Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis.
Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two-hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity-binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 +/- 11 nM and 125 +/- 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type-1 repeat (TSP-1) domain of CTGF binds to the exon 7-coded region of VEGF165 and that the COOH-terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full-length CTGF and the deletion mutant possessing the TSP-1 domain inhibited VEGF165-induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein-to-protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.
Inoki
Isao
I
Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-0016, Japan.
Shiomi
Takayuki
T
Hashimoto
Gakuji
G
Enomoto
Hiroyuki
H
Nakamura
Hiroyuki
H
Makino
Ken-ichi
K
Ikeda
Eiji
E
Takata
Shigeo
S
Kobayashi
Ken-ichi
K
Okada
Yasunori
Y
D000818:Animals; D001808:Blood Vessels / Q000737:chemistry / Q000187:drug effects / Q000254:growth & development; D002460:Cell Line; D055513:Connective Tissue Growth Factor; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007279:Injections, Subcutaneous; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012867:Skin / Q000098:blood supply / Q000737:chemistry / Q000187:drug effects; D020798:Two-Hybrid System Techniques; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014841:von Willebrand Factor / Q000032:analysis
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D016228:Endothelial Growth Factors; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014841:von Willebrand Factor; D055513:Connective Tissue Growth Factor