Rôle inattendu de la protéine des jonctions intercellulaires Zonula Occludens-1 (ZO-1) dans la régulation de l’ARN et de la prolifération des cellules endothéliales

Intercellular junctions between endothelial cells play a critical role in maintaining vascular integrity. ZO-1 is an essential protein that regulates important signaling pathways during angiogenesis. The basic mechanisms activated by growth factors that are involved in the regulation of ZO-1 function at the cell-cell junction are poorly studied. Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through the regulation of a plethora of signaling events in endothelial cells. VEGF and Ang-1, through their receptors VEGFR-2 and Tie-2 respectively, activates many common intracellular signaling pathways in endothelial cells to promote survival, proliferation and migration. However, these growth factors play antagonistic roles in the regulation of cellcell junctions. Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes at endothelial junctions conferring specific responses to each factor remain poorly defined. Our objective was to identify, using proteomics, novel intracellular mechanisms that are involved in important biological processes in endothelial cells in order to better understand the vascular development and angiogenesis. First, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in endothelial cells using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed a link between the junctional protein ZO-1 and the proliferation of endothelial cells. We found that the reduction of cellular ZO-1 levels is necessary for endothelial cell proliferation. This was shown in vitro and during vascular development of the mouse postnatal retina. Second, our phosphoproteomic study revealed that ZO-1 is phosphorylated on the Serine 617 when cells were treated with VEGF. Thus, we investigated the role of these residues of ZO-1 and we found that the Ser617 of ZO-1 is essential for its function on cell-cell junctions and the proliferation of endothelial cells. Third, we determined the interactome of ZO-1 in endothelial cells treated or not with VEGF using affinity purification coupled to mass spectrometry. Interestingly, we found that the junctional protein ZO-1 interacts with RNA binding proteins. VEGF treatment influences iv the interaction of ZO-1 with multiple RNA binding proteins. Our results show an intriguing link between the junctional protein ZO-1 and the RNA binding proteins involved in the regulation of the RNA machinery. In summary, we identified crucial signaling pathways in endothelial cells that contribute to a better understanding of vascular developement and angiogenesis. We uncovered ZO-1 as a central modulator of endothelial cell proliferation during angiogenesis. Moreover, we revealed a link between ZO-1 and RNA binding proteins. This allowed us to better understand the role of ZO-1 in endothelial cell biology.

Keyphrases: VEGF, Angiopoietin-1, ZO-1, Endothelial cell, Proteomics, Angiogenesis