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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 cell movement and DNA synthesis by the knockdown of Necl-5. NIH3T3 cells were transfected either with the siRNA vector against Necl-5 and the pEGFP-tub vector or with the control siRNA vector and the pEGFP-tub vector and cultured at the lower density for 48 h. The transfection efficiency was ∼20%. (A) Confirmation of the knockdown of Necl-5. (a) FACS analysis. The cells were stained with the anti–Necl-5 mAb-i and the EGFP-tub–positive cells were monitored. (b) Western blotting. The EGFP-tub–positive cells were sorted by FACS and then the lysate of the sorted cells was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (c) Immunofluorescence images. The cells were single stained with the anti–Necl-5 mAb-i. (c1) Necl-5; (c2) Necl-5 and EGFP-tub. Asterisks show the Necl-5 knockdown cells. Bar, 10 μm. (B) Measurement of cell movement by Boyden chamber assay. The transfected cells (∼20% transfection efficiency) were cultured at the lower density, directly replated in culture insert, and cultured for 9 h. The EGFP-tub–positive migrated cells were counted. The number of control EGFP-tub–positive migrated cells was consistent with that of control migrated cells precultured at lower density in Fig. 5 A (a) and Fig. 6 A, considering the transfection efficiency. *, P < 0.001. (C) Measurement of DNA synthesis by BrdU incorporation. The transfected cells (∼20% transfection efficiency) were directly cultured at the lower density for 18 h and incubated with BrdU for 2 h. The cells were double stained with the anti-BrdU mAb and DAPI. The EGFP-tub–positive cells were measured. (a) Control; (b) Necl-5 siRNA; (c) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results shown in A are representative of three independent experiments, and the results shown in B and C are the mean ± SEM of the three independent experiments.
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fig7: Reduction of cell movement and DNA synthesis by the knockdown of Necl-5. NIH3T3 cells were transfected either with the siRNA vector against Necl-5 and the pEGFP-tub vector or with the control siRNA vector and the pEGFP-tub vector and cultured at the lower density for 48 h. The transfection efficiency was ∼20%. (A) Confirmation of the knockdown of Necl-5. (a) FACS analysis. The cells were stained with the anti–Necl-5 mAb-i and the EGFP-tub–positive cells were monitored. (b) Western blotting. The EGFP-tub–positive cells were sorted by FACS and then the lysate of the sorted cells was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (c) Immunofluorescence images. The cells were single stained with the anti–Necl-5 mAb-i. (c1) Necl-5; (c2) Necl-5 and EGFP-tub. Asterisks show the Necl-5 knockdown cells. Bar, 10 μm. (B) Measurement of cell movement by Boyden chamber assay. The transfected cells (∼20% transfection efficiency) were cultured at the lower density, directly replated in culture insert, and cultured for 9 h. The EGFP-tub–positive migrated cells were counted. The number of control EGFP-tub–positive migrated cells was consistent with that of control migrated cells precultured at lower density in Fig. 5 A (a) and Fig. 6 A, considering the transfection efficiency. *, P < 0.001. (C) Measurement of DNA synthesis by BrdU incorporation. The transfected cells (∼20% transfection efficiency) were directly cultured at the lower density for 18 h and incubated with BrdU for 2 h. The cells were double stained with the anti-BrdU mAb and DAPI. The EGFP-tub–positive cells were measured. (a) Control; (b) Necl-5 siRNA; (c) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results shown in A are representative of three independent experiments, and the results shown in B and C are the mean ± SEM of the three independent experiments.

Mentions: We furthermore confirmed the inhibitory effects of the down-regulation of Necl-5 on cell movement and proliferation by knocking down Necl-5 using the siRNA method. Transfection of the siRNA vector against Necl-5 reduced the amount of cell surface Necl-5 as estimated by FACS analysis (Fig. 7 A, a). It also reduced the total amount of Necl-5 as estimated by Western blotting and immunofluorescence microscopy (Fig. 7 A, b–c2). The total amount of Necl-5 decreased to ∼20%. Movement and DNA synthesis of the Necl-5 knockdown cells decreased as compared with those of the control cells as estimated by the methods just mentioned (Fig. 7, B and C). These results indicate that the down-regulation of Necl-5 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)

Reduction of cell movement and DNA synthesis by the knockdown of Necl-5. NIH3T3 cells were transfected either with the siRNA vector against Necl-5 and the pEGFP-tub vector or with the control siRNA vector and the pEGFP-tub vector and cultured at the lower density for 48 h. The transfection efficiency was ∼20%. (A) Confirmation of the knockdown of Necl-5. (a) FACS analysis. The cells were stained with the anti–Necl-5 mAb-i and the EGFP-tub–positive cells were monitored. (b) Western blotting. The EGFP-tub–positive cells were sorted by FACS and then the lysate of the sorted cells was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (c) Immunofluorescence images. The cells were single stained with the anti–Necl-5 mAb-i. (c1) Necl-5; (c2) Necl-5 and EGFP-tub. Asterisks show the Necl-5 knockdown cells. Bar, 10 μm. (B) Measurement of cell movement by Boyden chamber assay. The transfected cells (∼20% transfection efficiency) were cultured at the lower density, directly replated in culture insert, and cultured for 9 h. The EGFP-tub–positive migrated cells were counted. The number of control EGFP-tub–positive migrated cells was consistent with that of control migrated cells precultured at lower density in Fig. 5 A (a) and Fig. 6 A, considering the transfection efficiency. *, P < 0.001. (C) Measurement of DNA synthesis by BrdU incorporation. The transfected cells (∼20% transfection efficiency) were directly cultured at the lower density for 18 h and incubated with BrdU for 2 h. The cells were double stained with the anti-BrdU mAb and DAPI. The EGFP-tub–positive cells were measured. (a) Control; (b) Necl-5 siRNA; (c) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results shown in A are representative of three independent experiments, and the results shown in B and C are the mean ± SEM of the three independent experiments.
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Related In: Results  -  Collection

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fig7: Reduction of cell movement and DNA synthesis by the knockdown of Necl-5. NIH3T3 cells were transfected either with the siRNA vector against Necl-5 and the pEGFP-tub vector or with the control siRNA vector and the pEGFP-tub vector and cultured at the lower density for 48 h. The transfection efficiency was ∼20%. (A) Confirmation of the knockdown of Necl-5. (a) FACS analysis. The cells were stained with the anti–Necl-5 mAb-i and the EGFP-tub–positive cells were monitored. (b) Western blotting. The EGFP-tub–positive cells were sorted by FACS and then the lysate of the sorted cells was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (c) Immunofluorescence images. The cells were single stained with the anti–Necl-5 mAb-i. (c1) Necl-5; (c2) Necl-5 and EGFP-tub. Asterisks show the Necl-5 knockdown cells. Bar, 10 μm. (B) Measurement of cell movement by Boyden chamber assay. The transfected cells (∼20% transfection efficiency) were cultured at the lower density, directly replated in culture insert, and cultured for 9 h. The EGFP-tub–positive migrated cells were counted. The number of control EGFP-tub–positive migrated cells was consistent with that of control migrated cells precultured at lower density in Fig. 5 A (a) and Fig. 6 A, considering the transfection efficiency. *, P < 0.001. (C) Measurement of DNA synthesis by BrdU incorporation. The transfected cells (∼20% transfection efficiency) were directly cultured at the lower density for 18 h and incubated with BrdU for 2 h. The cells were double stained with the anti-BrdU mAb and DAPI. The EGFP-tub–positive cells were measured. (a) Control; (b) Necl-5 siRNA; (c) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results shown in A are representative of three independent experiments, and the results shown in B and C are the mean ± SEM of the three independent experiments.
Mentions: We furthermore confirmed the inhibitory effects of the down-regulation of Necl-5 on cell movement and proliferation by knocking down Necl-5 using the siRNA method. Transfection of the siRNA vector against Necl-5 reduced the amount of cell surface Necl-5 as estimated by FACS analysis (Fig. 7 A, a). It also reduced the total amount of Necl-5 as estimated by Western blotting and immunofluorescence microscopy (Fig. 7 A, b–c2). The total amount of Necl-5 decreased to ∼20%. Movement and DNA synthesis of the Necl-5 knockdown cells decreased as compared with those of the control cells as estimated by the methods just mentioned (Fig. 7, B and C). These results indicate that the down-regulation of Necl-5 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