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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

Cdh2 expression does not alter the pluripotent status of mESCs.(A) mESCs transfected with an overexpression plasmid cording for mouse Cdh2 cDNA. Morphological feature of wild-type mESCs (WT), empty plasmid-transfected mESCs (Empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) are shown. The scale bars are 50 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2, Klf4, cMyc, as well as Cdh1 and Cdh2, in the wild-type ESCs (WT), empty plasmid-transfected ESCs (empty) and Cdh2-overexpressed ESCs (Cdh2-ESC). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WBs for Pou5f1, Nanog, Sox2, Klf4, Cdh1, Cdh2 and Actin in wild-type mESCs (WT) and Cdh2-overexpressed ESCs (Cdh2-ESC).
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f5: Cdh2 expression does not alter the pluripotent status of mESCs.(A) mESCs transfected with an overexpression plasmid cording for mouse Cdh2 cDNA. Morphological feature of wild-type mESCs (WT), empty plasmid-transfected mESCs (Empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) are shown. The scale bars are 50 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2, Klf4, cMyc, as well as Cdh1 and Cdh2, in the wild-type ESCs (WT), empty plasmid-transfected ESCs (empty) and Cdh2-overexpressed ESCs (Cdh2-ESC). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WBs for Pou5f1, Nanog, Sox2, Klf4, Cdh1, Cdh2 and Actin in wild-type mESCs (WT) and Cdh2-overexpressed ESCs (Cdh2-ESC).

Mentions: We also examined if Cdh2 affects the pluripotency of mESCs. We produced a Cdh2-overexpressing mESC-line (Cdh2-ESC) that formed typical dome-shaped mESC colonies (Fig. 5A) and proliferated at the same rate as the original mESC-line (data not shown). Our qRT-PCR and WB analyses revealed that Pou5f1, Nanog and Sox2 expression did not change in Cdh2-ESCs (Fig. 5B,C). Interestingly, overexpression of Cdh2 upregulated cMyc mRNA and protein (Fig. 5B,C)


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)

Cdh2 expression does not alter the pluripotent status of mESCs.(A) mESCs transfected with an overexpression plasmid cording for mouse Cdh2 cDNA. Morphological feature of wild-type mESCs (WT), empty plasmid-transfected mESCs (Empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) are shown. The scale bars are 50 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2, Klf4, cMyc, as well as Cdh1 and Cdh2, in the wild-type ESCs (WT), empty plasmid-transfected ESCs (empty) and Cdh2-overexpressed ESCs (Cdh2-ESC). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WBs for Pou5f1, Nanog, Sox2, Klf4, Cdh1, Cdh2 and Actin in wild-type mESCs (WT) and Cdh2-overexpressed ESCs (Cdh2-ESC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588589&req=5

f5: Cdh2 expression does not alter the pluripotent status of mESCs.(A) mESCs transfected with an overexpression plasmid cording for mouse Cdh2 cDNA. Morphological feature of wild-type mESCs (WT), empty plasmid-transfected mESCs (Empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) are shown. The scale bars are 50 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2, Klf4, cMyc, as well as Cdh1 and Cdh2, in the wild-type ESCs (WT), empty plasmid-transfected ESCs (empty) and Cdh2-overexpressed ESCs (Cdh2-ESC). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) WBs for Pou5f1, Nanog, Sox2, Klf4, Cdh1, Cdh2 and Actin in wild-type mESCs (WT) and Cdh2-overexpressed ESCs (Cdh2-ESC).
Mentions: We also examined if Cdh2 affects the pluripotency of mESCs. We produced a Cdh2-overexpressing mESC-line (Cdh2-ESC) that formed typical dome-shaped mESC colonies (Fig. 5A) and proliferated at the same rate as the original mESC-line (data not shown). Our qRT-PCR and WB analyses revealed that Pou5f1, Nanog and Sox2 expression did not change in Cdh2-ESCs (Fig. 5B,C). Interestingly, overexpression of Cdh2 upregulated cMyc mRNA and protein (Fig. 5B,C)

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