Functional relevance of urinary-type plasminogen activator receptor-α3β1 integrin association in proteinase regulatory pathways

Abstract

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin α3β1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of α3β1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of α3β1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/α3β1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered α3β1 integrins, the requirement for uPAR/α3β1 interaction in uPA regulation was assessed. Clustering of α3β1 in the presence of a peptide (α325) that disrupts uPAR/α3β1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin α3β1 clustering. These results were confirmed using a genetic strategy in which α3 null epithelial cells reconstituted with wild type α3 integrin, but not a mutant α3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/α3β1 binding using peptide α325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering of α3β1 integrin promotes uPAR/α3β1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.