Abstract
VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.
Highlights
From the ‡Department of Pharmacology, Faculty of Medicine, Universitede Montreal, Montreal, Quebec, Canada; §Department of Pathology and Cell Biology, Faculty of Medicine, Universitede Montreal, Montreal, Quebec, Canada; ¶Proteomics discovery platform, Institut de recherches cliniques de Montreal (IRCM), Montreal, Quebec, Canada
Phosphoproteome Profiling of VEGF or Ang-1 Stimulated endothelial cells (ECs)—To analyze in a comprehensive manner the phosphorylation events regulated by VEGF and Ang-1, we performed a phosphoproteomic profiling of BAECs stimulated with VEGF or Ang-1
Because VEGF and Ang-1 activate numerous signaling pathways implicated in cell survival, proliferation and migration through the phosphorylation of MAPK, Akt and eNOS, we first established that 10 min of stimulation was the optimal time point of these signaling intermediates in BAECs (Fig. 1A)
Summary
Ang-1, angiopoietin-1; BAEC, bovine aortic endothelial cell; EC, endothelial cell; FDR, false discovery rate; GO, gene ontology; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; DMEM, Dulbecco Modified Eagle Medium; pAb, polyclonal antibody; mAb, monoclonal antibody; EDTA, ethylenediaminetetraacetic; EGTA, ethyleneglycoltetraacetic; TFA, trifluoroacetic acid; ANOVA, Analysis of variance; CT, control; DTT, dithiothreitol; ACN, Acetonitrile; (q)RTPCR, quantitative reverse transcriptase – polymerase chain reaction; BrdU, bromodeoxyuridine; pH3, phospho-histone 3; P5, postnatal day 5; IB, immunoblot; IPA, ingenuity pathway analysis. In ECs, the adherens junction proteins -catenin and p120-catenin are known to elicit signaling pathways that induce proliferation when junctions are disrupted [21, 27]. Both proteins can translocate to the nucleus and act as modulator of gene expression through interaction with the TCF/LEF transcription factors for -catenin or by relieving the repressor activity of the transcription factor Kaiso for p120-catenin [28, 29]. Network analysis of the phosphoproteins regulated by VEGF and Ang-1 uncovered a cluster of cell-cell junction proteins unique to VEGF treatment, which is linked to activation of MAPK1 and promotion of EC proliferation. Our comparative phosphoproteomic analyses identified a regulatory signaling node, differentially engaged by VEGF over Ang-1, that controls EC proliferation
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