Inhibition of receptor-dependent urokinase signaling by specific Ser to Glu substitutions

We have previously reported that phosphorylation of human urokinase on Ser138/303 abolishes its catalytic-independent motogen and proadhesive abilities, whereas receptor binding is not affected. Here we show that substitution of the two relevant serines with glutamic acid residues impairs the ability of urokinase to mobilize a variety of human and mouse cell lines as well as human primary T lymphocytes. Accordingly, urokinase receptor-dependent signaling, leading to cytoskeletal rearrangements and paxillin re-distribution, does not occur in MCF-7 breast carcinoma cells exposed to 'phosphorylation-like' urokinase. Unlike the wild-type form, di-substituted urokinase is unable to induce the physical association of urokinase receptor with alphavbeta5 vitronectin receptor, which is required for MCF-7 urokinase-dependent cell migration. Finally, the di-substituted variant fails to activate p55fgr, a member of the Src tyrosine kinase family, which mediates cell migration and adhesion of U937 myelomonocytic cells. In conclusion, the finding that specific amino acid substitutions strongly interfere with the ability of urokinase to stimulate cell migration, and the associated intracellular events uncover a novel way to regulate urokinase receptor-dependent signaling.     

Receptor-mediated regulation of plasminogen activator function: plasminogen activation by two directly membrane-anchored forms of urokinase.

The generation of the broad specificity serine protease plasmin in the pericellular environment is regulated by binding of the urokinase-type plasminogen activator (uPA) to its specific glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor, uPAR. This interaction potentiates the reciprocal activation of the cell-associated zymogens pro-uPA and plasminogen. To further study the role of uPAR in this mechanism, we have expressed two directly membrane-anchored chimeric forms of uPA, one anchored by a C-terminal GPI-moiety (GPI-uPA), the other with a C-terminal transmembrane peptide (TM-uPA). These were expressed in the monocyte-like cell lines U937 and THP-1, which are excellent models for kinetic and mechanistic studies of cell-surface plasminogen activation. In both cell-lines, GPI-uPA activated cell-associated plasminogen with characteristics both qualitatively and quantitatively indistinguishable from those of uPAR-bound uPA. By contrast, TM-uPA activated cell-associated plasminogen less efficiently. This was due to effects on the K, for plasminogen activation (which was increased up to five-fold) and the efficiency of pro-uPA activation (which was decreased approximately four-fold). These observations suggest that uPAR serves two essential roles in mediating efficient cell-surface plasminogen activation. In addition to confining uPA to the cell-surface, the GPI-anchor plays an important role by increasing accessibility to substrate plasminogen and, thus, enhancing catalysis. However, the data also demonstrate that, in the presence of an alternative mechanism for uPA localization, uPAR is dispensable and, therefore, unlikely to participate in any additional interactions that may be necessary for the efficiency of this proteolytic system. In these experiments zymogen pro-uPA was unexpectedly found to be constitutively activated when expressed in THP-1 cells, suggesting the presence of an alternative plasmin-independent proteolytic activation mechanism in these cells.     

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR_88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR^S90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR^S90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR^S90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR^S90P. In conclusion, our findings indicate that Ser⁹⁰ is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR^S90E and uPAR^S90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.     

Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.     

Autocrine saturation of pro-urokinase receptors on human A431 cells

Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.     

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

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.     

Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide

The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.     

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.     

Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.     

Identification of a chemotactic epitope in human transforming growth factor-β1 spanning amino acid residues 368–374

TGF-b?1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-b?1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-b?1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-b?1 monomer were able to compete with [¹²⁵I]hrTGF-b?1 for binding to TGF-b? cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed. © 1995 Wiley-Liss, Inc.     

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR- complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down- modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.     

Soluble urokinase receptor promotes cell adhesion and requires tyrosine-92 for activation of p56/59(hck)

The urokinase plasminogen activator receptor (uPAR) plays an important role in the migration of leukocytes. It occurs as a membrane-bound form that contains a glycosylphosphatidylinositol (GPI) anchor and also as a soluble form (suPAR) that lacks the GPI anchor. Recently, a sequence of amino acids, SRSRYLE, within the receptor has been found to become unmasked on uPA binding or chymotrypsin cleavage. Exposure of the epitope results in the activation of p56/p59(hck) kinase and chemotaxis of myelomonocytic cells. Using an epitope-tagged suPAR molecule, we found that both three-domain and two-domain suPAR promote the adhesion of differentiated THP-1 cells to fibronectin and vitronectin, indicating that suPAR can modify cell adhesion as well as cell migration. In addition, we found that the amino acid sequence RYLE, within the chemotactic peptide, is conserved across species and that alanine substitution of Tyr 92 decreased the ability of the peptide to activate p56/59(hck).     

Urokinase plasminogen activator amino-terminal peptides inhibit development of the rat ventral prostate

The plasma membrane urokinase plasminogen activator receptor (uPAR) localizes and enhances activation of pro-uPA. Active uPA, in turn, promotes increased degradation of the extracellular matrix (ECM) by activation of plasminogen. uPAR binds to ECM molecules and integrins, which can affect cellular adhesion, signal transduction, and gene regulation. The current study examines the expression and function of uPAR in developing rat ventral prostates (VPs). We report that newborn VPs express uPAR mRNA and protein. In addition, the function of uPAR-bound uPA during in vitro prostatic development was studied by adding recombinant peptide competitive inhibitors of uPA-uPAR binding. Newborn VP explants were cultured in serum-free media for one week with 10(-8) M testosterone plus chimeric peptides containing a human immunoglobulin G Fc domain and either human uPA amino acids 1-138 (hu-uPA 1-138) as a control or mouse uPA amino acids 1-138 (mo-uPA 1-138) or 1-48 (mo-uPA 1-48). Hu-uPA 1-138-treated VPs underwent normal ductal branching morphogenesis and tissue differentiation. In contrast, VPs treated with mo-uPA 1-138 or mo-uPA 1-48 displayed a dose-dependent perturbation of ductal branching. Differentiation of both epithelial and mesenchymal tissues was also impaired. Mo-uPA 1-48-treated VPs contained significantly more apoptotic cells. These observations suggest that disruption of uPA binding to uPAR results in a retardation of the development of newborn VPs.     

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).     

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA^Glu mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA^Glu restores fluorescence in proportion to the amount of misacylated tRNA^Glu. This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA^Glu isodecoders on mistranslation and discuss the implications of our findings.     

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.     

An urokinase receptor antagonist that inhibits cell migration by blocking the formyl peptide receptor

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.     

Proteolytic processing and inactivation of CCL2/MCP-1 by meprins

Monocyte chemotactic protein 1 (CCL2/MCP-1) is a small chemokine involved in the recruitment and trafficking of mononuclear immune cells to inflammation sites. Our studies demonstrate that the metalloendopeptidases meprin A (purified from kidney cortex), recombinant meprin α, and recombinant meprin β can all process CCL2/MCP-1. The cleavage sites were determined by amino acid sequencing and mass spectrometry analysis of the generated products, and the biological activity of the products was evaluated by chemotactic migration assay using THP-1 cells. The cleavage sites generated by the meprin isoforms revealed that meprin A and meprin α cleaved the N-terminal domain of mouse CCL2/MCP-1 at the Asn⁶ and Ala⁷ bond, resulting in significant reduction in the chemotactic activity of the cleaved CCL2/MCP-1. Meprin β was unable to cleave the N-terminus of mouse CCL2/MCP-1 but cleaved the C-terminal region between Ser⁷⁴ and Glu⁷⁵. Human CCL2/MCP-1 that lacks the murine C-terminal region was also cleaved by meprin α at the N-terminus resulting in significant loss of CCL2/MCP-1 biological activity, whereas meprin β did not affect the biological activity. These studies suggest that meprin α and meprin β may play important roles in regulating the CCL2/MCP-1 chemokine activity during inflammation.