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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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Down-regulation of Necl-5 by its interaction with nectin-3. NIH3T3 cells were cultured at the lower density in the absence or presence of Nef-3, other recombinant proteins, and/or mAb for 1 h. (A) Immunofluorescence images. The cells were single or double stained with various combinations of the anti–Necl-5 mAb-i, the rhodamine-conjugated anti–human IgG Fc pAb, and the anti-Rab7 pAb. (a) Control; (b, f, and g) in the presence of 0.3 μM Nef-3; (c) in the presence of 0.3 μM Nef-1; (d) in the presence of 0.3 μM Nef-3 and 50 μg/ml of the anti–Necl-5 mAb-i; (e) in the presence of 0.3 μM Nef-3 and Nef-1. (a–e and g) Immunostaining with permeabilization; (f) immunostaining without permeabilization. Arrowheads show the signal for Necl-5 at the leading edges of the cells. Bars, 10 μm. (B) Western blotting of cell surface proteins in the absence or presence of 0.3 μM Nef-3. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5 in the absence or presence of 0.3 μM Nef-3. The cells were fixed with 1% formaldehyde before FACS analysis and stained with the anti–Necl-5 mAb-i. (D) Western blotting of endocytosed Necl-5 in the absence or presence of 0.3 μM Nef-3. Necl-5, which was biotinylated and endocytosed, was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb. The results shown are representative of three independent experiments.
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fig3: Down-regulation of Necl-5 by its interaction with nectin-3. NIH3T3 cells were cultured at the lower density in the absence or presence of Nef-3, other recombinant proteins, and/or mAb for 1 h. (A) Immunofluorescence images. The cells were single or double stained with various combinations of the anti–Necl-5 mAb-i, the rhodamine-conjugated anti–human IgG Fc pAb, and the anti-Rab7 pAb. (a) Control; (b, f, and g) in the presence of 0.3 μM Nef-3; (c) in the presence of 0.3 μM Nef-1; (d) in the presence of 0.3 μM Nef-3 and 50 μg/ml of the anti–Necl-5 mAb-i; (e) in the presence of 0.3 μM Nef-3 and Nef-1. (a–e and g) Immunostaining with permeabilization; (f) immunostaining without permeabilization. Arrowheads show the signal for Necl-5 at the leading edges of the cells. Bars, 10 μm. (B) Western blotting of cell surface proteins in the absence or presence of 0.3 μM Nef-3. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5 in the absence or presence of 0.3 μM Nef-3. The cells were fixed with 1% formaldehyde before FACS analysis and stained with the anti–Necl-5 mAb-i. (D) Western blotting of endocytosed Necl-5 in the absence or presence of 0.3 μM Nef-3. Necl-5, which was biotinylated and endocytosed, was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb. The results shown are representative of three independent experiments.

Mentions: We further confirmed this conclusion by another method using the recombinant protein of the extracellular fragment of nectin-3 fused to human IgG Fc portion (Nef-3), which interacts with cellular Necl-5 as well as nectin-1 and -3 (Honda et al., 2003; Sato et al., 2004). When NIH3T3 cells were incubated with Nef-3, Nef-3 reduced the cell surface signal for Necl-5 at the leading edges of the cells (Fig. 3 A, a [arrowheads] and b1), whereas Nef-1 did not do that for Necl-5 (Fig. 3 A, C, arrowheads). Nef-1 is the recombinant protein of the extracellular fragment of nectin-1, fused to human IgG Fc portion and interacts with nectin-1 and -3, but not with Necl-5 (Honda et al., 2003; Ikeda et al., 2003). The activity of Nef-3 was blocked by the anti–Necl-5 mAb-i (Fig. 3 A, d, arrowheads). The activity of Nef-3 was also blocked by Nef-1, which is known to interact with not only cellular nectin-1 and -3 but also Nef-3 (Honda et al., 2003; Ikeda et al., 2003; Fig. 3 A, e, arrowheads). The Nef-3–induced down-regulation of cell surface Necl-5 was confirmed by Western blotting and FACS analysis (Fig. 3, B and C). This effect of Nef-3 was dose dependent, and the minimum concentration of Nef-3 necessary for this effect was ∼7 nM. Together, these three lines of evidence indicate that the cell density–dependent down-regulation of Necl-5 is induced by the interaction of Necl-5 mainly with nectin-3.


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)

Down-regulation of Necl-5 by its interaction with nectin-3. NIH3T3 cells were cultured at the lower density in the absence or presence of Nef-3, other recombinant proteins, and/or mAb for 1 h. (A) Immunofluorescence images. The cells were single or double stained with various combinations of the anti–Necl-5 mAb-i, the rhodamine-conjugated anti–human IgG Fc pAb, and the anti-Rab7 pAb. (a) Control; (b, f, and g) in the presence of 0.3 μM Nef-3; (c) in the presence of 0.3 μM Nef-1; (d) in the presence of 0.3 μM Nef-3 and 50 μg/ml of the anti–Necl-5 mAb-i; (e) in the presence of 0.3 μM Nef-3 and Nef-1. (a–e and g) Immunostaining with permeabilization; (f) immunostaining without permeabilization. Arrowheads show the signal for Necl-5 at the leading edges of the cells. Bars, 10 μm. (B) Western blotting of cell surface proteins in the absence or presence of 0.3 μM Nef-3. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5 in the absence or presence of 0.3 μM Nef-3. The cells were fixed with 1% formaldehyde before FACS analysis and stained with the anti–Necl-5 mAb-i. (D) Western blotting of endocytosed Necl-5 in the absence or presence of 0.3 μM Nef-3. Necl-5, which was biotinylated and endocytosed, was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb. The results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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fig3: Down-regulation of Necl-5 by its interaction with nectin-3. NIH3T3 cells were cultured at the lower density in the absence or presence of Nef-3, other recombinant proteins, and/or mAb for 1 h. (A) Immunofluorescence images. The cells were single or double stained with various combinations of the anti–Necl-5 mAb-i, the rhodamine-conjugated anti–human IgG Fc pAb, and the anti-Rab7 pAb. (a) Control; (b, f, and g) in the presence of 0.3 μM Nef-3; (c) in the presence of 0.3 μM Nef-1; (d) in the presence of 0.3 μM Nef-3 and 50 μg/ml of the anti–Necl-5 mAb-i; (e) in the presence of 0.3 μM Nef-3 and Nef-1. (a–e and g) Immunostaining with permeabilization; (f) immunostaining without permeabilization. Arrowheads show the signal for Necl-5 at the leading edges of the cells. Bars, 10 μm. (B) Western blotting of cell surface proteins in the absence or presence of 0.3 μM Nef-3. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5 in the absence or presence of 0.3 μM Nef-3. The cells were fixed with 1% formaldehyde before FACS analysis and stained with the anti–Necl-5 mAb-i. (D) Western blotting of endocytosed Necl-5 in the absence or presence of 0.3 μM Nef-3. Necl-5, which was biotinylated and endocytosed, was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb. The results shown are representative of three independent experiments.
Mentions: We further confirmed this conclusion by another method using the recombinant protein of the extracellular fragment of nectin-3 fused to human IgG Fc portion (Nef-3), which interacts with cellular Necl-5 as well as nectin-1 and -3 (Honda et al., 2003; Sato et al., 2004). When NIH3T3 cells were incubated with Nef-3, Nef-3 reduced the cell surface signal for Necl-5 at the leading edges of the cells (Fig. 3 A, a [arrowheads] and b1), whereas Nef-1 did not do that for Necl-5 (Fig. 3 A, C, arrowheads). Nef-1 is the recombinant protein of the extracellular fragment of nectin-1, fused to human IgG Fc portion and interacts with nectin-1 and -3, but not with Necl-5 (Honda et al., 2003; Ikeda et al., 2003). The activity of Nef-3 was blocked by the anti–Necl-5 mAb-i (Fig. 3 A, d, arrowheads). The activity of Nef-3 was also blocked by Nef-1, which is known to interact with not only cellular nectin-1 and -3 but also Nef-3 (Honda et al., 2003; Ikeda et al., 2003; Fig. 3 A, e, arrowheads). The Nef-3–induced down-regulation of cell surface Necl-5 was confirmed by Western blotting and FACS analysis (Fig. 3, B and C). This effect of Nef-3 was dose dependent, and the minimum concentration of Nef-3 necessary for this effect was ∼7 nM. Together, these three lines of evidence indicate that the cell density–dependent down-regulation of Necl-5 is induced by the interaction of Necl-5 mainly with nectin-3.

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