Identification of CD36 molecular features required for its in vitro angiostatic activity.
Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.
Primo
Luca
L
Institute for Cancer Research and Treatment, Candiolo, and School of Medicine, University of Torino, Italy. luca.primo@ircc.it
Ferrandi
Chiara
C
Roca
Cristina
C
Marchiò
Serena
S
di Blasio
Laura
L
Alessio
Massimo
M
Bussolino
Federico
F
D000595:Amino Acid Sequence; D043165:Angiostatic Proteins / Q000737:chemistry*; D017209:Apoptosis; D018955:CD36 Antigens / Q000096:biosynthesis*; D002465:Cell Movement; D002469:Cell Separation; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009389:Neovascularization, Pathologic; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019700:Thrombospondin 1 / Q000378:metabolism / Q000502:physiology; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D043165:Angiostatic Proteins; D018955:CD36 Antigens; D018076:DNA, Complementary; D019012:Integrin beta1; D019700:Thrombospondin 1; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2