Coupling between the TRPC3 ion channel and the NCX1 transporter contributed to VEGF-induced ERK1/2 activation and angiogenesis in human primary endothelial cells.
It has been previously demonstrated that the bi-directional transporter Na+/Ca2+ exchanger (NCX) working in the reverse (Ca2+-influx) - mode promotes the activation of ERK1/2 in response to the key pro-angiogenic cytokine VEGF in human endothelial cells (ECs). However, the molecular event(s) that elicit NCX reversal in VEGF-stimulated ECs remain unclear. Here we investigated whether Na+ influx via the diacylglycerol (DAG) - activated non-selective cation channel TRPC3 was functionally associated with NCX and whether its activity was required for VEGF-induced ERK1/2 activation and angiogenesis. We provide evidence that TRPC3 inhibitors and siRNA attenuated ERK1/2 phosphorylation, reduced PKCα activity and partially suppressed Ca2+ transients in response to VEGF. Additionally, TRPC3 inhibitors and siRNA significantly suppressed endothelial tubular differentiation, an in vitro indicator of angiogenesis. We also report that simulating PLCγ activation downstream of VEGF receptor 2 by application of the cell-permeable DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was sufficient to activate ERK1/2 and enhance tubular differentiation. OAG-induced ERK1/2 activation and tubulogenesis were significantly suppressed by TRPC3 and reverse-mode NCX inhibitors and siRNA. Moreover, whilst both reverse-mode NCX and TRPC3 inhibitors attenuated OAG-induced Ca2+ transients, only TRPC3 antagonists blunted Na+ influx in response to OAG. Importantly, when Na+ was increased in ECs by inhibiting the Na+-K+-ATPase, TRPC3 activity was dispensable for OAG-induced ERK1/2 phosphorylation. Collectively, our research suggests that DAG generation downstream of VEGF receptors activatesTRPC3 causing Na+ influx with subsequent reversal of NCX, ERK1/2 activation and ultimately contributes to enhanced angiogenesis. Targeting reverse-mode NCX and its upstream initiator TRPC3 could be clinically relevant in conditions characterised by abnormal VEGF signalling.
['Andrikopoulos|Petros|P|', 'Eccles|Suzanne A|SA|', 'Yaqoob|Muhammad M|MM|']
D042783:Endothelial Cells D061307:Human Umbilical Vein Endothelial Cells D006801:Humans D020935:MAP Kinase Signaling System D018919:Neovascularization, Physiologic D010766:Phosphorylation D012964:Sodium D019831:Sodium-Calcium Exchanger D050052:TRPC Cation Channels D042461:Vascular Endothelial Growth Factor A
TRPC3 Angiogenesis VEGF Endothelium Extracellular-signal-regulated kinase (ERK) Sodium/calcium exchange