Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression.

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca concentration ([Ca]) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca] enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca] transiently increased the intracellular Ca concentration ([Ca]) due to activation of the calcium-sensing receptor (CaSR). The Ca-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca] decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca] enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca] and stimulation of Ca/calmodulin-dependent NFAT-signaling pathways.