Abstract
Bone mineralization is a carefully orchestrated process, regulated by a number of promoters and inhibitors that function to ensure effective hydroxyapatite formation. Here we sought to identify new regulators of this process through a time series microarray analysis of mineralising primary osteoblast cultures over a 27 day culture period. To our knowledge this is the first microarray study investigating murine calvarial osteoblasts cultured under conditions that permit both physiological extracellular matrix mineralization through the formation of discrete nodules and the terminal differentiation of osteoblasts into osteocytes. RT-qPCR was used to validate and expand the microarray findings. We demonstrate the significant up-regulation of >6,000 genes during the osteoblast mineralization process, the highest-ranked differentially expressed genes of which were those dominated by members of the PPAR-γ signalling pathway, namely Adipoq, Cd36 and Fabp4. Furthermore, we show that the inhibition of this signalling pathway promotes matrix mineralisation in these primary osteoblast cultures. We also identify Cilp, Phex, Trb3, Sox11, and Psat1 as novel regulators of matrix mineralization. Further studies examining the precise function of the identified genes and their interactions will advance our understanding of the mechanisms underpinning biomineralization.
Highlights
During endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the initial formation of crystalline hydroxyapatite (HA) in the sheltered interior of membrane-limited matrix vesicles (MVs) [2]
Transcripts significantly up-regulated during mineralization at both day 9 and day 27 compared to day 0 included key members of the peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling pathway, namely adiponectin (Adipoq), cluster of differentiation 36 (Cd36), and fatty acid binding protein 4 (Fabp4)
Whilst numerous in vivo and in vitro studies have examined the mechanisms of osteoblast differentiation and mineralization, few have incorporated the transition from osteoblasts to osteocytes
Summary
During endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the initial formation of crystalline hydroxyapatite (HA) in the sheltered interior of membrane-limited matrix vesicles (MVs) [2]. This is followed by the modulation of matrix composition to further promote propagation of HA outside of the MVs [Anderson et al, 1990]. This biphasic mineralization process is critically dependent on a regulated balance of a number of factors which include calcium and inorganic phosphate (Pi), ECM proteins, and the presence of mineralization inhibitors such as inorganic pyrophosphate (PPi), matrix gla protein, and osteopontin [Meyer, 1984; Sodek et al, 2000; Murshed et al, 2004; Johnson et al, 2000]. The bone specific phosphatase PHOSPHO1 is essential for the accumulation of Pi within MVs and bone mineralization.[13,14,15,16,17,18] we have found that PHOSPHO1 and TNAP have non-redundant functions in regulating HA formation and mineralization; genetic ablation of either produces a hypomineralised, functionally-impaired skeleton and spontaneous fracture, and simultaneous ablation, a complete lack of all skeletal mineralisation (yadav et al; huesa et al)
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