Hydroxyapatite Modulates mRNA Expression Profiles in Cultured Osteocytes

Osteocytes in vivo present a unique pattern of gene expression necessary for bone remodeling and mineral metabolism. Genes expressed in osteocytes such as Sost and fibroblast growth factor 23 (FGF23) are essential for load-driven bone formation and regulation of phosphate in the serum, respectively. However, their expression levels in standard cell cultures are significantly low and the mechanism of their transcriptional regulation is hardly examined. Since osteocytes reside in hydroxyapatite (HA)-surrounded lacunae, in this in vitro study we addressed a question whether they would exhibit mRNA expression profiles close to in vivo in the presence of HA. Our hypothesis was that HA would provide a three-dimensional (3D) growth environment important for the regulation of many of the osteocyte characteristic genes. To test this hypothesis, we grew MLO-A5 cells in a 3D collagen matrix with and without HA deposition and conducted a genome-wide expression analysis. The results revealed that deposition of HA markedly elevated the mRNA levels of Sost, dentin matrix protein 1 (DMP1), and FGF23. The microarray derived data indicated potential involvements of Wnt and PI3K signaling. Protein and mRNA expression analysis using pharmacological inhibitors such as LY294002 and pertussis toxin showed that HA-driven gene regulation was in part mediated by Akt in a PI3K pathway and G-protein linked receptors. In summary, we demonstrated herein that HA provided a growth environment that enabled osteocytes to stimulate their characteristic genes such as Sost, DMP1, and FGF23.