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 ␣3␤1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of ␣3␤1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of ␣3␤1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/␣3␤1 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/integrinmediated transcriptional activation. Because uPAR is redistributed to clustered ␣3␤1 integrins, the requirement for uPAR/␣3␤1 interaction in uPA regulation was assessed. Clustering of ␣3␤1 in the presence of a peptide (␣325) that disrupts uPAR/␣3␤1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin ␣3␤1 clustering. These results were confirmed using a genetic strategy in which ␣3 null epithelial cells reconstituted with wild type ␣3 integrin, but not a mutant ␣3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/␣3␤1 binding using peptide ␣325 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 ␣3␤1 integrin promotes uPAR/␣3␤1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Role of Integrins in Regulating Proteases to Mediate Extracellular Matrix Remodeling

“…Binding of an RGD-bearing ligand to integrin αVβ3 in metastatic murine mammary cancer cells transcriptionally up-regulates uPA expression through ILKdependent AP-1 activation [95]. In another case, ligandinduced clustering of integrin α3β1 promotes uPAR-α3β1 interaction and enhances uPA expression in a Src-ERKdependent pathway [96]. uPAR expression can also be upregulated by integrins.…”

“…Evidence has shown that uPAR localizes to integrin-containing adhesion complexes, and interacts with integrins [86]. Up to now, uPAR has been found to physically and functionally associated with several β1 [96,100], β2 [101,102], and αV [103,104] integrins, which can recruit uPA/uPAR to the cell surface [87,105]. A good example is that uPA and uPAR are associated with integrin αVβ3, which recruits them to the leading edge of migrating tumor cells for ECM degradation [106].…”

Integrin Regulation of β-Catenin Signaling in Ovarian Carcinoma

“…AlexaFluor 488 goat anti-mouse IgG (HϩL) was purchased from Molecular Probes (Eugene, OR). Antibodies to ␣2, ␣3, or ␤1 integrin and control IgG were passively absorbed onto polybead amino 3.0-m microspheres (2.59% solids latex; Polysciences, Inc., Warrington, PA) as described previously (30). Antibodies to ␤1 integrin were also passively absorbed onto Dynabeads M-450 epoxy magnetic beads (Invitrogen) using the procedure for coating of antibodies/proteins provided by the manufacturer.…”

“…Following a 10 -15 min incubation and a 5-min centrifugation (16,000 ϫ g at 4°C), the nuclear fraction was collected. Western blot analysis was done as described previously (30). Western blots were quantified using ImageJ (National Institutes of Health, Bethesda).…”

Integrin Regulation of Epidermal Functions in Wounds

“…104 While the extent to which the latter mechanism might render an activated growth factor available to a distal cellular compartment remains unclear, there are also examples of integrinmediated liberation and diffusion of ECM-bound growth factors. For example, certain integrins (i.e., a v b 6 and a 3 b 1 ) can induce expression of MMP-9, uPA, or other extracellular proteases 79,101,137,150 that can degrade ECM and release reservoirs of ECM-associated growth factors (i.e., VEGF) to promote angiogenesis. 151,152 In addition, degradation of laminins, collagens, or other ECM proteins by some of these extracellular proteases may lead to the generation of bioactive matrix fragments that can directly stimulate cell growth or motility.…”