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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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Enhancement of self-produced (autocrine) FGF function and the resultant proliferation of HDPCs by poly(P). (A) Cell proliferation after 21 and 45 hours of treatment with or without 1 mM poly(P) was measured by the MTS method. Relative cell growth was estimated by the absorbance of non-treated cells after 21 hours of incubation, which was set at 1. Open bars, relative cell growth of non-treated cells; gray bars, relative cell growth of poly(P)-treated (1 mM) cells. Significant differences between the relative cell growth rate for poly(P)-treated cells and the control cells were determined by Student's t test. Asterisk (*), p <0.01 to control (none). (B) Expression and localization of self-produced (autocrine) FGF-2 in HDPCs. Intracellular FGF-2 and cell surface receptor-bound FGF-2 were analyzed by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10 ng loaded); lane 2, intracellular FGF-2 in HDPCs; lane 3, receptor-bound FGF-2 of HDPCs. (C) Stabilization of FGF-2 located at cell surface receptors. Purified FGF-2 (40 ng/ml) was added to the cell culture medium and incubated for 20 or 40 hours. Cell surface receptor-bound FGF-2 was eluted by the method described in Materials and Methods. The amount of extracted FGF-2 was estimated by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10ng loaded); lane 2, receptor-bound FGF-2 of non-treated cells (20 hours after addition of FGF-2); lane 3, receptor-bound FGF-2 of poly(P)-treated cells (20 hours after addition of FGF-2); lane 4, receptor-bound FGF-2 of non-treated cells (40 hours after addition of FGF-2); lane 5, receptor-bound FGF-2 of poly(P)-treated cells (40 hours after addition of FGF-2).
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Figure 2: Enhancement of self-produced (autocrine) FGF function and the resultant proliferation of HDPCs by poly(P). (A) Cell proliferation after 21 and 45 hours of treatment with or without 1 mM poly(P) was measured by the MTS method. Relative cell growth was estimated by the absorbance of non-treated cells after 21 hours of incubation, which was set at 1. Open bars, relative cell growth of non-treated cells; gray bars, relative cell growth of poly(P)-treated (1 mM) cells. Significant differences between the relative cell growth rate for poly(P)-treated cells and the control cells were determined by Student's t test. Asterisk (*), p <0.01 to control (none). (B) Expression and localization of self-produced (autocrine) FGF-2 in HDPCs. Intracellular FGF-2 and cell surface receptor-bound FGF-2 were analyzed by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10 ng loaded); lane 2, intracellular FGF-2 in HDPCs; lane 3, receptor-bound FGF-2 of HDPCs. (C) Stabilization of FGF-2 located at cell surface receptors. Purified FGF-2 (40 ng/ml) was added to the cell culture medium and incubated for 20 or 40 hours. Cell surface receptor-bound FGF-2 was eluted by the method described in Materials and Methods. The amount of extracted FGF-2 was estimated by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10ng loaded); lane 2, receptor-bound FGF-2 of non-treated cells (20 hours after addition of FGF-2); lane 3, receptor-bound FGF-2 of poly(P)-treated cells (20 hours after addition of FGF-2); lane 4, receptor-bound FGF-2 of non-treated cells (40 hours after addition of FGF-2); lane 5, receptor-bound FGF-2 of poly(P)-treated cells (40 hours after addition of FGF-2).

Mentions: Since cell proliferation is one of the important steps for the initial phase of bone generation, we first investigated the effect of poly(P) on the proliferation of HDPCs. The addition of 1 mM poly(P) to the cell culture medium enhanced the proliferation of HDPCs up to 1.24 fold and 1.74 fold of that of non-treated cells after 21 and 45 hours of incubation, respectively (Fig. 2A).


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)

Enhancement of self-produced (autocrine) FGF function and the resultant proliferation of HDPCs by poly(P). (A) Cell proliferation after 21 and 45 hours of treatment with or without 1 mM poly(P) was measured by the MTS method. Relative cell growth was estimated by the absorbance of non-treated cells after 21 hours of incubation, which was set at 1. Open bars, relative cell growth of non-treated cells; gray bars, relative cell growth of poly(P)-treated (1 mM) cells. Significant differences between the relative cell growth rate for poly(P)-treated cells and the control cells were determined by Student's t test. Asterisk (*), p <0.01 to control (none). (B) Expression and localization of self-produced (autocrine) FGF-2 in HDPCs. Intracellular FGF-2 and cell surface receptor-bound FGF-2 were analyzed by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10 ng loaded); lane 2, intracellular FGF-2 in HDPCs; lane 3, receptor-bound FGF-2 of HDPCs. (C) Stabilization of FGF-2 located at cell surface receptors. Purified FGF-2 (40 ng/ml) was added to the cell culture medium and incubated for 20 or 40 hours. Cell surface receptor-bound FGF-2 was eluted by the method described in Materials and Methods. The amount of extracted FGF-2 was estimated by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10ng loaded); lane 2, receptor-bound FGF-2 of non-treated cells (20 hours after addition of FGF-2); lane 3, receptor-bound FGF-2 of poly(P)-treated cells (20 hours after addition of FGF-2); lane 4, receptor-bound FGF-2 of non-treated cells (40 hours after addition of FGF-2); lane 5, receptor-bound FGF-2 of poly(P)-treated cells (40 hours after addition of FGF-2).
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Figure 2: Enhancement of self-produced (autocrine) FGF function and the resultant proliferation of HDPCs by poly(P). (A) Cell proliferation after 21 and 45 hours of treatment with or without 1 mM poly(P) was measured by the MTS method. Relative cell growth was estimated by the absorbance of non-treated cells after 21 hours of incubation, which was set at 1. Open bars, relative cell growth of non-treated cells; gray bars, relative cell growth of poly(P)-treated (1 mM) cells. Significant differences between the relative cell growth rate for poly(P)-treated cells and the control cells were determined by Student's t test. Asterisk (*), p <0.01 to control (none). (B) Expression and localization of self-produced (autocrine) FGF-2 in HDPCs. Intracellular FGF-2 and cell surface receptor-bound FGF-2 were analyzed by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10 ng loaded); lane 2, intracellular FGF-2 in HDPCs; lane 3, receptor-bound FGF-2 of HDPCs. (C) Stabilization of FGF-2 located at cell surface receptors. Purified FGF-2 (40 ng/ml) was added to the cell culture medium and incubated for 20 or 40 hours. Cell surface receptor-bound FGF-2 was eluted by the method described in Materials and Methods. The amount of extracted FGF-2 was estimated by Western blotting. Lane 1, purified recombinant human FGF-2 standard (10ng loaded); lane 2, receptor-bound FGF-2 of non-treated cells (20 hours after addition of FGF-2); lane 3, receptor-bound FGF-2 of poly(P)-treated cells (20 hours after addition of FGF-2); lane 4, receptor-bound FGF-2 of non-treated cells (40 hours after addition of FGF-2); lane 5, receptor-bound FGF-2 of poly(P)-treated cells (40 hours after addition of FGF-2).
Mentions: Since cell proliferation is one of the important steps for the initial phase of bone generation, we first investigated the effect of poly(P) on the proliferation of HDPCs. The addition of 1 mM poly(P) to the cell culture medium enhanced the proliferation of HDPCs up to 1.24 fold and 1.74 fold of that of non-treated cells after 21 and 45 hours of incubation, respectively (Fig. 2A).

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