Abstract
BACKGROUND: Urokinase plasminogen activator (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to uPA is dependent on both the growth factor binding domain at the aminoterminal end and the kringle (K) domain of the molecule. uPA is readily degraded in vivo into these constitutive domains. The aim of this study was to examine cell signaling during the migration of smooth muscle cell in response to the kringle domain of urokinase. MATERIALS AND METHODS: Murine arterial smooth muscle cells were cultured in vitro. Migration assays were performed in the presence of K with and without the plasmin inhibitors (aprotinin and -aminocaproic acid), the Galphai inhibitor Pertussis toxin, the MMP inhibitor (GM6001), the PI3-K inhibitors, Wortmannin and LY294002, and the MAPK inhibitors PD98089 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor). Western blotting was performed for ERK 1/2 and p38(MAPK) phosphorylation after stimulation with K in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA (n = 6). RESULTS: The kringle domain (K) induced a plasmin-independent, MMP-dependent increase in cell migration (2-fold, P < 0.05) compared to control. This migratory response to K was Galphai mediated and dependent on both ERK 1/2 and p38(MAPK) activation. K induced time-dependent increases in the phosphorylation of ERK 1/2 (3-fold, P < 0.05) and p38(MAPK) (3-fold, P < 0.05). Activation of p38(MAPK) and ERK 1/2 was completely inhibited by the PI3-K inhibitors. We explored a potential role for the epidermal growth factor receptor (EGFR). K induced EGFR phosphorylation and the presence of AG1478, the EGFR inhibitor, inhibited both cell migration and akt activation in response to K. CONCLUSION: Kringle domain of uPA induces smooth muscle cell migration through a G-protein-coupled PI3-K-dependent process involving both ERK 1/2 and p38(MAPK) and is mediated in part through EGFR. Defining the differences in response to key molecular domains of uPA is vital to understand its role in vessel remodeling.