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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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Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The transfection efficiency was >90%. (A) Confirmation of the knockdown of nectin-3. (a) Western blotting. The cell lysates were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-3 pAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–nectin-3 mAb and the anti–N-cadherin pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. (B) Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. The siRNA-transfected cells were cultured at the lower or higher density. (a) 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 mAb-i, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–Necl-5 mAb-i and the anti–nectin-3 pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. The results shown are representative of three independent experiments.
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fig2: Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The transfection efficiency was >90%. (A) Confirmation of the knockdown of nectin-3. (a) Western blotting. The cell lysates were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-3 pAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–nectin-3 mAb and the anti–N-cadherin pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. (B) Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. The siRNA-transfected cells were cultured at the lower or higher density. (a) 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 mAb-i, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–Necl-5 mAb-i and the anti–nectin-3 pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. The results shown are representative of three independent experiments.

Mentions: We then performed knockdown of nectin-3 using the small interfering RNA (siRNA) method. Transfection of the synthetic siRNA against nectin-3 reduced the total amount of nectin-3 as estimated by Western blotting (Fig. 2 A, a). The total amount of nectin-1 or N-cadherin did not change (unpublished data). The amount of cell surface nectin-3, but not that of nectin-1 or N-cadherin, similarly decreased (Fig. 2 B, a). Immunofluorescence microscopy also showed that the signal for nectin-3 markedly decreased (Fig. 2 A, b1–c2). The signal for N-cadherin did not change, which might be attributable to the presence of nectin-1. When the nectin-3 knockdown cells were cultured at the lower or higher density, the down-regulation of cell surface Necl-5 at the higher density markedly decreased as estimated by Western blotting (Fig. 2 B, a). Immunofluorescence microscopy showed that the cell surface signal for Necl-5 did not decrease in the nectin-3 knockdown cells cultured at the higher density (Fig. 2 B, b1–c2). These results indicate that nectin-3 is a major molecule trans-interacting with Necl-5 and induces its down-regulation.


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)

Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The transfection efficiency was >90%. (A) Confirmation of the knockdown of nectin-3. (a) Western blotting. The cell lysates were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-3 pAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–nectin-3 mAb and the anti–N-cadherin pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. (B) Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. The siRNA-transfected cells were cultured at the lower or higher density. (a) 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 mAb-i, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–Necl-5 mAb-i and the anti–nectin-3 pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. The results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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fig2: Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The transfection efficiency was >90%. (A) Confirmation of the knockdown of nectin-3. (a) Western blotting. The cell lysates were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-3 pAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–nectin-3 mAb and the anti–N-cadherin pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. (B) Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. The siRNA-transfected cells were cultured at the lower or higher density. (a) 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 mAb-i, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–Necl-5 mAb-i and the anti–nectin-3 pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. The results shown are representative of three independent experiments.
Mentions: We then performed knockdown of nectin-3 using the small interfering RNA (siRNA) method. Transfection of the synthetic siRNA against nectin-3 reduced the total amount of nectin-3 as estimated by Western blotting (Fig. 2 A, a). The total amount of nectin-1 or N-cadherin did not change (unpublished data). The amount of cell surface nectin-3, but not that of nectin-1 or N-cadherin, similarly decreased (Fig. 2 B, a). Immunofluorescence microscopy also showed that the signal for nectin-3 markedly decreased (Fig. 2 A, b1–c2). The signal for N-cadherin did not change, which might be attributable to the presence of nectin-1. When the nectin-3 knockdown cells were cultured at the lower or higher density, the down-regulation of cell surface Necl-5 at the higher density markedly decreased as estimated by Western blotting (Fig. 2 B, a). Immunofluorescence microscopy showed that the cell surface signal for Necl-5 did not decrease in the nectin-3 knockdown cells cultured at the higher density (Fig. 2 B, b1–c2). These results indicate that nectin-3 is a major molecule trans-interacting with Necl-5 and induces its down-regulation.

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