VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation
VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation
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DOI：
10.1002/2211-5463.12918
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发表时间：
2020-07-12
期刊：
影响因子：
2.6
通讯作者：
Kawamoto, Tatsuo
中科院分区：
文献类型：
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作者：
Inoue, Asako;Nakao-Kuroishi, Kayoko;Kawamoto, Tatsuo
成骨细胞在机械应力后释放出细胞外的三磷酸腺苷（ATP）。虽然细胞外ATP通过P2受体[如嘌呤能受体P2 X7（P2 X7 R）和嘌呤能受体P2 Y2（P2 Y2 R）]影响骨代谢已被公认，但成骨细胞释放ATP的机制仍不清楚。最近，已经鉴定了在囊泡中保存ATP的囊泡核苷酸转运蛋白[VNUT，溶质载体家族17成员9（SLC 17 A9）]。本研究的目的是阐明VNUT在成骨细胞骨代谢中的作用。通过实时定量PCR（qPCR）和免疫组化证实VNUT的mRNA和蛋白质在小鼠骨和成骨细胞中的表达。接着，当通过离心对MC 3 T3-E1细胞施加压缩力时，Slc 17 a9、P2 x7 r和P2 y2 r的表达随着细胞外ATP水平的增加而增加。此外，压力降低了MC 3 T3-E1细胞的成骨分化能力。通过对成骨细胞标志物如Runx 2、Osterix和碱性磷酸酶（ALP）以及ALP活性的qPCR分析评估，MC 3 T3-E1细胞中Slc 17 a9的shRNA敲低降低了细胞外ATP的水平，并且还导致在施加压缩力后成骨细胞分化增加。与这些观察结果一致，通过siRNA敲低P2 x7 rorP 2 y2 r部分挽救了由机械负荷引起的成骨细胞分化标志物的下调。总之，我们的研究结果表明，VNUT在成骨细胞中表达，并且VNUT通过与ATP释放和P2 X7 R和/或P2 Y2 R活性相关的机制抑制成骨细胞对压缩力的分化。
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 ofSlc17a9,P2x7r, andP2y2rwas increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown ofSlc17a9in 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 ofP2x7rorP2y2rby 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.