Urokinase-type plasminogen activator regulates cranial neural crest cell migration in vitro

Proper migration and differentiation of neural crest (NC) cells are required for normal development of craniofacial structures, heart and great vessels, sensory and autonomic nervous systems, and other organs with vertebrate embryos. Serine-protease inhibitors reduce NC cell migration in vitro, suggesting the extracellular proteases are important mediators of NC cell migration. While plasminogen activator activity levels are high in NC cells relative to other embryonic tissue, its ability to regulate NC cell migration has not been specifically tested in vivo or in vitro through its ability to convert plasminogen to plasmin. Using a transfilter migration assay, NC cell migration was measured in the presence or absence of plasminogen. Our results showed that plasminogen significantly enhanced NC cell migration. This increase could not be attributed to differences in initial NC cell attachment or cytotoxicity and did not require a chemotactic gradient. The plasminogen-enhanced NC cell migration was blocked by aprotinin (a plasmin inhibitor) and was mimicked by the direct addition of plasmin to the NC cells, indicating that the plasminogen effect was mediated through plasmin generation. Furthermore, anticatalytic-uPA antibody blocked the plasminogen-enhanced NC cell migration showing that NC cell-associated uPA activity was required for this effect. Finally, decreasing NC-uPA activity by treating cells with transforming growth factor-Beta, also blocked the plasminogen-dependent increase in cell migration. These data show that in vitro, NC cell migration is regulated by NC-associated uPA activity suggesting that growth factor-regulation of this activity may play a major role in regulating NC cell migratory capacity in vivo.