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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 supports FGF2-induced proliferation and differentiation in mESCs.(A) Total cell numbers of wild-type mESCs (WT), empty plasmid-transfected mESCs (empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) after 0, 24 h (24 hours), 48 h (48 hours) and 72 h (72 hours) of FGF2 stimulation. The left panel shows the growth curve for cells in LIF+/FGF2− medium and the right panel shows the growth curve for cells in LIF−/FGF2+ medium. (B,C) FACS analysis for Oct4 deltaPE-GFP-positive cells showing the naïve-state specific undifferentiated status of the cells. The left panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF+/FGF2− medium and right panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF−/FGF2+ medium after 48 hours. (C) The percentages of Oct4 deltaPE-GFP-positive cells for each culture condition were obtained by FACS analysis. Bars represent the mean values of triplicates. Asterisks indicate significant differences with P < 0.05.
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f7: Cdh2 supports FGF2-induced proliferation and differentiation in mESCs.(A) Total cell numbers of wild-type mESCs (WT), empty plasmid-transfected mESCs (empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) after 0, 24 h (24 hours), 48 h (48 hours) and 72 h (72 hours) of FGF2 stimulation. The left panel shows the growth curve for cells in LIF+/FGF2− medium and the right panel shows the growth curve for cells in LIF−/FGF2+ medium. (B,C) FACS analysis for Oct4 deltaPE-GFP-positive cells showing the naïve-state specific undifferentiated status of the cells. The left panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF+/FGF2− medium and right panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF−/FGF2+ medium after 48 hours. (C) The percentages of Oct4 deltaPE-GFP-positive cells for each culture condition were obtained by FACS analysis. Bars represent the mean values of triplicates. Asterisks indicate significant differences with P < 0.05.

Mentions: FGF-Erk signaling induces differentiation of mESCs26. Thus, we examined if the overexpression of Cdh2 can accelerate FGF2-induced differentiation in mESCs. FGF2 is an important mitogenic cytokine for various types of cells including mEpiSCs2728. We observed cell proliferation of wild-type and Cdh2-overexpressing ESCs under FGF2 stimulation 24 h, 48 h and 72 h after changing the medium. In LIF-supplemented medium, there was no difference between wild-type mESCs and Cdh2-ESCs (Fig. 7A). In contrast, there was a significant increase in cell number at each time-point in Cdh2-ESC compared with wild-type ESCs when they were cultured in FGF2-supplemented medium (Fig. 7A). This suggests that Cdh2 enhances the response of mESCs to FGF2. Next, we observed the efficiency of FGF2-induced differentiation using mESCs transfected with an Oct4 deltaPE-GFP reported plasmid, which contains a distal enhancer and the Oct4 promoter for naïve-state-specific GFP expression. In the LIF-supplemented medium, GFP-positive mESCs were present at the same ratio in wild-type ESCs as in Cdh2-ESCs (Fig. 7B). However, in the FGF2-supplemented culture medium, the number of GFP-positive cells significantly decreased in Cdh2-ESCs (Fig. 7B,C). These results confirm that Cdh2 contributes to FGF2-mediated differentiation of mESCs.


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 supports FGF2-induced proliferation and differentiation in mESCs.(A) Total cell numbers of wild-type mESCs (WT), empty plasmid-transfected mESCs (empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) after 0, 24 h (24 hours), 48 h (48 hours) and 72 h (72 hours) of FGF2 stimulation. The left panel shows the growth curve for cells in LIF+/FGF2− medium and the right panel shows the growth curve for cells in LIF−/FGF2+ medium. (B,C) FACS analysis for Oct4 deltaPE-GFP-positive cells showing the naïve-state specific undifferentiated status of the cells. The left panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF+/FGF2− medium and right panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF−/FGF2+ medium after 48 hours. (C) The percentages of Oct4 deltaPE-GFP-positive cells for each culture condition were obtained by FACS analysis. Bars represent the mean values of triplicates. Asterisks indicate significant differences with P < 0.05.
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f7: Cdh2 supports FGF2-induced proliferation and differentiation in mESCs.(A) Total cell numbers of wild-type mESCs (WT), empty plasmid-transfected mESCs (empty) and Cdh2-overexpressed mESCs (Cdh2-ESC) after 0, 24 h (24 hours), 48 h (48 hours) and 72 h (72 hours) of FGF2 stimulation. The left panel shows the growth curve for cells in LIF+/FGF2− medium and the right panel shows the growth curve for cells in LIF−/FGF2+ medium. (B,C) FACS analysis for Oct4 deltaPE-GFP-positive cells showing the naïve-state specific undifferentiated status of the cells. The left panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF+/FGF2− medium and right panel shows the GFP-positive Oct4 deltaPE-GFP mESCs from LIF−/FGF2+ medium after 48 hours. (C) The percentages of Oct4 deltaPE-GFP-positive cells for each culture condition were obtained by FACS analysis. Bars represent the mean values of triplicates. Asterisks indicate significant differences with P < 0.05.
Mentions: FGF-Erk signaling induces differentiation of mESCs26. Thus, we examined if the overexpression of Cdh2 can accelerate FGF2-induced differentiation in mESCs. FGF2 is an important mitogenic cytokine for various types of cells including mEpiSCs2728. We observed cell proliferation of wild-type and Cdh2-overexpressing ESCs under FGF2 stimulation 24 h, 48 h and 72 h after changing the medium. In LIF-supplemented medium, there was no difference between wild-type mESCs and Cdh2-ESCs (Fig. 7A). In contrast, there was a significant increase in cell number at each time-point in Cdh2-ESC compared with wild-type ESCs when they were cultured in FGF2-supplemented medium (Fig. 7A). This suggests that Cdh2 enhances the response of mESCs to FGF2. Next, we observed the efficiency of FGF2-induced differentiation using mESCs transfected with an Oct4 deltaPE-GFP reported plasmid, which contains a distal enhancer and the Oct4 promoter for naïve-state-specific GFP expression. In the LIF-supplemented medium, GFP-positive mESCs were present at the same ratio in wild-type ESCs as in Cdh2-ESCs (Fig. 7B). However, in the FGF2-supplemented culture medium, the number of GFP-positive cells significantly decreased in Cdh2-ESCs (Fig. 7B,C). These results confirm that Cdh2 contributes to FGF2-mediated differentiation of mESCs.

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