title: The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization.
abstract: In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20-24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.
authors.affiliation: The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: Yuchuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Dian J
authors.initials: DJ
authors.lastname: Cao
authors.forename: Irma M
authors.initials: IM
authors.lastname: Sainz
authors.forename: Yan-Lin
authors.initials: YL
authors.lastname: Guo
authors.forename: Robert W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D017209:Apoptosis; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D006801:Humans; D007704:Kininogens / Q000494:pharmacology / Q000502:physiology*; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D007704:Kininogens; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D048049:Extracellular Signal-Regulated MAP Kinases; D014568:Urokinase-Type Plasminogen Activator