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...

To elucidate the precise physiological regulation of FGF-23, we characterized the mouse FGF-23 5Ј-flanking region and analyzed its promoter activity. The 5Ј-flanking region of the mouse FGF-23 gene contained a TFIID site (TATA box) and several putative transcription factor binding sites, including MZF1, GATA-1 and c-Ets-1 motifs, but it did not contain the typical sequences of the vitamin D response element. Plasmids encoding 554-bp (pGL/Ϫ0.6), 364-bp (pGL/Ϫ0.4) and 200-bp (pGL/Ϫ0.13) promoter regions containing the TFIID element and ϩ1-bp fragments drove the downstream expression of a luciferase reporter gene in transfection assays. We also found that FGF-23 mRNA was expressed in K-562 erythroleukemia cell lines but not in MC3T3-E1, Raji, or Hep G2 human carcinoma cells. Treatment with 1,25-dihydroxyvitamin D 3 in the presence of high phosphate markedly stimulated pGL/Ϫ0.6 activity, but calcium had no effect. In addition, the plasma FGF-23 levels were affected by the dietary and plasma inorganic phosphate concentrations. Finally, the levels of plasma FGF-23 in vitamin D receptor-null mice were significantly lower than in wild-type mice. The presents study demonstrated that vitamin D and the plasma phosphate level are important regulators of the transcription of the mouse FGF-23 gene. gene regulation; vitamin D receptor; phosphate homeostasis THE HOMEOSTASIS OF PLASMA PHOSPHATE is essential for many biological processes, including skeletal mineralization and energy metabolism. Recent investigations of diseases with abnormal phosphate homeostasis have revealed circulating phosphaturic hormones, collectively called phosphatonin (6, 23). Common clinical features, including hypophosphatemia resulting from renal phosphate wasting and impaired mineralization of bone are shared by X-linked hypophosphatemic rickets (XLH), tumor-induced osteomalacia (TIO), and autosomal-dominant hypophosphatemic rickets (ADHR; see Refs. 28,31,and 32), and the presence of a phosphatonin has been suspected in patients with these diseases because their plasma calcium and parathyroid hormone (PTH) levels are usually normal (8,9).Genetic studies of ADHR and TIO have identified fibroblast growth factor (FGF)-23 as a likely candidate for phosphatonin (28,31). In addition, continuous exposure to recombinant FGF-23 reproduces hypophosphatemic osteomalacia and the inappropriately low plasma levels of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ; see Ref. 28]. Also, a mutant form of FGF-23 (FGF-23R179Q), which is derived from disease-causing missense mutations of ADHR, is resistant to proteolytic processing that normally converts the biologically functional full-length polypeptide into inactive fragments (3, 33). Furthermore, elevated circulatory levels of FGF-23 occur not only in patients with TIO but also in those with XLH (35). These findings indicate that excess FGF-23 activity causes the hypophosphatemia and impaired mineralization of bone that is associated with phosphatonin (6, 21).In a previous study, we demonstrated that the mutant FGF-23 su...

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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.