Limits...
VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

Syx was identified as a downstream molecule of VEGF-A/NRP1 signaling and a RhoA activator that promoted DJM-1 cell proliferation. (A) The western blot for phospho-Akt and phospho-ERK of DJM-1 cell lysates. DJM-1 cells were treated with siRNAs (siControl, siVEGF-A or siNRP1, 20 nM respectively) under anchorage-independent conditions. The same proteins were re-immunoblotted with an anti-Akt or -ERK antibody to normalize the amounts of each phospho-protein. (B,C) The RhoA activity of DJM-1 cells under anchorage-independent conditions. (B) DJM-1 cells were treated with siControl or siVEGF-A and stimulated with (+) or without (−) VEGF-A (100 ng/ml) at the indicated time points. The siVEGF-A treatment (asterisk) decreased RhoA activity below that with the siControl treatment. (C) DJM-1 cells were treated with siVEGF-A, siNRP1, siGIPC1, and siSyx in the presence of VEGF-A (+) or its absence (−). (D) Structure of dominant negative Syx (Syx DN). An amino acid substitution of Leu 571 Glu in Syx DN prevented RhoA from interacting with the mutant. The V5 epitope was tagged at the N-terminus of Syx DN. (E) Lentiviral overexpression of Syx WT or Syx DN in DJM-1 cells. Virus infection amounts were adjusted for equal expression levels of Syx WT or Syx DN in the RhoA activity assay (F) and in the colony formation assay (G). Arrow shows Syx WT or Syx DN. (F) The RhoA activity assay for Syx DN-overexpressing DJM-1 cells under anchorage-independent conditions in the presence of VEGF-A (100 ng/ml) (+) or its absence (−). A 10% input was subsequently immunoblotted with an anti-V5 antibody to normalize the amounts of each protein. The arrow shows Syx WT or Syx DN. (G) The colony formation assay for DJM-1 cells that overexpressed Syx WT or Syx DN. These data represent the means±s.d. Percentages from each mean relative to the siControl (A), siControl (−)(B,C) or no infection (F,G) are shown below the graph. ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582117&req=5

BIO010918F5: Syx was identified as a downstream molecule of VEGF-A/NRP1 signaling and a RhoA activator that promoted DJM-1 cell proliferation. (A) The western blot for phospho-Akt and phospho-ERK of DJM-1 cell lysates. DJM-1 cells were treated with siRNAs (siControl, siVEGF-A or siNRP1, 20 nM respectively) under anchorage-independent conditions. The same proteins were re-immunoblotted with an anti-Akt or -ERK antibody to normalize the amounts of each phospho-protein. (B,C) The RhoA activity of DJM-1 cells under anchorage-independent conditions. (B) DJM-1 cells were treated with siControl or siVEGF-A and stimulated with (+) or without (−) VEGF-A (100 ng/ml) at the indicated time points. The siVEGF-A treatment (asterisk) decreased RhoA activity below that with the siControl treatment. (C) DJM-1 cells were treated with siVEGF-A, siNRP1, siGIPC1, and siSyx in the presence of VEGF-A (+) or its absence (−). (D) Structure of dominant negative Syx (Syx DN). An amino acid substitution of Leu 571 Glu in Syx DN prevented RhoA from interacting with the mutant. The V5 epitope was tagged at the N-terminus of Syx DN. (E) Lentiviral overexpression of Syx WT or Syx DN in DJM-1 cells. Virus infection amounts were adjusted for equal expression levels of Syx WT or Syx DN in the RhoA activity assay (F) and in the colony formation assay (G). Arrow shows Syx WT or Syx DN. (F) The RhoA activity assay for Syx DN-overexpressing DJM-1 cells under anchorage-independent conditions in the presence of VEGF-A (100 ng/ml) (+) or its absence (−). A 10% input was subsequently immunoblotted with an anti-V5 antibody to normalize the amounts of each protein. The arrow shows Syx WT or Syx DN. (G) The colony formation assay for DJM-1 cells that overexpressed Syx WT or Syx DN. These data represent the means±s.d. Percentages from each mean relative to the siControl (A), siControl (−)(B,C) or no infection (F,G) are shown below the graph. ***P<0.001.

Mentions: The MAPK and PI3K pathways are responsible for tumor malignancy and poor patient prognoses. The phosphorylation of MAPK (ERK) and Akt has been shown to contribute to cell proliferation and survival (Owonikoko and Khuri, 2013; Santarpia et al., 2012). However, siVEGF-A and siNRP1 did not significantly change the phosphorylation levels of either MAPK or Akt in DJM-1 cells from those in siControl cells (Fig. 5A). These results suggest that MAPK and Akt were not involved in VEGF-A/NRP1-induced DJM-1 cell proliferation.Fig. 5.


VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

Syx was identified as a downstream molecule of VEGF-A/NRP1 signaling and a RhoA activator that promoted DJM-1 cell proliferation. (A) The western blot for phospho-Akt and phospho-ERK of DJM-1 cell lysates. DJM-1 cells were treated with siRNAs (siControl, siVEGF-A or siNRP1, 20 nM respectively) under anchorage-independent conditions. The same proteins were re-immunoblotted with an anti-Akt or -ERK antibody to normalize the amounts of each phospho-protein. (B,C) The RhoA activity of DJM-1 cells under anchorage-independent conditions. (B) DJM-1 cells were treated with siControl or siVEGF-A and stimulated with (+) or without (−) VEGF-A (100 ng/ml) at the indicated time points. The siVEGF-A treatment (asterisk) decreased RhoA activity below that with the siControl treatment. (C) DJM-1 cells were treated with siVEGF-A, siNRP1, siGIPC1, and siSyx in the presence of VEGF-A (+) or its absence (−). (D) Structure of dominant negative Syx (Syx DN). An amino acid substitution of Leu 571 Glu in Syx DN prevented RhoA from interacting with the mutant. The V5 epitope was tagged at the N-terminus of Syx DN. (E) Lentiviral overexpression of Syx WT or Syx DN in DJM-1 cells. Virus infection amounts were adjusted for equal expression levels of Syx WT or Syx DN in the RhoA activity assay (F) and in the colony formation assay (G). Arrow shows Syx WT or Syx DN. (F) The RhoA activity assay for Syx DN-overexpressing DJM-1 cells under anchorage-independent conditions in the presence of VEGF-A (100 ng/ml) (+) or its absence (−). A 10% input was subsequently immunoblotted with an anti-V5 antibody to normalize the amounts of each protein. The arrow shows Syx WT or Syx DN. (G) The colony formation assay for DJM-1 cells that overexpressed Syx WT or Syx DN. These data represent the means±s.d. Percentages from each mean relative to the siControl (A), siControl (−)(B,C) or no infection (F,G) are shown below the graph. ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582117&req=5

BIO010918F5: Syx was identified as a downstream molecule of VEGF-A/NRP1 signaling and a RhoA activator that promoted DJM-1 cell proliferation. (A) The western blot for phospho-Akt and phospho-ERK of DJM-1 cell lysates. DJM-1 cells were treated with siRNAs (siControl, siVEGF-A or siNRP1, 20 nM respectively) under anchorage-independent conditions. The same proteins were re-immunoblotted with an anti-Akt or -ERK antibody to normalize the amounts of each phospho-protein. (B,C) The RhoA activity of DJM-1 cells under anchorage-independent conditions. (B) DJM-1 cells were treated with siControl or siVEGF-A and stimulated with (+) or without (−) VEGF-A (100 ng/ml) at the indicated time points. The siVEGF-A treatment (asterisk) decreased RhoA activity below that with the siControl treatment. (C) DJM-1 cells were treated with siVEGF-A, siNRP1, siGIPC1, and siSyx in the presence of VEGF-A (+) or its absence (−). (D) Structure of dominant negative Syx (Syx DN). An amino acid substitution of Leu 571 Glu in Syx DN prevented RhoA from interacting with the mutant. The V5 epitope was tagged at the N-terminus of Syx DN. (E) Lentiviral overexpression of Syx WT or Syx DN in DJM-1 cells. Virus infection amounts were adjusted for equal expression levels of Syx WT or Syx DN in the RhoA activity assay (F) and in the colony formation assay (G). Arrow shows Syx WT or Syx DN. (F) The RhoA activity assay for Syx DN-overexpressing DJM-1 cells under anchorage-independent conditions in the presence of VEGF-A (100 ng/ml) (+) or its absence (−). A 10% input was subsequently immunoblotted with an anti-V5 antibody to normalize the amounts of each protein. The arrow shows Syx WT or Syx DN. (G) The colony formation assay for DJM-1 cells that overexpressed Syx WT or Syx DN. These data represent the means±s.d. Percentages from each mean relative to the siControl (A), siControl (−)(B,C) or no infection (F,G) are shown below the graph. ***P<0.001.
Mentions: The MAPK and PI3K pathways are responsible for tumor malignancy and poor patient prognoses. The phosphorylation of MAPK (ERK) and Akt has been shown to contribute to cell proliferation and survival (Owonikoko and Khuri, 2013; Santarpia et al., 2012). However, siVEGF-A and siNRP1 did not significantly change the phosphorylation levels of either MAPK or Akt in DJM-1 cells from those in siControl cells (Fig. 5A). These results suggest that MAPK and Akt were not involved in VEGF-A/NRP1-induced DJM-1 cell proliferation.Fig. 5.

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus