Regulation of skeletal mineralisation by PHOSPHO1.
Houston, Dean Alexander
PHOSPHO1 is a skeletal specific phosphatase whose activity towards the lipid metabolites, phosphocholine (PCho) and phosphoethanolamine (PEth), results in the generation of inorganic phosphate (Pi) within matrix vesicles (MV). PHOSPHO1 activity is essential for the initiation of biomineralisation. The genetic ablation of Phospho1 results in severe hypomineralisation of the skeleton and dentition. Neutral sphingomyelinase 2 (nSMase2, encoded by the Smpd3 gene) catalyses the breakdown of the membrane lipid sphingomyelin to generate ceramide and PCho. Similar hypomineralisation of the skeleton is noted in the Smpd3-/- mouse. This observation led to the hypothesis that nSMase2 and PHOSPHO1 work in tandem for the generation of Pi within MV. Despite knowledge of the phenotype associated with the absence of Phospho1 or Smpd3, little is known about the expression profiles of these genes during the initiation of extracellular matrix (ECM) mineralisation, or the regulation of these genes. This thesis characterised the expression of Phospho1, Smpd3 and other key genes associated with ECM mineralisation in in vitro models of mineralisation under exogenous phosphatase substrate-free conditions. Additionally, building on preliminary work in osteocytes, the regulation of Phospho1 and Smpd3 by parathyroid hormone (PTH) was investigated both in vitro and in vivo. Characterisation of MC3T3 osteoblast-like cell cultures, primary calvarial osteoblast and embryonic metatarsal organ cultures similarly revealed simultaneous and striking increases in the expression of PHOSPHO1 and nSMase2 prior to the onset of ECM mineralisation. In Phospho1-/- cell and organ cultures, ECM mineralisation was markedly diminished, and nSMase2 expression was notably reduced. The parathyroid hormone (PTH) regulation of Phospho1 and Smpd3 in osteocytes was confirmed in MC3T3 osteoblast-like cell cultures. Phospho1 and Smpd3 mRNA expression was strongly and rapidly (within 15 minutes) inhibited by PTH. Experiments with cycloheximide revealed that this was a direct effect not requiring protein synthesis. Further experimentation utilising the adenylyl cylase agonist, Forskolin and the PKA inhibitor, PKI (5-24), identified the cAMP-PKA signalling pathway as the mediator of the effects of PTH on Phospho1 and Smpd3 expression. In contrast, however, primary calvarial osteoblasts, human subchondral bone osteoblasts and murine embryonic metatarsal cultures all displayed an upregulation of Phospho1 expression in response to a 24 h exposure to PTH. Although informative, these findings highlighted the need to investigate the PTH regulation of Phospho1 in vivo. The administration of PTH (80 μg/kg) enhanced the expression of Phospho1 and Smpd3 within 6 h and after 14 and 28-day intermittent exposure in the distal femur of male wild-type mice. The expression of the transcription factors, Runx2 and Trps1, which have been implicated in the regulation Phospho1 were similarly upregulated by these PTH exposures. I hypothesised that the upregulation of Phospho1 could provide a novel mechanism explaining the osteoanabolic effects of intermittent PTH (iPTH). Bone microarchitecture in response to iPTH was assessed in the tibiae of WT and Phospho1-/- mice by micro computed tomography. The absence of Phospho1 limited the anabolic effects of PTH in cortical bone but not in the metaphyseal trabecular bone. The work described within this thesis provides further evidence of the cooperative functions of nSMase2 and PHOSPHO1 in the initiation of skeletal mineralisation. The potent regulation of these enzymes in vivo by PTH offers an additional explanation of the anabolic effects of iPTH and forms part of an emerging body of evidence seeking to understand the regulation of these enzymes.