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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 reduction of cell movement and DNA synthesis by the down-regulation of Necl-5. (A) Measurement of cell movement by Boyden chamber assay. (a) Quantification of migrated cells. NIH3T3 cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (b) Reversion of the level of cell surface Necl-5. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, replated at the lower density, and cultured for 9 h. Biotinylated cell surface Necl-5 was collected before (0 h) or after (9 h) replating and subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (B) Measurement of DNA synthesis by BrdU incorporation. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) In the absence of the anti–Necl-5 mAb-i; (b and d) in the presence of the anti–Necl-5 mAb-i; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.
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fig5: Cell density–dependent reduction of cell movement and DNA synthesis by the down-regulation of Necl-5. (A) Measurement of cell movement by Boyden chamber assay. (a) Quantification of migrated cells. NIH3T3 cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (b) Reversion of the level of cell surface Necl-5. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, replated at the lower density, and cultured for 9 h. Biotinylated cell surface Necl-5 was collected before (0 h) or after (9 h) replating and subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (B) Measurement of DNA synthesis by BrdU incorporation. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) In the absence of the anti–Necl-5 mAb-i; (b and d) in the presence of the anti–Necl-5 mAb-i; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.

Mentions: We next examined the effect of the down-regulation of Necl-5 on cell movement and proliferation. NIH3T3 cells, which were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, were replated, and cell movement after 9 h was assayed by the Boyden chamber method. In the absence of this mAb-i, the amount of cell surface Necl-5 and the degree of movement of NIH3T3 cells precultured at the higher density decreased in a roughly parallel manner as compared with those of the cells precultured at the lower density (Fig. 5 A, a and b). The decreases of these parameters were blocked by the mAb-i (Fig. 5 A, a and b). The level of Necl-5 down-regulated in the cells cultured at the higher density did not return to the original level at 9 h after replating at the lower density, but returned to the original level at 24 h (Fig. 5 A, b; and not depicted). The cells were cultured at the lower or higher density in the presence or absence of the mAb-i, and DNA synthesis was assayed by measuring the incorporation of BrdU. In the absence of this mAb-i, the amount of cell surface Necl-5 and the degree of DNA synthesis of NIH3T3 cells cultured at the higher density decreased in a roughly parallel manner (Fig. 5, A [b] and B [a, c, and e]). The decreases of these parameters were inhibited by the mAb-i (Fig. 5, A [b] and B [b, d, and e]). In the nectin-3 knockdown cells, the cell density–dependent decreases of cell movement and DNA synthesis were similarly inhibited (Fig. 6, A and B). The levels of this inhibition were roughly similar to those of the inhibition of the cell density–dependent down-regulation of cell surface Necl-5 (Fig. 2). In addition, when the cells were starved for 24 h and cultured in the presence of serum and the mAb-i, the growth rate did not markedly change, but the cell density rose to slightly higher than that of the cells cultured in the absence of the mAb-i after they became confluent (Fig. 1 A, a, open blue squares). These results indicate that the interaction of Necl-5 with nectin-3 causes the down-regulation of Necl-5, which subsequently reduces cell movement and proliferation.


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 reduction of cell movement and DNA synthesis by the down-regulation of Necl-5. (A) Measurement of cell movement by Boyden chamber assay. (a) Quantification of migrated cells. NIH3T3 cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (b) Reversion of the level of cell surface Necl-5. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, replated at the lower density, and cultured for 9 h. Biotinylated cell surface Necl-5 was collected before (0 h) or after (9 h) replating and subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (B) Measurement of DNA synthesis by BrdU incorporation. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) In the absence of the anti–Necl-5 mAb-i; (b and d) in the presence of the anti–Necl-5 mAb-i; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.
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Related In: Results  -  Collection

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fig5: Cell density–dependent reduction of cell movement and DNA synthesis by the down-regulation of Necl-5. (A) Measurement of cell movement by Boyden chamber assay. (a) Quantification of migrated cells. NIH3T3 cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (b) Reversion of the level of cell surface Necl-5. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, replated at the lower density, and cultured for 9 h. Biotinylated cell surface Necl-5 was collected before (0 h) or after (9 h) replating and subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (B) Measurement of DNA synthesis by BrdU incorporation. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) In the absence of the anti–Necl-5 mAb-i; (b and d) in the presence of the anti–Necl-5 mAb-i; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.
Mentions: We next examined the effect of the down-regulation of Necl-5 on cell movement and proliferation. NIH3T3 cells, which were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, were replated, and cell movement after 9 h was assayed by the Boyden chamber method. In the absence of this mAb-i, the amount of cell surface Necl-5 and the degree of movement of NIH3T3 cells precultured at the higher density decreased in a roughly parallel manner as compared with those of the cells precultured at the lower density (Fig. 5 A, a and b). The decreases of these parameters were blocked by the mAb-i (Fig. 5 A, a and b). The level of Necl-5 down-regulated in the cells cultured at the higher density did not return to the original level at 9 h after replating at the lower density, but returned to the original level at 24 h (Fig. 5 A, b; and not depicted). The cells were cultured at the lower or higher density in the presence or absence of the mAb-i, and DNA synthesis was assayed by measuring the incorporation of BrdU. In the absence of this mAb-i, the amount of cell surface Necl-5 and the degree of DNA synthesis of NIH3T3 cells cultured at the higher density decreased in a roughly parallel manner (Fig. 5, A [b] and B [a, c, and e]). The decreases of these parameters were inhibited by the mAb-i (Fig. 5, A [b] and B [b, d, and e]). In the nectin-3 knockdown cells, the cell density–dependent decreases of cell movement and DNA synthesis were similarly inhibited (Fig. 6, A and B). The levels of this inhibition were roughly similar to those of the inhibition of the cell density–dependent down-regulation of cell surface Necl-5 (Fig. 2). In addition, when the cells were starved for 24 h and cultured in the presence of serum and the mAb-i, the growth rate did not markedly change, but the cell density rose to slightly higher than that of the cells cultured in the absence of the mAb-i after they became confluent (Fig. 1 A, a, open blue squares). These results indicate that the interaction of Necl-5 with nectin-3 causes the down-regulation of Necl-5, which subsequently reduces cell movement and proliferation.

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