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Cdh2 stabilizes FGFR1 and contributes to primed-state pluripotency in mouse epiblast stem cells.

Takehara T, Teramura T, Onodera Y, Frampton J, Fukuda K - Sci Rep (2015)

Bottom Line: Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs.Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation.Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, 377-2 Ohnohigashi, Osaka-sayama, Osaka, Japan 5898511.

ABSTRACT
The cell adhesion molecule Cadherin 2 (Cdh2) plays important roles in somatic cell adhesion, proliferation and migration. Cdh2 is also highly expressed in mouse epiblast stem cells (mEpiSCs), but its function in these cells is unknown. To understand the function of Cdh2 in mEpiSCs, we compared the expression of pluripotency-related genes in mEpiSCs and mouse embryonic stem cells (mESCs) after either Cdh2 knockdown or Cdh2 over-expression. Introduction of specific siRNA against Cdh2 led to attenuation of pluripotency-related genes. Pluripotent gene expression was not recovered by over-expression of Cdh1 following Cdh2 knockdown. Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs. Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation. Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

No MeSH data available.


Related in: MedlinePlus

Expression of Cdh2 in mESCs alters FGFR1 stability along with the phosphorylation status of Erk and Akt under FGF2 stimulation.(A) WB analysis for FGFR1 in the CHX-treated wild-type mESCs (WT+CHX), CHX-treated empty plasmid-transfected mESCs (Empty+CHX) and CHX-treated Cdh2-ESCs (Cdh2-ESC+CHX) 0, 5 min, 30 min, 1 hr (1 hours), 2 hr (2 hours), 4 hr (4 hours) and 8 hr (8 hours) after FGF2 stimulation. (B) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression of Actin. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WB analysis for phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin 0, 5 min, 6 hr and 24 hr after FGF2 stimulation. (D) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression total-Erk and -Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
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f6: Expression of Cdh2 in mESCs alters FGFR1 stability along with the phosphorylation status of Erk and Akt under FGF2 stimulation.(A) WB analysis for FGFR1 in the CHX-treated wild-type mESCs (WT+CHX), CHX-treated empty plasmid-transfected mESCs (Empty+CHX) and CHX-treated Cdh2-ESCs (Cdh2-ESC+CHX) 0, 5 min, 30 min, 1 hr (1 hours), 2 hr (2 hours), 4 hr (4 hours) and 8 hr (8 hours) after FGF2 stimulation. (B) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression of Actin. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WB analysis for phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin 0, 5 min, 6 hr and 24 hr after FGF2 stimulation. (D) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression total-Erk and -Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.

Mentions: To confirm that the overexpressed Cdh2 was functional, we observed the stability of FGFR1 in Cdh2-ESCs following treatment with CHX and FGF2. In wild-type mESCs, FGFR1 rapidly degraded. On the other hand, FGFR1 levels did not diminish in Cdh2-ESCs, even 8 hrs after FGF2 addition (Fig. 6A,B). These results clearly show that Cdh2 is important for FGFR1 stability. To verify Cdh2-mediated stabilization of FGFR1, we observed expression of phosphorylated Erk and Akt in Cdh2-ESCs after FGF2 stimulation. Phosphorylated Erk and Akt were significantly higher at all time-points in Cdh2-ESCs (Fig. 6C,D). These results support our hypothesis that Cdh2 is involved in the FGF-mediated signaling though interaction with FGFR1.


Cdh2 stabilizes FGFR1 and contributes to primed-state pluripotency in mouse epiblast stem cells.

Takehara T, Teramura T, Onodera Y, Frampton J, Fukuda K - Sci Rep (2015)

Expression of Cdh2 in mESCs alters FGFR1 stability along with the phosphorylation status of Erk and Akt under FGF2 stimulation.(A) WB analysis for FGFR1 in the CHX-treated wild-type mESCs (WT+CHX), CHX-treated empty plasmid-transfected mESCs (Empty+CHX) and CHX-treated Cdh2-ESCs (Cdh2-ESC+CHX) 0, 5 min, 30 min, 1 hr (1 hours), 2 hr (2 hours), 4 hr (4 hours) and 8 hr (8 hours) after FGF2 stimulation. (B) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression of Actin. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WB analysis for phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin 0, 5 min, 6 hr and 24 hr after FGF2 stimulation. (D) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression total-Erk and -Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588589&req=5

f6: Expression of Cdh2 in mESCs alters FGFR1 stability along with the phosphorylation status of Erk and Akt under FGF2 stimulation.(A) WB analysis for FGFR1 in the CHX-treated wild-type mESCs (WT+CHX), CHX-treated empty plasmid-transfected mESCs (Empty+CHX) and CHX-treated Cdh2-ESCs (Cdh2-ESC+CHX) 0, 5 min, 30 min, 1 hr (1 hours), 2 hr (2 hours), 4 hr (4 hours) and 8 hr (8 hours) after FGF2 stimulation. (B) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression of Actin. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WB analysis for phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin 0, 5 min, 6 hr and 24 hr after FGF2 stimulation. (D) Densitometry quantification of WBs for FGFR1 and Actin. Data are normalized to the expression total-Erk and -Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
Mentions: To confirm that the overexpressed Cdh2 was functional, we observed the stability of FGFR1 in Cdh2-ESCs following treatment with CHX and FGF2. In wild-type mESCs, FGFR1 rapidly degraded. On the other hand, FGFR1 levels did not diminish in Cdh2-ESCs, even 8 hrs after FGF2 addition (Fig. 6A,B). These results clearly show that Cdh2 is important for FGFR1 stability. To verify Cdh2-mediated stabilization of FGFR1, we observed expression of phosphorylated Erk and Akt in Cdh2-ESCs after FGF2 stimulation. Phosphorylated Erk and Akt were significantly higher at all time-points in Cdh2-ESCs (Fig. 6C,D). These results support our hypothesis that Cdh2 is involved in the FGF-mediated signaling though interaction with FGFR1.

Bottom Line: Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs.Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation.Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, 377-2 Ohnohigashi, Osaka-sayama, Osaka, Japan 5898511.

ABSTRACT
The cell adhesion molecule Cadherin 2 (Cdh2) plays important roles in somatic cell adhesion, proliferation and migration. Cdh2 is also highly expressed in mouse epiblast stem cells (mEpiSCs), but its function in these cells is unknown. To understand the function of Cdh2 in mEpiSCs, we compared the expression of pluripotency-related genes in mEpiSCs and mouse embryonic stem cells (mESCs) after either Cdh2 knockdown or Cdh2 over-expression. Introduction of specific siRNA against Cdh2 led to attenuation of pluripotency-related genes. Pluripotent gene expression was not recovered by over-expression of Cdh1 following Cdh2 knockdown. Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs. Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation. Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

No MeSH data available.


Related in: MedlinePlus