Submitted on November 30, 2007
Accepted on May 20, 2008
The Inhibitory Effect of HKa in Endothelial Cell Tube Formation Is Mediated by Disrupting the uPA-uPAR Complex and Inhibiting its Signaling and Internalization
Yuchuan Liu1, Dian J Cao1, Irma M. Sainz2, Yan-Lin Guo1, and Robert W Colman3*
1 Medicine, Temple University, Philadelphia, Pennsylvania, United States
2 Thrombosis, Temple University, philadelphia, Pennsylvania, United States
3 Thrombosis Research Center, Temple University Sch of Medicine, Philadelphia, Pennsylvania, United States
* To whom correspondence should be addressed. E-mail: colmanr{at}temple.edu.
In two-dimensional (2D) culture systems we have previously shown that cleaved high molecular weight kininogen (HKa) or its domain 5 (D5) induced apoptosis by disrupting the urokinase plasminogen activator receptor (uPAR)-integrin signal complex formation. We now use a three-dimensional (3D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3D system, HKa significantly inhibited tube and vacuole formation as low as 10nM, which represents 1.5% of the physiologic concentration of HK (660nM), without apparent apoptosis. However, HKa (300nM) completely inhibited tube formation and increased apoptotic cells about 2 fold by 20-24h incubation. The uPA-dependent extracellular signal-regulated kinase (ERK) activation and uPAR internalization regulate cell survival and migration. In 2D, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In 3D, we found that not only uPA-uPAR association, but also the activation of ERK, was inhibited by HKa. HKa disrupts the uPA-uPAR complex inhibiting their signaling pathways and also inhibits uPAR internalization and regeneration to the cell surface thereby interfering with uPAR-mediated cell migration, proliferation and survival. Thus, HKa inhibited tube formation at least partially through the suppression of the uPA-dependent ERK activation and uPAR internalization. We conclude that in this 3D 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.
