Regulation of skeletal mineralisation by PHOSPHO1
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Date
08/07/2017Author
Houston, Dean Alexander
Metadata
Abstract
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.