Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC-IP pathway and intracellular Ca signaling.

Intracellular Ca signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1-300 µM) increased intracellular Ca signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP ) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca -dependent mechanism via the PLC-IP pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.