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 (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.
Joshi
Manjunath B
MB
Basel University Hospital, Department of Biomedicine, Laboratory for Signal Transduction, ZLF 316, Hebelstrasse 20, CH 4031 Basel, Switzerland.
Kyriakakis
Emmanouil
E
Pfaff
Dennis
D
Rupp
Katharina
K
Philippova
Maria
M
Erne
Paul
P
Resink
Thérèse J
TJ
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
D043925:Angiogenesis Inducing Agents; D015820:Cadherins; C099972:H-cadherin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3