title: Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells.
abstract: 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 (DeltaI, DeltaII, DeltaIII, DeltaIV, DeltaV). Empty (E) lentivector-transduced cells served as control. Similarly to overexpression of native T-cad, cells expressing DeltaII, DeltaIII, or DeltaIV displayed elevated levels of p-Akt and p-GSK3beta and increased proliferation rates (for DeltaII, DeltaIII) vs. E. DeltaI- and DeltaV-transduced cells exhibited reduced levels of p-Akt and p-GSK3beta and retarded growth rates vs. E. Stimulatory effects of native T-cad overexpression on Akt and GSK3beta phosphorylation were dose dependently inhibited by coexpression of DeltaI or DeltaV. Subsequent functional analyses compared only DeltaI-, DeltaII-, and DeltaV-mutant constructs with E as a negative control. Unlike DeltaII cells, DeltaI and DeltaV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T-cad protein. In the wound assay, migration was increased for DeltaII cells but impaired for DeltaI and DeltaV cells. In endothelial cell-spheroid assay, angiogenic sprouting was augmented for DeltaII cells but inhibited for DeltaI and DeltaV cells. We conclude that EC1 and EC5 domains of T-cad are essential for its proangiogenic effects. DeltaI and DeltaV constructs may serve as dominant-negative mutants and as potential tools targeting excessive angiogenesis.
authors.affiliation: Basel University Hospital, Department of Biomedicine, Laboratory for Signal Transduction, ZLF 316, Hebelstrasse 20, CH 4031 Basel, Switzerland.
authors.forename: Manjunath B
authors.initials: MB
authors.lastname: Joshi
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Kyriakakis
authors.forename: Dennis
authors.initials: D
authors.lastname: Pfaff
authors.forename: Katharina
authors.initials: K
authors.lastname: Rupp
authors.forename: Maria
authors.initials: M
authors.lastname: Philippova
authors.forename: Paul
authors.initials: P
authors.lastname: Erne
authors.forename: Thérèse J
authors.initials: TJ
authors.lastname: Resink
mesh_terms: D043925:Angiogenesis Inducing Agents; D015820:Cadherins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000254:growth & development; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology; D017434:Protein Structure, Tertiary; D014945:Wound Healing / Q000502:physiology
chemical_list: D043925:Angiogenesis Inducing Agents; D015820:Cadherins; C099972:H-cadherin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3