Functional expression of the Ca2＋ signaling machinery in human embryonic stem cells  

作　　者：Ji-jun HUANG Yi-jie WANG Min ZHANG Peng ZHANG He LIANG Huajun BAI Xiu-jian YU Huang-tian YANG 

机构地区：[1]Heart Center, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China [2]Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai 200031, China [3]Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, China

出　　处：《Acta Pharmacologica Sinica》2017年第12期1663-1672,共10页中国药理学报（英文版）

基　　金：This work was supported by grants from the National Natu- ral Science of China （No 81520108004, 81470422 and 31030050 to HTY, No 31401167 to MZ）, the National Basic Research Program of China （No 2014CB965100 to Huang-tian YANG）, the National Science and Technology Major Project （No 2012ZX09501001 to Huang-tian YANG）, the National Key Research and Development Program （2016YFC1301200 to Huang-tian YANG）, and th.e Natural Science Foundation of Shanghai （No 17ZR1435500 to Jin-jun HUANG）. We thank WiCell Research Institute for providing the H7 and H9 hESCs and Dr He-ping CHENG （Peking University, Beijing, China） for providing the Flash Sniper software.

摘　　要：Emerging evidence suggests that Ca2＋ signals are important for the self-renewal and differentiation of human embryonic stem ceils （hESCs）. However, little is known about the physiological and pharmacological properties of the Ca2＋-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2＋-handling proteins; we Also used confocal Ca2＋ imaging and pharmacological approaches to determine the contribution of the Ca2＋-handling machinery to the regulation of Ca2＋ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2＋-handling-related Genes. ATP-induced Ca2＋ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor （IP3R） blocker 2-APB Or xestospongin C. In addition, Ca2＋ 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 Ca2＋ entry （SOCE） but not voltage-operated Ca2＋ channel-mediated Ca2＋ entry was observed. Inhibition of sarco/endoplasmic reticulum （ER） Ca2＋-ATPase （SERCA） by thapsigargin induced a significant increase in the cytosolic free Ca2＋ concentration （[Ca2＋]i）. For the Ca2＋ extrusion pathway. In hibition of plasma membrane Ca2＋ pumps （PMCAs） by carboxyeosin induced a slow increase in [Ca2＋]i, whereas the Na＋/Ca2＋ exchanger （NCX） inhibitor KBR7943 induced a rapid increase in [Ca2＋]i. Taken together, increased [Ca2＋]i is mainly mediated by Ca2＋ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2＋- Signaling machinery in hESCs; maintenance of low [Ca2＋]i is mediated by uptake of cytosolic Ca2＋ into the ER via SERCA and extrusion of Ca2＋ out of cells via NCX and PMCA in hESCs.Emerging evidence suggests that Ca2＋ signals are important for the self-renewal and differentiation of human embryonic stem ceils （hESCs）. However, little is known about the physiological and pharmacological properties of the Ca2＋-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2＋-handling proteins; we Also used confocal Ca2＋ imaging and pharmacological approaches to determine the contribution of the Ca2＋-handling machinery to the regulation of Ca2＋ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2＋-handling-related Genes. ATP-induced Ca2＋ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor （IP3R） blocker 2-APB Or xestospongin C. In addition, Ca2＋ 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 Ca2＋ entry （SOCE） but not voltage-operated Ca2＋ channel-mediated Ca2＋ entry was observed. Inhibition of sarco/endoplasmic reticulum （ER） Ca2＋-ATPase （SERCA） by thapsigargin induced a significant increase in the cytosolic free Ca2＋ concentration （[Ca2＋]i）. For the Ca2＋ extrusion pathway. In hibition of plasma membrane Ca2＋ pumps （PMCAs） by carboxyeosin induced a slow increase in [Ca2＋]i, whereas the Na＋/Ca2＋ exchanger （NCX） inhibitor KBR7943 induced a rapid increase in [Ca2＋]i. Taken together, increased [Ca2＋]i is mainly mediated by Ca2＋ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2＋- Signaling machinery in hESCs; maintenance of low [Ca2＋]i is mediated by uptake of cytosolic Ca2＋ into the ER via SERCA and extrusion of Ca2＋ out of cells via NCX and PMCA in hESCs.

关 键 词：Ca2＋ signaling Ca2＋ release Ca2＋ extrusion Ca2＋ channels Ca2＋ pumps human embryonic stem cells 

分 类 号：R0[医药卫生]