Elise H. Padbury, Štefan Bálint, Emanuela Carollo, David R. F. Carter, Esther B. E. Becker
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引用次数: 0
摘要
细胞外囊泡(EVs)对包括癌症进展在内的多种病理过程都有影响,但其生物发生的分子机制仍未完全阐明。基于四泛素的 pHluorin 报告器的开发使得在质膜上实时分析 EV 释放成为可能。在这里,我们利用 CD81-pHluorin 研究了卵巢癌(OC)细胞中 EV 释放的机制,并报告了钙离子渗透性瞬时受体电位(TRP)通道 TRPC3 在 EV 介导的通讯中的新作用。我们发现,特异性激活 TRPC3 会增加 SKOV3 细胞中的 Ca2+ 信号,并刺激 EV 释放的立即增加。Ca2+刺激物组胺和离子霉素同样诱导了EV释放,成像分析显示了不同刺激依赖的时间和空间释放动态。有趣的是,抑制 TRPC3 可减轻组胺刺激的 Ca2+ 进入和 EV 释放,这表明 TRPC3 可能是组胺信号在 EV 生物发生过程中的下游作用。此外,我们还发现,直接激活 TRPC3 以及应用来自 TRPC3 激活细胞的 EV 可增加 SKOV3 的增殖。我们的数据深入揭示了OC细胞中EV释放的分子机制和动力学,提出了TRPC3在EV生物发生中的关键作用。
TRPC3 signalling contributes to the biogenesis of extracellular vesicles
Extracellular vesicles (EVs) contribute to a wide range of pathological processes including cancer progression, yet the molecular mechanisms underlying their biogenesis remain incompletely characterized. The development of tetraspanin-based pHluorin reporters has enabled the real-time analysis of EV release at the plasma membrane. Here, we employed CD81-pHluorin to investigate mechanisms of EV release in ovarian cancer (OC) cells and report a novel role for the Ca2+-permeable transient receptor potential (TRP) channel TRPC3 in EV-mediated communication. We found that specific activation of TRPC3 increased Ca2+ signalling in SKOV3 cells and stimulated an immediate increase in EV release. Ca2+-stimulants histamine and ionomycin likewise induced EV release, and imaging analysis revealed distinct stimulation-dependent temporal and spatial release dynamics. Interestingly, inhibition of TRPC3 attenuated histamine-stimulated Ca2+-entry and EV release, indicating that TRPC3 is likely to act downstream of histamine signalling in EV biogenesis. Furthermore, we found that direct activation of TRPC3 as well as the application of EVs derived from TRPC3-activated cells increased SKOV3 proliferation. Our data provides insights into the molecular mechanisms and dynamics underlying EV release in OC cells, proposing a key role for TRPC3 in EV biogenesis.