Abstract
In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) 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 Ca(2+) 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.
Highlights
Different Ca2ϩ entry pathways co-exist in endothelial cells
We showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 isoforms are expressed, together with stromal interaction molecule-1 (STIM1) and Orai1, on EA.hy926 cells [20], an endothelial cell line derived from human umbilical vein endothelial cells (HUVECs) fused with human lung adenocarcinoma cell line A549 [21]
By using a siRNA approach, we showed that on EA.hy926 cells, the knockdown of TRPC3, TRPC4, or TRPC5 strongly decreased Matrigel-induced tube formation, whereas the silencing of STIM1 or Orai1 had no negative impact
Summary
Different Ca2ϩ entry pathways co-exist in endothelial cells. Results: On artificial basement membrane, endothelial cells displayed Ca2ϩ oscillations and formed tubes, both processes prevented after silencing TRPC channels, but not Orai. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai 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. We showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 isoforms are expressed, together with STIM1 and Orai, on EA.hy926 cells [20], an endothelial cell line derived from human umbilical vein endothelial cells (HUVECs) fused with human lung adenocarcinoma cell line A549 [21] In this cell line, massive store depletion achieved after thapsigargin stimulation induced Ca2ϩ entry that is mediated both by store-dependent STIM1/Orai and store-independent pathways involving TRPC3 [20]. We showed that on primary HUVEC, TRPC1, TRPC4, and STIM1, but not Orai, are required for tubulogenesis
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