus IX71 inverted microscope equipped with a cooled charge-coupled device camera, CoolSNAP HQ (Roper Scientific, Trenton, NJ), controlled by MetaMorph software (Universal Imaging, West Chester, PA) (51, 52). For dual-emission ratio imaging, we used a 440AF21 excitation filter, a 455DRLP dichroic mirror, and two emission filters, 480AF30 for CFP and 535AF25 for YFP (Omega Optical Inc., Brattleboro, VT). The cells were illuminated with a 75-watt xenon lamp through a 6% neutral density filter (Omega Optical Inc.) and a 60x oil immersion objective lens. Exposure times for 3 x 3 binning were 200 ms to obtain images of CFP and YFP, and 50 ms to obtain images of a differential interference contrast. After background subtraction, the ratio image of YFP/CFP was created with the MetaMorph software and used to represent FRET efficiency.

In Vivo Metastasis Assay—Female nude mice (BALB/c nu/nu) were purchased from Japan SLC Inc. (Shizuoka, Japan). Exponentially growing V12Ras-NIH3T3 or Necl-5-{Delta}CP-V12Ras-NIH3T3 cells were harvested, and each cell line (1 x 106 cells) in 0.1 ml of PBS was given 6-week-old nude mice by tail vein injection. The animals were killed on day 14, and tumor nodules in the lungs were counted. The animals and procedures used in this study were in accordance with the guidelines and approval of Osaka University Medical School Animal Care and Use Committee.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Enhancement of Motility of L Cells by Expression of Necl-5 and Necessity of Its Cytoplasmic Region—We first examined whether Necl-5 enhances motility of a single cell, which did not contact other cells. For this purpose, we used Necl-5-L cells (L cells stably expressing FLAG-Necl-5) and estimated their motility by the phagokinetic track motility assay on the colloidal gold-coated coverslips in the presence or absence of its specific interacting proteins: two anti-Necl-5 mAbs (the anti-Necl-5 mAb-i and mAb-s) and Nef-3 (the extracellular fragment of nectin-3 fused to the human IgG Fc). We have used the anti-Necl-5 mAb-i previously and shown that the anti-Necl-5-mAb-i reduces the intercellular motility-enhancing activity of Necl-5 by inhibiting the trans-interaction of Necl-5 with nectin-3 (26). The anti-Necl-5 mAb-s is newly made for the present study. In contrast to the anti-Necl-5-mAb-i, the anti-Necl-5 mAb-s does not inhibit the trans-interaction of Necl-5 with nectin-3 (data not shown). L cells express endogenous nectin-1 and -2, but not endogenous nectin-3 or Necl-5 (26, 42, 43, 53). A single wild-type L cell migrated moderately, but a single Necl-5-L cell more actively migrated (Fig. 1A, a1, a2, and b). When motility of Necl-5-L cells was assayed on the coverslips precoated with the anti-Necl-5 mAb-s, their motility was further enhanced (Fig. 1B, a1, a2, and c). When motility of Necl-5-L cells was assayed on the coverslips precoated with Nef-3, their motility was conversely inhibited (Fig. 1B, a1, a3, and c). This inhibitory effect of Nef-3 was restored by the addition of the anit-Necl-5 mAb-i into the medium (Fig. 1B, a3, a4, and c). The anti-Necl-5 mAb-i alone added into the medium or coated on the coverslips did not affect the motility of the cell (data not shown). The inhibitory effect of Nef-3 is not apparently consistent with the earlier observation that the dynamic trans-interaction of Necl-5 with cellular nectin-3 enhances intercellular motility (26), but the inhibitory effect may be just because of physical hindrance to the cell that stuck to the substratum through the stable interaction of cellular Necl-5 with Nef-3 fixed on the coverslips. These results indicate that Necl-5 indeed enhances motility of a single cell that does not contact other cells.



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  		FIG. 1.
Enhancement of motility of L cells by Necl-5. A, enhancement of motility of L cells by the cytoplasmic region of Necl-5. The cells were incubated on the colloidal gold-coated coverslips for 16 h. a, phase contrast images; a1, a wild-type L cell; a2, a Necl-5-L cell; a3, a Necl-5-{Delta}CP-L cell; a4, a Necl-5-{Delta}EC-L cell. b, the area free of gold particles around a single cell. *, p < 0.0001 versus wild-type L cells as determined by paired Student's t test; **, p < 0.0001 versus Necl-5-L cells as determined by paired Student's t test. B, further enhancement by the immobilized anti-Necl-5 mAb-s and reduction by stable interaction of Necl-5 with Nef-3 of motility of Necl-5-L cells. The cells were incubated on the colloidal gold-coated coverslips for 16 h. a and b, phase contrast images; a1, a Necl-5-L cell (control); a2, a Necl-5-L cell with the anti-Necl-5 mAb-s; a3, a Necl-5-L cell with Nef-3; a4, a Necl-5-L cell with Nef-3 in the presence of the anti-Necl-5 mAb-i; b1, a Necl-5-{Delta}EC-L cell (control); b2, a Necl-5-{Delta}EC-L cell with the anti-Necl-5 mAb-s; b3, a Necl-5-{Delta}EC-L cell with Nef-3; b4, a Necl-5-{Delta}EC-L cell with Nef-3 in the presence of the anti-Necl-5 mAb-i. c, the area free of gold particles around a single cell. *, p < 0.0001 versus the control as determined by paired Student's t test; **, p < 0.0003 versus the control as determined by paired Student's t test. Control, in the absence of additional reagents; mAb-s, the anti-Necl-5 mAb-s; mAb-i, the anti-Necl-5 mAb-i. Bars,50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. The results shown in Aa, Ba, and Bb are representative of five independent experiments, and the results shown in Ab and Bc are the means ± S.E. of five independent experiments.

 
By use of Necl-5-{Delta}CP-L cells (L cells expressing Necl-5, of which the cytoplasmic region except for the juxtamembrane 4 aa was deleted) and Necl-5-{Delta}EC-L cells (L cells expressing Necl-5, of which extracellular region except for the juxtamembrane 13 aa was deleted), we then examined whether the extracellular and/or cytoplasmic regions of Necl-5 are necessary for its cell motility-enhancing activity. A single Necl-5-{Delta}CP-L cell migrated moderately, and its motility was similar to that of a single wild-type L cell (Fig. 1A, a1, a3, and b). In contrast, a single Necl-5-{Delta}EC-L cell migrated more actively than a single Necl-5-L cell (Fig. 1A, a2, a4, and b). The anti-Necl-5 mAb-s or Nef-3 did not affect the motility of the Necl-5-{Delta}EC-L cell (Fig. 1B, b1-b4, and c), indicating that the stimulatory and inhibitory effects of the anti-Necl-5 mAb-s and Nef-3 on the motility of Necl-5-L cells, respectively, are mediated through its interaction with the extracellular region of Necl-5. These results indicate that the cytoplasmic region of Necl-5, but not the extracellular region, is essential for its cell motility-enhancing activity.

Involvement of Necl-5 in Directional Cell Migration and Necessity of Its Extracellular Region—Cultured cells migrate either randomly or directionally. We next examined whether Necl-5 enhances directional cell migration. For this purpose, a confluent culture of Necl-5-L cells was subjected to the wound healing assay. At the front row of the wound, the cells formed protrusions, such as ruffles, lamellipodia, and filopodia. Ruffles and lamellipodia were more frequently observed than filopodia. The immunofluorescence signal for Necl-5 was highly concentrated at these protrusions. Typical staining images of the cells stepped forward to the wound are shown (Fig. 2A, a1-a3). These cells with well developed protrusions were attached to the cells back from the wound. The time required for the wound healing of Necl-5-L cells was much shorter than that of wild-type L cells (Fig. 2B, a1-a3 and b1-b3). These results indicate that Necl-5 enhances directional cell migration.



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  		FIG. 2.
Involvement of Necl-5 in directional cell migration. A, localization of Necl-5 at the leading edges of Necl-5-L cells. Confluent cell layers were manually scratched with a 26-gauge needle, cultured for 6 h, and then doubly stained with various combinations of the anti-Necl-5 mAb-i, the anti-FLAG pAb, and rhodamine-phalloidin. These images are the cells at the front row of the wound. a, Necl-5-L cells; b, Necl-5-{Delta}EC-L cells. a1, Necl-5; b1, Necl-5-{Delta}EC; a2 and b2, F-actin; a3 and b3, merge. Arrowheads, edges of protrusions; double-headed arrows, the direction of the scratch. Bars, 10 µm. B, more rapid wound healing of Necl-5-L cells than wild-type L and Necl-5-{Delta}EC-L cells. Confluent cell layers were manually scratched with a 26-gauge needle, cultured for periods of time, and then stained with rhodamine-phalloidin. a, Necl-5-L cells; b, wild-type L cells; c, Necl-5-{Delta}EC-L cells. a1, b1, and c1, 0 h; a2, b2, and c2, 6 h; a3, b3, and c3, 9 h. Bars, 50 µm. The results shown are representative of three independent experiments.

 
We then examined whether the extracellular region of Necl-5 is necessary for its directional cell migration-enhancing activity. Confluent cultures of Necl-5-{Delta}EC-L cells were subjected to the wound healing assay. At the front row of Necl-5-{Delta}EC-L cells, the cells formed protrusions, such as ruffles, lamellipodia, and filopodia. These cells were attached to the cells back from the wound. The number of filopodia was roughly similar to that observed in Necl-5-L cells, but the numbers of ruffles and lamellipodia were far less than those observed in Necl-5-L cells. The signal for Necl-5-{Delta}EC was randomly distributed along the plasma membrane (Fig. 2A, b1-b3). The time required for the wound healing of Necl-5-{Delta}EC-L cells was similar to that of wild-type L cells and longer than that of Necl-5-L cells (Fig. 2B, c1-c3). Taken together the above results (see Fig. 1A, a1, a2, a4, and b), it is likely that the extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility.

Serum-dependent Cell Motility-enhancing Activity of Necl-5—All the experiments described above were done in the presence of serum in the culture medium. Therefore, we next examined whether serum is necessary for the motility of Necl-5-L cells. Neither Necl-5-L nor Necl-5-{Delta}EC-L cells showed active motility in the absence of serum in the medium as estimated by the phagokinetic track motility assay (Fig. 3, A1, A2, B1, B2, and C), indicating that activation of Necl-5 alone is not sufficient for the motility of Necl-5-L cells. Because the extracellular region is not essential for the cell motility-enhancing activity of Necl-5, it is likely that serum enhances cell motility through the cytoplasmic region of Necl-5 or that serum and the cytoplasmic region of Necl-5 synergistically enhance cell motility.



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  		FIG. 3.
Serum-dependent cell motility-enhancing activity of Necl-5. Necl-5-L or Necl-5-{Delta}EC-L cells were incubated on the colloidal gold-coated coverslips for 16 h. A and B, phase contrast images; A1, a Necl-5-L cell in the presence of serum; A2, a Necl-5-L cell in the absence of serum; B1, a Necl-5-{Delta}EC-L cell in the presence of serum; B2, a Necl-5-{Delta}EC-L cell in the absence of serum. C, the area free of gold particles around a single cell. *, p < 0.0001 versus in the presence of serum as determined by paired Student's t test. Bars, 50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. The results shown in A and B are representative of five independent experiments, and the results shown in C are the means ± S.E. of the five independent experiments.

 
Integrin-dependent Cell Motility-enhancing Activity of Necl-5—Integrins play crucial roles in cell motility (40, 54). Of many types of integrins, integrin {alpha}V{beta}3 has been shown to be expressed in L cells (55). This integrin has furthermore been shown to be concentrated at focal complexes of leading edges of migrating endothelial cells (56). Dynamic formation of focal complexes is known to be essential for cell motility (57). We therefore examined whether integrins are involved in the serum-induced, Necl-5-enhanced cell motility. When Necl-5 and integrin {alpha}V were stained with the anti-Necl-5 mAb-i and the anti-integrin {alpha}V mAb (clone 21), respectively, in Necl-5-L cells that were subjected to the wound healing assay, the immunofluorescence signals for both proteins were concentrated and colocalized at the protrusions, such as ruffles and lamellipodia, at the front row of the wound (Fig. 4A, a1-a3). These cells were attached to the cells back from the wound. When integrin {alpha}v or {beta}3 was stained with other Abs, the anti-integrin {alpha}V mAb (RMV-7), the anti-integrin {beta}3 mAb (2C9.G2), or the anti-integrin {beta}3 pAb, the essentially similar results were obtained (data not shown). When the wound healing assay of Necl-5-L cells was performed in the presence of echistatin, an inhibitor of integrin {alpha}V{beta}3 (58), the time required for wound healing was delayed (Fig. 4B, a1-a3 and b1-b3). Moreover, the morphology of the protrusions changed in the presence of echistatin; in the absence of it, typical ruffles and lamellipodia were observed, whereas in the presence of it, pseudopodia-like structures, but not typical ruffles or lamellipodia, were observed (Fig. 4B, a4-a6 and b4-b6). However, Necl-5 and integrin {alpha}V colocalized at edges of the pseudopodia-like structures (Fig. 4A, b1-b3). These results indicate that Necl-5 enhances serum-dependent cell motility in an integrin {alpha}V{beta}3-dependent manner.



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  		FIG. 4.
Integrin-dependent cell motility-enhancing activity of Necl-5. A, colocalization of Necl-5 and integrin {alpha}V{beta}3 at leading edges of Necl-5-L cells. Confluent cell layers were manually scratched with a 26-gauge needle, cultured for 6 h in the presence or absence of echistatin, an inhibitor of integrin {alpha}V{beta}3, and then doubly stained with the anti-Necl-5 mAb-i and the anti-integrin {alpha}V mAb (21). The signal for integrin {alpha}V was enhanced using Tyramide Signal Amplification kit. These images are the cells at the front row of the wound. a, Necl-5-L cells in the absence of echistatin; b, Necl-5-L cells in the presence of echistatin (370 nM). a1 and b1, Necl-5; a2 and b2, integrin {alpha}V; a3 and b3, merge. Arrowheads, edges of protrusions; double-headed arrows, the direction of the scratch. Bars,10 µm. B, delayed wound healing of Necl-5-L cells by echistatin. Confluent cell layers were manually scratched with a 26-gauge needle, cultured for periods of time, and then stained with rhodamine-phalloidin and/or the anti-Necl-5 mAb-i. a, Necl-5-L cells in the absence of echistatin; b, Necl-5-L cells in the presence of echistatin (370 nM). a1-a4 and b1-b4, F-actin; a5 and b5, Necl-5; a6, merge of a4 and a5; b6, merge of b4 and b5; a1 and b1, 0 h; a2 and b2, 6 h, a3 and b3, 9 h; a4-a6 and b4-b6, high magnification of the front row of the wound at 6 h. Bars, 50 µm. The results shown are representative of three independent experiments.

 
Vitronectin is an extracellular matrix molecule that binds to integrins {alpha}V{beta}1, {alpha}V{beta}3, {alpha}V{beta}5, and {alpha}IIb{beta}3 (54). We next examined the effect of vitronectin on the serum-dependent, Necl-5-enhanced motility of Necl-5-L cells. When vitronectin was added into the medium containing serum, it did not affect the motility of Necl-5-L cells as estimated by the phagokinetic track motility assay (data not shown). This might be just because of the presence of endogenous vitronectin in the serum (59). We then assayed the motility of Necl-5-L cells on the coverslips precoated with vitronectin. Vitronectin reduced the motility of Necl-5-L cells (Fig. 5, A1, A2, and C). Vitronectin also reduced the motility of Necl-5-{Delta}EC-L cells (Fig. 5, B1, B2, and C). The inhibitory effect of vitronectin was, however, relatively small (15-20%), but this small effect might be just because of vitronectin that was contained in serum and coated on the coverslips in our assay system. These results suggest that vitronectin does not inhibit the cell motility by directly binding to the extracellular region of Necl-5. The earlier observation, that the extracellular region of PVR/CD155 binds vitronectin (36), is apparently inconsistent with this result, but the reason for this inconsistency is currently unknown. Vitronectin is known to activate the integrin (59) but inhibited the motility of Necl-5-L cells. This result is consistent with the earlier observation that vitronectin precoated on the coverslips inhibits motility of keratinocytes as estimated by the phagokinetic track motility assay (60), although vitronectin precoated on the filters has been shown to enhance motility of cells, such as endothelial, melanoma, breast carcinoma, and fibrosarcoma cells, as estimated by the Boyden chamber assay (61, 62). The inhibitory effect of vitronectin may be merely the result of physical hindrance for the cells that stuck to the substratum through the stable interaction of the cellular integrin with vitronectin fixed on the coverslips. These results have provided another line of evidence for the involvement of integrin {alpha}V{beta}3 in the serum-induced, Necl-5-enhanced cell motility, and suggest that Necl-5 is functionally associated with this integrin.



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  		FIG. 5.
Reduction of Necl-5-enhanced cell motility by vitronectin. Necl-5-L or Necl-5-{Delta}EC-L cells were incubated on the colloidal gold-coated coverslips for 16 h. A and B, phase contrast images; A1, a Necl-5-L cell (control); A2, a Necl-5-L cell with vitronectin; B1, a Necl-5-{Delta}EC-L cell (control); B2, a Necl-5-{Delta}EC-L cell with vitronectin. C, the area free of gold particles around a single cell. *, p < 0.0001 versus the control as determined by paired Student's t test. Control, in the absence of vitronectin; VN, vitronectin. Bars, 50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. The results shown in A and B are representative of five independent experiments, and the results shown in C are the means ± S.E. of the five independent experiments.

 
Involvement of Cdc42 and Rac in the Cell Motility-enhancing Activity of Necl-5—The extension of protrusions, such as lamellipodia and filopodia, is essential for both directional and random cell motility (40). The formation of filopodia and lamellipodia requires activation of Cdc42 and Rac, respectively (63, 64). We therefore examined whether Cdc42 and Rac are involved in the serum-induced, Necl-5-enhanced cell motility. Expression of NWASP-CRIB, an inhibitor of Cdc42 (65), or N17Rac1, a dominant negative mutant of Rac (66), inhibited the motility of Necl-5-L cells as estimated by the phagokinetic track motility assay (Fig. 6A, a1-a3 and b). Moreover, Necl-5-L cells formed more prominent filopodia and lamellipodia than L cells (Fig. 6B, a and b). Necl-5-{Delta}EC-L cells similarly formed filopodia and lamellipodia, but Necl-5-{Delta}CP-L cells did not significantly form these protrusions (Fig. 6B, c and d). The formation of filopodia and lamellipodia in Necl-5-L cells was inhibited by expression of NWASP-CRIB or N17Rac1 (Fig. 6B, e and f). These experiments were performed in the presence of serum. In the absence of serum, the formation of neither filopodia nor lamellipodia was significantly enhanced in Necl-5-L cells as compared with that of L cells (data not shown). We further confirmed by the FRET imaging whether Cdc42 and Rac are activated by the action of Necl-5 in the presence of serum. The signals for activated Cdc42 and Rac were observed at the protrusions of Necl-5-L (Fig. 6C, a1 and a2) and Necl-5-{Delta}EC-L cells (data not shown), but not in Necl-5-{Delta}CP-L cells (Fig. 6C, b1 and b2). These results indicate that Necl-5 enhances serum-dependent formation of filopodia and lamellipodia through activation of Cdc42 and Rac, respectively, eventually leading to enhanced cell motility.



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  		FIG. 6.
Involvement of Cdc42 and Rac in cell motility-enhancing activity of Necl-5. A, reduction of motility of Necl-5-L cells by expression of NWASP-CRIB or N17Rac. Myc-NWASP-CRIB or Myc-N17Rac1 was transiently expressed in Necl-5-L cells. These cells were incubated on the colloidal gold-coated coverslips for 16 h. a, phase contrast images; a1, a Necl-5-L cell; a2, a Necl-5-L cell expressing Myc-NWASP-CRIB; a3, a Necl-5-L cell expressing Myc-N17Rac1. b, the area free of gold particles around a single cell. *, p < 0.0001 versus the control as determined by paired Student's t test. Control, Necl-5-L cells. Bars, 50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. B, formation of filopodia and lamellipodia by expression of Necl-5. Myc-NWASP-CRIB or Myc-N17Rac1 was transiently expressed in Necl-5-L cells. The cells were cultured for 16 h and then stained with rhodamine-phalloidin. a, a wild-type L cell; b, a Necl-5-L cell; c, a Necl-5-{Delta}EC-L cell; d, a Necl-5-{Delta}CP-L cell; e, a Necl-5-L cell expressing Myc-NWASP-CRIB; f, a Necl-5-L cell expressing Myc-N17Rac1. Bars, 10 µm. The cells expressing Myc-NWASP-CRIB or Myc-N17Rac1 were confirmed by immunofluorescence microscopy using the anti-Myc mAb. C, activation of Cdc42 and Rac by expression of Necl-5. Necl-5-L or Necl-5-{Delta}CP-L cells expressing either Raichu-Cdc42 or Raichu-Rac1 were cultured for 16 h. The cells were video-imaged for YFP and CFP. a, IMD images of Necl-5-L cell; b, IMD images of Necl-5-{Delta}CP-L cell; a1 and b1, activation of Cdc42; a2 and b2, activation of Rac. In IMD mode images, eight colors from red to blue are used to represent the YFP/CFP ratio, with the intensity of each color indicating the mean intensity of YFP and CFP. High YFP/CFP ratio shown in red color indicates high FRET efficiency of the probe, which reflects high GTP/GDP ratio of Cdc42 or Rac1. The upper and lower limits of ratio range are shown on the right. Bars, 10 µm. The results shown in Aa are representative of five independent experiments, the results shown in Ab are the means ± S.E. of the five independent experiments, and the results shown in B and C are representative of three independent experiments.

 
Involvement of Necl-5 in Motility of NIH3T3 Cells—All the experiments described above were performed using L cells overexpressing Necl-5 as a model cell. To gain the insight into the physiological role of Necl-5 in cell motility, we examined whether Necl-5 is involved in motility of NIH3T3 cells that express Necl-5 endogenously (26). For this purpose, we attempted to develop a dominant negative mutant of Necl-5. The above results, 1) that Necl-5 enhances serum-induced cell motility in an integrin {alpha}V{beta}3-dependent manner, 2) that the cytoplasmic region of Necl-5 is essential for its cell motility-enhancing activity, and 3) that the extracellular region of Necl-5 is not essential for its random cell motility-enhancing activity, have raised the possibility that Necl-5-{Delta}CP serves as a dominant negative mutant of Necl-5 and inhibits the cell motility-enhancing activity of Necl-5. We first assessed this possibility by use of non-tagged Necl-5-L and Necl-5-{Delta}EC-L cells. Transient expression of Necl-5-{Delta}CP in non-tagged Necl-5-L or Necl-5-{Delta}EC-L cells markedly reduced motility of non-tagged Necl-5-L, but not Necl-5-{Delta}EC-L cells, as estimated by the phagokinetic track motility assay (Fig. 7, A1, A2, B1, B2, and C), indicating that Necl-5-{Delta}CP indeed serves as a dominant negative mutant of Necl-5 and inhibits the cell motility-enhancing activity of Necl-5. We then examined by use of Necl-5-{Delta}CP whether Necl-5 is involved in motility of a singly migrating NIH3T3 cell. Transient expression of Necl-5-{Delta}CP in NIH3T3 cells reduced their motility (Fig. 8A, a1, a2, and c). We furthermore obtained NIH3T3 cells stably expressing Necl-5-{Delta}CP (Necl-5-{Delta}CPNIH3T3 cells). Consistently, the motility of a Necl-5-{Delta}CPNIH3T3 cell was reduced as compared with that of an NIH3T3 cell (data not shown). These results indicate that endogenous Necl-5 is at least partly involved in motility of NIH3T3 cells that do not contact other cells.



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  		FIG. 7.
Inhibition of Necl-5-enhanched cell motility by Necl-5- {Delta}CP. Reduction of motility of Necl-5-L cells by expression of Necl-5-{Delta}CP. FLAG-Necl-5-{Delta}CP or Necl-5-{Delta}CP was transiently expressed in non-tagged Necl-5-L or Necl-5-{Delta}EC-L cells, respectively. These cells were incubated on the colloidal gold-coated coverslips for 16 h. A and B, phase contrast images; A1, a non-tagged Necl-5-L cell; A2, a non-tagged Necl-5-L cell expressing FLAG-Necl-5-{Delta}CP; B1, a Necl-5-{Delta}EC-L cell; B2, a Necl-5-{Delta}EC-L cell expressing Necl-5-{Delta}CP. C, the area free of gold particles around a single cell. *, p < 0.0001 versus the control (non-tagged Necl-5-L cells) as determined by paired Student's t test. Bars,50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. The cells expressing FLAG-Necl-5-{Delta}CP or Necl-5-{Delta}CP were confirmed by immunofluorescence microscopy using the anti-FLAG mAb or the anti-Necl-5 mAb-i, respectively. The results shown in A and B are representative of five independent experiments, and the results shown in C are the means ± S.E. of the five independent experiments.

 



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  		FIG. 8.
Involvement of Necl-5 in motility of NIH3T3 and V12Ras-NIH3T3 cells. A, reduction of motility of NIH3T3 and V12Ras-NIH3T3 cells by expression of Necl-5-{Delta}CP. FLAG-Necl-5-{Delta}CP was transiently expressed in NIH3T3 or V12Ras-NIH3T3 cells. These cells were incubated on the colloidal gold-coated coverslips for 16 h. a and b, phase contrast images; a1, an NIH3T3 cell; a2, an NIH3T3 cell expressing FLAG-Necl-5-{Delta}CP; b1, a V12Ras-NIH3T3 cell; b2, a V12Ras-NIH3T3 cell expressing FLAG-Necl-5-{Delta}CP. c, the area free of gold particles around a single cell. *, p < 0.0001 versus the control (NIH3T3 cells) as determined by paired Student's t test; **, p < 0.0001 versus the control (V12Ras-NIH3T3 cells) as determined by paired Student's t test. Bars, 50 µm. Cell motility was analyzed by phase contrast microscopy as the area free of gold particles around a single cell. The cells expressing FLAG-Necl-5-{Delta}CP were confirmed by immunofluorescence microscopy using the anti-FLAG mAb. B, colocalization of Necl-5 and integrin {alpha}V{beta}3 at leading edges of V12Ras-NIH3T3 cells. The cells were cultured on the coverslips precoated with or without vitronectin for 16 h. Immunofluorescence images of V12Ras-NIH3T3 cells using the anti-Necl-5 mAb-i and the anti-integrin {beta}3 pAb. a, in the absence of vitronectin; b, in the presence of vitronectin; a1 and b1, Necl-5; a2 and b2, integrin {beta}3; a3 and b3, merge. Arrowheads, edges of protrusions. Bars, 10 µm. The results shown in Aa and Ab are representative of five independent experiments, the results shown in Ac are the means ± S.E. of the five independent experiments, and the results shown in B are representative of three independent experiments.

 
Involvement of Necl-5 in the PDGF-induced Motility of NIH3T3 Cells—As shown in Fig. 3, Necl-5-L and Necl-5-{Delta}EC-L cells required serum for their active motility. We therefore attempted to identify a serum factor that induced motility of NIH3T3 cells. Because PDGF has been shown by the Boyden chamber assay to enhance motility of NIH3T3 cells in an integrin {alpha}V{beta}3-dependent manner (49), we examined by the same assay method whether PDGF shows this activity. Consistently, PDGF enhanced motility of NIH3T3 cells, but this PDGF-enhanced motility was markedly reduced by stable expression of Necl-5-{Delta}CP (Fig. 9). These results indicate that PDGF is likely to be at least one of the serum factors that induce motility of NIH3T3 cells and that Necl-5 is at least involved in the PDGF-induced motility of NIH3T3 cells.



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  		FIG. 9.
Involvement of Necl-5 in the PDGF-induced motility of NIH3T3 cells. Motility of NIH3T3 and Necl-5-{Delta}CP-NIH3T3 cells was measured by the Boyden chamber method. Serum-starved NIH3T3 or Necl-5-{Delta}CP-NIH3T3 cells were incubated on the cell culture inserts coated with vitronectin or BSA and in the presence or absence of 30 ng/ml PDGF in the bottom well for 14 h. VN, vitronectin. *, p < 0.0001 versus the control (NIH3T3 cells) as determined by paired Student's t test. The results shown are the means ± S.E. of three independent experiments.

 
Involvement of Necl-5 in Motility of V12Ras-NIH3T3 Cells—We have previously shown that Necl-5 is up-regulated in V12Ras-NIH3T3 cells (NIH3T3 cells transformed by an oncogenic Ki-Ras) (26). We next examined by use of Necl-5-{Delta}CP whether motility of V12Ras-NIH3T3 cells is the result of up-regulated Necl-5. Transient expression of Necl-5-{Delta}CP in V12Ras-NIH3T3 cells reduced their motility (Fig. 8A, b1, b2, and c). We furthermore obtained V12Ras-NIH3T3 cells stably expressing Necl-5-{Delta}CP (Necl-5-{Delta}CP-V12Ras-NIH3T3 cells). Consistently, the motility of a Necl-5-{Delta}CP-V12Ras-NIH3T3 cell was reduced as compared with that of a V12Ras-NIH3T3 cell (data not shown). These results indicate that up-regulated Necl-5 is at least partly involved in enhanced motility of V12Ras-NIH3T3 cells that do not contact other cells.

We have shown above that Necl-5 colocalizes with integrin {alpha}V{beta}3 and is functionally associated with it in migrating Necl-5-L cells. We next attempted to confirm that Necl-5 colocalizes with integrin {alpha}V{beta}3 in a singly migrating V12Ras-NIH3T3 cell. Necl-5 and integrin {beta}3 were stained with the anti-Necl-5 mAb-i and the anti-integrin {beta}3 pAb, respectively. The immunofluorescence signal for Necl-5 was concentrated at edges of the protrusions that might be leading edges of a singly migrating V12Ras-NIH3T3 cell (Fig. 8B, a1). The signal for integrin {beta}3 was not concentrated at the leading edges (Fig. 8B, a2 and a3). However, when V12Ras-NIH3T3 cells were cultured on the coverslips precoated with vitronectin, the signals for both Necl-5 and integrin {beta}3 were concentrated and colocalized at the leading edges (Fig. 8B, b1, b2, and b3). When integrin {alpha}V was stained with the anti-integrin {alpha}V pAb, essentially similar results were obtained (data not shown). The reason the signal for integrin {alpha}V{beta}3 was not detected at the leading edges in the absence of vitronectin despite the presence of endogenous vitronectin in the serum may be the result of a lower concentration of integrin {alpha}V{beta}3 there and a low sensitivity of the Ab to this protein. The signal for Necl-5 was not detected at the leading edges of a singly migrating NIH3T3 cell irrespective of the presence and absence of vitronectin (data not shown). This might be the result of a low expression level of this protein and a low sensitivity of the Ab to this protein. These results have provided another line of evidence for the functional association of Necl-5 and integrin {alpha}V{beta}3 in migrating cells.

Involvement of Necl-5 in Metastasis of V12Ras-NIH3T3 Cells—In the last set of experiments, we examined whether Necl-5 is involved in metastasis of V12Ras-NIH3T3 cells. V12Ras-NIH3T3 cells have been shown to obtain metastatic ability (67, 68). V12Ras-NIH3T3 or Necl-5-{Delta}CP-V12Ras-NIH3T3 cells were injected into the tail vein of nude mice, and the numbers of tumor nodules in the lung were counted. The number of tumor nodules of Necl-5-{Delta}CP-V12Ras-NIH3T3 cells was much lower than that of V12Ras-NIH3T3 cells (Fig. 10, A and B). It may be noted that the sizes of the lungs metastasized by V12Ras-NIH3T3 cells were larger than those metastasized by Necl-5-{Delta}CP-V12Ras-NIH3T3 cells (Fig. 10A). This hypertrophy may be the result of more tumor nodules of V12Ras-NIH3T3 cells than those of Necl-5-{Delta}CP-V12Ras-NIH3T3 cells. These results indicate that up-regulated Necl-5 is at least partly involved in the metastasis of V12Ras-NIH3T3 cells.



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  		FIG. 10.
Involvement of Necl-5 in metastasis of V12Ras-NIH3T3 cells. Nude mice were injected intravenously with 1 x 106 V12Ras-NIH3T3 or Necl-5-{Delta}CP-V12Ras-NIH3T3 cells. The animals were killed on day 14, and tumor nodules in the lungs were counted. A, photographs of the lung of each group. B, the number of tumor nodules in the lung. The results shown are the means ± S.E. (n = 5). *, p < 0.000001, compared with the control V12Ras-NIH3T3 cells group. Bars, 5 mm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously found that Necl-5 does not homophilically trans-interact with Necl-5, but heterophilically trans-interacts selectively with nectin-3, and that Necl-5 is up-regulated in V12Ras-NIH3T3 cells and enhances intercellular motility through its heterophilic trans-interaction with nectin-3 (26). We have first shown here using L cells expressing various mutants of Necl-5 that Necl-5 shows cell motility-enhancing activity, which is dependent on serum or PDGF, but independent of nectin-3. The cytoplasmic region of Necl-5 is essential for the cell motility-enhancing activity of Necl-5. The extracellular region of Necl-5 is not necessary for random cell movement, but it is necessary for directional cell migration. We have then shown here that Necl-5 is functionally associated with integrin {alpha}V{beta}3 and that the serum-induced, Necl-5-enhanced cell motility is dependent on integrin {alpha}V{beta}3. This result is consistent with the earlier observation that PDGF enhances motility of NIH3T3 cells in an integrin {alpha}V{beta}3-dependent manner (49). We have moreover shown here that Cdc42 and Rac are involved in the serum-induced, integrin-dependent, Necl-5-enhanced cell motility through the respective formation of filopodia and lamellipodia. We have finally shown here that Necl-5 is indeed involved in motility of NIH3T3 cells that do not contact other cells, and that Necl-5 is furthermore involved in the enhanced motility and the metastasis of V12Ras-NIH3T3 cells.

The result, that the extracellular region of Necl-5 is not necessary for the serum-dependent cell motility, indicates that a cell motility-enhancing factor in serum exerts its action through its own membrane receptor. We have identified PDGF as a cell motility-enhancing factor contained in serum. It may be noted that the random motility of Necl-5-{Delta}EC-L cells (L cells expressing Necl-5 of which extracellular region is deleted) is more active than that of Necl-5-L cells. The extracellular region may inhibit the cell motility-enhancing activity of the cytoplasmic region, and that the interaction of the extracellular region with a factor contained in serum may release this inhibitory activity. Consistently, the anti-Necl-5 mAb-s, which binds to the extracellular region of Necl-5, further enhances the cell motility-enhancing activity of Necl-5. Necl-5 of a singly migrating cell used for the phagokinetic track motility assay is not likely to interact with any transmembrane proteins of other cells, because the cells do not contact each other. There may be a molecule that substitutes for this mAb in serum. Another possible mechanism is that deletion of the extracellular region of Necl-5 causes functionally stronger association of the cytoplasmic region of Necl-5 with the cytoplasmic region of the cell surface receptor of the serum factor, such as the PDGF receptor, and/or the cytoplasmic region of integrin {alpha}V{beta}3.

We have shown here that Necl-5 and integrin {alpha}V{beta}3 colocalize at leading edges of migrating cells. During the preparation of this manuscript, Mueller and Wimmer (38) reported that PVR/CD155 colocalizes with integrin {alpha}V{beta}3 on the surface of transfected mouse fibroblasts and at amniotic epithelial cell junctions, consistently with our present results. We have moreover shown here that the serum-induced, Necl-5-enhanced cell motility is inhibited by echistatin or vitronectin precoated on the coverslips. Echistatin is an inhibitor of integrin {alpha}V{beta}3 (58), and vitronectin is an extracellular matrix molecule that binds to integrins {alpha}V{beta}1, {alpha}V{beta}3, {alpha}V{beta}5, and {alpha}IIb{beta}3 (54). PDGF has been shown to enhance motility of NIH3T3 cells in an integrin {alpha}V{beta}3-dependent manner (49). Taken together, these results indicate that the serum- or PDGF-induced, Necl-5-enhanced cell motility is dependent on at least integrin {alpha}V{beta}3 and that Necl-5 and integrin {alpha}V{beta}3 are functionally associated with each other. It remains to be clarified how these two proteins are functionally associated with each other, but both the extracellular and cytoplasmic regions of Necl-5 and integrin {alpha}V{beta}3 may be involved it, because the cytoplasmic region, but not the extracellular region, is essential for cell motility-enhancing activity of Necl-5 and Necl-5-{Delta}CP inhibits motility of Necl-5-L cells, but not Necl-5-{Delta}EC-L cells. It may be noted that Necl-5 and integrin {alpha}V{beta}3 are highly concentrated and colocalize at the leading edges of V12Ras-NIH3T3 cells on the coverslips precoated with vitronectin. This result suggests that only the activated form of integrin {alpha}V{beta}3 is functionally associated with Necl-5.

It has been shown that some growth factor receptors, such as the PDGF receptor {beta} and the vascular endothelial growth factor receptor-2, enhance cell motility in cooperation with integrin {alpha}V{beta}3 (49, 69). It has furthermore been shown that the PDGF receptor {beta} or the vascular endothelial growth factor receptor-2 is co-immunoprecipitated with integrin {alpha}V{beta}3, suggesting the physical association between these growth factor receptors and integrin {alpha}V{beta}3 (49, 69, 70). Therefore, we attempted to obtain the evidence for the physical association between Necl-5 and integrin {alpha}V{beta}3. When Necl-5 was immunoprecipitated by the anti-Necl-5 mAb-i from the extract of Necl-5-L or V12Ras-NIH3T3 cells cultured in the presence or absence of serum, integrin {alpha}V or {beta}3 was not co-immunoprecipitated irrespective of serum (data not shown). When integrin {alpha}V{beta}3 was immunoprecipitated by the anti-integrin {alpha}V or {beta}3 pAbs from the extract of Necl-5-L or V12Ras-NIH3T3 cells cultured in the presence or absence of serum, Necl-5 was not co-immunoprecipitated irrespective of serum (data not shown). These results suggest that Necl-5 does not directly associate with integrin {alpha}V{beta}3, at least under the conditions used here. The association of these proteins is likely to be indirect and complex. The physical association between Necl-5 and the cell surface receptor of the serum factor, such as the PDGF receptor, is currently being investigated.

Dynamic extension of protrusions, such as filopodia and lamellipodia, and formation of focal complexes and focal adhesions as well as contraction of cell body and tail detachment are essential for cell migration (71). Filopodia and lamellipodia are formed by the action of Cdc42 and Rac, respectively (63, 64). Focal complexes are formed by the action of Rac at the lamellipodia of leading edges in a Rho-independent manner and focal adhesions are formed by maturation of focal complexes by the action of Rho (57). Integrin {alpha}V{beta}3 has been shown to be concentrated at focal complexes in endothelial cells (56). We have shown here that Cdc42 and Rac are activated by the action of serum, which is further enhanced by Necl-5, and that the Cdc42-dependent formation of filopodia and the Rac-dependent formation of lamellipodia are involved in the serum-dependent cell motility-enhancing activity of Necl-5. These results, together with the result that Necl-5 colocalizes and is functionally associated with integrin {alpha}V{beta}3, suggest that Necl-5 is involved in the formation of focal complexes through Rac in cooperation with integrin {alpha}V{beta}3. Cdc42 activated by the action of Necl-5 is likely to induce the formation of filopodia. Because it has been shown that activation of integrins induces activation of Cdc42 and Rac (72), integrin {alpha}V{beta}3 may also induce activation of these small G proteins independently of, or in cooperation with, Necl-5, although this activity of integrin {alpha}V{beta}3 remains unknown. Therefore, taken together, it is likely that the serum factor receptor, such as the PDGF receptor, and/or integrin {alpha}V{beta}3 transduces a signal to the cytoplasmic region of Necl-5, or alternatively the cytoplasmic region of Necl-5 transduces a signal to the serum factor receptor and/or integrin {alpha}V{beta}3, which then induces activation of Cdc42 and Rac and the subsequent formation of focal complexes, eventually leading to enhanced cell motility. It may be emphasized here that the functional association of Necl-5 with the integrin {alpha}V{beta}3 is of crucial importance, because Necl-5 heterophilically trans-interacts with nectin-3, which is associated with cadherins (10, 11). The cross-talk between the cell-cell and cell-matrix adhesions have been known for a long time to play important roles for regulation of cell migration, adhesion, and proliferation. The direct interaction of Necl-5 and nectin-3 may serve as connectors between integrins and cadherins and be involved in the cross-talk between the cell-cell and cell-matrix adhesions.

Transformation of cells causes increase of cell movement and loss of contact inhibition of cell movement and proliferation, eventually leading transformed cells to invasion into surrounding tissues and metastasis to other organs (4, 8). We have shown here that the enhanced motility and the metastasis of V12Ras-NIH3T3 cells are at least partly the result of Necl-5. Because Necl-5 is also up-regulated in human carcinomas (24-26, 32, 33), up-regulated Necl-5 may be also at least partly involved in the enhanced motility and the metastasis of human carcinomas. It remains unknown how Necl-5 is up-regulated by transformation of cells or whether up-regulated Necl-5 is related to loss of contact inhibition of movement of transformed cells. These issues should be addressed in the future for establishing the physiological and pathological roles of Necl-5.


   FOOTNOTES
 
* The work performed 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 and 2003). 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

To whom correspondence should be addressed. Tel.: 81-6-6879-3410; Fax: 81-6-6879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.

1 The abbreviations used are: AJ, adherens junction; aa, amino acid(s); PVR, poliovirus receptor; Necl, nectin-like molecule; V12Ras-NIH3T3 cells, NIH3T3 cells transformed by an oncogenic Ki-Ras; PDGF, platelet-derived growth factor; Nef-3, the extracellular fragment of nectin-3 fused to the human IgG Fc; DMEM, Dulbecco's modified Eagle's medium; non-tagged Necl-5-L cells, L cells stably expressing Necl-5; Necl-5-L cells, L cells stably expressing FLAG-Necl-5; Necl-5-{Delta}EC-L cells, L cells stably expressing Necl-5 of which extracellular region is deleted; Necl-5-{Delta}CP-L cells, L cells stably expressing Necl-5 of which cytoplasmic region is deleted; Necl-5-{Delta}CP-NIH3T3 cells, NIH3T3 cells stably expressing Necl-5 of which cytoplasmic region is deleted; Necl-5-{Delta}CP-V12Ras-NIH3T3 cells, V12Ras-NIH3T3 cells stably expressing Necl-5 of which cytoplasmic region is deleted; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; BSA, bovine serum albumin; FRET, fluorescent resonance energy transfer; PBS, phosphate-buffered saline; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein. Back


   ACKNOWLEDGMENTS
 
L cells were supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan), the cDNA of N17Rac1 was from Dr. A. Hall (University College London, London, United Kingdom), the pEF-BOS expression plasmid was from Dr. S. Nagata (Osaka University, Osaka, Japan), the cDNA of NWASP was from Drs. T. Takenawa and H. Miki (Tokyo University, Tokyo, Japan), and vitronectin was from Dr. K. Sekiguchi (Osaka University, Osaka, Japan). We thank these researchers for their generous gifts. We thank Drs. M. Matsuda and T. Nakamura (Osaka University, Osaka, Japan) for their generous gifts of pRaichu-Rac1 and pRaichu-Cdc42 and for their kind help for the experiments of the FRET imaging. We thank Y. Inoue and A. Hamaguchi (KAN Research Institute Inc.) for helpful assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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T. Wakayama, Y. Sai, A. Ito, Y. Kato, M. Kurobo, Y. Murakami, E. Nakashima, A. Tsuji, Y. Kitamura, and S. Iseki
Heterophilic Binding of the Adhesion Molecules Poliovirus Receptor and Immunoglobulin Superfamily 4A in the Interaction Between Mouse Spermatogenic and Sertoli Cells
Biol Reprod, June 1, 2007; 76(6): 1081 - 1090.
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M. Kajita, W. Ikeda, Y. Tamaru, and Y. Takai
Regulation of platelet-derived growth factor-induced Ras signaling by poliovirus receptor Necl-5 and negative growth regulator Sprouty2
Genes Cells, March 1, 2007; 12(3): 345 - 357.
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T. Umemoto, M. Yamato, Y. Shiratsuchi, M. Terasawa, J. Yang, K. Nishida, Y. Kobayashi, and T. Okano
Expression of Integrin beta3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype
J. Immunol., December 1, 2006; 177(11): 7733 - 7739.
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Y. Sakamoto, H. Ogita, T. Hirota, T. Kawakatsu, T. Fukuyama, M. Yasumi, N. Kanzaki, M. Ozaki, and Y. Takai
Interaction of Integrin {alpha}vbeta3 with Nectin: IMPLICATION IN CROSS-TALK BETWEEN CELL-MATRIX AND CELL-CELL JUNCTIONS
J. Biol. Chem., July 14, 2006; 281(28): 19631 - 19644.
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T. Fujito, W. Ikeda, S. Kakunaga, Y. Minami, M. Kajita, Y. Sakamoto, M. Monden, and Y. Takai
Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3
J. Cell Biol., October 10, 2005; 171(1): 165 - 173.
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Z. Yu, M.-K. Chan, P. O-charoenrat, D. P. Eisenberg, J. P. Shah, B. Singh, Y. Fong, and R. J. Wong
Enhanced Nectin-1 Expression and Herpes Oncolytic Sensitivity in Highly Migratory and Invasive Carcinoma
Clin. Cancer Res., July 1, 2005; 11(13): 4889 - 4897.
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T. Sato, K. Irie, T. Ooshio, W. Ikeda, and Y. Takai
Involvement of heterophilic trans-interaction of Necl-5/Tage4/PVR/CD155 with nectin-3 in formation of nectin- and cadherin-based adherens junctions
Genes Cells, September 1, 2004; 9(9): 791 - 799.
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S. Kakunaga, W. Ikeda, T. Shingai, T. Fujito, A. Yamada, Y. Minami, T. Imai, and Y. Takai
Enhancement of Serum- and Platelet-derived Growth Factor-induced Cell Proliferation by Necl-5/Tage4/Poliovirus Receptor/CD155 through the Ras-Raf-MEK-ERK Signaling
J. Biol. Chem., August 27, 2004; 279(35): 36419 - 36425.
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