Functional relevance of urinary-type plasminogen activator receptor-alpha3beta1 integrin association in proteinase regulatory pathways.
Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 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 alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 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 ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.
Ghosh
Supurna
S
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
Johnson
Jeff J
JJ
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
Sen
Ratna
R
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
Mukhopadhyay
Subhendu
S
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
Liu
Yueying
Y
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611.
Zhang
Feng
F
Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, California 94143.
Wei
Ying
Y
Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, California 94143.
Chapman
Harold A
HA
Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, California 94143.
Stack
M Sharon
MS
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611; Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611. Electronic address: mss130@northwestern.edu.
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D039201:Integrin alpha3beta1 / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes; D051379:Mice; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011554:Pseudopodia / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D039201:Integrin alpha3beta1; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011494:Protein Kinases; D014568:Urokinase-Type Plasminogen Activator