Department of Pathophysiology, Harbin Medical University, Harbin, China.
Differentiation. 2013 Jan;85(1-2):32-40. doi: 10.1016/j.diff.2012.11.002. Epub 2013 Jan 11.
The calcium-sensing receptor (CaSR), a G protein coupled receptor, is involved in a number of physiological and pathological processes. Embryonic stem cells (ESCs) have a potential role to differentiate into all types of cells. Whether CaSR is functionally expressed in ESCs is unclear. In this study, the expression and distribution of CaSR in 129 mouse ES-D3 cell lines were detected by Western blotting and immunofluorescence; and the intracellular calcium concentration ([Ca(2+)]i) was measured using Laser Confocal Scanning Microscopy. Mouse embryonic stem cells (mESCs) were cultured to embryoid bodies (EBs) and the differentiation of EBs into cardiomyocytes was induced by icariin (ICA). The cardiac specific proteins, a-Actinin and cardiac troponin-I (cTnI), were analyzed by immunofluorescence, and the differentiation rate was analyzed by flow cytometry. The expression of cardiac-specific transcription factors, Nkx2.5 and GATA-4, was detected by Western blotting. We found that the CaSR protein exists in both mESCs and mESC-derived cardiomyocytes (mESC-CMs). Increasing extracellular calcium or neomycin (an agonist of CaSR) increased [Ca(2+)]i and the differentiation rate. These effects were abolished by inhibition of CaSR, phospholipase C, IP3 receptor and Ca(2+) ATPase, or by depletion of the sarcoplasmic reticulum Ca(2+) store, respectively. Activation of CaSR up-regulated protein expression of Nkx2.5 and GATA4 in EBs at an early stage of ICA-induced differentiation. In conclusion, CaSR is functionally expressed in mESCs, and activation of CaSR is involved in the differentiation of mESCs into cardiomyocytes by facilitating the expression of NKx2.5 and GATA-4.
钙敏感受体（CaSR）是一种 G 蛋白偶联受体，参与许多生理和病理过程。胚胎干细胞（ESCs）具有分化为各种细胞的潜力。CaSR 是否在 ESCs 中具有功能性表达尚不清楚。在这项研究中，通过 Western blot 和免疫荧光检测了 129 只小鼠 ESC-D3 细胞系中 CaSR 的表达和分布；并通过激光共聚焦扫描显微镜测量了细胞内钙离子浓度([Ca(2+)]i)。将小鼠胚胎干细胞（mESCs）培养成类胚体（EBs），并用淫羊藿苷（ICA）诱导 EBs 分化为心肌细胞。通过免疫荧光分析心肌细胞特异性蛋白α-辅肌动蛋白和心肌肌钙蛋白 I（cTnI），并通过流式细胞术分析分化率。通过 Western blot 检测心脏特异性转录因子 Nkx2.5 和 GATA-4 的表达。我们发现 CaSR 蛋白存在于 mESCs 和 mESC 衍生的心肌细胞（mESC-CMs）中。增加细胞外钙或新霉素（CaSR 的激动剂）可增加[Ca(2+)]i 和分化率。这些作用分别被 CaSR、PLC、IP3 受体和 Ca(2+)ATP 酶的抑制，或肌浆网 Ca(2+)储存的耗竭所阻断。CaSR 的激活上调了 ICA 诱导分化早期 EB 中 Nkx2.5 和 GATA4 的蛋白表达。总之，CaSR 在 mESCs 中具有功能性表达，CaSR 的激活通过促进 NKx2.5 和 GATA-4 的表达，参与 mESCs 向心肌细胞的分化。
