uPAR expression controls cell migration.

<p>uPAR-293 and V-293 cells efficiently migrate toward serum growth factors or EGF (GF). However, uPAR, when expressed, recruits and bridges fMLFRs and β1 integrin at the cell surface, thus driving pro-migratory signaling (upper panels). In fact, anti-uPAR antibodies (anti-uPAR), FPR1 desensitization by the W peptide (W Pep), inhibition of uPAR/β1 integrin interaction by P-25 peptide (P-25) or of specific cell signalling mediators block migration of uPAR-293 cells without affecting migration of uPAR-negative V-293 control cells (lower panels).</p

uPAR recruits fMLF receptors and integrins on the cell membrane.

<p><b>A:</b> HEK-293 cells were seeded on glass coverslips and transiently transfected with EGFP-uPAR cDNA; after 24 h, cells were incubated for further 24 h in DMEM containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep) in DMEM. All samples were then fixed, incubated with the anti-FPR1 polyclonal antibody and with the mouse anti-β1 monoclonal antibody, stained with Cy5 or Alexa 594 conjugated secondary antibodies, and analyzed by confocal microscopy. <b>B:</b> The degree of co-localization of the fluorescent signals in panel A was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of pixels corresponding to co-localizing uPAR-FPR1 has been normalized to green (uPAR) or blue (FPR1) pixels and reported in 1^st and 2^nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-β1 integrin has been normalized to green (uPAR) or red (β1 Integrin) pixels and reported in 3^rd and 4^th set of columns, respectively. The number of pixels corresponding to co-localizing FPR1-β1 integrin has been normalized to blue (FPR1) or red (β1 Integrin) pixels and reported in 5^th and 6^th set of columns, respectively. (*) p≤0.05, as determined by the Student's <i>t</i> test. <b>C:</b> HEK-293 cells were seeded on glass coverslips and transiently transfected with the empty pEGFP-N1 vector; after 24 h, cells were incubated for further 24 h in DMEM containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep) in DMEM. All samples were then fixed, incubated with the anti-FPR1 polyclonal antibody and with the rabbit anti-β1 polyclonal antibody, stained with Cy5 or Alexa 594 conjugated secondary antibodies, and analyzed by confocal microscopy. <b>D:</b> The degree of co-localization of the fluorescent signals in panel C was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of pixels corresponding to co-localizing FPR1-β1 integrin has been normalized to blue (FPR1) or red (β1 integrin) pixels and reported in the graph.</p

FPR1 co-localizes with uPAR on the surface of uPAR-expressing HEK-293 cells.

<p>HEK-293 cells stably transfected with uPAR cDNA (uPAR-293 cells) were seeded on glass coverslips and transiently transfected with FPR1 cDNA. After 24 h, cells were incubated for further 24 h in culture medium (DMEM) containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep). All samples were then fixed, incubated with the anti-uPAR monoclonal R4 antibody and the anti-FPR1 polyclonal antibody (<b>A</b>) or with the anti-uPAR monoclonal R4 antibody and the rabbit anti-β1 polyclonal antibody (<b>B</b>), stained with Cy3 or Alexa 488 conjugated secondary antibodies, and analyzed by confocal microscopy. The insets show a 4× magnification for the indicated region in each merge image. <b>C:</b> The degree of co-localization of the fluorescent signals was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of yellow pixels, corresponding to co-localizing uPAR-FPR1, has been normalized to green (uPAR) or red (FPR1) pixels shown in A and reported in 1^st and 2^nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-β1 integrin has been normalized to green (uPAR) or red (β1 Integrin) pixels shown in B and reported in 3^rd and 4^th set of columns, respectively. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

fMLF receptors and β1 integrins are involved in uPAR capability to control cell migration.

<p>uPAR-293 cells (<b>A and C</b>) or V-293 cells (<b>B and D</b>) were pre-incubated with diluent (-) or W Peptide (W Pep) (<b>A and B</b>), or with diluent (-) or P-25 peptide (<b>C and D</b>). Cells were then plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test. Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR depletion or blocking impairs migration of uPAR-expressing cells.

<p><b>A:</b> Prostate carcinoma (PC3) cells were transfected with a uPAR-targeting siRNA or a non-targeting control siRNA; then, cells were partly lysed for Western blot analysis with a uPAR-specific antibody (left) and partly loaded in Boyden chamber and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (middle panel). The values are the mean±SD of a representative experiment performed in triplicate. Results of the migration assay are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractant (right). (*) p≤0.05, as determined by the Student's <i>t</i> test. <b>B:</b> PC3 cells were pre-incubated with nonimmune immunoglobulins (Ig) or anti-uPAR polyclonal antibodies, plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left). Results of the migration assay are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractant (right). The values are the mean±SD of three experiments. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR co-immunoprecipitates with fMLF-Rs.

<p><b>A:</b> Lysates of uPAR-293 cells or V-293 cells, transiently transfected with FPR1 cDNA, were analyzed by Western blot with a uPAR-specific monoclonal antibody or immunoprecipitated with anti-FPR1 polyclonal antibodies or nonimmune rabbit immunoglobulins and then analyzed by Western blot with the uPAR-specific monoclonal antibody (left panels). Lysates of FPR1- or vector-transfected uPAR-293 and V-293 cells were analyzed by Western blot with a FPR1-specific polyclonal antibody to assess the levels of FPR1 expression (right panel). Tubulin or actin were detected for loading control. <b>B:</b> Lysates of uPAR-293 cells, not transfected with FPR1 cDNA, were analyzed by Western blot with a uPAR-specific monoclonal antibody or immunoprecipitated with anti-FPR1 polyclonal antibodies or nonimmune rabbit immunoglobulins and then analyzed by Western blot with a uPAR-specific monoclonal antibody. <b>C:</b> Lysates of uPAR-293 cells, transiently transfected with FPR2 cDNA, were analyzed by Western blot with anti-uPAR antibodies or immunoprecipitated with an anti-FPR2 monoclonal antibody or nonimmune mouse immunoglobulins and analyzed by Western blot with a uPAR-specific polyclonal antibody (left). Lysates of FPR2- or vector- transfected uPAR-293 cells were analyzed by Western blot with anti-FPR2 rabbit or anti-actin mouse antibodies (as loading control) to assess the levels of FPR2 expression (right). <b>D:</b> Lysates of uPAR-293 cells, transiently transfected with FPR3 cDNA, were analyzed by Western blot with anti-uPAR antibodies or immunoprecipitated with anti-FPR3 polyclonal antibodies or nonimmune rabbit immunoglobulins and analyzed by Western blot with the uPAR-specific monoclonal antibody (left). Lysates of FPR3- or vector- transfected uPAR-293 cells were analyzed by Western blot with anti-FPR3 rabbit or anti-tubulin mouse antibodies (as loading control) to assess the levels of FPR3 expression (right).</p

uPAR-dependent cell migration is mediated by signaling mediators not involved in uPAR-independent cell migration.

<p>uPAR-293 cells (<b>A and C</b>) or V-293 cells (<b>B and D</b>) were pre-incubated with diluents (-) or inhibitors of Rho- or Rac1-dependent signaling pathways (<b>A and B</b>), or with diluents (-) or inhibitors of PI3K or ERK-MAPKs (<b>C and D</b>). Cells were then plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test. Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR expression controls cell migration toward serum.

<p>uPAR-293 cells (<b>A</b>) or V-293 cells (<b>B</b>) were pre-incubated with nonimmune immunoglobulins (Ig), anti-uPAR or anti-uPAR_84–95 polyclonal antibodies, plated in Boyden chambers and allowed to migrate toward 10% FBS. Migrated cells were fixed, stained with hematoxylin, and counted (left panels). The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test. Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

uPAR expression controls cell migration toward EGF.

<p>Stably-transfected uPAR-293 cells (<b>A, C, E</b>) or V-293 cells (<b>B, D, F</b>) were pre-incubated with nonimmune Ig (<b>-</b>) or anti-uPAR or anti-uPAR_84–95 polyclonal antibodies (<b>A and B</b>), with diluents (-) or P-25 or W (W Pep) peptides (<b>C and D</b>), with diluents (-) or inhibitors of Rho- or Rac1-dependent signaling pathways (<b>E and F, left panels</b>), with diluents (-) or inhibitors of PI3K or ERK-MAPKs (<b>E and F, right panels</b>). Cells were then plated in Boyden chambers and allowed to migrate toward 100 ng/ml EGF. Migrated cells were fixed, stained with hematoxylin, and counted; results are expressed as percentage of cells migrated towards EGF over the cells migrated without EGF; 100% values represent cell migration in the absence of chemoattractants. The values are the mean±SD of three experiments performed in triplicate. (*) p≤0.05, as determined by the Student's <i>t</i> test.</p

Mean values (and standard deviations in parentheses) relative to the flow cytometry analysis described in Figure 3.

<p>Mean values (and standard deviations in parentheses) relative to the flow cytometry analysis described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022800#pone-0022800-g003" target="_blank">Figure 3</a>.</p