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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

The NRP1 cytoplasmic region was essential for VEGF-A-induced cancer cell proliferation. (A) The western blot shows the expression of NRP1 in shControl- or NRP1 shRNA-treated DJM-1 clones (No. 12 and No. 13). (B) The comparison of NRP1 expression levels among lentivirus-overexpressed NRP1WT or cytoplasmic region deletion mutants (NRP1ΔSEA or NRP1ΔCyto) in shNRP1 DJM-1 No.13 clone (upper lanes). The same proteins were re-immunoblotted with an anti-actin antibody (lower lanes). (C) The colony formation assay of the lentivirus-overexpressed NRP1WT, NRP1ΔSEA or NRP1ΔCyto in the shNRP1 DJM-1 No.13 clone and shControl DJM-1 clone. (A,C) Percentages from each mean relative to the shControl are shown below the graph. (D) Co-immunoprecipitation assay with GIPC1 (a) or Syx (b). NRP1, GIPC1, and Syx were expressed in HEK293T cells and treated without (−) or with (+) VEGF-A (100 ng/ml) for 15 min. (a) Increased NRP1/GIPC1 and GIPC1/Syx interactions in the presence of VEGF-A are indicated by asterisks. (b) The Syx/GIPC1 interaction was increased in the presence of VEGF-A (asterisk). On the other hand, the NRP1/Syx interaction was decreased (asterisk). A 10% input as the loading control of NRP1, GIPC1, and Syx co-expressed in HEK293T cell lysates are shown in the right panels. Percentages from each blotted protein amount relative to “VEGF-A (−)” are shown below each lane. (E) Confirmation of the siRNA effects for GIPC1 or Syx. GIPC1 or Syx was overexpressed in HEK293T cells treated with 20 nM siControl, siGIPC1, or siSyx. The inhibitory efficiency of each siRNA on the expression of GIPC1 or Syx was compared to the siControl. The inhibitory percentages relative to the siControl are shown below each lane. (F) Colony formation assay in siNRP1, siGIPC1, or siSyx treated-DJM-1 cells in the presence or absence of exogenous VEGF-A (1 µg/ml) indicated as white columns (+) or black columns (−), respectively. Percentages from each mean relative to the siControl are shown below the graph. These data represent the means±s.d. N.S., not significant; **P<0.005; ***P<0.001.
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BIO010918F4: The NRP1 cytoplasmic region was essential for VEGF-A-induced cancer cell proliferation. (A) The western blot shows the expression of NRP1 in shControl- or NRP1 shRNA-treated DJM-1 clones (No. 12 and No. 13). (B) The comparison of NRP1 expression levels among lentivirus-overexpressed NRP1WT or cytoplasmic region deletion mutants (NRP1ΔSEA or NRP1ΔCyto) in shNRP1 DJM-1 No.13 clone (upper lanes). The same proteins were re-immunoblotted with an anti-actin antibody (lower lanes). (C) The colony formation assay of the lentivirus-overexpressed NRP1WT, NRP1ΔSEA or NRP1ΔCyto in the shNRP1 DJM-1 No.13 clone and shControl DJM-1 clone. (A,C) Percentages from each mean relative to the shControl are shown below the graph. (D) Co-immunoprecipitation assay with GIPC1 (a) or Syx (b). NRP1, GIPC1, and Syx were expressed in HEK293T cells and treated without (−) or with (+) VEGF-A (100 ng/ml) for 15 min. (a) Increased NRP1/GIPC1 and GIPC1/Syx interactions in the presence of VEGF-A are indicated by asterisks. (b) The Syx/GIPC1 interaction was increased in the presence of VEGF-A (asterisk). On the other hand, the NRP1/Syx interaction was decreased (asterisk). A 10% input as the loading control of NRP1, GIPC1, and Syx co-expressed in HEK293T cell lysates are shown in the right panels. Percentages from each blotted protein amount relative to “VEGF-A (−)” are shown below each lane. (E) Confirmation of the siRNA effects for GIPC1 or Syx. GIPC1 or Syx was overexpressed in HEK293T cells treated with 20 nM siControl, siGIPC1, or siSyx. The inhibitory efficiency of each siRNA on the expression of GIPC1 or Syx was compared to the siControl. The inhibitory percentages relative to the siControl are shown below each lane. (F) Colony formation assay in siNRP1, siGIPC1, or siSyx treated-DJM-1 cells in the presence or absence of exogenous VEGF-A (1 µg/ml) indicated as white columns (+) or black columns (−), respectively. Percentages from each mean relative to the siControl are shown below the graph. These data represent the means±s.d. N.S., not significant; **P<0.005; ***P<0.001.

Mentions: NRP1 does not have any known signaling motif in the short 44 amino acid cytoplasmic region; therefore, it currently remains unclear whether this domain is involved in signaling. We constructed a shNRP1 vector to abrogate the expression of NRP1 in DJM-1 cells. The sequence of shNRP1 was based on siNRP1 #3, which targeted NRP1 3′UTR. shNRP1 clones (No. 12 and No. 13) did not express NRP1 and also did not support DJM-1 cell proliferation (shControl: 100%, shNRP1-12: 35%, shNRP1-13: 24%, respectively) (Fig. 4A). In subsequent experiments, we used shNRP1 clone No. 13 and infected shNRP1-DJM-1 cell clones with NRP1WT, NRP1 lacking the 44 amino acid cytoplasmic region (NRP1ΔCyto), or NRP1 lacking the C-terminus amino acids, SEA (NRP1ΔSEA) (Fig. 4B). The growth of the shNRP1 clone was less than that of the shControl clone (40% of shControl) (Fig. 4C). The lentiviral overexpression of NRP1WT restored growth, whereas NRP1ΔSEA and NRP1ΔCyto did not (shNRP1+WT: 90%, shNRP1+ΔSEA: 27%, shNRP1+ΔCyto: 23%, respectively) (Fig. 4C). These results suggested that the NRP1 cytoplasmic region, containing SEA, was essential for VEGF-A-induced proliferation.Fig. 4.


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)

The NRP1 cytoplasmic region was essential for VEGF-A-induced cancer cell proliferation. (A) The western blot shows the expression of NRP1 in shControl- or NRP1 shRNA-treated DJM-1 clones (No. 12 and No. 13). (B) The comparison of NRP1 expression levels among lentivirus-overexpressed NRP1WT or cytoplasmic region deletion mutants (NRP1ΔSEA or NRP1ΔCyto) in shNRP1 DJM-1 No.13 clone (upper lanes). The same proteins were re-immunoblotted with an anti-actin antibody (lower lanes). (C) The colony formation assay of the lentivirus-overexpressed NRP1WT, NRP1ΔSEA or NRP1ΔCyto in the shNRP1 DJM-1 No.13 clone and shControl DJM-1 clone. (A,C) Percentages from each mean relative to the shControl are shown below the graph. (D) Co-immunoprecipitation assay with GIPC1 (a) or Syx (b). NRP1, GIPC1, and Syx were expressed in HEK293T cells and treated without (−) or with (+) VEGF-A (100 ng/ml) for 15 min. (a) Increased NRP1/GIPC1 and GIPC1/Syx interactions in the presence of VEGF-A are indicated by asterisks. (b) The Syx/GIPC1 interaction was increased in the presence of VEGF-A (asterisk). On the other hand, the NRP1/Syx interaction was decreased (asterisk). A 10% input as the loading control of NRP1, GIPC1, and Syx co-expressed in HEK293T cell lysates are shown in the right panels. Percentages from each blotted protein amount relative to “VEGF-A (−)” are shown below each lane. (E) Confirmation of the siRNA effects for GIPC1 or Syx. GIPC1 or Syx was overexpressed in HEK293T cells treated with 20 nM siControl, siGIPC1, or siSyx. The inhibitory efficiency of each siRNA on the expression of GIPC1 or Syx was compared to the siControl. The inhibitory percentages relative to the siControl are shown below each lane. (F) Colony formation assay in siNRP1, siGIPC1, or siSyx treated-DJM-1 cells in the presence or absence of exogenous VEGF-A (1 µg/ml) indicated as white columns (+) or black columns (−), respectively. Percentages from each mean relative to the siControl are shown below the graph. These data represent the means±s.d. N.S., not significant; **P<0.005; ***P<0.001.
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BIO010918F4: The NRP1 cytoplasmic region was essential for VEGF-A-induced cancer cell proliferation. (A) The western blot shows the expression of NRP1 in shControl- or NRP1 shRNA-treated DJM-1 clones (No. 12 and No. 13). (B) The comparison of NRP1 expression levels among lentivirus-overexpressed NRP1WT or cytoplasmic region deletion mutants (NRP1ΔSEA or NRP1ΔCyto) in shNRP1 DJM-1 No.13 clone (upper lanes). The same proteins were re-immunoblotted with an anti-actin antibody (lower lanes). (C) The colony formation assay of the lentivirus-overexpressed NRP1WT, NRP1ΔSEA or NRP1ΔCyto in the shNRP1 DJM-1 No.13 clone and shControl DJM-1 clone. (A,C) Percentages from each mean relative to the shControl are shown below the graph. (D) Co-immunoprecipitation assay with GIPC1 (a) or Syx (b). NRP1, GIPC1, and Syx were expressed in HEK293T cells and treated without (−) or with (+) VEGF-A (100 ng/ml) for 15 min. (a) Increased NRP1/GIPC1 and GIPC1/Syx interactions in the presence of VEGF-A are indicated by asterisks. (b) The Syx/GIPC1 interaction was increased in the presence of VEGF-A (asterisk). On the other hand, the NRP1/Syx interaction was decreased (asterisk). A 10% input as the loading control of NRP1, GIPC1, and Syx co-expressed in HEK293T cell lysates are shown in the right panels. Percentages from each blotted protein amount relative to “VEGF-A (−)” are shown below each lane. (E) Confirmation of the siRNA effects for GIPC1 or Syx. GIPC1 or Syx was overexpressed in HEK293T cells treated with 20 nM siControl, siGIPC1, or siSyx. The inhibitory efficiency of each siRNA on the expression of GIPC1 or Syx was compared to the siControl. The inhibitory percentages relative to the siControl are shown below each lane. (F) Colony formation assay in siNRP1, siGIPC1, or siSyx treated-DJM-1 cells in the presence or absence of exogenous VEGF-A (1 µg/ml) indicated as white columns (+) or black columns (−), respectively. Percentages from each mean relative to the siControl are shown below the graph. These data represent the means±s.d. N.S., not significant; **P<0.005; ***P<0.001.
Mentions: NRP1 does not have any known signaling motif in the short 44 amino acid cytoplasmic region; therefore, it currently remains unclear whether this domain is involved in signaling. We constructed a shNRP1 vector to abrogate the expression of NRP1 in DJM-1 cells. The sequence of shNRP1 was based on siNRP1 #3, which targeted NRP1 3′UTR. shNRP1 clones (No. 12 and No. 13) did not express NRP1 and also did not support DJM-1 cell proliferation (shControl: 100%, shNRP1-12: 35%, shNRP1-13: 24%, respectively) (Fig. 4A). In subsequent experiments, we used shNRP1 clone No. 13 and infected shNRP1-DJM-1 cell clones with NRP1WT, NRP1 lacking the 44 amino acid cytoplasmic region (NRP1ΔCyto), or NRP1 lacking the C-terminus amino acids, SEA (NRP1ΔSEA) (Fig. 4B). The growth of the shNRP1 clone was less than that of the shControl clone (40% of shControl) (Fig. 4C). The lentiviral overexpression of NRP1WT restored growth, whereas NRP1ΔSEA and NRP1ΔCyto did not (shNRP1+WT: 90%, shNRP1+ΔSEA: 27%, shNRP1+ΔCyto: 23%, respectively) (Fig. 4C). These results suggested that the NRP1 cytoplasmic region, containing SEA, was essential for VEGF-A-induced proliferation.Fig. 4.

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