EphrinB2 reverse signaling in endothelial cell migration and actin remodeling

Abstract

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is exaggerated in cancer. A ligand that may be involved in angiogenesis is ephrinB2, which upon binding to its cognate receptor on an adjacent cell, becomes phosphorylated on tyrosine residues and transduces an as yet uncharacterized signal inside the cell. To examine reverse signaling pathways mediated by ephrinB2, ephrinB2 mutants were generated in which five tyrosine residues in the cytoplasmic tail were mutated to phenylalanine by site directed mutagenesis to effectively block the putative signal transduction pathways. Upon overexpression of wildtype and mutant ephrinB2 in endothelial cells, cellular phenotype changed drastically, with impeded proliferation and severe impact on actin remodeling. Observed cytoskeletal changes suggested that the Rho-like GTPases were involved in the actin remodeling mediated by the cytoplasmic tail of ephrinB2. Thus, activation of Rac and Rho upon ephrinB2 stimulation with soluble EphB6, a cognate receptor of ephrinB2, was examined. Adenoviral vectors encoding wildype and mutant ephrinB2 were also generated and used to infect endothelial cells. Wounding assays were used to monitor cell migration of ephrinB2 overexpressing cells to characterize the role of ephrinB2 in this process. Our data suggest that ephrinB2 may play an important role in the migration of endothelial cells during angiogenesis and future work will identify the signaling components of this pathway.