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Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate.

Kawazoe Y, Katoh S, Onodera Y, Kohgo T, Shindoh M, Shiba T - Int. J. Biol. Sci. (2008)

Bottom Line: The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated.Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR.The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Regenetiss Inc., 1-9-4, Asahigaoka, Hino, Tokyo 191-0065, Japan.

ABSTRACT
Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

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Elevated phosphorylation level of ERK1/2 in HDPCs treated with poly(P). Phosphorylation levels of ERK1/2 after poly(P) treatment were estimated by ELISA. Relative phosphorylation levels of ERK1/2 were estimated by dividing the amount of phosphorylated ERK1/2 by the total amount of ERK1/2. Open bars, relative phosphorylation level of ERK1/2 in non-treated cells; closed bars, level in 10 ng/ml FGF-2-treated cells; shaded bars, level in 0.2 mM poly(P)-treated cells; gray bars, level in 1 mM poly(P)-treated cells. Each value is the average of three independent assays. Significant differences between the values of poly(P)-treated cells and the control cells (None) were determined by Student's t test. Asterisk (*), p <0.01 to control (none).
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Figure 3: Elevated phosphorylation level of ERK1/2 in HDPCs treated with poly(P). Phosphorylation levels of ERK1/2 after poly(P) treatment were estimated by ELISA. Relative phosphorylation levels of ERK1/2 were estimated by dividing the amount of phosphorylated ERK1/2 by the total amount of ERK1/2. Open bars, relative phosphorylation level of ERK1/2 in non-treated cells; closed bars, level in 10 ng/ml FGF-2-treated cells; shaded bars, level in 0.2 mM poly(P)-treated cells; gray bars, level in 1 mM poly(P)-treated cells. Each value is the average of three independent assays. Significant differences between the values of poly(P)-treated cells and the control cells (None) were determined by Student's t test. Asterisk (*), p <0.01 to control (none).

Mentions: To confirm whether FGF-2 that is stabilized by poly(P) and located at the cell surface receptor is physiologically active, the phosphorylation level of mitogen-activated protein kinase (MAPK) ERK1/2, which is involved in the FGF-induced cell signaling pathway 26, was evaluated in the presence and absence of poly(P). As shown in Fig. 3, ERK1/2 phosphorylation in poly(P)-treated HDPCs was slightly enhanced after 24 hours (1.3- and 1.2-times enhanced in 0.2 and 1 mM poly(P)-treated cells, respectively, compared to that in non-treated cells), while the enhancement in FGF-2-treated cells was about 2.2-times greater than that in non-treated cells. However, after 48 hours, the phosphorylation level in poly(P)-treated (0.2 mM) cells was further increased to the almost same level as that of FGF-2-treated cells (1.7-times greater than that in non-treated cells). In 1 mM poly(P)-treated cells, ERK1/2 phosphorylation was also enhanced by about 1.5-times compared to that in non-treated cells after 48 hours of incubation. These results suggest that poly(P) activates the FGF-mediated cell signaling pathway by stabilizing FGF-2 bound at the cell surface receptors.


Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate.

Kawazoe Y, Katoh S, Onodera Y, Kohgo T, Shindoh M, Shiba T - Int. J. Biol. Sci. (2008)

Elevated phosphorylation level of ERK1/2 in HDPCs treated with poly(P). Phosphorylation levels of ERK1/2 after poly(P) treatment were estimated by ELISA. Relative phosphorylation levels of ERK1/2 were estimated by dividing the amount of phosphorylated ERK1/2 by the total amount of ERK1/2. Open bars, relative phosphorylation level of ERK1/2 in non-treated cells; closed bars, level in 10 ng/ml FGF-2-treated cells; shaded bars, level in 0.2 mM poly(P)-treated cells; gray bars, level in 1 mM poly(P)-treated cells. Each value is the average of three independent assays. Significant differences between the values of poly(P)-treated cells and the control cells (None) were determined by Student's t test. Asterisk (*), p <0.01 to control (none).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238184&req=5

Figure 3: Elevated phosphorylation level of ERK1/2 in HDPCs treated with poly(P). Phosphorylation levels of ERK1/2 after poly(P) treatment were estimated by ELISA. Relative phosphorylation levels of ERK1/2 were estimated by dividing the amount of phosphorylated ERK1/2 by the total amount of ERK1/2. Open bars, relative phosphorylation level of ERK1/2 in non-treated cells; closed bars, level in 10 ng/ml FGF-2-treated cells; shaded bars, level in 0.2 mM poly(P)-treated cells; gray bars, level in 1 mM poly(P)-treated cells. Each value is the average of three independent assays. Significant differences between the values of poly(P)-treated cells and the control cells (None) were determined by Student's t test. Asterisk (*), p <0.01 to control (none).
Mentions: To confirm whether FGF-2 that is stabilized by poly(P) and located at the cell surface receptor is physiologically active, the phosphorylation level of mitogen-activated protein kinase (MAPK) ERK1/2, which is involved in the FGF-induced cell signaling pathway 26, was evaluated in the presence and absence of poly(P). As shown in Fig. 3, ERK1/2 phosphorylation in poly(P)-treated HDPCs was slightly enhanced after 24 hours (1.3- and 1.2-times enhanced in 0.2 and 1 mM poly(P)-treated cells, respectively, compared to that in non-treated cells), while the enhancement in FGF-2-treated cells was about 2.2-times greater than that in non-treated cells. However, after 48 hours, the phosphorylation level in poly(P)-treated (0.2 mM) cells was further increased to the almost same level as that of FGF-2-treated cells (1.7-times greater than that in non-treated cells). In 1 mM poly(P)-treated cells, ERK1/2 phosphorylation was also enhanced by about 1.5-times compared to that in non-treated cells after 48 hours of incubation. These results suggest that poly(P) activates the FGF-mediated cell signaling pathway by stabilizing FGF-2 bound at the cell surface receptors.

Bottom Line: The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated.Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR.The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Regenetiss Inc., 1-9-4, Asahigaoka, Hino, Tokyo 191-0065, Japan.

ABSTRACT
Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

Show MeSH
Related in: MedlinePlus