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Extracellular Cadherin repeat domains EC1 and EC5 of T‐cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells
Author(s) -
Joshi Manjunath B.,
Kyriakakis Emmanouil,
Pfaff Dennis,
Rupp Katharina,
Philippova Maria,
Erne Paul,
Resink Thérèse J.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.09-133611
Subject(s) - angiogenesis , cadherin , microbiology and biotechnology , biology , protein kinase b , endothelial stem cell , cancer research , chemistry , phosphorylation , cell , biochemistry , in vitro
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 (ΔΙ, ΔΠ, ΔΠΙ, ΔIV, ΔV). Empty (E) lentivector‐transduced cells served as control. Similarly to overexpression of native T‐cad, cells expressing ΔΙΙ, ΔΙΙΙ, or ΔΙV displayed elevated levels of p‐Akt and p‐GSK3β and increased proliferation rates (for ΔΙΙ, ΔΙΙΙ) vs. E. ΔΙ‐ and ΔV‐transduced cells exhibited reduced levels of p‐Akt and p‐GSK3β and retarded growth rates vs. E. Stimulatory effects of native T‐cad overexpression on Akt and GSK3β phosphorylation were dose dependently inhibited by coexpression of ΔΙ or ΔV. Subsequent functional analyses compared only ΔΙ‐, ΔΙΙ‐, and ΔV‐mutant constructs with E as a negative control. Unlike ΔΙΙ cells, ΔΙ and ΔV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T‐cad protein. In the wound assay, migration was increased for ΔΙΙ cells but impaired for ΔΙ and ΔV cells. m endothelial cell‐spheroid assay, angiogenic sprouting was augmented for ΔΙΙ cells but inhibited for ΔΙ and ΔV cells. We conclude that EC1 and EC5 domains of T‐cad are essential for its proan‐giogenic effects. ΔΙ and ΔV 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. FASEBJ. 23, 4011–4021 (2009). www.fasebj.org