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J Dent Res 83(8): 613-618, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Induction of Calcification in MC3T3-E1 Cells by Inorganic Polyphosphate

Y. Kawazoe1,2,3, T. Shiba1,2,5,*, R. Nakamura4, A. Mizuno2, K. Tsutsumi4,{dagger}, T. Uematsu5, M. Yamaoka5, M. Shindoh3, and T. Kohgo3

1 Regenetiss Co., Ltd., 1-5-17, Akabane, Okaya, Nagano 394-0002, Japan;
2 Frontier Research Division, Fujirebio Inc., Hachioji, Tokyo, Japan;
3 Graduate School of Dental Medicine, and
4 Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido, Japan; and
5 Matsumoto Dental University, Shiojiri, Nagano, Japan;

* corresponding author, shiba{at}regenetiss.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ß-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.

KEY WORDS: calcification • osteoblasts • osteopontin • osteocalcin • polyphosphate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic polyphosphates [poly(P)s] are linear polymers consisting of many orthophosphate (Pi) residues linked by energy-rich phosphoanhydride bonds. They have been found in a wide range of organisms, including bacteria, fungi, algae, mosses, insects, and protozoa, and also in the tissues of higher plants and animals (Kulaev, 1979; Wood and Clark, 1988; Kornberg, 1995; Kulaev et al., 1999). The biological functions of poly(P) in micro-organisms have been extensively investigated, but there have been relatively few studies in eukaryotic cells, particularly in mammals. The presence of poly(P) has been demonstrated in rat brain and liver, human peripheral blood mononuclear cells, human erythrocytes, human gingival fibroblasts, human osteoblasts, and human blood plasma, and the levels in human osteoblasts have been shown to be relatively high (Kumble and Kornberg, 1995; Leyhausen et al., 1998; Schröder et al., 2000). High levels of exopolyphosphatase (PPX) activity have been detected, in addition to endopolyphosphatase (PPN) activity, in human osteoblasts. PPX catalyzes the hydrolysis of terminal phosphates from poly(P), and PPN catalyzes the distributive cleavage of poly(P) to release intermediate-size chains during the course of the reaction. Although these findings indicate that there is a causal relationship between bone differentiation and poly(P) functions (Leyhausen et al., 1998; Schröder et al., 2000), the role of poly(P) in bone differentiation is still not clear. Here we report the effect of poly(P) on cell calcification in vitro using the established cell line MC3T3-E1, derived from newborn mice calvaria (Sudo et al., 1983), which displays time-dependent and sequential expression of osteoblast characteristics analogous to that in in vivo bone formation (Quarles et al., 1992).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
MC3T3-E1 and Balb/c 3T3 cells were purchased from Riken Cell Bank (Tsukuba, Japan). Dulbecco’s modified Eagle’s minimal essential medium (D-MEM) and Eagle’s minimum essential medium alpha modification ({alpha}-MEM), L-glutamine, fetal bovine serum (FBS), and sodium phosphate glass Type 65 (with average chain lengths of 65 phosphate residues) were obtained from Sigma. All non-radioactive poly(P)s used in the experiments were sodium phosphate glass Type 65, and the contamination level of pyrophosphate in this poly(P) is less than 0.01%. Radioactive poly(P) represented as [32P]poly(P) (with a chain length of more than 700 phosphate residues) was synthesized with the use of purified E. coli polyphosphate kinase with [{gamma}32P]-ATP and purified as described previously (Ahn and Kornberg, 1990). Polyethyleneimine thin-layer chromatography (PEI-TLC) plates were obtained from Merck. Recombinant yeast-soluble exopolyphosphatase (rPPX1) was overproduced in E. coli and purified as described previously (Wurst et al., 1995). Additional chemicals were purchased from Wako Pure Chemicals Co. (Osaka, Japan). The concentrations of poly(P) are represented in terms of phosphate residues.

Induction of Cell Calcification
MC3T3-E1 cells were plated on 60-mm plastic Petri dishes at a density of 1 x 105 cells/dish and maintained in {alpha}-MEM supplemented with 50 µg/mL of kanamycin and 10% FBS at 37°C in a humidified atmosphere of 5% CO2 in air. After the cells had become confluent, the medium was replaced with {alpha}-MEM supplemented with either 0.5% FBS and 50 µg/mL kanamycin or with 0.1, 0.5, or 1 mM poly(P), 0.1, 0.5 or 1 mM sodium phosphate buffer or 10 mM of ß-glycerophosphate (ß-GP) and 50 µg/mL of ascorbic acid (AA). The cells were further cultured with or without stimulants, and the medium was replaced every fourth day.

Quantification of mRNA Levels by Real-time PCR
Total RNA was purified with the use of an SV Total RNA Isolation System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Reverse-transcription reactions were performed with the use of an oligo dT (20 mer) primer and ReverTra AceTM (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. The synthesized cDNAs were then used for quantitative PCR analysis with primers for the exon/intron junctions. The sequences of the designed primers and the basic procedures used for quantitative PCR are described in the legend of Fig. 1Go.



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  		Figure 1. Quantitation of OPN and OC expression levels by real-time PCR. Relative levels of OPN (A,B,C) and OC (D,E,F) mRNA were measured by real-time PCR and standardized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels, which were used as an internal control. The primer sequences for quantitative PCR are as follows: 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC, and 5'-ACTTTCACTCCAATCGTCCCTACA-3' and 5'-GGCATCAGGATACTGTTCATCAGA-3' for OPN. Quantification of GAPDH mRNA (internal control) was performed with TaqMan® Rodent GAPDH Control Reagents (VICTM Probe) (Applied Biosystems, Foster City, CA, USA). Detection of OPN and OC mRNAs was performed with the use of TaqMan® FAM-MGB probes with the following sequences: 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. Quantitative PCR analysis was performed with the use of an ABI Prism 7000 Sequence Detection System and TaqMan® Universal PCR Master Mix (Applied Biosystems) for 40 cycles of 95°C for 15 sec and 60°C for 1 min as described in the manufacturer’s protocol. We calculated cellular mRNA levels as relative values, dividing each mRNA level by the GAPDH mRNA level of each sample as internal control. Relative mRNA levels—without treatment (open circles), following treatment with 0.1 mM (C and F), 0.5 mM (B and E), and 1 mM (A and D) Na-PO4 buffer (closed triangles), and following treatment with poly(P) (closed squares)—are shown. Error bars represent the mean ± SD of three independent analyses.

 
Alizarin Red Staining
Cells were washed once with distilled water and then fixed with phosphate-buffered formalin for 20 min. The fixed cells were also washed once with distilled water and subsequently stained with 1% alizarin red S solution for 5 min. The remaining dye was washed out twice with distilled water, and the cells were washed once more and then dehydrated with ethanol.

Preparation of Cell Extracts and Determination of Protein Concentration
Cells were trypsinized, collected in micro-tubes, and washed once with 20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl (TBS). The cell pellets were re-suspended in the appropriate buffers used in each assay, and the cells were then ultrasonicated on ice for the preparation of whole-cell extracts. Protein concentrations of both extracts and supernatant fractions were determined by BCA assays (Smith et al., 1987) and Bradford assays (Bradford, 1976), respectively.

Assay for Alkaline Phosphatase (ALPase) Activity
The reaction mixtures contained 50 mM Tris-HCl (pH 8.8), 10 mM MgCl2, 20 mM of p-nitrophenylphosphate (p-NPP), and whole-cell extract (20 µg). Following incubation for appropriate times at 37°C, the absorbance at 405 nm was measured by spectrophotometry, and the specific activity was then determined. One OD405 unit corresponds to a decomposition of 66.6 nM of p-NPP, and one unit represents a decomposition of 1 µmol of p-NPP per min.

Assay for Polyphosphatase [poly(P)ase] Activities
Each reaction mixture (20 µL) contained 20 mM Tris-HCl (pH 7.5), 20 mM (NH4)2SO4, 5 mM MgSO4, 2.4 mM of [32P]poly(P), and a supernatant fraction of ultrasonicated cell extract (20 µg). Following incubation for 8 hrs at 37°C, 2 µL of each reaction mixture was applied to a PEI-TLC plate and developed with 2 M LiCl and 1 M HCOOH.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Osteoblast Differentiation Marker Expression by Poly(P)
During cell calcification, specific marker genes are expressed, and the resulting proteins are secreted from the cells and play important roles in the binding of osteoblasts to extracellular matrices. Osteopontin (OPN) and osteocalcin (OC) are matrix GLA proteins that are well-known osteoblast differentiation markers (Stein et al., 1996). OPN expression is generally induced in MC3T3-E1 cells 10 to 20 days following induction of cell calcification, and a high level of OC expression is observed after enhancement of OPN expression (Beck et al., 2000). As shown in Figs. 1AGo and 1BGo, the level of OPN mRNA expression was about 3.5- to 10-fold higher in treated cells, after 10 days’ exposure to both 0.5 and 1 mM poly(P), than in control cells. No appreciable enhancement of OPN expression was observed when the cells were exposed to 0.1 mM poly(P) (Fig. 1CGo), suggesting that a poly(P) concentration between 0.1 and 0.5 mM is the critical point for OPN expression enhancement. Two- to four-fold higher OC mRNA expression was observed in cells following poly(P) induction than in control cells at 27 days following the start of treatment (Figs. 1DGo, 1EGo, 1FGo), and a poly(P) concentration of less than 1 mM proved to be the most effective for OC expression enhancement.

Induction of ALPase Activity by Poly(P)
ALPase activity is commonly used as an indicator of calcification in the matrix maturation phase (Beck et al., 1998), and an experiment was carried out in this study to determine whether poly(P) has an effect on ALPase activity. The level of ALPase activity in poly(P)-treated cells did indeed increase with incubation time, but the magnitude of this increase was less than that in cells treated with ß-GP and AA (a positive control for cell calcification) (Fig. 2Go). The fact that no increase in ALPase activity was observed in phosphate-buffer-treated cells or non-treated cells (negative controls for cell calcification) indicates that poly(P) has a positive effect on the induction of ALPase activity.



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  		Figure 2. Effects of poly(P) on ALP activities. Open circles: ALP activities in cells that were not subjected to any treatment. Open triangles: cells that were treated with 1 mM Na-PO4 buffer. Closed squares: cells that were treated with 1 mM poly(P). Open diamonds: cells that were treated with both 10 mM ß-GP and 50 µg/mL AA. Error bars represent the mean ± SD of three independent analyses.

 
Visualization of Cell Calcification Induced by Poly(P)
For visualization of cell calcification, alizarin red staining was performed, and cells treated with poly(P) were strongly stained (Fig. 3AGo), and the presence of calcium precipitation was clearly observed. Additionally, the level of calcification of poly(P)-treated cells was much greater than that of the cells treated with ß-GP and AA, with no calcification being observed in negative control cells.



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  		Figure 3. Visualization of matrix calcification by alizarin red staining. (A) Cells that had been treated with either 1 mM of poly(P), 10 mM of ß-GP and 50 µg/mL of AA, or with 1 mM Na-PO4 for 31 days and control cells were stained with alizarin red. (B) Differences between the levels of poly(P)-induced calcification in MC3T3-E1 cells (top) and in Balb/c 3T3 cells (bottom). MC3T3-E1 cells and Balb/c 3T3 cells were cultured in {alpha}-MEM and D-MEM supplemented with 10% FBS, respectively. Confluent cells were further cultured in media with 0.5% FBS containing 1 mM poly(P). The medium was replaced every third day, and the cells were stained by alizarin red S at the indicated timepoints.

 
To confirm that the effect of poly(P) on cell calcification is specific for MC3T3-E1 osteoblast-like cells, we cultured Balb/c 3T3 cells, which do not have the ability to differentiate into osteoblast-like cells. The results in Fig. 3BGo show that calcification occurs only in MC3T3-E1 cells, and that it increased with the duration of poly(P) treatment. In contrast, no cell calcification was observed in Balb/c 3T3 cells.

Enhancement of Poly(P)ase Activity by Poly(P)
Since relatively high levels of PPX and endopolyphosphatase (PPN) activity were detected in human osteoblasts (Schröder et al., 2000), we measured the levels of poly(P)ase activity in MC3T3-E1 cells that were treated with poly(P), phosphate buffer, and ß-GP and AA. Fig. 4AGo shows the results of TLC analysis of poly(P)ase activity during cell calcification treatment. Whole-cell extracts were prepared, and poly(P)ase activities were determined with [32P]poly(P) used as a substrate. In cells treated with poly(P), poly(P)ase activity was enhanced, whereas no enhancement of activity was observed in extracts from cells treated with other reagents. Extracts from poly(P)-treated cells had strong poly(P)ase activities that resulted in the production of low-molecular-weight products and orthophosphate after 11 days of treatment. The maximum level of induced activity was up to six-fold greater than that in non-treated cells, and the levels were gradually decreased but were detectable until 21 days after the start of poly(P) treatment. These results indicate that poly(P) induces poly(P)ase activities, including those of both PPX and PPN, in MC3T3-E1 cells.



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  		Figure 4. Enhancement of poly(P)ase activities following poly(P) treatment. (A) Detection of polyphosphatase activities by TLC analysis. Whole-cell extracts were prepared from cultured cells that had been treated with either poly(P), Na-PO4, or ß-GP + AA and untreated control cells. Poly(P)ase activities were detected by TLC analysis of reaction mixtures containing these cell extracts and with [32P]poly(P) as a substrate. Substrate [32P]poly(P) (long chain) remained at the origin of the TLC plate, and low-molecular-weight products, corresponding to short-chain poly(P) species, migrated to the top of the TLC plate following development by 1 M HCOOH and 2 M LiCl. These reactions were performed in triplicate and developed in three lanes. (B) Identification of low-molecular-weight labeled products by purified poly(P)ase (rPPX1) treatment. Poly(P)-treated cell extracts (day 11) were incubated with [32P]poly(P) under the same conditions as described in panel A and were further incubated with or without purified rPPX1 (2.2 x 103 units) (Wurst et al., 1995) for 1 hr at 37°C. The reaction products were also analyzed by TLC. (C) Identification of low-molecular-weight labeled products by longer-TLC plate. Poly(P)-treated cell extracts (day 16) were incubated with [32P]poly(P) under the same conditions as described in panel A and were analyzed by longer-TLC (20 cm). Lanes: 1, [32P]poly (P) hydrolyzed by 10 mM HCl for 5 min at 90°C; 2, [32P] orthophosphate; 3, degradation products of [32P]poly(P) treated with cell extract (day 16). Radioactive images of the TLC plates were visualized in a BAS2000 image analyzer (FUJIX, Tokyo, Japan).

 
Since the enhancement of these activities was observed in poly(P)-treated cells, but not in ß-GP/AA-treated cells, this suggests that poly(P)ase activity plays a role in cell calcification induced by poly(P), but not by ß-GP and AA. The mechanism of cell calcification induced by poly(P) may therefore be different from other mechanisms that have been previously reported. Since a slight induction of poly(P)ase activities was also detected at 16 and 23 days after phosphate- buffer treatment, phosphate levels may also cause induction of poly(P)ases, although poly(P) enhancement was far greater.

To determine that the low-molecular-weight products detected between poly(P) (origin) and orthophosphate during TLC analysis (Fig. 4AGo, days 11, 16, and 23 in poly(P)-treated cell extracts) are short-chain poly(P) species, we further treated the products of these reactions with purified rPPX1 (Wurst et al., 1995). TLC analysis of reaction products treated with or without rPPX1 showed that the low-molecular-weight products had completely disappeared, and the orthophosphate spot became more pronounced following rPPX1 treatment (Fig. 4BGo).

For further identification of the low-molecular-weight products, the products were separated by longer TLC. A pyrophosphate spot (P2) migrated a little below the position of the orthophosphate spot (Pi) (Fig. 4CGo). We did not observe any obvious spot in the area corresponding to the pyrophosphate spot in the [32P]poly(P) sample treated with cell extract (day 16) (lane 3). This suggests that the main components of the low-molecular-weight products were larger than tripolyphosphate (P3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since the developmental stages of MC3T3-E1 cells are similar to those of normal osteoblasts in bone tissue, and since MC3T3-E1 cells are widely used as an in vitro model of osteoblast development (Quarles et al., 1992), evaluation of the effect of poly(P) on cell calcification with MC3T3-E1 cells is important for determination of the effects of poly(P) on osteoblasts. In this study, it was found that poly(P) induced calcification of MC3T3-E1 cells in tandem with induction of OPN, OC, and ALP expression. Calcium-phosphate precipitation was observed in poly(P)-treated MC3T3-E1 cells but not in poly(P)-treated Balb/c 3T3 cells, suggesting that poly(P)-induced cell calcification is specific to osteoblast-like cells. We also found that cell calcification was induced by poly(P) in normal human osteoblast and odontoblast cells (manuscript in preparation). Taken together, these results suggest that poly(P) is a calcification inducer in normal osteoblast-like cells.

ALPase activity was also induced during poly(P) treatment but at lower levels than in cells treated with ß-GP and AA. Since Schröder et al.(2000) demonstrated that only the intestinal isoform of ALPase is able to degrade poly(P), whereas placenta-type and tissue-non-specific isoenzymes which are present in bone did not, ALPase may not be involved in poly(P) metabolism in MC3T3-E1 cells. We also demonstrated that PPX and PPN activities were enhanced by poly(P) treatment, which may result in the supply of phosphate to cells for calcification. It had previously been shown that when cells are treated with ß-GP and AA, ß-GP is degraded by the induced ALPase activity (Stein et al., 1996). Elevation of the level of ALPase activity has also been shown to result in the formation of phosphate molecules and the concomitant induction of OPN expression, since high phosphate levels trigger the induction of OPN (Beck et al., 2000). Therefore, poly(P) may work as a phosphate source for the induction of OPN expression in place of ß-GP. Elevated poly(P)ase activity generates free phosphate from poly(P), whereas elevated ALPase activity produces phosphate from ß-GP, and enhancement of poly(P)ase activities may thus compensate for ALPase activity, the level of which is less in poly(P)-treated cells than in ß-GP- and AA-treated cells. Based on this possibility, it can be speculated that poly(P) acts as a phosphate source for cell calcification itself. Since we found that most of the poly(P) in the culture medium binds to the cells (data not shown), polyphosphate may be concentrated and degraded at the surfaces of or inside the cells, and the phosphate concentrations in the cells may be increased. Concentrated poly(P) inside the cell or on the cell surface may cause the condition similar to that when the cells were treated with a large amount of phosphate in culture medium. This condition may induce calcification of MC3T3-E1 cells. In addition, since poly(P)ase can also produce pyrophosphate (PPi), the balance of poly(P), PPi, and Pi levels might be crucial for regulating cell calcification (Terkeltaub, 2001).

We found, in a recent in vitro study, that poly(P) dosage enhanced fibroblast cell growth by stabilizing FGF-1 and FGF-2 and by promoting the binding between FGFs and their receptors (FGFRs) (Shiba et al., 2003). FGFs are also important factors in cell calcification, because both they and their cell-surface receptors are widely distributed in osteoblast and pre-osteoblast cells. Hurley et al. have demonstrated that parathyroid hormone induces the expression of FGF-2 and FGFRs in MC3T3-E1 cells and in osteoblasts in mice calvaria (Hurley et al., 1999). It has also been shown that FGF-2 induced new long-bone formation when injected into bone marrow (Tanaka et al., 1997). These findings suggest that enhancement of FGF activity by poly(P) may be responsible for the promotion of cell calcification.


   ACKNOWLEDGMENTS
 
We thank Drs. Y. Takahashi, H. Tanaka, K. Ooi, and H. Itoh for technical assistance and valuable discussions. This work was supported by a Grant-in-Aid for Creation of Innovations through the Business-Academic-Public Sector Cooperation and a Grant-in-Aid for Scientific Research on Priority Areas (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported by the Creation and Support Program for Start-ups from Universities from the Japan Science and Technology for Matsumoto Dental University. New Energy and Industrial Technology Development Organization (NEDO) supported this work through Regenetiss Co., Ltd.


   FOOTNOTES
 
{dagger} present address, Department of Radiological Technology, College of Medical Technology, Hokkaido University, Sapporo, Hokkaido, Japan; Back

Received September 17, 2003; Last revision April 17, 2004; Accepted May 24, 2004


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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Ahn K, Kornberg A (1990). Polyphosphate kinase from Escherichia coli. J Biol Chem 265:11734–11739.[Abstract/Free Full Text]

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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.[Abstract/Free Full Text]

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.[ISI][Medline]

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.[ISI][Medline]

Kornberg A (1995). Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J Bacteriol 177:491–496.[Abstract/Free Full Text]

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

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