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Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Fujito T, Ikeda W, Kakunaga S, Minami Y, Kajita M, Sakamoto Y, Monden M, Takai Y - J. Cell Biol. (2005)

Bottom Line: This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis.Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells.These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

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Cell density–dependent down-regulation of Necl-5. (A) Cell density–dependent down-regulation of Necl-5. NIH3T3 cells were seeded, starved of serum, and cultured in the medium containing serum in the presence or absence of the anti–Necl-5 mAb-i. The cells were sampled at each indicated time. (a) Cell growth curves, amounts of cell surface Necl-5, and levels of Necl-5 mRNA. The relative amounts of cell surface Necl-5 and the level of Necl-5 mRNA at 0 h were set to 1. (b) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (B–E) Down-regulation of Necl-5 in the cells cultured at the higher density. NIH3T3 cells were cultured at the lower or higher density for 24 h. (B) Western blotting of cell surface proteins in the absence or presence of the anti–Necl-5 mAb-i. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5. The cells were stained with the anti–Necl-5 mAb-i. (D) Immunofluorescence images. The cells were double stained with various combinations of the anti–Necl-5 mAb-i, the anti–nectin-3 mAb, the anti–N-cadherin pAb, and the antiactin mAb. Bars, 10 μm. (E) The levels of Necl-5 mRNA. The level of Necl-5 mRNA at the lower cell density was set to 1. The results shown in A and E are the mean ± SEM of the three independent experiments, and the results shown in B, C, and D are representative of three independent experiments.
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fig1: Cell density–dependent down-regulation of Necl-5. (A) Cell density–dependent down-regulation of Necl-5. NIH3T3 cells were seeded, starved of serum, and cultured in the medium containing serum in the presence or absence of the anti–Necl-5 mAb-i. The cells were sampled at each indicated time. (a) Cell growth curves, amounts of cell surface Necl-5, and levels of Necl-5 mRNA. The relative amounts of cell surface Necl-5 and the level of Necl-5 mRNA at 0 h were set to 1. (b) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (B–E) Down-regulation of Necl-5 in the cells cultured at the higher density. NIH3T3 cells were cultured at the lower or higher density for 24 h. (B) Western blotting of cell surface proteins in the absence or presence of the anti–Necl-5 mAb-i. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5. The cells were stained with the anti–Necl-5 mAb-i. (D) Immunofluorescence images. The cells were double stained with various combinations of the anti–Necl-5 mAb-i, the anti–nectin-3 mAb, the anti–N-cadherin pAb, and the antiactin mAb. Bars, 10 μm. (E) The levels of Necl-5 mRNA. The level of Necl-5 mRNA at the lower cell density was set to 1. The results shown in A and E are the mean ± SEM of the three independent experiments, and the results shown in B, C, and D are representative of three independent experiments.

Mentions: NIH3T3 cells were starved for 24 h and cultured in the presence of serum. The cells continued to proliferate until they became confluent (Fig. 1 A, a, closed blue squares). At various periods of time, the cell surface proteins containing Necl-5 were labeled with biotin and the amount of cell surface Necl-5 was measured. The amount of cell surface Necl-5 gradually decreased as cell density increased (Fig. 1 A, a [closed red circles] and b), whereas the amount of cell surface nectin-1, nectin-3, or N-cadherin did not change. Expression of nectin-2, Necl-1, or Necl-2 was not detected by Western blotting in NIH3T3 cells (Shingai et al., 2003; unpublished data). When NIH3T3 cells were cultured at two different cell densities for 24 h, the amount of cell surface Necl-5 decreased in the cells cultured at a higher density as compared with that in the cells cultured at a lower density (Fig. 1 B), whereas the amount of cell surface nectin-1, nectin-3, or N-cadherin did not change at these different cell densities. Similar results were obtained by FACS analysis (Fig. 1 C). The immunofluorescence signal for Necl-5 was concentrated presumably at the leading edges of the cells cultured at the lower density (Fig. 1 D, a1–a3). The signal for nectin-3 did not concentrate anywhere (Fig. 1 D, b1–b3). The signal for N-cadherin was mainly detected at the cytoplasmic and perinuclear regions and slightly detected at the entire plasma membrane (Fig. 1 D, c1–c3). When the cells were cultured at the higher density, the signals for nectin-3 and N-cadherin were concentrated at the cell–cell contact sites (Fig. 1 D, d2, d3, and e1–e3), but the signal for Necl-5 was hardly detected there (Fig. 1 D, d1 and d3). These results indicate that Necl-5 is down-regulated in a cell density–dependent manner. We observed the similar down-regulation of Necl-5 in other cell lines, including Swiss3T3 cells (unpublished data) and mouse embryonic fibroblasts (MEFs; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501090/DC1).


Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Fujito T, Ikeda W, Kakunaga S, Minami Y, Kajita M, Sakamoto Y, Monden M, Takai Y - J. Cell Biol. (2005)

Cell density–dependent down-regulation of Necl-5. (A) Cell density–dependent down-regulation of Necl-5. NIH3T3 cells were seeded, starved of serum, and cultured in the medium containing serum in the presence or absence of the anti–Necl-5 mAb-i. The cells were sampled at each indicated time. (a) Cell growth curves, amounts of cell surface Necl-5, and levels of Necl-5 mRNA. The relative amounts of cell surface Necl-5 and the level of Necl-5 mRNA at 0 h were set to 1. (b) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (B–E) Down-regulation of Necl-5 in the cells cultured at the higher density. NIH3T3 cells were cultured at the lower or higher density for 24 h. (B) Western blotting of cell surface proteins in the absence or presence of the anti–Necl-5 mAb-i. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5. The cells were stained with the anti–Necl-5 mAb-i. (D) Immunofluorescence images. The cells were double stained with various combinations of the anti–Necl-5 mAb-i, the anti–nectin-3 mAb, the anti–N-cadherin pAb, and the antiactin mAb. Bars, 10 μm. (E) The levels of Necl-5 mRNA. The level of Necl-5 mRNA at the lower cell density was set to 1. The results shown in A and E are the mean ± SEM of the three independent experiments, and the results shown in B, C, and D are representative of three independent experiments.
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Related In: Results  -  Collection

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fig1: Cell density–dependent down-regulation of Necl-5. (A) Cell density–dependent down-regulation of Necl-5. NIH3T3 cells were seeded, starved of serum, and cultured in the medium containing serum in the presence or absence of the anti–Necl-5 mAb-i. The cells were sampled at each indicated time. (a) Cell growth curves, amounts of cell surface Necl-5, and levels of Necl-5 mRNA. The relative amounts of cell surface Necl-5 and the level of Necl-5 mRNA at 0 h were set to 1. (b) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (B–E) Down-regulation of Necl-5 in the cells cultured at the higher density. NIH3T3 cells were cultured at the lower or higher density for 24 h. (B) Western blotting of cell surface proteins in the absence or presence of the anti–Necl-5 mAb-i. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5. The cells were stained with the anti–Necl-5 mAb-i. (D) Immunofluorescence images. The cells were double stained with various combinations of the anti–Necl-5 mAb-i, the anti–nectin-3 mAb, the anti–N-cadherin pAb, and the antiactin mAb. Bars, 10 μm. (E) The levels of Necl-5 mRNA. The level of Necl-5 mRNA at the lower cell density was set to 1. The results shown in A and E are the mean ± SEM of the three independent experiments, and the results shown in B, C, and D are representative of three independent experiments.
Mentions: NIH3T3 cells were starved for 24 h and cultured in the presence of serum. The cells continued to proliferate until they became confluent (Fig. 1 A, a, closed blue squares). At various periods of time, the cell surface proteins containing Necl-5 were labeled with biotin and the amount of cell surface Necl-5 was measured. The amount of cell surface Necl-5 gradually decreased as cell density increased (Fig. 1 A, a [closed red circles] and b), whereas the amount of cell surface nectin-1, nectin-3, or N-cadherin did not change. Expression of nectin-2, Necl-1, or Necl-2 was not detected by Western blotting in NIH3T3 cells (Shingai et al., 2003; unpublished data). When NIH3T3 cells were cultured at two different cell densities for 24 h, the amount of cell surface Necl-5 decreased in the cells cultured at a higher density as compared with that in the cells cultured at a lower density (Fig. 1 B), whereas the amount of cell surface nectin-1, nectin-3, or N-cadherin did not change at these different cell densities. Similar results were obtained by FACS analysis (Fig. 1 C). The immunofluorescence signal for Necl-5 was concentrated presumably at the leading edges of the cells cultured at the lower density (Fig. 1 D, a1–a3). The signal for nectin-3 did not concentrate anywhere (Fig. 1 D, b1–b3). The signal for N-cadherin was mainly detected at the cytoplasmic and perinuclear regions and slightly detected at the entire plasma membrane (Fig. 1 D, c1–c3). When the cells were cultured at the higher density, the signals for nectin-3 and N-cadherin were concentrated at the cell–cell contact sites (Fig. 1 D, d2, d3, and e1–e3), but the signal for Necl-5 was hardly detected there (Fig. 1 D, d1 and d3). These results indicate that Necl-5 is down-regulated in a cell density–dependent manner. We observed the similar down-regulation of Necl-5 in other cell lines, including Swiss3T3 cells (unpublished data) and mouse embryonic fibroblasts (MEFs; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501090/DC1).

Bottom Line: This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis.Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells.These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

Show MeSH
Related in: MedlinePlus