Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells

T-cadherin (T-cad) promotes survival, proliferation, and migration of endothelial cells and induces angiogenesis. We aimed to identify domains of T-cad functionally relevant to its effects on endothelial cell behavior. To specifically target the functional properties of the 5 cadherin repeat domains (EC1-EC5) of T-cad, endothelial cells were transduced with lentivectors containing specific T-cad-domain-deletion mutant constructs (Delta I, Delta II, Delta III, Delta IV, Delta V). Empty (E) lentivector-transduced cells served as control. Similarly to overexpression of native T-cad, cells expressing Delta II, Delta III, or Delta IV displayed elevated levels of p-Akt and p-GSK3 beta and increased proliferation rates (for Delta II, Delta III) vs. E. Delta I- and Delta V-transduced cells exhibited reduced levels of p-Akt and p-GSK3 beta and retarded growth rates vs. E. Stimulatory effects of native T-cad overexpression on Akt and GSK3 beta phosphorylation were dose dependently inhibited by coexpression of Delta I or Delta V. Subsequent functional analyses compared only Delta I-, Delta II-, and Delta V-mutant constructs with E as a negative control. Unlike Delta II cells, Delta I and Delta V cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T-cad protein. In the wound assay, migration was increased for Delta II cells but impaired for Delta I and Delta V cells. In endothelial cell-spheroid assay, angiogenic sprouting was augmented for Delta II cells but inhibited for Delta I and Delta V cells. We conclude that EC1 and EC5 domains of T-cad are essential for its proangiogenic effects. Delta I and Delta V constructs may serve as dominant-negative mutants and as potential tools targeting excessive angiogenesis.-Joshi, M. B., Kyriakakis, E., Pfaff, D., Rupp, K., Philippova, M., Erne, P., Resink, T. J. Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells. FASEB J. 23, 4011-4021 (2009). www.fasebj.org