Urokinase-type plasminogen activator and the plasminogen activator inhibitor-1 (uPA-PAI) complex promote cell migration and axonal outgrowth in the developing optic tectum.

BARBER, MARIANA; RAPACIOLI, MELINA; TERUEL, LUISA; DUARTE, SANTIAGO; FLORES, VLADIMIR; SANCHEZ, VIVIANA

Buzios, Brasil

Congreso; I CONGRESS IBRO/LARC OF NEUROSCIENCES FOR LATIN AMERICA, CARIBBEAN AND IBERIAN PENINSULA; 2008

Sociedad Argentina de Neurociencias

Objectives: The aim of this work is to investigate the rol of the urokinase-type plasminogen activator - plasminogen activator inhibitor-1 (uPA-PAI1) complex and the uPA receptor in cell migration and neuritogenesis using the avian optic tectum (OT). Methods: OT obtained from embryos of 6 days were dissected. Each portion was grown in DMEM with glutamine and N2, incubated at 37 ºC for 20 hours and then treated with uPA, PAI1 or both at a final equimolar concentration of 0.74 nM. After 4 hours, the explants were observed by phase-contrast microscopy and photographed. The images were assembled and two different variables were measured: cell migration pattern and axonal length. Inmunohistochemistry: After the treatment explants were fixed in 4% paraformaldehyde in 0.1M phosphate buffer, blocked with 5% normal goat serum and incubated with anti-PAI-1 (Calbiochem®, final concentration 25 µg/ml) or with the primary antibody anti- urokinase-type plasminogen activator receptor (uPAR, Santa Cruz®, final dilution 0.4 mg/ml) for 48 hours at 4ºC. After incubation, the primary antibodies were removed and explants were incubated with the appropriate secondary antibodies Alexa Fluor (Molecular Probes®) in order to detect uPAR and PAI1 or with phalloidin-rhodamine (Sigma®, 1/1000) to detect actin cytoskeleton. Results: The results show a significant increase in the number of cells (48.16% +/-16%) and the covered distance (19% +/-1.8%) of migrating cells as well as an increase in the number of axons (13.3 %+/-1.9%) in the explants treated with uPA-PAI1 0.74 nM compared with the control explants. The immunohistochemistry shows an increase in the number of stress fibres in treated explants compared with the controls and the same expression pattern of PAI1 and uPAR Conclusion: These results indicate that uPA-PAI1 complex participates actively in the process of cell migration and neuritogenesis. The appearance of stress fibres in treated explants suggests that the reorganization of the actin cytoskeleton is the result of the activation of an intracellular signaling pathway mediated by the complex uPA-PAI- uPAR.