Diabetic Kidney Disease Centre, Renal Unit, Barts Health NHS Trust, The Royal London Hospital, Whitechapel Road, London E1 1BB, UK; Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, UK.
Division of Cancer Therapeutics, The Institute of Cancer Research, London SW7 3RP, UK.
Cell Signal. 2017 Sep;37:12-30. doi: 10.1016/j.cellsig.2017.05.013. Epub 2017 May 20.
It has been previously demonstrated that the bi-directional transporter Na/Ca exchanger (NCX) working in the reverse (Ca-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 Ca 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 Ca 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.
先前已经证明，双向转运体钠/钙交换器（NCX）以反向（钙内流）模式工作时，可促进人内皮细胞（EC）中关键的促血管生成细胞因子 VEGF 引起 ERK1/2 的激活。然而，在 VEGF 刺激的 EC 中引起 NCX 反转的分子事件尚不清楚。在这里，我们研究了二酰基甘油（DAG）激活的非选择性阳离子通道 TRPC3 是否通过 Na 流入与 NCX 功能相关，以及其活性是否是 VEGF 诱导的 ERK1/2 激活和血管生成所必需的。我们提供的证据表明，TRPC3 抑制剂和 siRNA 减弱了 ERK1/2 的磷酸化，降低了 PKCα 的活性，并部分抑制了 VEGF 引起的 Ca 瞬变。此外，TRPC3 抑制剂和 siRNA 显著抑制了内皮管状分化，这是血管生成的体外指标。我们还报告说，通过应用细胞通透的 DAG 类似物 1-油酰基-2-乙酰基-sn-甘油（OAG）模拟 VEGF 受体 2 下游的 PLCγ 激活足以激活 ERK1/2 并增强管状分化。OAG 诱导的 ERK1/2 激活和管状形成明显受到 TRPC3 和反向模式 NCX 抑制剂和 siRNA 的抑制。此外，虽然反向模式 NCX 和 TRPC3 抑制剂均减弱了 OAG 诱导的 Ca 瞬变，但只有 TRPC3 拮抗剂可减弱 OAG 诱导的 Na 内流。重要的是，当通过抑制 Na-K-ATP 酶增加 EC 中的 Na 时，TRPC3 活性对于 OAG 诱导的 ERK1/2 磷酸化是可有可无的。总之，我们的研究表明，VEGF 受体下游的 DAG 生成激活了 TRPC3，导致 Na 内流，随后 NCX 反转，ERK1/2 激活，并最终促进了增强的血管生成。靶向反向模式 NCX 及其上游启动子 TRPC3 在以异常 VEGF 信号为特征的情况下可能具有临床相关性。
