Abstract
The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail.We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/β1 integrin and FPR1/β1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/β1 integrin co-localization. By contrast, a low FPR1/β1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations.The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells.Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum.Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking.We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and β1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.
Highlights
To reach their final destination or their workplace, cells must move through the extracellular matrix (ECM) and, sometimes, between each other
We and others previously observed a cross-talk between cellanchored uPAR and fMLF-Rs [18,20,21,22]; their association on the cell membrane has never been explored in detail
Since Human embrional kidney 293 (HEK-293) cells mainly express FPR1-b1 integrin has been normalized to blue (FPR1) [18], we focused our studies on uPAR association to this fMLF-R. uPAR-293 cells were transiently transfected with the FPR1 cDNA, to reinforce FPR1 expression, and analyzed by confocal microscopy with uPAR- and FPR1specific antibodies; the analysis confirmed the expression of both receptors on the cell surface and showed their co-localization (Fig. 1A, top panel)
Summary
To reach their final destination or their workplace, cells must move through the extracellular matrix (ECM) and, sometimes, between each other. The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) serine-protease has been considered crucial in cell migration processes since it concentrates uPA proteolytic activity at the cell surface, allowing localized ECM degradation [2]. UPAR acts as a high affinity receptor for vitronectin (VN), an ECM component, abundant in ECM associated to tumor tissues [5]. Both uPA and VN, which require full-length uPAR for binding, are able to activate intracellular signaling pathways, leading to cell proliferation, survival, adhesion and migration, in spite of the absence of a transmembrane and a cytosolic region in the uPAR molecule [6]. UPAR-integrin association has been shown by co-immunolocalization, co-immunoprecipitation, FRET and by in vitro binding
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