Limits...
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 is important for maintenance of pluripotency in mEpiSCs.(A) SSEA-1 expression in control mEpiSCs (non-treated control: NTC) after treatment with ADH-1, control siRNA (siScramble) or Cdh2 siRNA (siCdh2). White dotted lines demarcate the colony regions. The scale bars are 100 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in non-treated mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). Data are normalized to the expression of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) 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 mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). (D) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt 24 hours after FGF2 treatment. Data are normalized to the expression levels of total-ErK and total-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

License
getmorefigures.php?uid=PMC4588589&req=5

f2: Cdh2 is important for maintenance of pluripotency in mEpiSCs.(A) SSEA-1 expression in control mEpiSCs (non-treated control: NTC) after treatment with ADH-1, control siRNA (siScramble) or Cdh2 siRNA (siCdh2). White dotted lines demarcate the colony regions. The scale bars are 100 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in non-treated mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). Data are normalized to the expression of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) 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 mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). (D) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt 24 hours after FGF2 treatment. Data are normalized to the expression levels of total-ErK and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.

Mentions: To determine the relationship between Cdh2 and mEpiSCs pluripotency, we either disrupted Cdh2 function using ADH-1 (also known as Exherin), which is a selective inhibitor for Cdh2, or suppressed Cdh2 expression using specific siRNA to Cdh2 (siCdh2). ADH-1 and siCdh2 had no effect on colony size and viability (data not shown), but both treatments induced differentiation in mEpiSCs, as indicated by a decrease in SSEA-1, which is a pluripotent state-specific glycoprotein in mouse cells (Fig. 2A). We next analyzed the expression levels of other pluripotency-related genes in the ADH-1- and siCdh2-treated cells. A knockdown efficiency of ~80% occurred for siCdh2 compared with scrambled siRNA-transfected cells, as determined by qRT-PCR. The ADH-1 and the siCdh2 treatments both suppressed the expression of the Pou5f1, Sox2 and cMyc genes at both the RNA and protein levels. Although Nanog mRNA expression did not decrease in siCdh2-treated cells, Nanog protein levels were lower in both ADH-1- and siCdh2-treated cells compared to non-treated cells. Expression of Eomes, which is an important transcription factor for neuronal and trophoblast differentiation, was upregulated by the ADH-1 and siCdh2 treatments (Fig. 2B). To elucidate the mechanism by which pluripotency-related genes are down-regulated by inhibition of Cdh2, we observed phosphorylation of Erk and Akt, both of which are important molecular mediators of pluripotency in primed-state stem cells232425. WB analysis revealed that disruption of Cdh2 inhibited Erk and Akt phosphorylation (Fig. 2C,D).


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 is important for maintenance of pluripotency in mEpiSCs.(A) SSEA-1 expression in control mEpiSCs (non-treated control: NTC) after treatment with ADH-1, control siRNA (siScramble) or Cdh2 siRNA (siCdh2). White dotted lines demarcate the colony regions. The scale bars are 100 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in non-treated mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). Data are normalized to the expression of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) 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 mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). (D) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt 24 hours after FGF2 treatment. Data are normalized to the expression levels of total-ErK and total-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

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

f2: Cdh2 is important for maintenance of pluripotency in mEpiSCs.(A) SSEA-1 expression in control mEpiSCs (non-treated control: NTC) after treatment with ADH-1, control siRNA (siScramble) or Cdh2 siRNA (siCdh2). White dotted lines demarcate the colony regions. The scale bars are 100 μm. (B) qRT-PCR for the pluripotency-related genes Pou5f1, Nanog, Sox2 and cMyc, the differentiation marker Eomes and Cdh2 in non-treated mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). Data are normalized to the expression of Lamina. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) 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 mEpiSCs (non-treated control: NTC), ADH-1-treated mEpiSCs, control siRNA-treated mEpiSCs (siScramble) and Cdh2 siRNA-treated mEpiSCs (siCdh2). (D) Densitometry quantification of WBs for phosphorylated-Erk and phosphorylated-Akt 24 hours after FGF2 treatment. Data are normalized to the expression levels of total-ErK and total-Akt. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05.
Mentions: To determine the relationship between Cdh2 and mEpiSCs pluripotency, we either disrupted Cdh2 function using ADH-1 (also known as Exherin), which is a selective inhibitor for Cdh2, or suppressed Cdh2 expression using specific siRNA to Cdh2 (siCdh2). ADH-1 and siCdh2 had no effect on colony size and viability (data not shown), but both treatments induced differentiation in mEpiSCs, as indicated by a decrease in SSEA-1, which is a pluripotent state-specific glycoprotein in mouse cells (Fig. 2A). We next analyzed the expression levels of other pluripotency-related genes in the ADH-1- and siCdh2-treated cells. A knockdown efficiency of ~80% occurred for siCdh2 compared with scrambled siRNA-transfected cells, as determined by qRT-PCR. The ADH-1 and the siCdh2 treatments both suppressed the expression of the Pou5f1, Sox2 and cMyc genes at both the RNA and protein levels. Although Nanog mRNA expression did not decrease in siCdh2-treated cells, Nanog protein levels were lower in both ADH-1- and siCdh2-treated cells compared to non-treated cells. Expression of Eomes, which is an important transcription factor for neuronal and trophoblast differentiation, was upregulated by the ADH-1 and siCdh2 treatments (Fig. 2B). To elucidate the mechanism by which pluripotency-related genes are down-regulated by inhibition of Cdh2, we observed phosphorylation of Erk and Akt, both of which are important molecular mediators of pluripotency in primed-state stem cells232425. WB analysis revealed that disruption of Cdh2 inhibited Erk and Akt phosphorylation (Fig. 2C,D).

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