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The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² internal sequence.To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² sequence.In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr⁹² chemotactic sequence, the maximal effect being reached at 10 nmol L⁻¹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) β₃ vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2.Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² chemotactic sequence.

Functional assembly of the plasminogen-dependent proteolytic system on the cell surface requires multiple interactions involving urokinase (uPA), urokinase receptor (uPAR), plasminogen activator inhibitors, and other molecules that mediate cell migration and adhesion. We analyzed the in vitro interaction of uPAR-containing particulate cell fractions with the amino-terminal fragment (ATF) of human urokinase and the matrix-like form of vitronectin. Binding and cross-linking of 125I-labeled ATF to crude membrane extracts from LB6-19 mouse cells overexpressing human uPARs in the presence of 25 nM urea-denatured vitronectin led to the formation of Mr 137,000, 92, 000, and 82,000 covalent complexes. Immunoprecipitation of the preformed cross-linked 125I-labeled complexes with anti-vitronectin, anti-uPA, or anti-uPAR antibodies revealed that the Mr 82,000 and 92, 000 species do contain ATF and vitronectin and identified the Mr 137, 000 species as a ternary complex formed by ATF, uPAR, and vitronectin. A similar electrophoretic pattern was displayed by acid-pretreated membranes extracted from MCF-7 breast carcinoma or HT1080 fibrosarcoma cell lines, as well as a ductal breast carcinoma specimen; the latter exhibited complex formation at concentrations of vitronectin lower than 10 nM. Finally, uPAR-vitronectin interaction was further documented by the decreased reactivity of an anti-uPAR polyclonal antibody to acid-pretreated sections of 10 breast carcinomas that had been preincubated with vitronectin. Our findings highlight the ability of uPAR to interact simultaneously with vitronectin and uPA in breast cancer, supporting a dynamic coupling of the molecular mechanisms underlying plasminogen-dependent matrix degradation and cell adhesion.

Current evidence regarding the regulation of urokinase-dependent extracellular proteolysis indicates that plasminogen activation is a surface-associated process. We have compared the histological localization of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) in breast cancer sections using a panel of monoclonal antibodies. First, the ability of six different anti-uPA monoclonal antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator inhibitor type 1 was compared. Then the reactivity of the anti-uPAR antibodies was tested, and the occurrence of an uPA receptor of about M(r) 55,000 in samples from breast carcinoma was assessed by immunoprecipitating the uPA receptor from an in vitro 125I-labeled tumor extract. Immunocytochemical data from adjacent sections of 10 tumor specimens showed that antibodies recognizing free and bound uPA mostly stain the cytoplasm and the membrane of epithelial tumor cells in confined areas of the tumor and some fibroblast-like stromal cells. Acid pretreatment of tumor sections, which removes receptor-bound uPA, causes a strong reduction of the immunocytochemical reactivity of epithelial tumor cells, whereas staining of fibroblast-like cells is not considerably affected. Consistent with these results, epithelial tumor cells were mostly unreactive to anti-uPAR antibodies unless pretreated with acidic buffer, whereas fibroblast-like stromal cells showed a faint but acid-resistant staining with all anti-uPARs. In conclusion, these results show that occupied uPA receptors are definitely present on the membrane of epithelial tumor cells and suggest the occurrence of uPA-uPAR-dependent proteolytic activity on the surface of breast cancer cells.

We measured the tissue concentration of human urokinase receptor (uPAR) in 22 breast carcinomas and 9 benign breast lesions using in vitro quantitative autoradiography. Tissue sections were incubated with increasing concentrations of 125I-pro-urokinase in the presence or absence of unlabeled competitor. Breast carcinomas were found to contain 5 times more uPAR than benign breast lesions with respect to their protein content [523 +/- 72 versus 108 +/- 20 (SE) fmol/mg (P0.001)]. Simultaneous quantitation of urokinase (uPA) by immunoenzymatic assay on tissue extracts from the same specimens showed that breast carcinomas also contain 19 times more uPA than benign tumors (611 +/- 134 versus 32 +/- 8 fmol/mg) (P0.01). The reliability of quantitative autoradiography measurements was confirmed by uPAR cross-linking assay on membrane fraction from either U937 histiocytic lymphoma cells or breast carcinomas and immunoperoxidase staining with an anti-uPAR antibody on tumor sections. Also, immunoperoxidase staining with an anti-uPA monoclonal antibody showed that uPA is indeed localized on the plasma membrane of epithelial tumor cells in confined areas of breast carcinomas whereas cells from benign breast lesions were devoid of uPA under the same experimental conditions. In conclusion, our findings support the hypothesis that uPAR plays a central role in the acquisition of an invasive phenotye and favor its potential use as a prognostic factor in patients with breast carcinoma.

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.

The purified amino-terminal fragment (ATF) of human urokinase plasminogen activator (residues 1-135), which is not required for activation of plasminogen, binds with high affinity to specific plasma membrane receptors on U937 monocytes. Intact urokinase efficiently competes for 125I-labeled ATF binding; 50% competition occurs with 1 nM urokinase. A large part of receptor-bound urokinase remains on the cell surface for at least 2 hr at 37 degrees C. Differentiation of U937 monocytes into macrophage-like cells specifically increases ATF binding 10- to 20-fold. These results suggest an important role for urokinase in monocyte/macrophage biology: the native enzyme binds to the cells with the amino-terminal domain; the catalytic, carboxyl-terminal domain remains exposed on the cell surface to stimulate localized proteolysis and facilitate cell migration.

Many human cells and cell lines possess a specific receptor that binds urokinase plasminogen activator (uPA) with an affinity of about 10(-10) M. Bound enzyme is not internalized, is slowly dissociated, and retains its enzymatic activity. The amino acid sequence of uPA responsible for receptor binding is located within the first 35 aminoterminal residues, ie, in the growth factor domain. Binding, however, is not competed for by other proteins that contain the growth factor domain (including epidermal growth factor). Cells that produce uPA secrete the pro-uPA form, which subsequently binds to the receptor. A431 cells, in fact, have their receptors completely saturated with pro-uPA. It is proposed that uPA:uPA-receptor interaction plays a direct role in physiological and pathological processes that require cell migration.

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.

Previous studies have shown that the region of human urokinase-type plasminogen activator (uPA) responsible for receptor binding resides in the amino-terminal fragment (ATF, residues 1-135) (Stoppelli, M.P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R.K. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 4939-4943). The area within ATF responsible for specific receptor binding has now been identified by the ability of different synthetic peptides corresponding to different regions of the amino terminus of uPA to inhibit receptor binding of 125I-labeled ATF. A peptide corresponding to human [Ala19]uPA-(12-32) resulted in 50% inhibition of ATF binding at 100 nM. Peptides uPA-(18-32) and [Ala13]uPA-(9-20) inhibit at 100 and 2000 microM, respectively. The human peptide uPA-(1-14) and the mouse peptide [Ala20]uPA-(13-33) have no effect on ATF receptor binding. This region of uPA is referred to as the growth factor module since it shares partial amino acid sequence homology (residues 14-33) to epidermal growth factor (EGF). Furthermore, this region of EGF is responsible for binding of EGF to its receptor (Komoriya, A. Hortsch, M., Meyers, C., Smith, M., Kanety, H., and Schlessinger, J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1351-1355). However, EGF does not inhibit ATF receptor binding. Comparison of the sequences responsible for receptor binding of uPA and EGF indicate that the region of highest homology is between residues 13-19 and 14-20 of human uPA and EGF, respectively. In addition, there is a conservation of the spacings of four cysteines in this module whereas there is no homology between residues 20-30 and 21-33 of uPA and EGF. Thus, residues 20-30 of uPA apparently confer receptor binding specificity, and residues 13-19 provide the proper conformation to the adjacent binding region.

Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.

Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [125I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser88-Arg-Ser-Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH2 (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe–dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an αv integrin–dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/αv association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein–expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy. [Mol Cancer Ther 2009;8(9):2708–17]

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser 88 -Arg-Ser-Arg-Tyr 92 (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH 2 ) shares the same binding site with SRSRY and competes with N -formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N -formyl-peptide receptor (FPR). pERERY-NH 2 is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERY-NH 2 is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.