Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation.

即使在基因一致的细胞群体中，均匀的细胞外刺激也可触发具有细胞特异性的钙信号模式。然而，介导细胞间异质性的潜在机制仍未明确。我们利用荧光IP3传感器，监测了表现出不同组胺诱导钙振荡模式的单个HeLa细胞的胞浆肌醇1,4,5-三磷酸（IP3）浓度变化，这些钙振荡模式以钙峰振幅衰减的时间常数与钙振荡频率作为区分依据。经组胺刺激的HeLa细胞，其钙信号的时间模式存在显著差异，我们发现IP3动力学具有细胞特异性，且与钙信号模式密切相关。逆转录聚合酶链式反应（RT-PCR）与蛋白质印迹（Western Blot）分析显示，HeLa细胞中磷脂酶C（PLC）-β1、-β3、-β4、-γ1、-δ3及-ε均呈较高水平表达。通过小干扰RNA（siRNA）介导的PLC同工酶沉默实验表明，PLC-β1与PLC-β4特异性参与了HeLa细胞中组胺诱导的IP3升高过程。敲低或过表达PLC-β1、PLC-β4以调控IP3动力学，可使钙振荡的特征（如钙峰振幅的时间变化常数与钙振荡频率）发生特异性改变，且变化范围处于野生型细胞群体的细胞间异质性区间内。上述研究结果表明，并非IP3诱导的钙释放过程，而是IP3生成过程中的异质性，可导致钙信号模式的细胞间差异，且PLC-β1与PLC-β4参与介导了G蛋白偶联受体（GPCR）刺激所诱发的细胞特异性钙信号。