u-PA promoted neuronal migration and neuritogenesis are associated with u-PA-uPAR-integrin complex formation and FAK phosphorylation

N. LINO; C. ALONSO; N. MENGHINI; J. DI NAPOLI; M. RAPACIOLI; V. FLORES; V. N. SANCHEZ

San Diego, CA, USA

Congreso; 40th Annual Meeting of the Society for Neuroscience; 2010

Society for Nueroscience

Previously we demonstrated that uPA (urokinase-type plasminogen activator) promotes neuronal migration and neuritogenesis in the developing nervous system. This response is doses-dependent and it is unrelated with its proteolytic activity. The membrane receptor of uPA (uPAR) is a GPI-anchored protein and more than one hypothetical transmembrane adapter has been proposed including integrins. Recent studies have shown that uPAR mediates the phosphorylation of focal adhesion kinase (FAK) in cultured cells. FAK becomes rapidly phorphorylated upon coupling of integrins and is involved in cell adhesion and/or migration. The present work explored: 1) whether a ab integrin heterodimmer is associated with u-PA:uPAR in uPA stimulated postmitotic neurons and 2) whether uPA:uPAR complex participates in the FAK phosphorylation during neuronal migration and neuritogenesis. Chicken embryos of 61/2 days were decapitated and the cephalic portions of optic tecta were dissected. The tissue was cut into small pieces and transferred to culture dishes treated with polilysine and laminin. The explants were incubated with DMEM, glutamine and N2 for 16 hours and submitted to a 10 nM uPA pulse for 2 minutes and fixed in paraphormaldehyde-sacarose in 0.1M phosphate buffer. The explants were blocked with normal goat serum and incubated with anti uPAR and anti a5; a6 and b1 integrin subunits  antibodies, anti FAK or pFAK. Western blots were carried out with whole cell lysates in buffer containing protease and phosphatase inhibitors. The confocal laser microscopy analysis for integrin subunits  and uPAR showed a co-localization of a5/uPAR and b1/uPAR and the co-immunoreactivity was higher in the uPA stimulated than in control conditions. The pFAK distribution in control explants was restricted to the somas however when he postmitotic neurons were stimulated with 10 nM uPA the pFAK wxpression appeared in neuronal bodies and neuritis with a high immunoreactivity in growth cones. Analysis of total and phosphorylated FAK by immunoprecipitation and western blot showed a significant increase of pFAK in uPA stimulated neurons. These results suggest that a5b1 integrin could act as the uPAR co-receptor and that uPA:uPAR:integrin complex could promote the activation of cell signaling pathways in postmitotic neurons.  Funded by UBA.