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The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation.

Yamana S, Tokiyama A, Mizutani K, Hirata K, Takai Y, Rikitake Y - PLoS ONE (2015)

Bottom Line: We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells.When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor.When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

ABSTRACT
Contact inhibition of cell movement and proliferation is critical for proper organogenesis and tissue remodeling. We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells. When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor. This interaction inhibited the tyrosine-phosphorylation of the VEGF receptor through protein-tyrosine phosphatase, non-receptor type 13 (PTPN13), eventually reducing cell movement and proliferation. When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement. Necl-4 further facilitated the activation of extracellular signal-regulated kinase 1/2, eventually enhancing cell proliferation. Thus, Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation cooperatively with the VEGF receptor and PTPN13.

No MeSH data available.


Related in: MedlinePlus

Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.A, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (C) or VEGFR2 (D) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.
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pone.0124259.g003: Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.A, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (C) or VEGFR2 (D) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.

Mentions: Of the two VEGF receptors, VEGFR1 and VEGFR2, the responses to VEGF for movement and proliferation are mainly mediated by VEGFR2 [31,32]. Because Necl-4 was localized at cell—cell contact sites in confluently cultured ECs, we hypothesised that Necl-4 might interact with VEGFR2 and coordinate its activation, signaling, and cellular responses under confluent conditions. We therefore first examined whether Necl-4 could interact with VEGFR1 and VEGFR2. In human embryonic kidney (HEK) 293 cells where FLAG-Necl-4 was co-expressed with VEGFR1 or VEGFR2, both VEGFR1 and VEGFR2 were co-immunoprecipitated with FLAG-Necl-4 (Fig 3A). Consistent with this, endogenous Necl-4 was co-immunoprecipitated with endogenous VEGFR2 in ECs, as VE-cadherin was co-immunoprecipitated (Fig 3B). Compared with sparse conditions, the amount of co-immunoprecipitated Necl-4 was decreased under confluent conditions (Fig 3B), although the amount of PTPN13 co-immunoprecipitated with VEGFR2 was increased (data not shown). In HEK293 cells where various Necl-4 mutants were co-expressed with VEGFR1 or VEGFR2, both VEGFR1 and VEGFR2 were co-immunoprecipitated with FLAG-Necl-4 and FLAG-Necl-4-ΔCP, but hardly with FLAG-Necl-4-ΔEC (Fig 3C and 3D). These results indicate that Necl-4 interacts with VEGFR2 through the extracellular region.


The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation.

Yamana S, Tokiyama A, Mizutani K, Hirata K, Takai Y, Rikitake Y - PLoS ONE (2015)

Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.A, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (C) or VEGFR2 (D) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4404054&req=5

pone.0124259.g003: Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.A, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (C) or VEGFR2 (D) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.
Mentions: Of the two VEGF receptors, VEGFR1 and VEGFR2, the responses to VEGF for movement and proliferation are mainly mediated by VEGFR2 [31,32]. Because Necl-4 was localized at cell—cell contact sites in confluently cultured ECs, we hypothesised that Necl-4 might interact with VEGFR2 and coordinate its activation, signaling, and cellular responses under confluent conditions. We therefore first examined whether Necl-4 could interact with VEGFR1 and VEGFR2. In human embryonic kidney (HEK) 293 cells where FLAG-Necl-4 was co-expressed with VEGFR1 or VEGFR2, both VEGFR1 and VEGFR2 were co-immunoprecipitated with FLAG-Necl-4 (Fig 3A). Consistent with this, endogenous Necl-4 was co-immunoprecipitated with endogenous VEGFR2 in ECs, as VE-cadherin was co-immunoprecipitated (Fig 3B). Compared with sparse conditions, the amount of co-immunoprecipitated Necl-4 was decreased under confluent conditions (Fig 3B), although the amount of PTPN13 co-immunoprecipitated with VEGFR2 was increased (data not shown). In HEK293 cells where various Necl-4 mutants were co-expressed with VEGFR1 or VEGFR2, both VEGFR1 and VEGFR2 were co-immunoprecipitated with FLAG-Necl-4 and FLAG-Necl-4-ΔCP, but hardly with FLAG-Necl-4-ΔEC (Fig 3C and 3D). These results indicate that Necl-4 interacts with VEGFR2 through the extracellular region.

Bottom Line: We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells.When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor.When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.

ABSTRACT
Contact inhibition of cell movement and proliferation is critical for proper organogenesis and tissue remodeling. We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells. When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor. This interaction inhibited the tyrosine-phosphorylation of the VEGF receptor through protein-tyrosine phosphatase, non-receptor type 13 (PTPN13), eventually reducing cell movement and proliferation. When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement. Necl-4 further facilitated the activation of extracellular signal-regulated kinase 1/2, eventually enhancing cell proliferation. Thus, Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation cooperatively with the VEGF receptor and PTPN13.

No MeSH data available.


Related in: MedlinePlus