Mechanisms of initiation of skeletal mineralisation

Lead Research Organisation: University of Edinburgh
Department Name: UNLISTED

Abstract

We will test this hypothesis by affecting the first and second steps of MV-mediated minerlisation using a genetuc and pharmacological approach. Experimentally we will characterize the mineralisation abnormalities and related metabolic changes in mice deficient in PHospho 1 expression compared to AKp2 mice and assess the effect of the simultaneous inactivation of the Ohosph1 and Akp2 genes on skeletal minerlisation. we will also study the effects of ablating or inhibiting OHOSPHO1 and/or TNAP activity on the ability of osteoblast-derived MVs to initiate and propagate derived MVs to initiate and propagate calcification in vitro. Our work will provide fundamental insights into the mechanisms of normal bone minerlisation. Our project will also produce valuable tools and reagents that will facilitate future studies aimed at understanding the devlopment of diverse bone minerlisation and soft tissue ossification abnormalities that include some diseases of great public health concern e.g osteoarthritis, osteoporosis and arterial calcification.

Publications

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Ciancaglini P (2010) Kinetic analysis of substrate utilization by native and TNAP-, NPP1-, or PHOSPHO1-deficient matrix vesicles. in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

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Roberts S (2007) Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization. in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

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Yadav MC (2011) Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification. in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

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Yadav MC (2014) Ablation of osteopontin improves the skeletal phenotype of phospho1(-/-) mice. in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

 
Description First, we showed 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. We also demonstrated that isolated MVs contain PHOSPHO1. Phosphoethanolamine hydrolase activity was observed in sonicated MVs from Akp2!/! osteoblasts but not intact MVs in agreement with the intra-vesicular localization of PHOSPHO1. Inhibitors to PHOSPHO1 were identified (namely lanzoprazole,
ebselen and SCH-202676), characterized and used to show that inhibiting PHOSPHO1 function further inhibited the mineralization ability of MVs from Ak2 null osteoblasts. We also demonstrated the existence of a tightly regulated pattern of PHOSPHO1 and TNAP expression preceding mineralization, in chick limb bud mesenchymal micromass cultures
Next, we generated and characterized Phospho1 knockout mice. Phospho1-/- mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia and scoliosis in early life. We found that long bones from Phospho1-/- mice did not fracture during 3- point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1-/- tibia were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1-/- tibia. The N-terminal propeptide and C-terminal telopeptide of Type I collagen, which are markers for bone formation and resorption respectively, were both increased in Phospho1-/- mice and we associated this with increased bone remodeling during fracture repair or an attempt to remodel a mechanically competent bone capable of
withstanding physiological load. Primary cultures of Phospho1-/- tibial growth plate
chondrocytes and chondrocyte-derived MVs show reduced mineralizing ability and plasma samples of Phospho1-/- mice show reduced levels of TNAP and elevated PPi concentrations. However, transgenic overexpression of TNAP did not correct the bone phenotype in Phospho1-/- mice despite normalization of their plasma PPi levels. In contrast, double ablation of PHOSPHO1 and TNAP function lead to the complete absence of skeletal mineralization and perinatal lethality. This suggested that the availability of free inorganic Pi is not sufficient to initiate mineralization and that TNAP must be involved in generating Pi in the immediate vicinity of MVs.
Consequently, we studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated WT as well as TNAP-, NPP1- and PHOSPHO1- deficient MVs. We found that TNAP is an efficient ATPase in addition to its established role as a pyrophosphatase (PPiase) in the MV compartment. We also found that in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, NPP1 does not have a major PPi-generating activity at the level of MVs, but acts as both as an ATPase and as a PPiase.

In order to help understand the complex enzymatic interaction between TNAP, NPP1 and PHOSPHO1 in the MV compartment, we screened small molecule compound libraries
to identify more specific inhibitors of PHOSPHO1 since lanzoprazole, ebselen and SCH-202676, all have off-target effects, as well as more potent inhibitors of TNAP.
Exploitation Route This work shows conclusively the importance of the enzyme PHOSPHO1 in the maintenance of bone. In its absence the bone is soft and poorly mineralized. These findings have helped us and others understand the physiological processes of bone formation better and others have used this information to also advance or knowledge of pathological mineralization which occurs in blood vessel mineralisation
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our findings have been used to advance our knowledge of physiological and pathological mineralizaton. Such knowledge has allowed inclusive models to be postulated that will help targeted therapies to be identified that will treat conditions of great and economic importance such as osteoporosis, arthritis and coronary heart disease
First Year Of Impact 2010
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic