Transient Receptor Potential Canonical Channels Are Required for in Vitro Endothelial Tube Formation
Transient Receptor Potential Canonical Channels Are Required for in Vitro Endothelial Tube Formation
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DOI：
10.1074/jbc.m111.295733
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发表时间：
2012-02-17
影响因子：
4.8
通讯作者：
Frieden, Maud
中科院分区：
文献类型：
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作者：
Antigny, Fabrice;Girardin, Nathalie;Frieden, Maud
在内皮细胞中，钙离子内流是细胞增殖或血管生成等过程中发生的钙信号的重要组成部分。Ca~(2+)内流通过钙离子内流途径发生，包括基质相互作用分子-1(STIM1)和Orai1，但也通过第二信使调节的通道，如瞬时受体潜伏期通道(TRPC)。人脐静脉来源的内皮细胞株EA.hy926表达STIM1和Orai1以及几个TRPC通道。通过使这些分子中的每一个失效，我们证明了TRPC3、TRPC4和TRPC5对于EA.hy926细胞在Matrigel上培养后观察到的管状结构的形成是必不可少的。相反，沉默STIM1或Orai1并不能阻止小管的形成。在Matrigel上电镀后不久，细胞显示出自发的钙振荡，这种振荡可以被针对TRPC3、TRPC4或TRPC5的siRNA强烈地减少，但不能处理针对STIM1或Orai1的siRNA。此外，我们还发现，针对TRPC3、TRPC5和Orai1通道的siRNA处理降低了细胞的增殖，而STIM1的敲除没有影响。在原代培养的人脐静脉内皮细胞上，TRPC1、TRPC4和STIM1参与了管道的形成，而Orai1则没有作用。这些数据表明，无论是在内皮细胞系还是在原代内皮细胞上，TRPC通道对于体外小管形成都是必不可少的。
In endothelial cells Ca2+ entry is an essential component of the Ca2+ signal that takes place during processes such as cell proliferation or angiogenesis. Ca2+ influx occurs via the store-operated Ca2+ entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca2+ oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.