title: Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β1-integrin and VEGFR2.
abstract: How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser(473)) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser(473)) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser(473)), in SIN1(-/-) cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser(473)). A peptide of the integrin-binding site on uPAR, a β1-integrin peptide that binds uPAR, antibody 6S6 to β1-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1-HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser(473)). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser(473)), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Gretchen A
authors.initials: GA
authors.lastname: Larusch
authors.forename: Alona
authors.initials: A
authors.lastname: Merkulova
authors.forename: Fakhri
authors.initials: F
authors.lastname: Mahdi
authors.forename: Zia
authors.initials: Z
authors.lastname: Shariat-Madar
authors.forename: Robert G
authors.initials: RG
authors.lastname: Sitrin
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cines
authors.forename: Alvin H
authors.initials: AH
authors.lastname: Schmaier
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D055293:Receptors, Urokinase Plasminogen Activator / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D046509:Tissue Culture Techniques; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing
chemical_list: D002352:Carrier Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D019012:Integrin beta1; D019679:Kininogen, High-Molecular-Weight; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D040301:Vascular Endothelial Growth Factor Receptor-2; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; C561937:Mapkap1 protein, mouse; D048868:Adaptor Proteins, Signal Transducing
keywords: epidermal growth factor receptors; high-molecular-weight kininogen; single-chain urokinase; urokinase plasminogen activator receptor; vascular epidermal growth factor receptor 2