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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

Overexpression of Cdh1 does not restore the function of Cdh2 in mEpiSCs.(A) The mEpiSC-line was transfected with an overexpression plasmid cording for mouse Cdh1 cDNA. Cdh1-expressing mEpiSCs (Cdh1-EpiSC) highly expressed the Cdh1 protein. NTC indicates control mEpiSCs and Empty indicates empty plasmid-transfected mEpiSCs. The right panel shows the localization of Cdh1. The scale bar is 5 μm. (B) SSEA-1 expression in Cdh1-EpiSCs after treatment with ADH-1 (Cdh1-EpiSC+ADH-1), control siRNA (Cdh1-EpiSC+siScramble) or Cdh2 siRNA (Cdh1-EpiSC+siCdh2). The white dotted lines demarcate the colony regions. Scale bars are 100 μm. (C) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in the non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). Data are normalized to the expression level of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (D) WB analysis for Pou5f1, Nanog, Sox2, cMyc, phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin in non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). (E) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt. Data are normalized to the expression total-Erk and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
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f3: Overexpression of Cdh1 does not restore the function of Cdh2 in mEpiSCs.(A) The mEpiSC-line was transfected with an overexpression plasmid cording for mouse Cdh1 cDNA. Cdh1-expressing mEpiSCs (Cdh1-EpiSC) highly expressed the Cdh1 protein. NTC indicates control mEpiSCs and Empty indicates empty plasmid-transfected mEpiSCs. The right panel shows the localization of Cdh1. The scale bar is 5 μm. (B) SSEA-1 expression in Cdh1-EpiSCs after treatment with ADH-1 (Cdh1-EpiSC+ADH-1), control siRNA (Cdh1-EpiSC+siScramble) or Cdh2 siRNA (Cdh1-EpiSC+siCdh2). The white dotted lines demarcate the colony regions. Scale bars are 100 μm. (C) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in the non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). Data are normalized to the expression level of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (D) WB analysis for Pou5f1, Nanog, Sox2, cMyc, phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin in non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). (E) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt. Data are normalized to the expression total-Erk and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.

Mentions: To identify the specific function of Cdh2 and its significance in determining primed state pluripotency, we examined if Cdh1 could compensate for a loss of Cdh2 function in mEpiSCs. For this experiment, we produced a mEpiSC line (Cdh1-EpiSC) that stably expressed Cdh1 and maintained pluripotency in standard mEpiSC medium. Cdh1 localized to the surface of Cdh1-EpiSCs (Fig. 3A). Interestingly, even though Cdh1-EpiSCs strongly expressed cell surface Cdh1, some cells lost SSEA-1 expression after treatment with either ADH-1 or siCdh2 (Fig. 3B). To investigate this phenomenon in detail, we performed qRT-PCR and WB assays for pluripotency-related genes. ADH-1 suppressed the expression of Nanog, Sox2 and cMyc, although Cdh1 expression rescued Pou5f1. Furthermore, consistent with the results from wild-type mEpiSCs, Eomes was upregulated by ADH-1 treatment. Treatment with siCdh2 had similar effects on Sox2, cMyc and Eomes expression (Fig. 3C). In addition, we found that siCdh2 treatment suppressed Erk and Akt phosphorylation in Cdh1-EpiSCs (Fig. 3D,E). These results suggest that Cdh2 is important for Erk and Akt phosphorylation and that suppression of Cdh2 decreases the expression of pluripotency-related genes.


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)

Overexpression of Cdh1 does not restore the function of Cdh2 in mEpiSCs.(A) The mEpiSC-line was transfected with an overexpression plasmid cording for mouse Cdh1 cDNA. Cdh1-expressing mEpiSCs (Cdh1-EpiSC) highly expressed the Cdh1 protein. NTC indicates control mEpiSCs and Empty indicates empty plasmid-transfected mEpiSCs. The right panel shows the localization of Cdh1. The scale bar is 5 μm. (B) SSEA-1 expression in Cdh1-EpiSCs after treatment with ADH-1 (Cdh1-EpiSC+ADH-1), control siRNA (Cdh1-EpiSC+siScramble) or Cdh2 siRNA (Cdh1-EpiSC+siCdh2). The white dotted lines demarcate the colony regions. Scale bars are 100 μm. (C) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in the non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). Data are normalized to the expression level of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (D) WB analysis for Pou5f1, Nanog, Sox2, cMyc, phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin in non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). (E) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt. Data are normalized to the expression total-Erk and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
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f3: Overexpression of Cdh1 does not restore the function of Cdh2 in mEpiSCs.(A) The mEpiSC-line was transfected with an overexpression plasmid cording for mouse Cdh1 cDNA. Cdh1-expressing mEpiSCs (Cdh1-EpiSC) highly expressed the Cdh1 protein. NTC indicates control mEpiSCs and Empty indicates empty plasmid-transfected mEpiSCs. The right panel shows the localization of Cdh1. The scale bar is 5 μm. (B) SSEA-1 expression in Cdh1-EpiSCs after treatment with ADH-1 (Cdh1-EpiSC+ADH-1), control siRNA (Cdh1-EpiSC+siScramble) or Cdh2 siRNA (Cdh1-EpiSC+siCdh2). The white dotted lines demarcate the colony regions. Scale bars are 100 μm. (C) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in the non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). Data are normalized to the expression level of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (D) WB analysis for Pou5f1, Nanog, Sox2, cMyc, phosphorylated-Erk (p-Erk), total-Erk (t-Erk), phosphorylated-Akt (p-Akt), total-Akt (t-Akt), Cdh2 and Actin in non-treated Cdh1-EpiSCs (non-treated control: NTC), ADH-1-treated Cdh1-EpiSCs, control siRNA-treated Cdh1-EpiSCs (siScramble) and Cdh2 siRNA-treated Cdh1-EpiSCs (siCdh2). (E) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt. Data are normalized to the expression total-Erk and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
Mentions: To identify the specific function of Cdh2 and its significance in determining primed state pluripotency, we examined if Cdh1 could compensate for a loss of Cdh2 function in mEpiSCs. For this experiment, we produced a mEpiSC line (Cdh1-EpiSC) that stably expressed Cdh1 and maintained pluripotency in standard mEpiSC medium. Cdh1 localized to the surface of Cdh1-EpiSCs (Fig. 3A). Interestingly, even though Cdh1-EpiSCs strongly expressed cell surface Cdh1, some cells lost SSEA-1 expression after treatment with either ADH-1 or siCdh2 (Fig. 3B). To investigate this phenomenon in detail, we performed qRT-PCR and WB assays for pluripotency-related genes. ADH-1 suppressed the expression of Nanog, Sox2 and cMyc, although Cdh1 expression rescued Pou5f1. Furthermore, consistent with the results from wild-type mEpiSCs, Eomes was upregulated by ADH-1 treatment. Treatment with siCdh2 had similar effects on Sox2, cMyc and Eomes expression (Fig. 3C). In addition, we found that siCdh2 treatment suppressed Erk and Akt phosphorylation in Cdh1-EpiSCs (Fig. 3D,E). These results suggest that Cdh2 is important for Erk and Akt phosphorylation and that suppression of Cdh2 decreases the expression of pluripotency-related genes.

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