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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 is localized at cell—cell contact sites and leading edges of ECs depending on cell density.A, Expression of Necls in cultured mouse ECs. Cell lysates were subjected to Western blotting using anti-Necl-4 mAb. MLEC, primary-cultured mouse lung EC; MS1, mouse pancreatic islet EC line; bEnd.3, mouse brain EC line. HEK293 cells transfected with each Necl-expressing plasmid were used as controls. B, Expression of Necl-4 in cultured human ECs. Cell lysates were subjected to Western blotting using the indicated antibodies. HAEC, human aortic EC; HMVEC, human lung microvascular EC; HBMEC, human brain microvascular EC; HUVEC, human umbilical vein EC. C, Expression of Necls in mouse coronary artery. Sections were stained with the antibodies against Necls. D, Expression of Necl-4 in mouse blood vessels. Sections were stained with anti-Necl-4 pAb and isolectin B4. E, Localization of Necl-4 at the leading edge. HUVECs were cultured in the presence or absence of 50 ng/ml VEGF and stained with anti-Necl-4 mAb. Representative low (left lane) and high (right lane) magnification images are shown (n = 3). F, Localization of Necl-4 confluent conditions. HUVECs were cultured until they formed cell—cell contact and double-stained with anti-Necl-4 mAb and anti-VE-cadherin pAb. Representative images are shown (n = 3).
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pone.0124259.g001: Necl-4 is localized at cell—cell contact sites and leading edges of ECs depending on cell density.A, Expression of Necls in cultured mouse ECs. Cell lysates were subjected to Western blotting using anti-Necl-4 mAb. MLEC, primary-cultured mouse lung EC; MS1, mouse pancreatic islet EC line; bEnd.3, mouse brain EC line. HEK293 cells transfected with each Necl-expressing plasmid were used as controls. B, Expression of Necl-4 in cultured human ECs. Cell lysates were subjected to Western blotting using the indicated antibodies. HAEC, human aortic EC; HMVEC, human lung microvascular EC; HBMEC, human brain microvascular EC; HUVEC, human umbilical vein EC. C, Expression of Necls in mouse coronary artery. Sections were stained with the antibodies against Necls. D, Expression of Necl-4 in mouse blood vessels. Sections were stained with anti-Necl-4 pAb and isolectin B4. E, Localization of Necl-4 at the leading edge. HUVECs were cultured in the presence or absence of 50 ng/ml VEGF and stained with anti-Necl-4 mAb. Representative low (left lane) and high (right lane) magnification images are shown (n = 3). F, Localization of Necl-4 confluent conditions. HUVECs were cultured until they formed cell—cell contact and double-stained with anti-Necl-4 mAb and anti-VE-cadherin pAb. Representative images are shown (n = 3).

Mentions: We first analyzed the expression in cultured ECs of Necls other than Necl-5 for which expression was previously reported [23,30]. Necl-4, but not Necl-1, Necl-2 or Necl-3, was expressed to various degrees in cultured mouse ECs (Fig 1A). Cross-reaction was not seen among the antibodies used (S1 Fig). Necl-4 was also expressed in various cultured human ECs (Fig 1B). The immunofluorescence signals for Necl-4 and Necl-5, but not for Necl-1, Necl-2 or Necl-3, were observed in mouse intramural coronary arteries (Fig 1C). The signal for Necl-4 was co-localized with that for the endothelial marker isolectin B4 in various vessels, suggesting Necl-4 expression in ECs (Fig 1D). Thus, in addition to Necl-5 [23,30], Necl-4 is also expressed in ECs in vitro and in vivo.


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 is localized at cell—cell contact sites and leading edges of ECs depending on cell density.A, Expression of Necls in cultured mouse ECs. Cell lysates were subjected to Western blotting using anti-Necl-4 mAb. MLEC, primary-cultured mouse lung EC; MS1, mouse pancreatic islet EC line; bEnd.3, mouse brain EC line. HEK293 cells transfected with each Necl-expressing plasmid were used as controls. B, Expression of Necl-4 in cultured human ECs. Cell lysates were subjected to Western blotting using the indicated antibodies. HAEC, human aortic EC; HMVEC, human lung microvascular EC; HBMEC, human brain microvascular EC; HUVEC, human umbilical vein EC. C, Expression of Necls in mouse coronary artery. Sections were stained with the antibodies against Necls. D, Expression of Necl-4 in mouse blood vessels. Sections were stained with anti-Necl-4 pAb and isolectin B4. E, Localization of Necl-4 at the leading edge. HUVECs were cultured in the presence or absence of 50 ng/ml VEGF and stained with anti-Necl-4 mAb. Representative low (left lane) and high (right lane) magnification images are shown (n = 3). F, Localization of Necl-4 confluent conditions. HUVECs were cultured until they formed cell—cell contact and double-stained with anti-Necl-4 mAb and anti-VE-cadherin pAb. Representative images are shown (n = 3).
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pone.0124259.g001: Necl-4 is localized at cell—cell contact sites and leading edges of ECs depending on cell density.A, Expression of Necls in cultured mouse ECs. Cell lysates were subjected to Western blotting using anti-Necl-4 mAb. MLEC, primary-cultured mouse lung EC; MS1, mouse pancreatic islet EC line; bEnd.3, mouse brain EC line. HEK293 cells transfected with each Necl-expressing plasmid were used as controls. B, Expression of Necl-4 in cultured human ECs. Cell lysates were subjected to Western blotting using the indicated antibodies. HAEC, human aortic EC; HMVEC, human lung microvascular EC; HBMEC, human brain microvascular EC; HUVEC, human umbilical vein EC. C, Expression of Necls in mouse coronary artery. Sections were stained with the antibodies against Necls. D, Expression of Necl-4 in mouse blood vessels. Sections were stained with anti-Necl-4 pAb and isolectin B4. E, Localization of Necl-4 at the leading edge. HUVECs were cultured in the presence or absence of 50 ng/ml VEGF and stained with anti-Necl-4 mAb. Representative low (left lane) and high (right lane) magnification images are shown (n = 3). F, Localization of Necl-4 confluent conditions. HUVECs were cultured until they formed cell—cell contact and double-stained with anti-Necl-4 mAb and anti-VE-cadherin pAb. Representative images are shown (n = 3).
Mentions: We first analyzed the expression in cultured ECs of Necls other than Necl-5 for which expression was previously reported [23,30]. Necl-4, but not Necl-1, Necl-2 or Necl-3, was expressed to various degrees in cultured mouse ECs (Fig 1A). Cross-reaction was not seen among the antibodies used (S1 Fig). Necl-4 was also expressed in various cultured human ECs (Fig 1B). The immunofluorescence signals for Necl-4 and Necl-5, but not for Necl-1, Necl-2 or Necl-3, were observed in mouse intramural coronary arteries (Fig 1C). The signal for Necl-4 was co-localized with that for the endothelial marker isolectin B4 in various vessels, suggesting Necl-4 expression in ECs (Fig 1D). Thus, in addition to Necl-5 [23,30], Necl-4 is also expressed in ECs in vitro and in vivo.

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