Wajdner Hannah E, Farrington Jasmine, Barnard Claire, Peachell Peter T, Schnackenberg Christine G, Marino Joseph P, Xu Xiaoping, Affleck Karen, Begg Malcolm, Seward Elizabeth P
Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK.
Academic Unit of Respiratory Medicine, University of Sheffield The Royal Hallamshire Hospital, Sheffield, UK.
Physiol Rep. 2017 Mar;5(5). doi: 10.14814/phy2.13166.
Inappropriate activation of mast cells via the FcRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KKEE - TRPC1 gating mutant) failed to alter FcRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcRI-mediated calcium influx through Orai is necessary for histamine and TNF release but is differentially involved in the generation of cytokines and eicosanoids.
通过FcRI受体不适当激活肥大细胞会导致炎症介质的释放和过敏性疾病症状。钙内流是肥大细胞信号传导的关键调节因子，是预形成介质胞吐作用以及类花生酸、细胞因子和趋化因子合成所必需的。对啮齿动物和人类肥大细胞的研究已确定Orai钙通道是FcRI引发的介质释放的关键因素。然而，到目前为止，TRPC钙通道在FcRI介导的人类肥大细胞信号传导中的作用尚未见报道。在这里，我们展示了原代人肺肥大细胞和LAD2人肥大细胞系中Orai 1、2和3以及TRPC1和6表达的证据，但我们仅发现Orai而非TRPC通道对FcRI介导的钙内流有功能贡献的证据。利用Orai选择性拮抗剂（Synta66）进行的钙成像实验显示了Orai对人类肥大细胞中FcRI介导的信号传导的贡献。尽管使用TRPC3/6选择性拮抗剂和激动剂（分别为GSK-3503A和GSK-2934A）未发现TRPC6对人类肥大细胞中FcRI介导的钙信号传导有贡献的证据。同样，在人类肥大细胞中使STIM1调节的TRPC1失活（通过用STIM1-KKEE - TRPC1门控突变体转染细胞进行测试）未能改变LAD2人肥大细胞中FcRI介导的钙信号传导。介质释放试验证实，通过Orai的FcRI介导的钙内流是组胺和TNF释放所必需的，但在细胞因子和类花生酸的产生中参与程度不同。
