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J. Biol. Chem., Vol. 282, Issue 21, 15872-15883, May 25, 2007

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Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity^*

William N. Addison¹, Fereshteh Azari, Esben S. Sørensen, Mari T. Kaartinen^¶2, and Marc D. McKee^||23

From the Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada, the Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, and the Departments of ^¶Medicine and ^||Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada

Inorganic pyrophosphate (PP_i) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P_i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP_i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP_i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP_i. This inhibition could be reversed by Tnap, but not if PP_i was bound to mineral. PP_i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP_i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP_i, hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP_i inhibits Tnap-mediated P_i release from -glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP_i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

Received for publication, February 6, 2007 , and in revised form, March 21, 2007.

^* This work was supported in part by operating grants from the Canadian Institutes of Health Research (to M. D. M. and M. T. K.) and the Danish Dairy Board (to E. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a graduate student award from the Canadian Institutes of Health Research Skeletal Health Training program.

² Supported by scholarship awards from the Fonds de la Recherche en Santé du Québec. Members of the McGill Centre for Bone and Periodontal Research, the McGill Centre for Biorecognition and Biosensors, the McGill Institute for Advanced Materials, and the Fonds de la Recherche en Santé du Québec Network for Oral and Bone Health Research.

³ Member of the Canadian Arthritis Network. To whom correspondence should be addressed: Faculty of Dentistry, McGill University, 3640 University St., Montreal, Quebec H3A 2B2, Canada. Tel.: 514-398-7203, Ext. 00041; Fax: 514-398-8900; E-mail: marc.mckee{at}mcgill.ca.

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