The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan.
Human interferon-inducible protein 10 (IP-10; HGMW-approved gene symbol CXCL10) is an ELR(-) CXC chemokine that contains binding domains for both the chemokine receptor CXCR3 and glycosaminoglycans. IP-10 has been recently demonstrated to be a potent angiostatic protein in vivo. Whether IP-10 exerts its angiostatic function through binding to CXCR3, glycosaminoglycans, or both, is not clear. To clarify this issue, we created expression constructs for mutants of IP-10 that exhibit partial (IP-10C) or total (IP-10C22) loss of binding to CXCR3 or loss of binding to glycosaminoglycans (IP-10H and IP-10C22H). The A375 human melanoma cell line was transfected with these expression vectors, and stable clones were selected and inoculated subcutaneously into nude mice. As expected, tumor cells secreting wild-type IP-10 showed remarkable reduction in tumor growth compared to control vector-transfected tumor cells. Surprisingly, mutation of IP-10 resulting in partial loss of receptor binding (IP-10C), or loss of GAG binding (IP-10H), did not significantly alter the ability to inhibit tumor growth. This tumor growth inhibition was associated with a reduction in microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis. In contrast, expression of the IP-10C22 mutant failed to inhibit melanoma tumor growth. These data suggest that CXCR3 receptor binding, but not glycosaminoglycan binding, is essential for the tumor angiostatic activity of IP-10. We conclude that the arginine 22 amino acid residue of IP-10 is essential for both CXCR3 binding and angiostasis.
Yang
Jinming
J
Veterans Affairs Medical Center and Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Richmond
Ann
A
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D020533:Angiogenesis Inhibitors / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002196:Capillaries / Q000737:chemistry; D045744:Cell Line, Tumor; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D011485:Protein Binding; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000378:metabolism*; D014162:Transfection
D020533:Angiogenesis Inhibitors; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; C515188:Cxcr3 protein, mouse; D006025:Glycosaminoglycans; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine