Division of Orofacial Functions and Orthodontics, Department of Health Improvement, Kyushu Dental University, Kitakyushu-shi, Japan.
Division of Anatomy, Department of Health Improvement, Kyushu Dental University, Kitakyushu-shi, Japan.
FEBS Open Bio. 2020 Aug;10(8):1612-1623. doi: 10.1002/2211-5463.12918. Epub 2020 Jul 12.
Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.
成骨细胞在受到机械压力后会将三磷酸腺苷（ATP）从细胞内释放出来。尽管已经证实细胞外 ATP 通过 P2 受体（如嘌呤能受体 P2X7（P2X7R）和嘌呤能受体 P2Y2（P2Y2R））影响骨代谢，但成骨细胞中 ATP 的释放机制仍不清楚。最近，已经鉴定出一种囊泡核苷酸转运体[VNUT，溶质载体家族 17 成员 9（SLC17A9）]，它可将 ATP 保存在囊泡中。本研究旨在阐明 VNUT 在成骨细胞骨代谢中的作用。通过定量实时 PCR（qPCR）和免疫组织化学，在小鼠骨骼和成骨细胞中证实了 VNUT 的 mRNA 和蛋白表达。接下来，当通过离心对 MC3T3-E1 细胞施加压缩力时，Slc17a9、P2x7r 和 P2y2r 的表达增加，同时细胞外 ATP 水平升高。此外，压缩力降低了 MC3T3-E1 细胞的成骨细胞分化能力。MC3T3-E1 细胞中 Slc17a9 的 shRNA 敲低降低了细胞外 ATP 水平，并在施加压缩力后导致成骨细胞分化增加，这可以通过 qPCR 分析成骨细胞标志物（如 Runx2、Osterix 和碱性磷酸酶（ALP））以及 ALP 活性来评估。与这些观察结果一致，通过 siRNA 敲低 P2x7r 或 P2y2r 部分挽救了机械加载引起的成骨细胞分化标志物的下调。总之，我们的结果表明，VNUT 在成骨细胞中表达，并且通过与 ATP 释放和 P2X7R 和/或 P2Y2R 活性相关的机制，VNUT 抑制成骨细胞对压缩力的分化。
