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

The oligopeptide that inhibited the GIPC1 and Syx interaction suppressed RhoA activity and the proliferation of DJM-1 cells. (A) A schematic of the construct that contained TAT, EGFP, and the Gly insertion prior to the Targeted peptide sequence (STLTASEV; Syx C terminus sequence). The Scrambled peptide amino acid sequence is also shown in the lower case (EASTSLVT). (B) Confirmation of the peptide incorporation into DJM-1 cells. DJM-1 cells were treated with the Scrambled or Targeted peptide (500 nM each) for 1 h. Confocal images indicated the Scrambled or Targeted peptide in the intracellular region of DJM-1 cells (green). Nuclei in the same position were shown in the upper panels (blue). Scale bar: 30 μm. (C) The co-immunoprecipitation assay with the Target peptide. HA-tagged GIPC1 was overexpressed in HEK293T cells. The Scrambled or Targeted peptide was mixed with the cell lysate and co-immunoprecipitated with GIPC1 after a 1 h rotation at 4°C. The same lysates (10% input) were immunoblotted with anti-GFP or anti-GIPC1 antibodies to normalize the amounts of the peptide and GIPC1. Percentages from each relative to the Scrambled are shown below the graph. (D) NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells, which were subsequently treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A induced the GIPC1/Syx interaction in the presence of the Scrambled peptide (asterisk). On the other hand, the Targeted peptide abrogated the GIPC1/Syx interaction (asterisk). Percentages from each protein level [GIPC1 or V5 (Syx)] compared to the lane of Scrambled (−) are indicated below the lane. The same lysates (10% input) were immunoblotted with anti-GIPC1 or V5 antibodies to normalize the amounts of each protein. (E) The RhoA activity assay. DJM-1 cells were treated with the Targeted or Scrambled peptide and stimulated with (+) or without (−) VEGF-A (100 ng/ml) under anchorage-independent conditions. The same lysates (10% input) were immunoblotted with an anti-RhoA antibody to normalize the protein amounts with each treatment. Percentages from each relative to the Scrambled (−) are shown below the graph. (F) The colony formation assay. DJM-1 cells were treated with 500 nM of the Targeted or Scrambled peptide. The Targeted peptide inhibited DJM-1 cell proliferation, whereas the Scrambled peptide did not. These data represent the means±s.d. Percentages from each mean relative to the no addition control are shown below the graph. ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

BIO010918F7: The oligopeptide that inhibited the GIPC1 and Syx interaction suppressed RhoA activity and the proliferation of DJM-1 cells. (A) A schematic of the construct that contained TAT, EGFP, and the Gly insertion prior to the Targeted peptide sequence (STLTASEV; Syx C terminus sequence). The Scrambled peptide amino acid sequence is also shown in the lower case (EASTSLVT). (B) Confirmation of the peptide incorporation into DJM-1 cells. DJM-1 cells were treated with the Scrambled or Targeted peptide (500 nM each) for 1 h. Confocal images indicated the Scrambled or Targeted peptide in the intracellular region of DJM-1 cells (green). Nuclei in the same position were shown in the upper panels (blue). Scale bar: 30 μm. (C) The co-immunoprecipitation assay with the Target peptide. HA-tagged GIPC1 was overexpressed in HEK293T cells. The Scrambled or Targeted peptide was mixed with the cell lysate and co-immunoprecipitated with GIPC1 after a 1 h rotation at 4°C. The same lysates (10% input) were immunoblotted with anti-GFP or anti-GIPC1 antibodies to normalize the amounts of the peptide and GIPC1. Percentages from each relative to the Scrambled are shown below the graph. (D) NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells, which were subsequently treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A induced the GIPC1/Syx interaction in the presence of the Scrambled peptide (asterisk). On the other hand, the Targeted peptide abrogated the GIPC1/Syx interaction (asterisk). Percentages from each protein level [GIPC1 or V5 (Syx)] compared to the lane of Scrambled (−) are indicated below the lane. The same lysates (10% input) were immunoblotted with anti-GIPC1 or V5 antibodies to normalize the amounts of each protein. (E) The RhoA activity assay. DJM-1 cells were treated with the Targeted or Scrambled peptide and stimulated with (+) or without (−) VEGF-A (100 ng/ml) under anchorage-independent conditions. The same lysates (10% input) were immunoblotted with an anti-RhoA antibody to normalize the protein amounts with each treatment. Percentages from each relative to the Scrambled (−) are shown below the graph. (F) The colony formation assay. DJM-1 cells were treated with 500 nM of the Targeted or Scrambled peptide. The Targeted peptide inhibited DJM-1 cell proliferation, whereas the Scrambled peptide did not. These data represent the means±s.d. Percentages from each mean relative to the no addition control are shown below the graph. ***P<0.001.

Mentions: We designed a membrane-penetrating peptide targeted to inhibit complex formation between GIPC1 and Syx (Fig. 7A). The 30 kDa targeted peptide consisted of TAT, a cell penetrating sequence of the HIV virus, EGFP, and eight amino acid residues that included the Syx C-terminal amino acid sequence (STLTASEV). The Syx C-terminal amino acid sequence was important for recognizing the GIPC1 PDZ domain in the GIPC1/Syx interaction; therefore, the targeted peptide acted as a competitive inhibitor. The incorporation of these peptides into DJM-1 cells was confirmed through the detection of a green fluorescent protein linked to the peptides after a 1 h treatment (Fig. 7B). In order to establish whether the Targeted peptide interacted with GIPC1, HA-tagged GIPC1 was overexpressed in HEK293T cells and the cell lysate was incubated with either the Scrambled peptide or Targeted peptide. Binding of the Targeted peptide with GIPC1 was 3-fold greater than that with the Scrambled peptide (Fig. 7C). In order to evaluate whether the Targeted peptide inhibited the interaction between GIPC1 and Syx, NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells and these cells were then treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A/NRP1 induced GIPC1/Syx complex formation in the presence of the Scrambled peptide (Fig. 7D, asterisk). On the other hand, the Targeted peptide abrogated the VEGF-A/NRP1 signal-induced GIPC1/Syx interaction (Fig. 7D, asterisk). In addition, the Targeted peptide more strongly prevented the activation of RhoA than the Scrambled peptide in the absence and presence of VEGF-A (Fig. 7E). Additionally, DJM-1 cell proliferation was inhibited by the Targeted peptide (Scramble peptide: 99%, Targeted peptide: 43%) (Fig. 7F). These results demonstrated that, in the VEGF-A/NRP1 signaling pathway, the GIPC1 and Syx interaction was necessary for the activation of RhoA in order to promote the proliferation of cancer cells.Fig. 7.


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 oligopeptide that inhibited the GIPC1 and Syx interaction suppressed RhoA activity and the proliferation of DJM-1 cells. (A) A schematic of the construct that contained TAT, EGFP, and the Gly insertion prior to the Targeted peptide sequence (STLTASEV; Syx C terminus sequence). The Scrambled peptide amino acid sequence is also shown in the lower case (EASTSLVT). (B) Confirmation of the peptide incorporation into DJM-1 cells. DJM-1 cells were treated with the Scrambled or Targeted peptide (500 nM each) for 1 h. Confocal images indicated the Scrambled or Targeted peptide in the intracellular region of DJM-1 cells (green). Nuclei in the same position were shown in the upper panels (blue). Scale bar: 30 μm. (C) The co-immunoprecipitation assay with the Target peptide. HA-tagged GIPC1 was overexpressed in HEK293T cells. The Scrambled or Targeted peptide was mixed with the cell lysate and co-immunoprecipitated with GIPC1 after a 1 h rotation at 4°C. The same lysates (10% input) were immunoblotted with anti-GFP or anti-GIPC1 antibodies to normalize the amounts of the peptide and GIPC1. Percentages from each relative to the Scrambled are shown below the graph. (D) NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells, which were subsequently treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A induced the GIPC1/Syx interaction in the presence of the Scrambled peptide (asterisk). On the other hand, the Targeted peptide abrogated the GIPC1/Syx interaction (asterisk). Percentages from each protein level [GIPC1 or V5 (Syx)] compared to the lane of Scrambled (−) are indicated below the lane. The same lysates (10% input) were immunoblotted with anti-GIPC1 or V5 antibodies to normalize the amounts of each protein. (E) The RhoA activity assay. DJM-1 cells were treated with the Targeted or Scrambled peptide and stimulated with (+) or without (−) VEGF-A (100 ng/ml) under anchorage-independent conditions. The same lysates (10% input) were immunoblotted with an anti-RhoA antibody to normalize the protein amounts with each treatment. Percentages from each relative to the Scrambled (−) are shown below the graph. (F) The colony formation assay. DJM-1 cells were treated with 500 nM of the Targeted or Scrambled peptide. The Targeted peptide inhibited DJM-1 cell proliferation, whereas the Scrambled peptide did not. These data represent the means±s.d. Percentages from each mean relative to the no addition control 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

BIO010918F7: The oligopeptide that inhibited the GIPC1 and Syx interaction suppressed RhoA activity and the proliferation of DJM-1 cells. (A) A schematic of the construct that contained TAT, EGFP, and the Gly insertion prior to the Targeted peptide sequence (STLTASEV; Syx C terminus sequence). The Scrambled peptide amino acid sequence is also shown in the lower case (EASTSLVT). (B) Confirmation of the peptide incorporation into DJM-1 cells. DJM-1 cells were treated with the Scrambled or Targeted peptide (500 nM each) for 1 h. Confocal images indicated the Scrambled or Targeted peptide in the intracellular region of DJM-1 cells (green). Nuclei in the same position were shown in the upper panels (blue). Scale bar: 30 μm. (C) The co-immunoprecipitation assay with the Target peptide. HA-tagged GIPC1 was overexpressed in HEK293T cells. The Scrambled or Targeted peptide was mixed with the cell lysate and co-immunoprecipitated with GIPC1 after a 1 h rotation at 4°C. The same lysates (10% input) were immunoblotted with anti-GFP or anti-GIPC1 antibodies to normalize the amounts of the peptide and GIPC1. Percentages from each relative to the Scrambled are shown below the graph. (D) NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells, which were subsequently treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A induced the GIPC1/Syx interaction in the presence of the Scrambled peptide (asterisk). On the other hand, the Targeted peptide abrogated the GIPC1/Syx interaction (asterisk). Percentages from each protein level [GIPC1 or V5 (Syx)] compared to the lane of Scrambled (−) are indicated below the lane. The same lysates (10% input) were immunoblotted with anti-GIPC1 or V5 antibodies to normalize the amounts of each protein. (E) The RhoA activity assay. DJM-1 cells were treated with the Targeted or Scrambled peptide and stimulated with (+) or without (−) VEGF-A (100 ng/ml) under anchorage-independent conditions. The same lysates (10% input) were immunoblotted with an anti-RhoA antibody to normalize the protein amounts with each treatment. Percentages from each relative to the Scrambled (−) are shown below the graph. (F) The colony formation assay. DJM-1 cells were treated with 500 nM of the Targeted or Scrambled peptide. The Targeted peptide inhibited DJM-1 cell proliferation, whereas the Scrambled peptide did not. These data represent the means±s.d. Percentages from each mean relative to the no addition control are shown below the graph. ***P<0.001.
Mentions: We designed a membrane-penetrating peptide targeted to inhibit complex formation between GIPC1 and Syx (Fig. 7A). The 30 kDa targeted peptide consisted of TAT, a cell penetrating sequence of the HIV virus, EGFP, and eight amino acid residues that included the Syx C-terminal amino acid sequence (STLTASEV). The Syx C-terminal amino acid sequence was important for recognizing the GIPC1 PDZ domain in the GIPC1/Syx interaction; therefore, the targeted peptide acted as a competitive inhibitor. The incorporation of these peptides into DJM-1 cells was confirmed through the detection of a green fluorescent protein linked to the peptides after a 1 h treatment (Fig. 7B). In order to establish whether the Targeted peptide interacted with GIPC1, HA-tagged GIPC1 was overexpressed in HEK293T cells and the cell lysate was incubated with either the Scrambled peptide or Targeted peptide. Binding of the Targeted peptide with GIPC1 was 3-fold greater than that with the Scrambled peptide (Fig. 7C). In order to evaluate whether the Targeted peptide inhibited the interaction between GIPC1 and Syx, NRP1, GIPC1, and Syx vectors were transfected and expressed in HEK293T cells and these cells were then treated with the Targeted or Scrambled peptide for 16 h. After a 10 min stimulation with (+) or without (−) VEGF-A (100 ng/ml), the cells were lysed and the indicated proteins in the cell lysates were co-immunoprecipitated with V5-tagged Syx (left panels). VEGF-A/NRP1 induced GIPC1/Syx complex formation in the presence of the Scrambled peptide (Fig. 7D, asterisk). On the other hand, the Targeted peptide abrogated the VEGF-A/NRP1 signal-induced GIPC1/Syx interaction (Fig. 7D, asterisk). In addition, the Targeted peptide more strongly prevented the activation of RhoA than the Scrambled peptide in the absence and presence of VEGF-A (Fig. 7E). Additionally, DJM-1 cell proliferation was inhibited by the Targeted peptide (Scramble peptide: 99%, Targeted peptide: 43%) (Fig. 7F). These results demonstrated that, in the VEGF-A/NRP1 signaling pathway, the GIPC1 and Syx interaction was necessary for the activation of RhoA in order to promote the proliferation of cancer cells.Fig. 7.

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