The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization

• Published in: American Journal of Physiology: Cell Physiology 2008-07, Vol.295 (1), p.C257-C267
• Main Authors: Liu, Yuchuan, Cao, Dian J, Sainz, Irma M, Guo, Yan-Lin, Colman, Robert W
• Format: Article
• Language: English
• Subjects: Apoptosis; Cell Adhesion; Cell culture; Cell Movement; Cells; Cells, Cultured; Endothelial Cells - drug effects; Endothelial Cells - physiology; Endothelium, Vascular - cytology; Extracellular Signal-Regulated MAP Kinases - physiology; Humans; Kinases; Kininogens - pharmacology; Kininogens - physiology; Molecules; Phosphorylation; Proteins; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Three dimensional imaging; Urokinase-Type Plasminogen Activator - metabolism; Vascular Biology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00569.2007

1 The Sol Sherry Thrombosis Research Center and 2 Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania Submitted 30 November 2007 ; accepted in final form 20 May 2008 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. urokinase plasminogen activator; urokinase plasminogen activator receptor; angiogenesis; extracellular signal-regulated kinase Address for reprint requests and other correspondence: R. W. Colman, The Sol Sherry Thrombosis Research Center, Temple Univ. School of Medicine, 3400 N. Broad St., Rm. 418 OMS, Philadelphia, PA 19140 (e-mail: colmanr{at}temple.edu )