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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

RhoA activity was essential for the DJM-1 cell proliferation signal to induce p27kip1 protein degradation. (A,B) The colony formation assay for DJM-1 cells. (A) DJM-1 cells were treated with siRNAs (siControl, siVEGF-A, 20 nM each) and C3 exoenzyme (2 μg/ml) in the presence or absence of exogenous VEGF-A (1 µg/ml), indicated as white columns (+) or black columns (−), respectively. (B) DJM-1 cells were treated with the ROCK inhibitor, Y27632 (10 or 20 μM). (C) An increase in the lentiviral infection of RhoA constitutively active form (RhoA CA) enhanced the RhoA active form in DJM-1 cells. (D) The colony formation assay for siRNAs (siControl or siVEGF-A, 20 nM each) treated-DJM-1 cells with (+) or without (−) RhoA CA expression. Percentages from each relative to the siControl (−)(A,D) or no addition (B) are shown below the graph. (E) DJM-1 cells were treated with siControl, siVEGF-A, or siNRP1 (20 nM each) under anchorage-independent conditions and total cell lysates were immunoblotted with an anti-p27 antibody. Percentages from the p27 level in each siRNA treated-cell lysate relative to siNRP1 are indicated below the lane. The same proteins were re-immunoblotted with an anti-actin antibody to normalize the amounts of each protein. These data represent the means±s.d. **P<0.005; ***P<0.001.
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BIO010918F6: RhoA activity was essential for the DJM-1 cell proliferation signal to induce p27kip1 protein degradation. (A,B) The colony formation assay for DJM-1 cells. (A) DJM-1 cells were treated with siRNAs (siControl, siVEGF-A, 20 nM each) and C3 exoenzyme (2 μg/ml) in the presence or absence of exogenous VEGF-A (1 µg/ml), indicated as white columns (+) or black columns (−), respectively. (B) DJM-1 cells were treated with the ROCK inhibitor, Y27632 (10 or 20 μM). (C) An increase in the lentiviral infection of RhoA constitutively active form (RhoA CA) enhanced the RhoA active form in DJM-1 cells. (D) The colony formation assay for siRNAs (siControl or siVEGF-A, 20 nM each) treated-DJM-1 cells with (+) or without (−) RhoA CA expression. Percentages from each relative to the siControl (−)(A,D) or no addition (B) are shown below the graph. (E) DJM-1 cells were treated with siControl, siVEGF-A, or siNRP1 (20 nM each) under anchorage-independent conditions and total cell lysates were immunoblotted with an anti-p27 antibody. Percentages from the p27 level in each siRNA treated-cell lysate relative to siNRP1 are indicated below the lane. The same proteins were re-immunoblotted with an anti-actin antibody to normalize the amounts of each protein. These data represent the means±s.d. **P<0.005; ***P<0.001.

Mentions: In order to determine whether the activation of RhoA promoted DJM-1 cell proliferation, DJM-1 cells were treated with C3 exoenzyme, a specific inhibitor of RhoA. C3 exoenzyme completely suppressed DJM-1 cell proliferation in the absence and presence of exogenous VEGF-A (2% and 1% of siControl, respectively) (Fig. 6A). Y27632, a ROCK inhibitor that is a downstream effector of RhoA, suppressed DJM-1 cell proliferation (no addition: 100%, 10 µM: 51%, 20 µM: 50%, respectively) (Fig. 6B). Proliferation was recovered (31% to 82%) when RhoA constitutively active form (RhoA CA) was overexpressed in siVEGF-A-treated DJM-1 cells (Fig. 6C,D). p27 was degraded by the activation of RhoA, thereby leading to cell proliferation (S-phase entry). p27 is an inhibitor of G1 cyclin-dependent kinase and regulates cell proliferation downstream of RhoA. Under anchorage-independent conditions, the accumulation of p27 was greater in siVEGF-A- and siNRP1-treated DJM-1 cells than in siControl-treated DJM-1 cells (Fig. 6E). Taken together, these results demonstrated that VEGF-A/NRP1 signaling activated RhoA activity via a GIPC1/Syx complex to inhibit the accumulation of p27.Fig. 6.


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)

RhoA activity was essential for the DJM-1 cell proliferation signal to induce p27kip1 protein degradation. (A,B) The colony formation assay for DJM-1 cells. (A) DJM-1 cells were treated with siRNAs (siControl, siVEGF-A, 20 nM each) and C3 exoenzyme (2 μg/ml) in the presence or absence of exogenous VEGF-A (1 µg/ml), indicated as white columns (+) or black columns (−), respectively. (B) DJM-1 cells were treated with the ROCK inhibitor, Y27632 (10 or 20 μM). (C) An increase in the lentiviral infection of RhoA constitutively active form (RhoA CA) enhanced the RhoA active form in DJM-1 cells. (D) The colony formation assay for siRNAs (siControl or siVEGF-A, 20 nM each) treated-DJM-1 cells with (+) or without (−) RhoA CA expression. Percentages from each relative to the siControl (−)(A,D) or no addition (B) are shown below the graph. (E) DJM-1 cells were treated with siControl, siVEGF-A, or siNRP1 (20 nM each) under anchorage-independent conditions and total cell lysates were immunoblotted with an anti-p27 antibody. Percentages from the p27 level in each siRNA treated-cell lysate relative to siNRP1 are indicated below the lane. The same proteins were re-immunoblotted with an anti-actin antibody to normalize the amounts of each protein. These data represent the means±s.d. **P<0.005; ***P<0.001.
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Related In: Results  -  Collection

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BIO010918F6: RhoA activity was essential for the DJM-1 cell proliferation signal to induce p27kip1 protein degradation. (A,B) The colony formation assay for DJM-1 cells. (A) DJM-1 cells were treated with siRNAs (siControl, siVEGF-A, 20 nM each) and C3 exoenzyme (2 μg/ml) in the presence or absence of exogenous VEGF-A (1 µg/ml), indicated as white columns (+) or black columns (−), respectively. (B) DJM-1 cells were treated with the ROCK inhibitor, Y27632 (10 or 20 μM). (C) An increase in the lentiviral infection of RhoA constitutively active form (RhoA CA) enhanced the RhoA active form in DJM-1 cells. (D) The colony formation assay for siRNAs (siControl or siVEGF-A, 20 nM each) treated-DJM-1 cells with (+) or without (−) RhoA CA expression. Percentages from each relative to the siControl (−)(A,D) or no addition (B) are shown below the graph. (E) DJM-1 cells were treated with siControl, siVEGF-A, or siNRP1 (20 nM each) under anchorage-independent conditions and total cell lysates were immunoblotted with an anti-p27 antibody. Percentages from the p27 level in each siRNA treated-cell lysate relative to siNRP1 are indicated below the lane. The same proteins were re-immunoblotted with an anti-actin antibody to normalize the amounts of each protein. These data represent the means±s.d. **P<0.005; ***P<0.001.
Mentions: In order to determine whether the activation of RhoA promoted DJM-1 cell proliferation, DJM-1 cells were treated with C3 exoenzyme, a specific inhibitor of RhoA. C3 exoenzyme completely suppressed DJM-1 cell proliferation in the absence and presence of exogenous VEGF-A (2% and 1% of siControl, respectively) (Fig. 6A). Y27632, a ROCK inhibitor that is a downstream effector of RhoA, suppressed DJM-1 cell proliferation (no addition: 100%, 10 µM: 51%, 20 µM: 50%, respectively) (Fig. 6B). Proliferation was recovered (31% to 82%) when RhoA constitutively active form (RhoA CA) was overexpressed in siVEGF-A-treated DJM-1 cells (Fig. 6C,D). p27 was degraded by the activation of RhoA, thereby leading to cell proliferation (S-phase entry). p27 is an inhibitor of G1 cyclin-dependent kinase and regulates cell proliferation downstream of RhoA. Under anchorage-independent conditions, the accumulation of p27 was greater in siVEGF-A- and siNRP1-treated DJM-1 cells than in siControl-treated DJM-1 cells (Fig. 6E). Taken together, these results demonstrated that VEGF-A/NRP1 signaling activated RhoA activity via a GIPC1/Syx complex to inhibit the accumulation of p27.Fig. 6.

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