Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes
Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes
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DOI：
10.1016/j.ceca.2021.102363
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发表时间：
2021-02-05
期刊：
影响因子：
4
通讯作者：
Price, Christopher
中科院分区：
文献类型：
--
作者：
McDonough, Ryan C.;Gilbert, Rachel M.;Price, Christopher
钙信号的时间动态是软骨细胞稳态和软骨发生的关键调节因子。钙振荡调节软骨细胞及其前体的分化和合成代谢过程。控制软骨细胞钙信号的尝试已经通过机械扰动和合成离子通道调节剂实现。然而，当唤起钙信号时，这种刺激可能缺乏局部和全局特异性和精确性。合成信号平台可以更精确和选择性地激活钙信号，从而改善对软骨细胞行为中细胞内钙（[Ca 2 +]（i））作用的剖析。一个这样的平台是hM 3Dq，一种化学发生DREADD（设计者受体，仅由设计者药物激活），其在施用氯氮平N-氧化物（CNO）时通过G α（q）-PLC β-IP 3-ER途径激活钙信号传导。我们先前描述了hM 3Dq在软骨细胞样ATDC 5细胞中精确介导靶向合成钙信号的首次使用。在这里，我们产生了稳定表达hM 3Dq-ATDC 5细胞，以深入研究G alpha（q）-GPCR钙信号转导的动力学。CNO以温度和浓度依赖性（1 pM-100 μ M）的方式驱动强有力的钙反应，并引发高于10 nM的振荡钙信号水平升高。ATDC 5细胞中hM 3Dq介导的钙振荡依赖于ER钙储备的启动和维持，CNO刺激后hM 3Dq的下调和恢复动力学与传统报道的GPCR再循环动力学一致。这项研究成功地产生了一个稳定的hM 3Dq细胞系，以精确地驱动ATDC 5细胞中G alpha（q）-GPCR介导的和ER依赖的振荡钙信号，并建立了一种新的工具来阐明GPCR介导的钙信号在软骨细胞生物学，软骨病理学和软骨组织工程中的作用。
The temporal dynamics of calcium signaling are critical regulators of chondrocyte homeostasis and chondrogenesis. Calcium oscillations regulate differentiation and anabolic processes in chondrocytes and their precursors. Attempts to control chondrocyte calcium signaling have been achieved through mechanical perturbations and synthetic ion channel modulators. However, such stimuli can lack both local and global specificity and precision when evoking calcium signals. Synthetic signaling platforms can more precisely and selectively activate calcium signaling, enabling improved dissection of the roles of intracellular calcium ([Ca2+](i)) in chondrocyte behavior. One such platform is hM3Dq, a chemogenetic DREADD (Designer Receptors Exclusively Activated by Designer Drugs) that activates calcium signaling via the G alpha(q)-PLC beta-IP3-ER pathway upon administration of clozapine N-oxide (CNO). We previously described the first-use of hM3Dq to precisely mediate targeted, synthetic calcium signals in chondrocyte-like ATDC5 cells. Here, we generated stably expressing hM3Dq-ATDC5 cells to investigate the dynamics of G alpha(q)-GPCR calcium signaling in depth. CNO drove robust calcium responses in a temperature- and concentration-dependent (1 pM-100 mu M) manner and elicited elevated levels of oscillatory calcium signaling above 10 nM. hM3Dq-mediated calcium oscillations in ATDC5 cells were reliant on ER calcium stores for both initiation and sustenance, and the downregulation and recovery dynamics of hM3Dq after CNO stimulation align with traditionally reported GPCR recycling kinetics. This study successfully generated a stable hM3Dq cell line to precisely drive G alpha(q)-GPCR-mediated and ER-dependent oscillatory calcium signaling in ATDC5 cells and established a novel tool to elucidate the role that GPCR-mediated calcium signaling plays in chondrocyte biology, cartilage pathology, and cartilage tissue engineering.