Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana.
Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
J Cell Physiol. 2020 Feb;235(2):1723-1732. doi: 10.1002/jcp.29091. Epub 2019 Jul 12.
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.
细胞内 Ca 信号对于干细胞功能至关重要，并在分化过程中发挥重要作用。牙髓干细胞（DPSCs）是干细胞的潜在来源；然而，控制细胞分化的机制在很大程度上仍然未知。利用大鼠 DPSCs，我们研究了三磷酸腺苷（ATP）对成骨细胞分化的影响，并通过实时 Ca 成像分析来表征其作用机制。我们的结果表明，ATP 通过茜素红 S 染色增强了细胞外基质中的 Ca 沉积，从而增强了成骨作用。这与成骨细胞基因 BMP2、Mmp13、Col3a1、Ctsk、Flt1 和 Bgn 的上调一致。用 ATP（1-300μM）刺激 DPSCs 以浓度依赖性方式增加细胞内 Ca 信号，而组胺、乙酰胆碱、精氨酸加压素、卡巴胆碱和基质细胞衍生因子-1α 则不能。用 thapsigargin 耗尽内质网中的细胞内 Ca 库会使 ATP 反应消失，但在细胞外 Ca 自由条件下仍能检测到该反应。此外，磷脂酶 C（PLC）抑制剂 U73122 和三磷酸肌醇（IP ）受体抑制剂 2-氨基乙氧基二苯硼酸盐抑制了 Ca 信号。我们的研究结果提供了更好的理解，即 ATP 如何控制 DPSCs 中的成骨作用，这涉及通过 PLC-IP 途径的 Ca 依赖性机制。这一知识可能有助于改善骨结构组织再生的成骨分化方案。
