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Induction of Calcification in MC3T3-E1 Cells by Inorganic Polyphosphate

Y. Kawazoe^1,2,3, T. Shiba^1,2,5,*, R. Nakamura⁴, A. Mizuno², K. Tsutsumi^4,, 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;

View larger version (39K): [in a new window]   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.

View larger version (20K): [in a new window]   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.

View larger version (76K): [in a new window]   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 -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.

View larger version (44K): [in a new window]   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).