uPAR Binding Research Articles

OC 41.1 Structural Identification of the Factor XII - uPAR Binding Interface for Selective Drug Development

OC 41.1 Structural Identification of the Factor XII - uPAR Binding Interface for Selective Drug Development

Small-Molecule Inhibition of the uPAR ⋅ uPA Interaction by Conformational Selection.

The urokinase receptor (uPAR) is a cell surface receptor that binds to the serine protease urokinase-type plasminogen activator (uPA) with high affinity. This interaction is beneficial for extravascular fibrin clearance, but it has also been associated with a broad range of pathological conditions including cancer, atherosclerosis, and kidney disease. Here, starting with a small molecule that we previously discovered by virtual screening and cheminformatics analysis, we design and synthesize several derivatives that were tested for binding and inhibition of the uPAR ⋅ uPA interaction. To confirm the binding site and establish a binding mode of the compounds, we carried out biophysical studies using uPAR mutants, among them uPARH47C-N259C , a mutant previously developed to mimic the structure of uPA-bound uPAR. Remarkably, a substantial increase in potency is observed for inhibition of uPARH47C-N259C binding to uPA compared to wild-type uPAR, consistent with our use of the structure of uPAR in its uPA-bound state to design small-molecule uPAR ⋅ uPA antagonists. Combined with the biophysical studies, molecular docking followed by extensive explicit-solvent molecular dynamics simulations and MM-GBSA free energy calculations yielded the most favorable binding pose of the compound. Collectively, these results suggest that potent inhibition of uPAR binding to uPA with small molecules will likely only be achieved by developing small molecules that exhibit high-affinity to solution apo structures of uPAR, rather than uPA-bound structures of the receptor.

Inhibiting the urokinase‐type plasminogen activator receptor system recovers STZ‐induced diabetic nephropathy

The urokinase‐type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR ‐(s)uPAR‐ from circulation) is to regulate podocyte function through αvβ3 integrin/Rac‐1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)‐induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac‐1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ‐induced up‐regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac‐1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen‐plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR‐targeting approaches.

A Cross Talk between Neuronal Urokinase-type Plasminogen Activator (uPA) and Astrocytic uPA Receptor (uPAR) Promotes Astrocytic Activation and Synaptic Recovery in the Ischemic Brain

Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin on the cell surface. Our previous studies indicate that uPA and uPAR expression increase in the ischemic brain during the recovery phase from an acute ischemic injury and that uPA binding to uPAR promotes neurological recovery after an acute ischemic stroke. Here, we used male mice genetically deficient on either uPA (uPA-/-) or uPAR (uPAR-/-) or with a four-amino acid substitution into the growth factor domain of uPA that abrogates its binding to uPAR (PlatGFDhu/GFDhu) to investigate the mechanism whereby uPA promotes neurorepair in the ischemic brain. We found that neurons release uPA and astrocytes recruit uPAR to their plasma membrane during the recovery phase from a hypoxic injury and that binding of neuronal uPA to astrocytic uPAR induces astrocytic activation by a mechanism that does not require plasmin generation, but instead is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-regulated phosphorylation of the signal transducer and activator of transcription 3 (STAT3). We report that uPA/uPAR binding is necessary and sufficient to induce astrocytic activation in the ischemic brain and that astrocytes activated by neuronal uPA promote synaptic recovery in neurons that have suffered an acute hypoxic injury via a mechanism mediated by astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). In summary, we show that uPA/uPAR-induced astrocytic activation mediates a cross talk between astrocytes and injured neurons that promotes synaptic recovery in the ischemic brain.SIGNIFICANCE STATEMENT To date, there is no therapeutic strategy to promote synaptic recovery in the injured brain. Here, we show that neurons release urokinase-type plasminogen activator (uPA) and astrocytes recruit the uPA receptor (uPAR) to their plasma membrane during the recovery phase from a hypoxic injury. We found that binding of neuronal uPA to astrocytic uPAR promotes astrocytic activation and that astrocytes activated by uPA-uPAR binding promote synaptic recovery in neurons that have suffered a hypoxic injury by a mechanism that does not require plasmin generation, but instead is mediated by ERK1/2-regulated STAT3 phosphorylation, astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). Our work unveils a new biological function for uPA-uPAR as mediator of a neuron-astrocyte cross talk that promotes synaptic recovery in the ischemic brain.

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

Membrane tension drives ligand-independent integrin signaling.

Integrins anchor cells to the extracellular matrix (ECM) and control a multitude of essential cellular functions by activating a variety of signaling pathways. In this issue of The EMBO Journal, a study conducted by Ferraris and colleagues report that binding of urokinase-type plasminogen activator receptor (uPAR) to vitronectin is sufficient to trigger ligand-independent β1 and β3 integrin signaling. The coupling of uPAR and integrins occurs independent of lateral interaction between the two receptors, but relies on membrane tension (Ferraris et al, 2014).

Correction: Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

Correction: Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

Correction: Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

Correction: Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

A site for direct integrin αvβ6·uPAR interaction from structural modelling and docking

Integrin αvβ6 is an epithelially-restricted heterodimeric transmembrane glycoprotein, known to interact with the urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression. While the X-ray crystallographic structures of segments of other integrin heterodimers are known, there is no structural information for the complete αvβ6 integrin to assess its direct interaction with uPAR. We have performed structural analysis of αvβ6·uPAR interactions using model data with docking simulations to pinpoint their interface, in accord with earlier reports of the β-propeller region of integrin α-chain interacting with uPAR. Interaction of αvβ6·uPAR was demonstrated by our previous study using immunoprecipitation coupled with proteomic analysis by mass spectrometry. Recently this interaction was validated with proximity ligation assays and peptide arrays. The data suggested that two potential peptide regions from domain II and one peptide region from domain III of uPAR, interact with αvβ6 integrin. Only the peptide region from domain III is consistent with the three-dimensional interaction site proposed in this study. The molecular basis of integrin αvβ6·uPAR binding using structural data is discussed for its implications as a potential therapeutic target in cancer management.

Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC50 values of 3.6 and 1.2 μmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga-NODAGA-AE105-NH(2) 60 min postinjection. The use of (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the (64)Cu-labeled version ((64)Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH(2)) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and (68)Ga-NODAGA-AE105-NH(2) is the favored tracer as it provides the highest tumor-to-background ratio.

Abstract 5280: PET imaging of proteolysis: Evaluation of 68Ga-DOTA and 68Ga-NODAGA chelates of an uPAR-specific peptide in a human glioblastoma xenograft model

Abstract Background &amp; Aim: The urokinase-type plasminogen activator receptor (uPAR) has been shown to facilitate cancer cell invasion and metastasis. uPAR is over-expressed in various cancers, including human breast, prostate and colorectal cancer, and over-expression has been shown to correlate with poor prognosis. The established correlation between uPAR expression and disease progression provides an opportunity to develop an in vivo imaging agent, which could identify cancer patients at risk. In the present study, we produced and compared two new PET tracers based on 68Ga, the chelators DOTA and NODAGA and the uPAR antagonist peptide AE105 in a human glioblastoma xenograft model using microPET/CT. Method: Both compounds were synthesized using standard Fmoc solid-phase peptide chemistry and subsequently N-terminal conjugated with either DOTA or NODAGA. The conjugated peptides were radiolabeled with 68Ga using a 68Ge/68Ga generator system, which render the production of the tracers independent of a onsite cyclotron. The compounds were evaluated in vitro by surface plasmon resonance against human uPAR and in vivo using micro-positron emission tomography in a mouse model bearing human glioblastoma U87MG xenografts. All findings were validated using uPAR ELISA on tumor tissue and uPAR immnohistochemistry on tumor slides. Results: Both compounds were labeled under mild conditions with high yield and radiochemical purity (&amp;gt;95%). Both compounds showed excellent uPAR binding (IC50 values of 10-15 nM) in vitro, which was identical to the un-conjugated peptide AE105. In vivo, they displayed different tumor uptake, with 68Ga-DOTA-AE105 and 68Ga-NODAGA-AE105 having an uptake of 0.48±0.3 and 2.58±0.02 %ID/g 0.5 hr post injection, respectively. Besides tumor uptake, high uptake in the kidneys was also observed for both compounds. Dynamic PET scans illustrated higher stability in vivo for 68Ga-NODAGA-AE105 compared to 68Ga-DOTA-AE105, thus generating the best tumor-to-background contrast. Conclusion: A new 68Ga-based PET tracer for tumor imaging of human uPAR has been developed. Based on head-to-head comparison, NODAGA seems to be the optimal chelator, giving the highest uptake in tumor tissue. The use of generator-produced 68Ga could further support translation into the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2011-5280

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC 50 1 = 0.8 nM, IC 50 2 = 33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR’s native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10–100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC 50 >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA.

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation

The urokinase plasminogen activator receptor (uPAR) has emerged as a potential regulator of cell adhesion, cell migration, proliferation, differentiation, and cell survival in multiple physiologic and pathologic contexts. The urokinase plasminogen activator (uPA) was the first identified ligand for uPAR, but elucidation of the specific functions of the uPA-uPAR interaction in vivo has been difficult because uPA has important physiologic functions that are independent of binding to uPAR and because uPAR engages multiple ligands. Here, we developed a new mouse strain (Plau(GFDhu/GFDhu)) in which the interaction between endogenous uPA and uPAR is selectively abrogated, whereas other functions of both the protease and its receptor are retained. Specifically, we introduced 4 amino acid substitutions into the growth factor domain (GFD) of uPA that abrogate uPAR binding while preserving the overall structure of the domain. Analysis of Plau(GFDhu/GFDhu) mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing inflammation secondary to fibrin deposition. In contrast, leukocyte recruitment and tissue regeneration were unaffected by the loss of uPA binding to uPAR. This study identifies a principal in vivo role of the uPA-uPAR interaction in cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation and provides a valuable model for further exploration of this multifunctional receptor.

Antagonistic Anti-urokinase Plasminogen Activator Receptor (uPAR) Antibodies Significantly Inhibit uPAR-mediated Cellular Signaling and Migration

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.

A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines

BackgroundThe urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines.ResultsA DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension.ConclusionThis doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477–490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN CS) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.