The role of phosphatases in the initiation of skeletal mineralization

Abstract

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PPi), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl(-/-)) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the Pi needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.

Fig. 1

Model of initiation of skeletal mineralization including the function of PHOSPHO1, TNAP, NPP1, and phosphate transporters. The first step of MV-mediated mineralization involves the convergence of two independent biochemical pathways: intravesicular P_i generation by the enzymatic action of PHOSPHO1 and influx of P_i, generated in the perivesicular space by the activities of TNAP and NPP1, via P_i transporters. PC phosphocholine, PEA phosphoethanolamine, MV matrix vesicle, P_iT phosphate transporter 1, HA hydroxyapatite, ECM extracellular matrix, P_i inorganic phosphate, PP_i inorganic pyrophosphate

Fig. 2

Summary of findings and how they can be explained by the unified model of initiation of skeletal mineralization. Original data were published in Yadav et al. [38]. Wild-type mice the first step of MV-mediated mineralization involves intravesicular P_i generation by PHOSPHO1 and influx of P_i, generated extravesicularly by TNAP and NPP1. Extravesicular propagation occurs on collagen scaffolds facilitated by the pyrophosphatase function of TNAP and NPP1. Phospho1^−/− mice a growth plate and skeletal phenotype is apparent, but HA is still found inside MVs because P_iT-mediated transport of P_i, generated by TNAP and NPP1, is unaffected. Akp2^−/− mice rickets and osteomalacia are prominent, due to increases in extracellular PP_i. HA crystals are still found inside MVs due to the P_i-generating ability of PHOSPHO1. P_iT-mediated influx of P_i is greatly diminished except where NPP1 activity is high (axial skeleton). [Phospho1^−/−; Alpl^−/−] mice complete absence of skeletal mineralization can be explained by the absence of intravesicular P_i generation by PHOSPHO1, the lack of extravesicular P_i generation by TNAP needed for P_iT-mediated influx, and accumulation of PP_i in the ECM. Abbreviations as in Fig. 1