A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

Abstract

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.

FIGURE 1.

Vitronectin adhesion and cytoskeletal rearrangements in HEK cells expressing wild-type and mutated uPAR. A, adhesion of uPAR transfected cells on a reconstituted vitronectin matrix. Mock-transfected (Mock) cells or cells expressing uPAR_WT or uPAR mutant proteins were seeded in vitronectin-coated culture wells in the presence of EDTA and allowed to adhere during a 1-h incubation period at 37 °C. After washing, adherent cells were quantified by thiazolyl blue tetrazolium bromide assay. Each column represents the mean of a triple determination. The standard deviations are indicated. B, morphological changes and cytoskeletal rearrangements. Mock-transfected cells or cells expressing uPAR_WT or uPAR mutant proteins were cultured for 5 days on vitronectin-coated coverslips. The cells were then fixed and permeabilized followed by FITC-phalloidin staining and examination by fluorescence microscopy. Note the exclusive appearance of lamellipodia and cytoskeletal extensions in the uPAR_WT-transfected cells. C, quantification of fields with lamellipodia-positive cells. Cells were cultured, stained, and examined by fluorescence microscopy as in B. Each cell type was assigned an arbitrary designation, after which five randomly selected microscope fields for each cell type were scored blindly by four investigators for lamellipodia-positive cells (see “Experimental Procedures”). The cumulative score is represented for each sample.

FIGURE 2.

Effect of pro-uPA on vitronectin adhesion and cell morphology. Vitronectin adhesion and morphological properties of transfected HEK cells were analyzed as in Fig. 1, except that pro-uPA (100 nm) was added to the samples where indicated. Symbols designating the four transfectant cell types are indicated. A, cell adhesion on a vitronectin matrix in the presence or absence of pro-uPA. Mock, mock-transfected. B, cytoskeletal rearrangements as a consequence of the addition of pro-uPA during cell culture. After fixation, permeabilization, and FITC-phalloidin staining, cells were examined by fluorescence microscopy. C, occurrence of lamellipodia-positive cells in the presence or absence of pro-uPA. FITC-phalloidin-stained cell samples were scored blindly as described in the legend for Fig. 1 (panel C), after which the cumulative score for each type of sample was calculated.

FIGURE 3.

Dependence on different uPAR ligands and the Rac pathway. A and B, adhesion (A) and lamellipodia-positive cells (B), analyzed as in Fig. 2, A and C, but in the presence of the indicated ligands. Ligands were added at final concentrations of 100 nm (pro-uPA, ATF, and GFD) or 1 μm (synthetic peptide AE120), respectively. Mock, cells transfected with vector alone. C, inactivation of the Rac pathway. Cells were transfected with the dominant-negative Rac expression plasmid, N17Rac, or with vector alone, as indicated, and cultured in the presence of 20 nm pro-uPA. After fixation, permeabilization, and FITC-phalloidin staining, randomly selected microscope fields representing at least 50 cells were scored for the percentage of protrusion-positive cells.