Padbury Elise H, Bálint Štefan, Carollo Emanuela, Carter David R F, Becker Esther B E
Nuffield Department of Clinical Neurosciences University of Oxford Oxford UK.
Kavli Institute for Nanoscience Discovery University of Oxford Oxford UK.
J Extracell Biol. 2023 Dec 25;3(1):e132. doi: 10.1002/jex2.132. eCollection 2024 Jan.
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 Ca-permeable transient receptor potential (TRP) channel TRPC3 in EV-mediated communication. We found that specific activation of TRPC3 increased Ca signalling in SKOV3 cells and stimulated an immediate increase in EV release. Ca-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 Ca-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.
细胞外囊泡（EVs）参与包括癌症进展在内的多种病理过程，但其生物发生的分子机制仍未完全明确。基于四跨膜蛋白的pHluorin报告基因的开发，使得在质膜上实时分析EV释放成为可能。在此，我们利用CD81-pHluorin来研究卵巢癌细胞（OC）中EV释放的机制，并报道了钙通透瞬时受体电位（TRP）通道TRPC3在EV介导的细胞通讯中的新作用。我们发现，TRPC3的特异性激活增加了SKOV3细胞中的钙信号，并刺激EV释放立即增加。钙刺激剂组胺和离子霉素同样诱导EV释放，成像分析揭示了不同的刺激依赖性时间和空间释放动态。有趣的是，TRPC3的抑制减弱了组胺刺激的钙内流和EV释放，表明TRPC3可能在EV生物发生中组胺信号的下游起作用。此外，我们发现TRPC3的直接激活以及应用来自TRPC3激活细胞的EVs均可增加SKOV3的增殖。我们的数据为OC细胞中EV释放的分子机制和动态提供了见解，提出了TRPC3在EV生物发生中的关键作用。
