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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 stabilizes FGFR1.(A) qRT-PCR for the FGFR family genes Fgfr1, Fgfr2 and Fgfr3 in mESCs and mEpiSCs. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (B) qRT-PCR for pluripotency-related genes in the negative control siRNA-treated mEpiSCs (siNC) and the Fgfr1 siRNA-treated mEpiSCs (siFgfr1). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) Co-immunoprecipitation (Co-IP) assay showing interaction between FGFR1 and Cdh1 in Cdh1-mEpiSCs and FGFR1 and Cdh2 in the wild-type mEpiSCs (WT). (D) WBs for FGFR1, Cdh2 and Actin in siRNA- and cycloheximide (CHX)-treated mEpiSCs 0, 5 min, 30 min, 1 hr, 2 hr and 4 hr after FGF2 stimulation. (E) Densitometry quantification of WBs for FGFR1, Cdh2 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.
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f4: Cdh2 stabilizes FGFR1.(A) qRT-PCR for the FGFR family genes Fgfr1, Fgfr2 and Fgfr3 in mESCs and mEpiSCs. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (B) qRT-PCR for pluripotency-related genes in the negative control siRNA-treated mEpiSCs (siNC) and the Fgfr1 siRNA-treated mEpiSCs (siFgfr1). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) Co-immunoprecipitation (Co-IP) assay showing interaction between FGFR1 and Cdh1 in Cdh1-mEpiSCs and FGFR1 and Cdh2 in the wild-type mEpiSCs (WT). (D) WBs for FGFR1, Cdh2 and Actin in siRNA- and cycloheximide (CHX)-treated mEpiSCs 0, 5 min, 30 min, 1 hr, 2 hr and 4 hr after FGF2 stimulation. (E) Densitometry quantification of WBs for FGFR1, Cdh2 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.

Mentions: Since Cdh2 disruption suppresses Akt and Erk phosphorylation, we focused our attention on the FGF signaling cascade, a pathway in which both Akt and Erk act as mediators. Since it is most likely for other molecules on the cell surface to interact with Cdh2, we hypothesized that Cdh2 may regulate FGF receptors (FGFRs). To test this hypothesis, we observed expression levels of three FGFRs in mEpiSCs. FGFR1 showed the highest expression in both mESCs and mEpiSCs. FGFR2 and FGFR3 expression levels were significantly lower than FGFR1 expression levels in mEpiSCs. The expression levels of FGFR2 and FGFR3 were 0.1-fold that of the FGFR1 level and were almost undetectable (Fig. 4A). Next, we examined the significance of FGFR1 in mEpiSCs pluripotency by introducing specific siRNA for Fgfr1 (siFgfr1). According to qRT-PCR, the siFgfr1 achieved a knockdown efficiency of 90% compared with scrambled siRNA. Treatment with siFgfr1 resulted in reduced expression of pluripotency-related genes (Fig. 4B). We next performed co-immunoprecipitation to confirm the interaction of Cdh2 with FGFR1 and to demonstrate that Cdh2 bound to FGFR1 in mEpiSCs (Fig. 4C). Furthermore, we determined if Cdh2 stabilized FGFR1 during FGF2 stimulation by analyzing FGFR1 degradation efficiency in cells treated with cycloheximide (CHX), which is an inhibitor of de novo protein synthesis. FGFR1 protein levels decreased in a time-dependent manner following CHX treatment, while Cdh2 expression did not change. The siCdh2-treatment accelerated FGFR1 protein degradation in these cells (Fig. 4D,E).


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 stabilizes FGFR1.(A) qRT-PCR for the FGFR family genes Fgfr1, Fgfr2 and Fgfr3 in mESCs and mEpiSCs. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (B) qRT-PCR for pluripotency-related genes in the negative control siRNA-treated mEpiSCs (siNC) and the Fgfr1 siRNA-treated mEpiSCs (siFgfr1). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) Co-immunoprecipitation (Co-IP) assay showing interaction between FGFR1 and Cdh1 in Cdh1-mEpiSCs and FGFR1 and Cdh2 in the wild-type mEpiSCs (WT). (D) WBs for FGFR1, Cdh2 and Actin in siRNA- and cycloheximide (CHX)-treated mEpiSCs 0, 5 min, 30 min, 1 hr, 2 hr and 4 hr after FGF2 stimulation. (E) Densitometry quantification of WBs for FGFR1, Cdh2 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Cdh2 stabilizes FGFR1.(A) qRT-PCR for the FGFR family genes Fgfr1, Fgfr2 and Fgfr3 in mESCs and mEpiSCs. Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (B) qRT-PCR for pluripotency-related genes in the negative control siRNA-treated mEpiSCs (siNC) and the Fgfr1 siRNA-treated mEpiSCs (siFgfr1). Bars represent the mean normalized values of triplicates. Asterisks indicate significant differences with P < 0.05. (C) Co-immunoprecipitation (Co-IP) assay showing interaction between FGFR1 and Cdh1 in Cdh1-mEpiSCs and FGFR1 and Cdh2 in the wild-type mEpiSCs (WT). (D) WBs for FGFR1, Cdh2 and Actin in siRNA- and cycloheximide (CHX)-treated mEpiSCs 0, 5 min, 30 min, 1 hr, 2 hr and 4 hr after FGF2 stimulation. (E) Densitometry quantification of WBs for FGFR1, Cdh2 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.
Mentions: Since Cdh2 disruption suppresses Akt and Erk phosphorylation, we focused our attention on the FGF signaling cascade, a pathway in which both Akt and Erk act as mediators. Since it is most likely for other molecules on the cell surface to interact with Cdh2, we hypothesized that Cdh2 may regulate FGF receptors (FGFRs). To test this hypothesis, we observed expression levels of three FGFRs in mEpiSCs. FGFR1 showed the highest expression in both mESCs and mEpiSCs. FGFR2 and FGFR3 expression levels were significantly lower than FGFR1 expression levels in mEpiSCs. The expression levels of FGFR2 and FGFR3 were 0.1-fold that of the FGFR1 level and were almost undetectable (Fig. 4A). Next, we examined the significance of FGFR1 in mEpiSCs pluripotency by introducing specific siRNA for Fgfr1 (siFgfr1). According to qRT-PCR, the siFgfr1 achieved a knockdown efficiency of 90% compared with scrambled siRNA. Treatment with siFgfr1 resulted in reduced expression of pluripotency-related genes (Fig. 4B). We next performed co-immunoprecipitation to confirm the interaction of Cdh2 with FGFR1 and to demonstrate that Cdh2 bound to FGFR1 in mEpiSCs (Fig. 4C). Furthermore, we determined if Cdh2 stabilized FGFR1 during FGF2 stimulation by analyzing FGFR1 degradation efficiency in cells treated with cycloheximide (CHX), which is an inhibitor of de novo protein synthesis. FGFR1 protein levels decreased in a time-dependent manner following CHX treatment, while Cdh2 expression did not change. The siCdh2-treatment accelerated FGFR1 protein degradation in these cells (Fig. 4D,E).

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