Mouse uPA Research Articles

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

Disruption of Water Networks is the Cause of Human/Mouse Species Selectivity in Urokinase Plasminogen Activator (uPA) Inhibitors Derived from Hexamethylene Amiloride (HMA).

The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising antimetastasis target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and antikaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenograft mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5-N,N-hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as antimetastasis drugs.

Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models.

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

Reduction of mouse atherosclerosis by urokinase inhibition or with a limited-spectrum matrix metalloproteinase inhibitor.

Elevated activity of urokinase plasminogen activator (uPA) and MMPs in human arteries is associated with accelerated atherosclerosis, aneurysms, and plaque rupture. We used Apoe-null mice with macrophage-specific uPA overexpression (SR-uPA mice; a well-characterized model of protease-accelerated atherosclerosis) to investigate whether systemic inhibition of proteolytic activity of uPA or a subset of MMPs can reduce protease-induced atherosclerosis and aortic dilation. SR-uPA mice were fed a high-fat diet for 10 weeks and treated either with an antibody inhibiting mouse uPA (mU1) or a control antibody. mU1-treated mice were also compared with PBS-treated non-uPA-overexpressing Apoe-null mice. Other SR-uPA mice were treated with one of three doses of a limited-spectrum synthetic MMP inhibitor (XL784) or vehicle. mU1 reduced aortic root intimal lesion area (20%; P = 0.05) and aortic root circumference (12%; P = 0.01). All XL784 doses reduced aortic root intimal lesion area (22-29%) and oil-red-O-positive lesion area (36-42%; P < 0.05 for all doses and both end points), with trends towards reduced aortic root circumference (6-10%). Neither mU1 nor XL784 significantly altered percent aortic surface lesion coverage. Several lines of evidence identified MMP-13 as a mediator of uPA-induced aortic MMP activity. Pharmacological inhibition of either uPA or selected MMPs decreased atherosclerosis in SR-uPA mice. uPA inhibition decreased aortic dilation. Differential effects of both agents on aortic root vs. distal aortic atherosclerosis suggest prevention of atherosclerosis progression vs. initiation. Systemic inhibition of uPA or a subset of MMPs shows promise for treating atherosclerosis.

Abstract 701: In vivo anti-metastatic effects of uPAR retargeted oncolytic measles virus

Abstract The urokinase receptor (uPAR) is a clinically relevant target for novel biological therapies. Here, we investigated the antimetastatic effects of oncolytic measles virus retargeting uPAR (MV-uPA) in breast cancer metastases models. Species specificities of recombinant viruses retargeted against human (MV-h-uPA) or mouse uPA (MV-m-uPA) were demonstrated in vitro in human and murine breast cancer cell lines. MV-h-uPA and MV-m-uPA induced in vitro cytotoxicity and replicated in human breast cancer cells (MDA-MB231, MDA-MB436 and MCF-7) and murine mammary cancer cells (4T1), respectively. A syngeneic model of mammary cancer metastases was established by intravenous injection of 4T1 cells (5x10e4 per mouse) expressing highly efficient firefly luciferase into immunocompetent BALB/C mice. Seven days after tumor cell inoculation, MV-m-uPA or vehicle was administered into tumor-bearing mice via the tail vein (virus dose: 1×10e6 TCID50 per injection, three injections, every other day). Metastases progression was assessed by bioluminescence analysis. In this highly aggressive syngeneic model, MV-m-uPA treatment was associated with delay in metastases progression and improvement in survival, compared to controls. These effects were associated with detection of viable viral particles from lungs of mice treated with MV-m-uPA, as well as detection of MV-N staining in lung samples in tumor bearing treated mice. Finally, we validated the above effects in a model of human breast cancer metastases. This model was established by injection of 1×10e6 MDA-MB231-luc2 cells via tail vein into female nude mice. Ten days after tumor cell injection, mice were treated with three IV injections (every other day) of PBS or MV-h-uPA (1×10e6 TCID50). Metastases progression (by bioluminescence evaluation) was markedly inhibited in treated mice, compared to controls, resulting in improvement in survival. In conclusion, systemic administration of MV-uPA is effective in the treatment of breast cancer metastases in immune-competent and immune-deficient model, suggesting that measles virus mediated virotherapy may present a novel and promising strategy for treatment against metastatic breast cancer. Citation Format: Yuqi Jing, Julia Zaias, Jaime Merchan. In vivo anti-metastatic effects of uPAR retargeted oncolytic measles virus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2014-701

Abstract 4593: Targeted therapy of pancreatic cancer by intraperitoneal delivery of uPAR-targeted theranostic nanoparticles

Abstract The leading challenge in pancreatic cancer treatment is inability to deliver sufficient amounts of therapeutic agents into cancer cells due to the presence of extensive tumor stroma. Increasing evidence shows that pancreatic tumor microenvironment promotes proliferation, metastasis, and chemoresistance of the cancer cells. Urokinase plasminogen activator receptor (uPAR) is highly expressed in pancreatic tumor cells and tumor stroma but is not found in the normal pancreas. Therapeutic agents targeting to uPAR may offer a means to break down the tumor stromal barrier and enhance drug delivery into cancer cells. In this study, we engineered a near infrared dye NIR-830 labeled amino-terminal fragment (ATF) of mouse uPA conjugated magnetic iron oxide nanoparticles (IONP) carrying chemotherapy drug cisplatin (Cis) without or with PEG-coating (ATF-IONP-Cis ATF-PEG-IONP-Cis). To determine the effect of PEG-coating on non-specific uptake of the IONPs by enriched macrophages in the peritoneal cavity and its consequent effect on targeted delivery of the theranostic IONPs, we established a mouse pancreatic tumor model derived from the PANC02 cell line with simultaneous carrying a subcutaneous and an orthotopic tumor. First, we examined the best delivery method for this animal tumor model and found that the i.p. delivery of mouse uPAR targeted ATF-PEG-IONPs resulted in stronger optical signal in the orthotopic cancers with much lesser signals in the liver compared that of the i.v. delivery. Next, the effects of the i.p. delivery of the ATF-IONP-Cis, ATF-PEG-IONP-Cis, and non-targeted ATF-IONP-Cis were evaluated in the mouse pancreatic cancer model. We found that i.p. delivery of 5 mg/kg of Cisplatin equivalent ATF-IONP-Cis or ATF-PEG IONP-Cis every 3 day for 4 injections significantly inhibited tumor growth (Control vs ATF-PEG-IONP-cis: p=0.008; ATF-IONP-Cis: p=0.016). It seemed that PEG-coating didn't affect the inhibitory effects of ATF-IONP-Cis nanoparticles on tumor growth. However, free cisplatin and non-targeted-IONP-Cis treated groups didn't show significant anti-tumor effects. We also observed that the mice treated with ATF-IONP-Cis had much less amounts of ascites (average 0.5ml/mouse) compared to the mice treated with free cisplatin (an average of 5 ml/mouse). Additionally, markedly systemic toxicity effect was seen in the mice treated conventional cisplatin but this was not observed in the mice treated with ATF-IONP-cis. We further confirmed targeted delivery of ATF-IONP-cis into the tumor using Prussian blue staining on frozen tissue sections and chemical analysis to quantify amount of IONPs in tumor tissues. Taken together, the ATF-PEG-IONP-Cis theranostic nanoparticles and the i.p. delivery approach developed in this study have great potential for the development of novel targeted and image-guided treatment of pancreatic cancer Citation Format: Ning Gao, Erica Bozeman, Weiping Qian, Charles Staley, Andrew Wang, Hui Mao, Lily Yang. Targeted therapy of pancreatic cancer by intraperitoneal delivery of uPAR-targeted theranostic nanoparticles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2014-4593

SLURP-1 modulates corneal homeostasis by serving as a soluble scavenger of urokinase-type plasminogen activator.

Our previous study revealed the immunomodulatory property of the secreted lymphocyte antigen (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1), abundantly expressed in the cornea and associated with the hyperkeratotic disorder Mal de Meleda. Here, we test the hypothesis that SLURP1 modulates the functions of membrane-tethered uPAR by acting as a soluble scavenger of its ligand urokinase-type plasminogen activator (uPA). Human corneal limbal epithelial (HCLE) and mouse corneal stromal fibroblast MK/T-1 cells were employed to examine the effect of SLURP1 on cell proliferation and migration. Human corneal limbal epithelial cell clones stably expressing SLURP1 under the control of cytomegalovirus (CMV) promoter were generated using lentiviral vectors. Recombinant 6× His-mouse Slurp1 and maltose-binding protein (MBP)-mouse uPA were expressed in Escherichia coli and partially purified using nickel-ion and amylose columns, respectively. Slurp1 interaction with uPA was detected using ligand blots, ELISA, pull-down assays, and immunofluorescent staining. Stable expression of SLURP1 in HCLE cells was confirmed by immunoblots and immunofluorescent staining. Human corneal limbal epithelial and MK/T-1 cell proliferation and migration rates were suppressed by exogenous SLURP1. Ligand blots, ELISA, and pull-down assays indicated that Slurp1 efficiently interacts with uPA. Immunofluorescent staining demonstrated that exogenous SLURP1 decreased the amount of cell surface-bound uPA in the leading edges of migrating cells. In gap-filling assays, wild-type HCLE cells responded to uPA by increasing their velocity and closing larger area, while the SLURP1-expressing HCLE cells failed to do so. SLURP1 modulates corneal homeostasis by serving as a soluble scavenger of uPA and regulating the uPA-dependent functions of uPAR.

Abstract 138: Treatment of Atherosclerosis by Systemic Protease Inhibition

Background Elevated activity of urokinase plasminogen activator (uPA) and matrix metalloproteases (MMPs) in human arteries is associated with accelerated atherosclerosis (athero), aneurysms, and plaque rupture. Apoe -null mice with macrophage-targeted uPA overexpression (SR-uPA mice) have elevated vascular uPA and MMP activity, accelerated athero, dilated aortic roots, and increased histologic features of plaque rupture. SR-uPA mice are therefore a useful experimental setting for beginning to investigate whether inhibition of uPA or MMPs can prevent athero progression, aortic dilation, and plaque rupture. Hypothesis Treatment with a murine monoclonal antibody inhibiting mouse uPA (mU1), or a limited-spectrum synthetic MMP inhibitor (XL784) will reduce athero in SR-uPA mice. Methods SR-uPA mice were fed a high-fat diet for 10 wk beginning at 5 - 6 wk of age. Fat-fed mice were injected with mU1 or control antibody twice per wk (for 10 wk), or were given either XL784 (125, 250, or 500 mg/kg) or vehicle by daily oral gavage. Mice were then anesthetized, formalin-perfused, and athero was measured in isolated aortic roots and pinned aortas. Groups were compared by t-test and ANOVA. Results None of the treatments altered peripheral blood monocyte counts or plasma cholesterol. mU1 reduced aortic root intimal lesion area by 20% ( P &lt; 0.05) and aortic root circumference by 12% ( P = 0.01). All 3 doses of XL784 significantly decreased oil-red O-positive lesion area (36 - 42%; P &lt; 0.05 for all). All XL784 doses also reduced aortic root intimal lesion area (22 - 29%; P &lt; 0.05 for low dose only). The highest XL784 dose also reduced total macrophage-positive lesion area (33%; P &lt; 0.05). All XL784 doses yielded trends towards decreased aortic root circumference (5 - 10%). Neither mU1 nor XL784 significantly altered percent aortic surface lesion coverage. Conclusions In SR-uPA mice, pharmacologic inhibition of either uPA or selected MMPs significantly decreased aortic root athero and limited aortic dilation. Differential effects of both agents on aortic root versus surface athero suggest prevention of athero progression rather than initiation. These approaches to protease inhibition show promise for preventing the progression and complications of established athero.

UPAR as Anti-Cancer Target: Evaluation of Biomarker Potential, Histological Localization, and Antibody-Based Therapy

Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted action of several proteolytic systems in generation of a high proteolytic potential required for tissue remodeling processes. uPAR is additionally cleaved by uPA on the cell surface, liberating domain I, resulting in abrogated pericellular proteolysis. The expression of both uPAR and uPA is significantly up-regulated during cancer progression and is primarily confined to the tumor-associated stromal compartment. Furthermore, both uPAR and uPA have proven to be prognostic markers in several types of cancer; high levels indicating poor survival. The cleaved forms of uPAR are also prognostic markers, and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to uPAR on the cell surface and/or by direct inhibition of the catalytic activity of uPA. Both strategies have been pursued and inhibition of these functions has shown effect in xenogenic cancer models. Pericellular proteolysis has also been inhibited in vivo in mouse models of wound healing and hepatic fibrinolysis using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models.

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

BackgroundProteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds.Methodology/Principal FindingsTo evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure.Conclusions/SignificanceOur findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.

Abstract 4401: Antibody-mediated targeting of the uPA proteolytic activity neutralizes uPA functions in vivo

Abstract Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes, during both normal physiological conditions and cancer progression. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the acute and specific interference with uPA functionality in adult mice, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by ELISA, Western blotting, SPR and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We provide evidence that acute disruption of uPA activity in the adult mouse was effective with mU1, but not mU3, because only mU1 rescued mice treated with a uPA-activable anthrax pro-toxin. Moreover, systemic administration of mU1 (i) delayed wound healing in tissue-type plasminogen activator (tPA)-deficient mice, and (ii) impaired uPA-mediated hepatic fibrinolysis in tPA-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double-deficient mice. Importantly, these results demonstrate the specific antagonist-directed targeting of mouse uPA at the enzyme activity level in normal physiological processes in vivo. Preliminary results indicate that wild-type mice, which have been transplanted with murine Lewis lung carcinoma cells, display significant reduction in primary tumour growth, when treated with mU1 as compared to placebo-treated (i.e. PBS-treated) littermates. We thus suggest that mU1 is an excellent mAb candidate for therapeutic intervention studies in mouse cancer models. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4401.

Induction of Tissue Factor by Urokinase in Lung Epithelial Cells and in the Lungs

Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates. This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs). Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA(-/-)) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity. uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA(-/-) mice exposed to LPS failed to induce TF. uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.

The Urokinase Receptor Promotes Cancer Metastasis Independently of Urokinase-Type Plasminogen Activator in Mice

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

Chemotactic effect of urokinase-type plasminogen activator on mouse spermatozoa in vitro

The aim of this study is to investigate the chemotactic effect of urokinase-type plasminogen activator (uPA) on mouse spermatozoa. Capillary assays were applied to study the chemotactic activity of ascending and descending gradients of uPA. Firstly, the chemotactic effect of an ascending gradient of uPA on mouse spermatozoa was observed by counting the number of spermatozoa that migrated into the capillary after incubation with uPA for 5, 10, 20, and 30 min, respectively, compared with that after incubation with F10. Twenty minutes was a suitable incubation time to obtain a plateau of sperm accumulation. Meanwhile, to confirm the specific effect of uPA on mouse sperm chemotaxis, uPA inhibitor (PAI-1) and anti-uPAR rabbit IgG were added to the test solution containing 20 U/mL uPA, respectively. To exclude the possibility that PAI-1 and anti-uPAR rabbit IgG may affect sperm accumulation nonspecifically, PAI-1 and anti-uPAR rabbit IgG were added to F10, respectively. It was found that the chemotactic effect of uPA was neutralized completely by PAI-1 and anti-uPAR rabbit IgG. PAI-1 and anti-uPAR rabbit IgG had no neutralizing effect on the sperm chemotactic effect. Lastly, the sperm chemotaxis response to a descending gradient of uPA was also observed. Taken together, the results suggest that uPA can induce sperm chemotaxis in vitro by binding to its receptor on the sperm membrane and may act as a chemoattractant in precontacting sperm-egg communication thereby increasing the chance encounter of spermatozoa and eggs.

ID: 262 Inhibition of mouse uPA activity by mouse monoclonal antibodies

ID: 262 Inhibition of mouse uPA activity by mouse monoclonal antibodies

Binding of Urokinase to Low Density Lipoprotein-related Receptor (LRP) Regulates Vascular Smooth Muscle Cell Contraction

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA(-/-) mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA(-/-) mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR(-/-)) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA(-/-) mice were prevented by antibody to low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA(-/-) mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.

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.

Urokinase expression and binding activity associated with the transforming growth factor ?1-induced migratory and invasive phenotype of mouse epidermal keratinocytes

Transforming growth factor beta1(TGF-beta1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF-beta1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial-fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous-spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF-beta1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI-1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF-beta1-induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF-beta1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas.

Transcriptional Regulation of the Murine Urokinase-type Plasminogen Activator Gene in Skeletal Myoblasts

We have previously shown that urokinase-type plasminogen activator (uPA) is highly expressed in murine C2C12 myoblasts and that antibodies against uPA are able to block both myoblast fusion and differentiation. Here we show the characterization of cis-acting elements in the mouse uPA promoter in vitro which are involved in uPA gene expression in C2C 12 myoblast cells. DNase I hypersensitive (HS) site analysis revealed the presence of three HS sites in myoblasts. Deletion analysis of stably transfected uPA-promoter constructs revealed that at least two of the three HS sites accounted for the high transcriptional expression in C2C12 cells. One was located at -2.4 kb and corresponded to a known PEA3/AP1A element and the other one was located at -4.9 kb and contained a CArG box and a CRE element. So far, no regulatory function had been assigned to this CRE/CArG element. Both HS sites alone were able to activate transcription of a heterologous promoter and showed a cooperative effect when placed together. Electrophoretic mobility-shift assays using myoblast nuclear extracts and specific antibodies demonstrated that cJun, JunD and ATF2 bound to the PEA3/AP1A element, whereas the CRE/CArG element bound SRF. Altogether, these results suggest that high uPA expression in myoblasts is dependent on the cooperation of two regulatory sites in the uPA promoter.