How to build a bone: the role of PHOSPHO1 in biomineralisation of the developing skeleton

The vertebrate skeleton is a hugely complex organ which performs varied and diverse functions encompassing its action as a biomechanical and protective scaffold in conjunction with the musculature, its role in calcium and phosphate ion homeostasis, and recent evidence demonstrating its capacity as an endocrine organ involved with energy homeostasis. The skeleton consists of a multitude of bones distributed throughout the body, and many of which exhibit diverse morphologies which are critical to their functions. Many of these functions are dependent upon the hierarchical structure of bone, however the fine details of this structure at the nanoscale and the initial steps regulating its formation are unclear. This thesis will examine the role of orphan phosphatase 1 (PHOSPHO1) in embryonic bone development and investigate the pathway through which PHOSPHO1 directs bone biomineralisation. This thesis has confirmed the expression of PHOSPHO1 at the mineralising surfaces of long bones and calvaria in the mouse during skeletal development and its co-localisation with the established mineralisation marker tissue non-specific alkaline phosphatase (TNAP). The phenotype of the Phospho1-/-mouse during development was characterised revealing a significant loss of mineralised bone throughout the skeleton. The ultrastructure of Phospho1-null bone examined using focussed ion beam-scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM) revealed a hypomineralised fibrous structure containing small electron dense particles which may represent matrix vesicles (MVs) which have failed to nucleate mineral. Having established a critical role for PHOSPHO1 in embryonic bone mineralisation, the biochemical mechanism providing substrates for hydrolysis of PHOSPHO1 inside extracellular MVs was interrogated. The skeletal phenotype of the Enpp6-/-mouse was characterised to investigate its proposed function upstream of PHOSPHO1, revealing a transient hypomineralisation of both trabecular and cortical bone in young animals which recovered over time. This phenotype was confirmed by backscattered scanning electron microscopy, demonstrating small electron dense particles in Enpp6-/- trabeculae which may represent a failure of mineralisation foci to propagate and fuse. To further interrogate the biochemistry of MVs a primary osteoblast cell culture model of MV generation and isolation was characterised relative to the more commonly used MC3T3 cell line. Primary osteoblasts generated vesicles were largely consistent with those from MC3T3s and contained both PHOSPHO1 and TNAP. These data confirm that primary osteoblasts represent a suitable model for the investigation of MVs. This model was further used to characterise the protein and lipid cargo of MVs to investigate both their biogenesis and to examine whether the biochemical mechanism hypothesised to deliver PHOSPHO1 substrates within vesicles, and therefore the generation of intravesicular phosphate, is disrupted in its absence. Proteomics data implicated the role of depolymerisation in the osteoblast cytoskeleton during the release of MVs into the extracellular matrix, and also indicated that vesicle biogenesis may be mediated by GTPase signalling. Lipidomic analysis of wild-type and Phospho1-/- vesicles furthermore revealed perturbations in the pattern of lipids present in the absence of PHOSPHO1. The data set out here has for the first time confirmed the critical role of PHOSPHO1 during biomineralisation of the developing skeleton and strongly implicates its function inside extracellular MVs. These data provide a promising avenue of investigation into the fundamental mechanisms regulating bone biomineralisation and skeletal development.