title: 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.
abstract: VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) 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 Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), 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 Ca(2+) 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 PKCα 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 Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, McElwain Laboratories, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, United Kingdom; Division of Cell and Molecular Biology, Neuroscience Solutions to Cancer Research Group, Imperial College London, London SW7 2AZ, United Kingdom; Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom. Electronic address: p.andrikopoulos@qmul.ac.uk.
authors.forename: Petros
authors.initials: P
authors.lastname: Andrikopoulos
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871.
authors.forename: Akemichi
authors.initials: A
authors.lastname: Baba
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871.
authors.forename: Toshio
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Division of Cell and Molecular Biology, Neuroscience Solutions to Cancer Research Group, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Mustafa B A
authors.initials: MBA
authors.lastname: Djamgoz
authors.affiliation: Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom.
authors.forename: Muhammad M
authors.initials: MM
authors.lastname: Yaqoob
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, McElwain Laboratories, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, United Kingdom. Electronic address: sue.eccles@icr.ac.uk.
authors.forename: Suzanne A
authors.initials: SA
authors.lastname: Eccles
mesh_terms: D002118:Calcium / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D020868:Gene Silencing; D006801:Humans; D007477:Ions; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009389:Neovascularization, Pathologic; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D007477:Ions; D019831:Sodium-Calcium Exchanger; D042461:Vascular Endothelial Growth Factor A; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium