Huang Jijun, Zhang Min, Zhang Peng, Liang He, Ouyang Kunfu, Yang Huang-Tian
Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, China.
Second Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, 310009, China.
Purinergic Signal. 2016 Sep;12(3):465-78. doi: 10.1007/s11302-016-9512-9. Epub 2016 Apr 20.
Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca(2+) signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca(2+) mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca(2+) responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca(2+) transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca(2+) signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca(2+) transients only in hESCs but not in CVPCs. Furthermore, IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP3R2 and IP3R3 contributed to UTP-induced Ca(2+) responses while ATP-induced Ca(2+) responses were more dependent on IP3R2 in the CVPCs. In conclusion, a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca(2+) mobilization between these cells.
由P2受体（P2Rs）介导的嘌呤能信号传导在胚胎和干细胞发育中发挥重要作用。然而，其如何在人胚胎干细胞（hESCs）和衍生的心血管祖细胞（CVPCs）中介导Ca(2+)信号仍不清楚。在此，我们旨在确定P2Rs在介导这些细胞Ca(2+)动员中的作用。通过我们最近建立的方法将hESCs诱导分化为CVPCs。通过定量/RT-PCR分析P2Rs和肌醇1,4,5-三磷酸受体（IP3Rs）的基因表达。分别通过shRNA或TALEN介导的基因操作建立IP3R3敲低（KD）或IP3R2敲除（KO）的hESCs。共聚焦成像显示，CVPCs对ATP和UTP的Ca(2+)反应比hESCs更敏感、更强。一致地,除P2Y1外，大多数P2YRs的基因表达水平在CVPCs中升高。苏拉明或PPADS阻断了hESCs中ATP诱导的Ca(2+)瞬变，但仅部分抑制了CVPCs中的Ca(2+)瞬变。此外，P2Y1受体特异性拮抗剂MRS2279消除了hESCs中大多数ATP诱导的Ca(2+)信号，但在CVPCs中没有。P2Y1受体特异性激动剂MRS2365仅在hESCs中诱导Ca(2+)瞬变，而在CVPCs中没有。此外，IP3R2KO而非IP3R3KD降低了对MRS2365有反应的hESCs比例。相反，在CVPCs中，IP3R2和IP3R3都参与UTP诱导的Ca(2+)反应，而ATP诱导的Ca(2+)反应更依赖于IP3R2。总之，P2Y1受体在hESCs中的主要作用以及衍生CVPCs中P2Y-IP3R偶联的转变是这些细胞之间Ca(2+)动员差异的原因。
