Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA; Interdisciplinary Program of Biomedical Sciences, Graduate School, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA.
Arch Oral Biol. 2021 Aug;128:105169. doi: 10.1016/j.archoralbio.2021.105169. Epub 2021 May 27.
The aim of this study was to elucidate the molecular mechanism by which antidiuretic hormone (ADH) inhibited osteogenesis in dental follicle stem cells.
Rat dental follicle stem cells were cultured in osteogenic differentiation medium supplemented with ADH. Alkaline phosphatase enzyme activity, Alizarin Red S staining, MTT assay and RT-qPCR was used to examine ADH's impact on cell mineralization, viability, and osteogenic gene expression. Real-time calcium imaging analysis was performed to identify the ADH receptor and its mechanism of action.
ADH supplementation to the osteogenic differentiation medium inhibited cell mineralization without compromising cell viability and downregulated the expression of key osteogenic genes: DCN (Decorin), RUNX2 (Runt-related transcription factor 2) and BSP (Bone sialoprotein). Real-time calcium imaging analysis revealed that ADH (1-1000 nM) increased intracellular calcium in a concentration-dependent manner. Pretreatment of cells with V2255, a V1a receptor blocker, inhibited the calcium signals, but not with the V1b (Nelivaptan) or V2 (Tolvaptan). V2255 also reversed the inhibitory effect of ADH on osteogenesis. Furthermore, U73122, a Phospholipase C (PLC) inhibitor, 2-APB, an Inositol Triphosphate (IP) receptor blocker, and depletion of endoplasmic reticulum calcium stores abolished the calcium signals by ADH.
Our results demonstrated that ADH activates V1a receptors and the PLC-IP pathway to stimulate intracellular calcium signals, which inhibits cell mineralization and osteogenic gene expression. These findings uncovered a novel function for ADH as a negative regulator of osteogenesis in dental follicle stem cells. The role of ADH in the pathogenesis of bone diseases remains to be determined.
本研究旨在阐明抗利尿激素（ADH）抑制牙周膜干细胞成骨的分子机制。
将大鼠牙周膜干细胞培养在添加 ADH 的成骨分化培养基中。使用碱性磷酸酶酶活性、茜素红 S 染色、MTT 测定和 RT-qPCR 检测 ADH 对细胞矿化、活力和成骨基因表达的影响。进行实时钙成像分析以鉴定 ADH 受体及其作用机制。
ADH 补充到成骨分化培养基中抑制细胞矿化而不影响细胞活力，并下调关键成骨基因的表达：DCN（Decorin）、RUNX2（Runt 相关转录因子 2）和 BSP（骨涎蛋白）。实时钙成像分析显示 ADH（1-1000 nM）以浓度依赖的方式增加细胞内钙。用 V2255（V1a 受体阻滞剂）预处理细胞可抑制钙信号，但 V1b（Nelivaptan）或 V2（Tolvaptan）则不行。V2255 还逆转了 ADH 对成骨的抑制作用。此外，PLC 抑制剂 U73122、三磷酸肌醇（IP）受体阻滞剂 2-APB 和内质网钙库耗竭消除了 ADH 引起的钙信号。
我们的结果表明 ADH 激活 V1a 受体和 PLC-IP 途径以刺激细胞内钙信号，从而抑制细胞矿化和成骨基因表达。这些发现揭示了 ADH 作为牙周膜干细胞成骨的负调控因子的新功能。ADH 在骨病发病机制中的作用仍有待确定。
