BACKGROUND In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

中文翻译：

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N-甲基-D-天冬氨酸（NMDA）受体的表达和功能是早期软骨形成所必需的。

背景技术体外软骨形成取决于许多信号传导途径的协同作用，其中许多对细胞内Ca 2+浓度的变化敏感。N-甲基-D-天冬氨酸（NMDA）谷氨酸受体是对Ca2 +具有高渗透性的阳离子通道。尽管现在有越来越多的证据表明NMDA受体在包括成熟软骨和骨骼在内的非神经组织中的表达和功能，但尚未阐明谷氨酸信号对软骨形成调控的作用。方法我们使用单细胞荧光钙成像，膜片钳，瞬时基因沉默和蛋白质印迹研究了谷氨酸能信号传导在体外软骨形成过程中在高密度软骨细胞培养过程中的作用。结果在这里，我们表明，在主要的鸡软骨形成模型系统中，在体外软骨形成过程中，谷氨酸能信号传导途径的关键成分起作用。我们还介绍了这些培养物的完整谷氨酸受体亚基mRNA和蛋白质表达谱。这是第一个报道NMDA介导的信号传导可能引起胚胎肢芽芽软骨形成培养的关键因素的研究，因为它引起细胞内Ca2 +瞬变，而GluN2B亚基特异性抑制剂ifenprodil消除了这种瞬变。NMDAR的功能对于软骨形成至关重要，因为使用ifenprodil或GRIN1 siRNA的功能性敲除可暂时阻断软骨生成细胞的分化。随着GluN1蛋白的重新表达，软骨形成得以完全恢复。