Tie-2–dependent activation of RhoA and Rac1 participates in endothelial cell motility triggered by angiopoietin-1

Abstract

IntroductionAngiogenesis is a crucial event in embryonic development and plays a critical role in many pathologic processes. 1 Sprouting of new blood vessels from pre-existing ones is a multistep process that requires digestion of extracellular matrix, and migration and proliferation of endothelial cells (ECs). 1 Finally, cells change their shape and fold up to form capillaries that are surrounded by pericytes, necessary for the cells' stabilization. 2 A cascade of molecules whose biologic activities are partially overlapped drives this process. Among them, vascular endothelial growth factor (VEGF) and angiopoietin (Ang) families have restricted activities on ECs. Through VEGF receptor-1 and -2, VEGFs primarily regulate migration, proliferation, and survival of ECs. By analysis of genetic and pathologic models, Ang-1, a ligand of Tie-2 receptor, is instead supposed to stabilize EC networks, presumably by stimulating interactions between ECs and pericytes. [3][4][5] In vitro experiments show that Ang-1 causes ECs to form capillary networks 6 and sprouts from preassembled spheroids. 7 Ang-1 induces EC chemotaxis 8,9 and chemokinesis, 10,11 which require the activation of the downstream effector phosphoinositide 3-OH kinase (PI 3-kinase) and the phosphorylation of focal adhesion kinase and paxillin. 10,12 Such morphogenetic events are dependent on cell motility and shape rearrangement, which imply dramatic changes in cytoskeletal dynamics; this suggests that Ang-1 could be directly implicated in the regulation of the motility machinery in migrating cells.Rho guanosine triphosphatases (Rho GTPases) are major regulators of cell polarization and motility. In fibroblasts, Cdc42 mediates filipodia extension, Rac1 lamellipodia formation and membrane ruffling, and RhoA stress fiber formation. 13 Besides Rac1, RhoA is involved in ruffling as well. 14 Nevertheless, considering that GTPase activities have antagonist effects in the regulation of different steps of cell motility 15 and discrete subcellular localization, 16-18 the above model seems to be more complex. Recent evidence suggests that Rho GTPases participate to the EC signaling pathways triggered by angiogenic inducers [19][20][21] and, more in general, regulate biologic responses requiring changes in EC shape. [22][23][24][25] Here we analyze the spatiotemporal modulation and localization of Rac1 and RhoA during EC locomotion induced by Ang-1 and we investigate the motility of ECs carrying RhoA and Rac1 dominant-negative molecules. Furthermore we studied the signaling pathway from Tie-2 receptor to these enzymes through PI 3-kinase activity. Materials and methods ReagentsRecombinant Ang-1 was produced in a baculovirus expression system as described and was able to phosphorylate Tie-2 in ECs. 11 Glutathione-Stransferase (GST)-Pak 1B binding domain (GST-PBD), GST-rhotekin binding domain (GST-RBD), and GST-Wiskott-Aldrich syndrome protein (WASP) binding domain (GST-WBD) fusion proteins were purified as described. 26 GST-PBD, GST-RBD, and GST-WBD bind the bound form o...

Adaptor ShcA Protein Binds Tyrosine Kinase Tie2 Receptor and Regulates Migration and Sprouting but Not Survival of Endothelial Cells

“…PI 3-kinase is generally involved in cell migration and survival (47) and has been demonstrated to be necessary for Ang-1-stimulated motility (11,46) as well as for its cytoprotective effect (5, 10 -12). The overexpression of SchA-SH2 mutant partially reduces the Tyr phosphorylation of the regulatory p85 subunit of PI 3-kinase induced by Ang-1.…”

“…Expression of ShcSH2 Partially Inhibits the Ang-1-dependent Tyrosine Phosphorylation of the PI 3-Kinase Regulatory Subunit p85-Because PI 3-kinase has been demonstrated to be involved in Tie2-dependent signaling pathway (11,46) and play a role in both cell migration and survival (47), we studied the effect of the expression of ShcSH2 on Tyr phosphorylation of p85 regulatory subunit upon Ang-1 stimulation of ECs. Fig.…”

Rho signalling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas

“…PA-deleted pancreatic explants were incubated with RDB-GST purified protein and subsequently processed for immunofluorescence with anti-GST antibody (Cascone et al, 2003). Open arrowheads indicate localisation of active Rho-GTP bound (GST; green) in Ecad + pancreatic cells (red) in Stard13 PA-deleted embryos.…”

“…3A). Second, to visualise GTP-bound Rho proteins we used an immunolocalisation assay (Cascone et al, 2003) on cultures of E11.5 pancreas (Fig. 3C) (Horb and Slack, 2000;Petzold and Spagnoli, 2012;Puri and Hebrok, 2007).…”

“…Pull-down (GTP-Rho; arrow) and total lysates were immunoblotted with anti-Rho antibody. WT lysate pre-loaded with the non-hydrolysable GTP analogue GTPγS (WT+GTPγS) (Cascone et al, 2003) and His-tagged RhoA fusion protein were used as positive controls in the assay. The band present above Rho is caused by non-specific binding to the RDB-GST-beads.…”