VEGF and Angiopoietin - 1 exert opposing effects on cell junctions by regulating the Rho GEF Syx Siu

Regulation of the paracellular permeability of the endothelial cell (EC) monolayer is essential for the normal function of the vascular system, and its impairment has severe pathological effects. VEGF and Ang1 (Angiopoietin-1) play essential but opposite roles in the regulation of EC junctions and vessel permeability. The molecular mechanisms through which these ligands affect vessel permeability are known partially. VEGF increases vessel permeability by disrupting intercellular junctions through a signaling pathway that includes Src tyrosine kinase (Weis and Cheresh, 2005). Ang1, on the other hand, opposes the effect of VEGF by sequestering Src (Gavard et al., 2008) and stabilizing intercellular junctions. In epithelial cells, junction stability is modulated by the apicobasal polarity complexes CRB (Crumbs–Pals–Patj), PAR (Par3–Par6–atypical PKC), and SCRIB (Scribble–Dlg–Lgl; Tepass, 1996; Qin et al., 2005; Dow and Humbert, 2007). The underlying molecular mechanism and the role of these polarity complexes in EC junction maintenance are unknown. Rho GTPases constitute a major class of polarity protein and intercellular adhesion effectors (Fukata et al., 2003; Hall, 2005; Iden and Collard, 2008). Junction homeostasis appears to require a precise level of RhoA activity: both hyperand hypoactivation of RhoA increased paracellular permeability (Braga et al., 1997; Popoff and Geny, 2009; Spindler et al., 2010). Furthermore, the effect of RhoA on cell junctions depends on the agonist: RhoA stabilized junctions in response to Ang1 but destabilized them in response to VEGF (Gavard et al., 2008). The regulation of RhoA by polarity complexes and its signaling at cell junctions are poorly understood. Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoAspecific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx/ mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function. VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx