Functional expression of the Ca2+ signaling machinery in human embryonic stem cells
Functional expression of the Ca2+ signaling machinery in human embryonic stem cells
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人胚胎干细胞中Ca2信号传导机制的功能表达
DOI：
10.1038/aps.2017.29
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发表时间：
2017-12-01
影响因子：
8.2
通讯作者：
Yang, Huang-tian
中科院分区：
文献类型：
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作者：
Huang, Ji-Jun;Wang, Yi-Jie;Yang, Huang-tian
近年来的研究表明，钙信号在人胚胎干细胞（human embryonic stem cells，hESCs）的自我更新和分化过程中起着重要作用。然而，很少有人知道的生理和药理学特性的钙处理机制在hESC。本研究采用RT-PCR和Western blotting技术分析了hESC中Ca 2+调控蛋白基因的表达谱，并采用共聚焦Ca 2+成像和药理学方法研究了hESC中Ca 2+调控机制对Ca 2+信号转导的调控作用。我们发现hESCs表达多能标记物和各种Ca 2+处理相关基因。几乎所有hESC中ATP诱导的Ca 2+瞬变都可被1，4，5-三磷酸肌醇受体（IP 3R）阻断剂2-APB或xestospongin C抑制另外，RyR激活剂咖啡因可诱导10%~ 15%的hESCs产生Ca 2+瞬变，RyR可阻断咖啡因的Ca 2+瞬变，而咖啡因和ATP则无相加效应。此外，还观察到钙池操纵的钙内流（SOCE），而电压操纵的钙通道不介导的钙内流。Thapsigargin抑制肌质内质网Ca 2+-ATP酶（SERCA），可使胞浆游离Ca 2+浓度（[Ca 2+] i）显著升高。在Ca 2+外排途径中，羧化肌球蛋白抑制质膜Ca 2+泵（PMCAs）可使[Ca 2+] i缓慢增加，而Na+/Ca 2+交换器（NCX）抑制剂KBR 7943则使[Ca 2+] i迅速增加。[Ca 2+] i的增加主要是由胞内钙库通过IP 3Rs释放Ca 2+介导的。此外，RyR在部分hESC中起作用，表明hESC中Ca 2+信号机制的异质性;低[Ca 2+] i的维持是通过SERCA将胞浆Ca 2+摄取到ER中以及通过NCX和PMCA将Ca 2+挤出细胞而介导的。
Emerging evidence suggests that Ca 2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca 2+-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca 2+-handling proteins; we also used confocal Ca 2+ imaging and pharmacological approaches to determine the contribution of the Ca 2+-handling machinery to the regulation of Ca 2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca 2+-handling-related genes. ATP-induced Ca 2+ transients in almost all hESCs were inhibited by the inositol-1, 4, 5-triphosphate receptor (IP 3 R) blocker 2-APB or xestospongin C. In addition, Ca 2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%–15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca 2+ entry (SOCE) but not voltage-operated Ca 2+ channel-mediated Ca 2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca 2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca 2+ concentration ([Ca 2+] i). For the Ca 2+ extrusion pathway, inhibition of plasma membrane Ca 2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca 2+] i, whereas the Na+/Ca 2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca 2+] i. Taken together, increased [Ca 2+] i is mainly mediated by Ca 2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca 2+-signaling machinery in hESCs; maintenance of low [Ca 2+] i is mediated by uptake of cytosolic Ca 2+ into the ER via SERCA and extrusion of Ca 2+ out of cells via NCX and PMCA in hESCs.