Induction of Calcification in MC3T3-E1 Cells by Inorganic Polyphosphate

Abstract

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 μM) 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 ~ 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 β-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.

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References

Ahn K, Kornberg A (1990). Polyphosphate kinase from Escherichia coli. J Biol Chem 265:11734–11739.

Beck GR Jr, Sullivan EC, Moran E, Zerler B (1998). Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts. J Cell Biochem 68:269–280.

Beck GR Jr, Zerler B, Moran E (2000). Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci USA 97:8352–8357.

Bradford MM (1976). A rapid and sensitive method for the quantitation for microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.

Hurley MM, Tetradis S, Huang YF, Hock J, Kream BE, Raisz LG, et al. (1999). Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells. J Bone Miner Res 14:776–783.

Kornberg A (1995). Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J Bacteriol 177:491–496.

Kulaev IS (1979). The biochemistry of inorganic polyphosphates. New York, NY, USA: John Wiley & Sons, Inc.

Kulaev IS, Vogabov VM, Kulakovskaya T (1999). New aspects of inorganic polyphospate metabolism and function. J Biosci Bioeng 88:111–129.

Kumble KD, Kornberg A (1995). Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270:5818–5822.

10.

Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Muller WEG, et al. (1998). Inorganic polyphosphate in human osteoblast-like cells. J Bone Miner Res 13:803–812.

11.

Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ (1992). Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 7:683–692.

12.

Schröder HC, Kurz L, Muller WEG, Lorenz B (2000). Polyphosphate in bone. Biochemistry (Moscow) 65:353–361.

13.

Shiba T, Nishimura D, Kawazoe Y, Onodera Y, Tsutsumi K, Nakamura R, et al. (2003). Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate. J Biol Chem 278:26788–26792.

14.

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. (1987). Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.

15.

Stein GS, Lian JB, Stein JL, van Wijnen AJ, Montecino M (1996). Transcriptional control of osteoblast growth and differentiation. Physiol Rev 76:593–629.

16.

Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983). In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198.

17.

Tanaka M, Amizuka N, Kim KJ, Itoh T, Ozawa H (1997). Histochemical examination of ectopic bone formation induced in rat bone marrow. J Bone Miner Metab 15:67–76.

18.

Terkeltaub RA (2001). Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol 281:C1–C11.

19.

Wood HG, Clark JE (1988). Biological aspects of inorganic polyphosphates. Ann Rev Biochem 57:235–260.

20.

Wurst H, Shiba T, Kornberg A (1995). The gene for a major exopolyphosphatase of Saccharomyces cerevisiae. J Bacteriol 177:898–906.