Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β1-integrin and VEGFR2.

Pubmed ID: 23709605

Pubmed Central ID: PMC3742872

Journal: American journal of physiology. Heart and circulatory physiology

Publication Date: Aug. 1, 2013

Affiliation: Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.

MeSH Terms: Humans, Cell Proliferation, Cells, Cultured, Extracellular Signal-Regulated MAP Kinases, Phosphorylation, Proto-Oncogene Proteins c-akt, Animals, Time Factors, Carrier Proteins, Signal Transduction, Endothelial Cells, Mice, Neovascularization, Physiologic, Transfection, Mice, Knockout, Binding Sites, Kininogen, High-Molecular-Weight, Models, Molecular, Protein Kinase Inhibitors, Protein Structure, Tertiary, RNA Interference, Receptors, Urokinase Plasminogen Activator, Tissue Culture Techniques, Urokinase-Type Plasminogen Activator, Vascular Endothelial Growth Factor Receptor-2, Integrin beta1

Grants: HD-057355, HL-052775-18, HL-090697, HL-112666A, NS-064447, P30 CA016520

Authors: Mahdi F, Schmaier AH, Larusch GA, Merkulova A, Shariat-Madar Z, Sitrin RG, Cines DB

Cite As: Larusch GA, Merkulova A, Mahdi F, Shariat-Madar Z, Sitrin RG, Cines DB, Schmaier AH. Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β1-integrin and VEGFR2. Am J Physiol Heart Circ Physiol 2013 Aug 1;305(3):H305-20. Epub 2013 May 24.

Studies:

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.