Abstract
Phosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process. Induced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1mM inorganic phosphate, or 3mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation. Osteogenic differentiation and mineralization were promoted in the 3mM Pi group compared to those in the GP and 1mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3mM Pi group on day 7. WNT11 mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, including WNT5b, WNT11 and ROR2 mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3mM Pi group on day 7 by the inhibition of WNT5b. Pi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.
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