The authors state that they have no conflicts of interest.
Functional Involvement of PHOSPHO1 in Matrix Vesicle–Mediated Skeletal Mineralization†
Article first published online: 18 JAN 2007
Copyright © 2007 ASBMR
Journal of Bone and Mineral Research
Volume 22, Issue 4, pages 617–627, April 2007
How to Cite
Roberts, S., Narisawa, S., Harmey, D., Millán, J. L. and Farquharson, C. (2007), Functional Involvement of PHOSPHO1 in Matrix Vesicle–Mediated Skeletal Mineralization. J Bone Miner Res, 22: 617–627. doi: 10.1359/jbmr.070108
- Issue published online: 4 DEC 2009
- Article first published online: 18 JAN 2007
- Manuscript Accepted: 10 JAN 2007
- Manuscript Revised: 14 DEC 2006
- Manuscript Received: 29 SEP 2006
- growth plate;
- alkaline phosphatase
PHOSPHO1 is a phosphatase highly expressed in bone. We studied its functional involvement in mineralization through the use of novel small molecule inhibitors. PHOSPHO1 expression was present within matrix vesicles, and inhibition of enzyme action caused a decrease in the ability of matrix vesicles to calcify.
Introduction: The novel phosphatase, PHOSPHO1, belongs to the haloacid dehalogenase superfamily of hydrolases and is capable of cleaving phosphoethanolamine (PEA) and phosphocholine to generate inorganic phosphate. Our aims in this study were to examine the expression of PHOSPHO1 in murine mineralizing cells and matrix vesicles (MV) and to screen a series of small-molecule PHOSPHO1-specific inhibitors for their ability to pharmacologically inhibit the first step of MV-mediated mineralization.
Materials and Methods: q-PCR and immunohistochemistry were used to study the expression and localization profiles of PHOSPHO1. Inhibitors of PHOSPHO1's PEA hydrolase activity were discovered using high-throughput screening of commercially available chemical libraries. To asses the efficacy of these inhibitors to inhibit MV mineralization, MVs were isolated from TNAP-deficient (Akp2−/−) osteoblasts and induced to calcify in their presence.
Results: q-PCR revealed a 120-fold higher level of PHOSPHO1 expression in bone compared with a range of soft tissues. The enzyme was immunolocalized to the early hypertrophic chondrocytes of the growth plate and to osteoblasts of trabecular surfaces and infilling primary osteons of cortical bone. Isolated MVs also contained PHOSPHO1. PEA hydrolase activity was observed in sonicated MVs from Akp2−/− osteoblasts but not intact MVs. Inhibitors to PHOSPHO1 were identified and characterized. Lansoprazole and SCH202676 inhibited the mineralization of MVs from Akp2−/− osteoblasts by 56.8% and 70.7%, respectively.
Conclusions: The results show that PHOSPHO1 localization is restricted to mineralizing regions of bone and growth plate and that the enzyme present within MVs is in an active state, inhibition of which decreases the capacity of MVs to mineralize. These data further support our hypothesis that PHOSPHO1 plays a role in the initiation of matrix mineralization.