Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells
Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells
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DOI：
10.1074/jbc.m111.251777
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发表时间：
2011-11-04
影响因子：
4.8
通讯作者：
Eccles, Suzanne A.
中科院分区：
文献类型：
--
作者：
Andrikopoulos, Petros;Baba, Akemichi;Eccles, Suzanne A.
VEGF是一种关键的血管生成细胞因子，也是抗血管生成治疗策略的主要靶点。在内皮细胞（EC）中，VEGF结合VEGF受体并通过磷脂酶γ（PLC γ）-PKC α-B-Raf途径激活ERK 1/2。我们以前的工作表明，细胞外Ca 2+的流入是VEGF诱导的ERK 1/2激活所必需的，我们假设这可能通过反向模式（Ca 2+输入和Na+输出）Na+-Ca 2+交换（NCX）发生。然而，NCX活性在VEGF信号传导和内皮细胞血管生成功能中的作用以前没有被描述过。在这里，使用人脐静脉内皮细胞（HUVECs），我们报告说，细胞外Ca 2+是VEGF诱导的ERK 1/2激活所必需的，并且在没有细胞外Ca 2+的情况下，仅从细胞内储存释放Ca 2+不足以激活ERK 1/2。此外，抑制剂的反向模式NCX抑制VEGF诱导的ERK 1/2的激活在时间和剂量依赖性的方式和衰减VEGF诱导的Ca 2+瞬变。通过siRNA敲低NCX 1（HUVEC中的主要NCX同种型）证实了药理学数据。一组NCX抑制剂也显著降低VEGF诱导的B-Raf活性，并抑制PKC α易位至质膜和原位总PKC活性。最后，在体外替代血管生成功能测定中，NCX抑制剂减少了VEGF诱导的HUVEC增殖、迁移和肾小管分化。我们提出，通过反向模式NCX的Ca 2+内流是激活和靶向PKC α到质膜所需的，这是VEGF诱导的ERK 1/2磷酸化和血管生成中下游EC功能的重要步骤。
VEGF is a key angiogenic cytokine and a major target in antiangiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase gamma (PLC gamma)-PKC alpha-B-Raf pathway. Our previous work suggested that influx of extracellular Ca2+ is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca2+ in and Na+ out) Na+-Ca2+ exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca2+ is required for VEGF-induced ERK1/2 activation and that release of Ca2+ from intracellular stores alone, in the absence of extracellular Ca2+, is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time-and dose-dependent manner and attenuated VEGF-induced Ca2+ transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKC alpha translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca2+ influx through reverse mode NCX is required for the activation and the targeting of PKC alpha to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.