title: An integrin-binding N-terminal peptide region of TIMP-2 retains potent angio-inhibitory and anti-tumorigenic activity in vivo.
abstract: Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin α3β1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to α3β1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the α3β1-binding domain and the growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43-Ala66, which shows α3β1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to α3β1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that possesses both angio-inhibitory and, using a murine xenograft model of Kaposi's sarcoma, anti-tumorigenic activity in vivo. Thus, both the α3β1-binding and the angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B-C loop).
authors.affiliation: Radiation Oncology Branch, Advanced Technology Center, Center for Cancer Research, National Cancer Institute, NIH, 8717 Grovemont Circle, Bethesda, MD 20892, USA.
authors.forename: Dong-Wan
authors.initials: DW
authors.lastname: Seo
authors.forename: W Carl
authors.initials: WC
authors.lastname: Saxinger
authors.forename: Liliana
authors.initials: L
authors.lastname: Guedez
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Cantelmo
authors.forename: Adriana
authors.initials: A
authors.lastname: Albini
authors.forename: William G
authors.initials: WG
authors.lastname: Stetler-Stevenson
mesh_terms: D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000378:metabolism; D010455:Peptides / Q000008:administration & dosage / Q000138:chemical synthesis / Q000494:pharmacology; D011485:Protein Binding; D012514:Sarcoma, Kaposi / Q000473:pathology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D039201:Integrin alpha3beta1; D010455:Peptides; C560822:TIMP1 protein, human; C560823:TIMP2 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; D019716:Tissue Inhibitor of Metalloproteinase-2