The proliferation of normal human fibroblast cells was enhanced by the addition of inorganic polyphosphate (poly(P)) into culture media. The mitogenic activities of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) were also enhanced by poly(P). A physical interaction between poly(P) and FGF-2 was observed, and FGF-2 was both physically and functionally stabilized by poly(P). Furthermore, poly(P) facilitated the FGF-2 binding to its cell surface receptors. Because poly(P) is widely distributed in mammalian tissues, it may be a spontaneous modulator of FGFs. Inorganic polyphosphates (poly(P))1 are linear polymers of many tens or hundreds of orthophosphate residues linked by high energy phosphoanhydride bonds that have been found in a wide range of organisms including bacteria, fungi, algae, mosses, insects, and protozoa and in the tissues of higher plants and animals (1-4). The biological functions of poly(P) have been investigated mostly in microorganisms, and the following functions have been proposed: (i) storage substance of energy or orthophosphate; (ii) chelator of metal cations; (iii) donor for sugar and adenylate kinase; (iv) buffer against alkaline stress; (v) structural element in competence for DNA entry and transformation; and (vi) a regulatory factor of gene expression (1-4). Although the presence of poly(P) has been demonstrated in the rat brain, rat liver, human peripheral blood mononuclear cells, human erythrocytes, human gingival fibroblasts, human osteoblasts, and human plasma and intracellularly in the nucleus, the mitochondria, lysosomes and plasma membrane (5), little is known regarding the functions of poly(P) and the effects of poly(P) on mammalian cells. Recently, the involvement of poly(P) in apoptosis and in modulation of the mineralization process in bone tissue (5, 6) has been suggested.Because there has been no report concerning the direct effect of poly(P) on mammalian cells and because poly(P) is widely distributed in mammalian tissues and plasma (5), we speculated that poly(P) has some physiological effect on cells. Based on this idea, we first studied in this report the effect of poly(P) on mammalian cell growth or proliferation in vitro and revealed the novel poly(P) functions concerning the modulation of mitogenic activity of fibroblast growth factors (FGF) (8). EXPERIMENTAL PROCEDURESMaterials-Normal dermal fibroblasts (NHDF) isolated from adult human were purchased from BioWhittaker, Inc. Normal human gingival fibroblasts (HGF) isolated from adult human were provided by Dr. Nishimura (Osaka Dental University). Balb/c 3T3 cells were from Riken Cell Bank (Tsukuba, Japan). Poly(P) type 65 (sodium salts with average chain length of 65 phosphate residues) was purchased from Sigma. Concentrations of poly(P) are given in terms of phosphate residues. As the control of poly(P), NaPO 4 buffer (orthophosphate) was used. The pH of the NaPO 4 buffer was adjusted to 7.0 by mixing the same concentrations of Na 2 HPO 4 and NaH 2 PO 4 solution. MTS (3-(4,5-dime...

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 microM) have been found in normal osteoblasts. The effect of poly(P) with an average chain length of 65 phosphate residues on cell calcification was therefore investigated with the use of MC3T3-E1 cells. Expression of both osteopontin and osteocalcin was induced by poly(P) (0.1 approximately 1 mM), and cells treated with poly(P) were strongly stained by alizarin red. In addition, the level of alkaline phosphatase activity induced in poly(P)-treated cells was two-fold higher than that in either orthophosphate-treated or control cells but not higher than that in cells treated with beta-glycerophosphate and ascorbic acid. In contrast, however, polyphosphatase activities were activated by poly(P) treatment to levels up to six-fold greater than that in controls. MC3T3-E1 cells may utilize poly(P) as a phosphate source for calcification rather than phosphate sources that are mainly produced by ALPase. Poly(P)-dependent induction of polyphosphatase activities may therefore promote calcification in MC3T3-E1 cells.

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.