Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC–IP3 pathway and intracellular Ca2+ signaling

Intracellular Ca²⁺ 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 ²⁺ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca ²⁺ 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 ²⁺ 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 ²⁺ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca ²⁺ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP ₃) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca ²⁺ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca ²⁺-dependent mechanism via the PLC-IP ₃ pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.