Limits...
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.

Show MeSH

Related in: MedlinePlus

Changes in the relative mRNA levels of MMP-1, OPN and OC genes during poly(P) treatment. The mRNA levels of MMP-1 (A and B), OPN (C and D) and OC (E and F) in HDPCs (A, C and E) and hMSCs (B, D and F) were determined by quantitative real-time PCR using an ABI Prism 7000 Sequence Detection System. The sequences of the designed primers are 5'-CATCCAAGCCATATATGGACGTTCCC-3' and 5'-AGAACATCACTTCTCCCCGAATCGTAG-3' for MMP-1, 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC and 5'-ACTTTCACTCCAATCGTCCCTACA -3' and 5'-GGCATCAGGATACTGTTCATCAGA -3' for OPN, and the sequences of TaqMan® FAM-MGB probes are 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. The basic procedures used for quantitative PCR are described in Materials and Methods. Open bars, relative gene expression levels in non-treated cells; gray bars, levels in 1 mM poly(P)-treated cells. Relative expression levels of each genes were calculated when the expression level of day 0 was set at 1. Each value is the average ± SD of three independent experiments. Significant differences between the values of the poly(P)-treated group and control group (none) at the same time points (days) were determined by Student's t test. Asterisk (*), p <0.01 to control (none). Double asterisk (**), p <0.05 to control (none).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2238184&req=5

Figure 5: Changes in the relative mRNA levels of MMP-1, OPN and OC genes during poly(P) treatment. The mRNA levels of MMP-1 (A and B), OPN (C and D) and OC (E and F) in HDPCs (A, C and E) and hMSCs (B, D and F) were determined by quantitative real-time PCR using an ABI Prism 7000 Sequence Detection System. The sequences of the designed primers are 5'-CATCCAAGCCATATATGGACGTTCCC-3' and 5'-AGAACATCACTTCTCCCCGAATCGTAG-3' for MMP-1, 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC and 5'-ACTTTCACTCCAATCGTCCCTACA -3' and 5'-GGCATCAGGATACTGTTCATCAGA -3' for OPN, and the sequences of TaqMan® FAM-MGB probes are 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. The basic procedures used for quantitative PCR are described in Materials and Methods. Open bars, relative gene expression levels in non-treated cells; gray bars, levels in 1 mM poly(P)-treated cells. Relative expression levels of each genes were calculated when the expression level of day 0 was set at 1. Each value is the average ± SD of three independent experiments. Significant differences between the values of the poly(P)-treated group and control group (none) at the same time points (days) were determined by Student's t test. Asterisk (*), p <0.01 to control (none). Double asterisk (**), p <0.05 to control (none).

Mentions: Total RNA was obtained using an SV Total RNA Isolation System (Promega), following the manufacturer's instructions. Reverse transcription reactions were performed using an oligo dT (20-mer) primer and ReverTra AceTM (TOYOBO) for 30 minutes at 42˚C. The synthesized cDNAs were used for quantitative PCR analysis with specific primers designed to hybridize to the exon/ intron junctions. Quantification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (as an internal control for gene expression in the cells) was performed using TaqMan® Human GAPDH Control Reagents (with VICTM Probe, Applied Biosystems). The expression of matrix metalloproteinase-1 (MMP-1) mRNA was detected using SYBR® Green. Detection of osteopontin (OPN) and osteocalcin (OC) mRNAs was performed using TaqMan® FAM-MGB probes. The sequences of the designed primers for detection of MMP1, OPN and OC and the sequences of the TaqMan® FAM-MGB probes for OPN and OC are shown in the legend of Figure 5. Quantitative PCR analysis was performed using an ABI Prism 7000 Sequence Detection System and TaqMan® Universal PCR Master Mix for detection of OPN and OC mRNA and using SYBR® Green PCR Master Mix for detection of MMP-1 mRNA (both provided by Applied Biosystems) with 40 cycles of 95˚C for 15 seconds and 60˚C for 1 minute, following the manufacturer's protocol. Each mRNA expression level was normalized by the expression level of GAPDH, and the relative expression level of each gene is shown as a relative value when the expression level of day 0 was set at 1.


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)

Changes in the relative mRNA levels of MMP-1, OPN and OC genes during poly(P) treatment. The mRNA levels of MMP-1 (A and B), OPN (C and D) and OC (E and F) in HDPCs (A, C and E) and hMSCs (B, D and F) were determined by quantitative real-time PCR using an ABI Prism 7000 Sequence Detection System. The sequences of the designed primers are 5'-CATCCAAGCCATATATGGACGTTCCC-3' and 5'-AGAACATCACTTCTCCCCGAATCGTAG-3' for MMP-1, 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC and 5'-ACTTTCACTCCAATCGTCCCTACA -3' and 5'-GGCATCAGGATACTGTTCATCAGA -3' for OPN, and the sequences of TaqMan® FAM-MGB probes are 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. The basic procedures used for quantitative PCR are described in Materials and Methods. Open bars, relative gene expression levels in non-treated cells; gray bars, levels in 1 mM poly(P)-treated cells. Relative expression levels of each genes were calculated when the expression level of day 0 was set at 1. Each value is the average ± SD of three independent experiments. Significant differences between the values of the poly(P)-treated group and control group (none) at the same time points (days) were determined by Student's t test. Asterisk (*), p <0.01 to control (none). Double asterisk (**), p <0.05 to control (none).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Changes in the relative mRNA levels of MMP-1, OPN and OC genes during poly(P) treatment. The mRNA levels of MMP-1 (A and B), OPN (C and D) and OC (E and F) in HDPCs (A, C and E) and hMSCs (B, D and F) were determined by quantitative real-time PCR using an ABI Prism 7000 Sequence Detection System. The sequences of the designed primers are 5'-CATCCAAGCCATATATGGACGTTCCC-3' and 5'-AGAACATCACTTCTCCCCGAATCGTAG-3' for MMP-1, 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC and 5'-ACTTTCACTCCAATCGTCCCTACA -3' and 5'-GGCATCAGGATACTGTTCATCAGA -3' for OPN, and the sequences of TaqMan® FAM-MGB probes are 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. The basic procedures used for quantitative PCR are described in Materials and Methods. Open bars, relative gene expression levels in non-treated cells; gray bars, levels in 1 mM poly(P)-treated cells. Relative expression levels of each genes were calculated when the expression level of day 0 was set at 1. Each value is the average ± SD of three independent experiments. Significant differences between the values of the poly(P)-treated group and control group (none) at the same time points (days) were determined by Student's t test. Asterisk (*), p <0.01 to control (none). Double asterisk (**), p <0.05 to control (none).
Mentions: Total RNA was obtained using an SV Total RNA Isolation System (Promega), following the manufacturer's instructions. Reverse transcription reactions were performed using an oligo dT (20-mer) primer and ReverTra AceTM (TOYOBO) for 30 minutes at 42˚C. The synthesized cDNAs were used for quantitative PCR analysis with specific primers designed to hybridize to the exon/ intron junctions. Quantification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (as an internal control for gene expression in the cells) was performed using TaqMan® Human GAPDH Control Reagents (with VICTM Probe, Applied Biosystems). The expression of matrix metalloproteinase-1 (MMP-1) mRNA was detected using SYBR® Green. Detection of osteopontin (OPN) and osteocalcin (OC) mRNAs was performed using TaqMan® FAM-MGB probes. The sequences of the designed primers for detection of MMP1, OPN and OC and the sequences of the TaqMan® FAM-MGB probes for OPN and OC are shown in the legend of Figure 5. Quantitative PCR analysis was performed using an ABI Prism 7000 Sequence Detection System and TaqMan® Universal PCR Master Mix for detection of OPN and OC mRNA and using SYBR® Green PCR Master Mix for detection of MMP-1 mRNA (both provided by Applied Biosystems) with 40 cycles of 95˚C for 15 seconds and 60˚C for 1 minute, following the manufacturer's protocol. Each mRNA expression level was normalized by the expression level of GAPDH, and the relative expression level of each gene is shown as a relative value when the expression level of day 0 was set at 1.

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