anti-uPA Antibody Research Articles

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Deficiency of Urokinase Plasminogen Activator May Impair β Cells Regeneration and Insulin Secretion in Type 2 Diabetes Mellitus.

Background: The relationship between urokinase-type plasminogen activator (uPA) and the development of type 2 diabetes mellitus (T2DM) was investigated in the study by using mice and cell models, as well as patients with T2DM. Methods: In mice models, wild-type and uPA knockout (uPA-/-) BALB/c mice were used for induction of T2DM. In cell models, insulin secretion rate and β cell proliferation were assessed in normal and high glucose after treating uPA siRNA, uPA, or anti-uPA antibody. In our clinical study, patients with T2DM received an oral glucose-tolerance test, and the relationship between uPA and insulin secretion was assessed. Results: Insulin particles and insulin secretion were mildly restored one month after induction in wild-type mice, but not in uPA-/- mice. In cell models, insulin secretion rate and cell proliferation declined in high glucose after uPA silencing either by siRNA or by anti-uPA antibody. After treatment with uPA, β cell proliferation increased in normal glucose. In clinical study, patients with T2DM and higher uPA levels had better ability of insulin secretion than those with lower uPA levels. Conclusion: uPA may play a substantial role in insulin secretion, β cell regeneration, and progressive development of T2DM. Supplementation of uPA might be a novel approach for prevention and treatment of T2DM in the future.

Urokinase plasminogen activator secreted by cancer-associated fibroblasts induces tumor progression via PI3K/AKT and ERK signaling in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are believed to influence tumor behavior and clinical outcomes. We previously showed that conditioned medium (CM) from CAFs induces proliferation and motility of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the molecular mechanisms by which the CAF-secreted proteins induce ESCC development and progression. Using antibody arrays, we identified urokinase plasminogen activator (uPA) as one of the main proteins whose release was increased in CAFs compared to normal fibroblasts (NFs). Immunohistochemical analysis of pathological sections showed that uPA-positive cells were localized at the boundaries of tumor and stroma tissues, in stroma between tumor nests, and within the tumors. Increased stromal uPA levels (132/146 cases) correlated with tumor invasion (p < 0.05) and overall survival of ESCC patients (p < 0.05). In vitro assays showed that uPA promotes ESCC cell proliferation, migration, and invasion via PI3K/AKT and ERK signaling pathways. In vivo, anti-uPA antibody suppressed tumor growth in ESCC xenografts. These results suggest that uPA released from stroma, and especially from CAFs, might be a predictive marker for ESCC diagnosis and prognosis, as well as an effective therapeutic target.

A8, an anti-uPA agonistic antibody, promotes metastasis of cancer cells via ERK pathway.

Urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) are expressed in many tumors and have been reported to be correlated to protein expression and poor prognosis in malignant tumors. In a previous study, we reported on the selection of human single-chain variable fragment (scFv) A8 specific to uPA from phage-displayed human naïve scFv library. In this study, scFv A8 was converted to minibody form and evaluated for its functional ability on the uPA system involved in cellular signaling and cancer cell metastasis. A8 minibody increased enzyme activity of uPA and enhanced the migration and invasion of HT1080 colon cancer cells in a dose-dependent manner. A8 increased ERK phosphorylation, and enhanced migration was blocked by U0126, but not by LY0294002, SB2203580, and SP600125. A8 minibody also enhanced migration of MDA-MB231 by mediated expressing surface uPA, but not that of MCF-7 non-expressing surface uPA. Taken together, the A8 anti-uPA antibody is a uPA agonistic antibody, enhancing migration and invasion of cancer cells that express uPA via activation of ERK pathway.

Tumor necrosis factor α stimulates expression and secretion of urokinase plasminogen activator in human dental pulp cells

Plasminogen activator (PA) is the enzyme responsible for converting plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. PA exists in two forms: urokinase-type PA (uPA) and tissue-type PA (tPA). Here we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNF-α) on PA production and secretion in human dental pulp cells. When the cells were stimulated with TNF-α (10 ng/mL), PA activity in the medium clearly increased in a time-dependent manner, and this activity was reduced after immunoprecipitation with anti-uPA antibody, but not with anti-tPA antibody. In TNF-α-stimulated cells, the expression of uPA mRNA was enhanced, but was lower than that of tPA mRNA. The expression of uPA mRNA and PA secretion stimulated by TNF-α were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFκB inhibitor pyrrolidine dithiocarbamate, but were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. In the presence of another inflammatory cytokine, interleukin 1β (IL-1β, 100 pg/mL), TNF-α-stimulated expression of uPA mRNA and secretion of uPA were enhanced. These observations suggest that TNF-α stimulates uPA production and secretion, and that this effect is regulated via activation of NFκB and tyrosine phosphorylation, apparently in conjunction with IL-1β, during inflammation in human dental pulp.

Sensitivity of Acute Myeloid Leukemia (AML) cells to a urokinase‐activated recombinant anthrax toxin

We tested efficacy of a urokinase‐activated recombinant anthrax toxin (PrAgU2/FP59) on 11 human AML cell lines. PrAgU2/FP59 consists of PrAgU2, the cell binding domain in which the furin activation site is replaced by a urokinase plasminogen activator (uPA) activation site and FP59, the catalytic domain, consisting of Pseudomonas aeruginosa exotoxin A. PrAgU2 binds cells, is cleaved by uPA, heptamerizes, binds 3 molecules of FP59 and undergoes endocytosis releasing FP59 in the cytosol. FP59 ADP ribosylates EF2 causing protein synthesis inhibition and cell death. Introduction of the uPA activation site selectively targets this toxin to tumor cells expressing an active uPA system.7/11 AML cell lines were highly sensitive to PrAgU2/FP59 (IC50 = 0.5–33 pM, % cell kill &gt; 95%) while 4/11 cell lines were moderately sensitive to PrAgU2/FP59 (IC50 = 160–410 pM, % cell kill &gt; 70%). Addition of pro‐uPA increased AML sensitivity to PrAgU2/FP59 by an average of 40 fold. Addition of a neutralizing anti‐uPA antibody decreased toxicity of PrAgU2/FP59 to AML cells by an average of 14 fold, demonstrating requirement for active uPA for selective activation of PrAgU2/FP59.AML cells are sensitive to the cytotoxic effects of a urokinase‐targeted recombinant anthrax toxin with sensitivity depending on the expression of active uPA. PrAgU2/FP59 is a promising tumor selective therapeutic for the potential treatment of AML.

Abstract 520: Inhibition of uPAR and uPA reduces invasion and degradative potential in papillary thyroid carcinoma cells

Abstract Papillary thyroid cancer (PTC) is the most prevalent endocrine and thyroid malignancy. The ability of PTC to invade and migrate great distances contributes to distant metastases, which represent the most common cause of PTC-related death. The urokinase plasminogen activator (uPA) and the urokinase plasminogen receptor (uPAR) are key mediators of tumor invasion. Upon binding to uPAR, pro-uPA is converted to its active form, which is then capable of cleaving plasminogen to plasmin. Plasmin can then degrade components of the basement membrane and extracellular matrix, a prerequisite for tumor cell invasion and metastasis. The binding of uPA by uPAR also seems to mediate several signaling events that contribute to a migratory phenotype, as well as various growth signals. In this study, we analyzed uPA and uPAR expression in PTC and normal thyroid tissue, and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, as well as the functional consequences of inhibiting this system. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and western blotting demonstrated increased active uPA secreted by BCPAP cells compared to NTHY-Ori-3-1. Fluorimetric assays revealed that BCPAP CM was able to activate plasminogen, resulting in measureable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. The in vitro invasion phenotype of BCPAP cells was augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. Cells transfected with siRNA against uPAR demonstrated decreased migratory and invasive potentials as measured by Matrigel invasion assays. Finally, uPAR-knockdown cells exhibited decreased proliferation when compared to non-targeting siRNA transfectant cells. These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets. 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 520.

Protein Kinase C Synergistically Stimulates Tumor Necrosis Factor-α–Induced Secretion of Urokinase-Type Plasminogen Activator in Human Dental Pulp Cells

Plasminogen activator (PA) is the enzyme converting plasminogen to its active form, plasmin, involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). When human dental pulp cells were stimulated by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), PA activity in the conditioned medium was increased, indicating that TNF-alpha provoked PA secretion. The TNF-alpha-induced PA release was significantly enhanced in the presence of phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. The PKC inhibitor Ro31-8220 abolished the effect of PMA on the PA release. The activity of PA secreted from the cells stimulated by TNF-alpha and PMA was reduced by immunoprecipitation using anti-uPA antibody. PMA failed to enhance the TNF-alpha-induced expression of uPA mRNA. These results suggest that protein kinase C synergistically enhances the secretion of uPA in TNF-alpha-stimulated human dental pulp cells.

IL-1β stimulates urokinase-type plasminogen activator expression and secretion in human dental pulp cells

Plasminogen activator (PA) is the enzyme that converts plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated the effect of the inflammatory cytokine interleukin-1beta (IL-1beta) on PA secretion in human dental pulp cells. When the cells were stimulated by IL-1beta, PA activity in the medium was clearly increased in a time- and dose-dependent manner. This PA activity in the medium was reduced after immunoprecipitation with anti-uPA antibody, and uPA protein was detected in the immunoprecipitated fraction by Western blotting. However, no such effect was observed with anti-tPA antibody. In the IL-1beta-stimulated cells, expression of uPA mRNA was enhanced whereas expression of tPA mRNA was less. The IL-1beta-stimulated uPA mRNA expression and PA activities in the cell lysate and medium were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFkappaB inhibitor pyrolidinedithiocarbamate, and were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. These observations suggest that IL-1beta stimulates uPA production via activation of NFkappaB and tyrosine phosphorylation, and also secretion of the enzyme, and that the uPA/plasmin system appears to be involved in inflammation in human dental pulp.

Mechanism of the Inhibitory Effect of Tranexamic Acid on Melanogenesis in Cultured Human Melanocytes in the Presence of Keratinocyte-conditioned Medium

Tranexamic acid (TA) reduces hyperpigmentation in melasma patients, but the mechanism of its action is unknown. We have investigated the action of TA in human melanocyte cultures with or without keratinocyte-conditioned medium (KCM). In melanocyte cultures without KCM, TA in the concentration range of 0.5 mM to 5 mM did not reduce the activity of tyrosinase, a key enzyme of melanin synthesis, whereas it reduced the tyrosinase activity in the presence of KCM. These results indicate that TA inhibits melanin synthesis of melanocytes not by acting directly on melanocytes, but by inhibiting melanocyte activators contained in KCM. In fractionation studies of KCM, the stimulatory activity was predominantly contained in the fractions with an apparent molecular weight of 54000. Inhibition of the urokinase-type plasminogen activator (uPA) in KCM with specific anti-uPA antibody significantly decreased the KCM-induced increase of tyrosinase activity and inhibited the KCM-induced morphological changes of melanocytes. Our results suggest that TA inhibits melanin synthesis in melanocytes by interfering with the interaction of melanocytes and keratinocytes through inhibition of the plasminogen/plasmin system.

Hypoxia Regulates Cross-talk between Syk and Lck Leading to Breast Cancer Progression and Angiogenesis

Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer.

Stimulation of human breast carcinoma cell invasiveness and urokinase plasminogen activator activity by glucose deprivation

Glucose deprivation has been shown to increase the invasive and metastatic potential of tumour cells. In the present study, we determined whether the enhanced tumour cell invasiveness resulting from glucose deprivation is linked to increased activity of enzymes required for extracellular matrix degradation. Results of in vitro invasion assays revealed that the invasiveness of human MDA-MB-231 and MCF-7 breast carcinoma cells and MCF-10A1 normal breast cells was, respectively, 3.9-, 2.9-, and 2.1-fold higher when they were incubated under glucose-deprivation (0.2 mM glucose) than when incubated under physiological blood glucose levels (5 mM). This effect of glucose deprivation on invasion correlated with increased urokinase plasminogen activator (uPA) and plasmin activity. Glucose deprivation did not increase the levels of gelatinase and plasminogen activator inhibitor-1 secretion, or the expression of cell-associated uPA receptor. To determine whether the increased invasiveness resulting from glucose deprivation is causally linked to increased uPA activity, invasion assays were conducted using MDA-MB-231 cells incubated in 0.2 mM or 5 mM glucose in the presence of a neutralising anti-uPA antibody. Results revealed that the anti-uPA antibody significantly inhibited invasion in a dose-dependent manner and to a much greater extent in cells incubated in 0.2 mM glucose than in cells incubated in 5 mM glucose. These results suggest that low glucose levels in malignant cancers increase tumour cell invasiveness by stimulating uPA and plasmin activity.

Human thyroid carcinoma cell invasion is controlled by the low density lipoprotein receptor-related protein-mediated clearance of urokinase plasminogen activator

The low density lipoprotein receptor-related protein (LRP), a large scavenger receptor reported to mediate the uptake and degradation of various ligands, emerges as a promising receptor for targeting the invasive behaviour of human cancer cells. However, the accurate function of LRP during tumor invasion seems to be highly dependent on cellular context and remains controversial. The expression patterns of both this receptor and the main proteolytic systems involved in cell invasion were examined in two follicular thyroid carcinoma cell lines exhibiting different invasive phenotypes. We established that a low expression of LRP at the cell surface was associated to elevated extracellular MMP2 and urokinase plasminogen activator (uPA) activities as well as to high invasiveness properties. Surprisingly, neither exogenously added receptor-associated protein, an antagonist of LRP, nor LRP blocking antibodies significantly modified the amount of extracellular MMP2. Furthermore, the invasive phenotype of thyroid carcinoma cells was not related to their matrix metalloproteinases amount since different specific inhibitors of these proteases failed to affect the invasive properties of both cell lines. Additionally, blocking LRP-mediated clearance led to a further increase of the uPA amount and activities and to increased invasiveness in both cell lines. Finally thyroid carcinoma cells aggressiveness was widely increased by exogenous uPA; and anti-uPA antibodies treatments abolished both basal and receptor-associated protein-induced thyroid cell invasion. Overall our results identified the LRP-mediated clearance of uPA as one of the mechanisms involved during the control of human thyroid carcinoma cell invasion.

Deflazacort modulates the fibrinolytic pattern and reduces uPA-dependent chemioinvasion and proliferation in rheumatoid arthritis synoviocytes

Extracellular fibrinolysis, controlled by the cell-associated fibrinolytic system (urokinase plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor type-1, PAI-1), is involved in cartilage damage generation and in rheumatoid arthritis (RA) synovitis. Since steroids reduce the rate of radiological progression of RA, we planned to evaluate in healthy and RA synoviocytes the effects of the steroid deflazacort on uPA, uPAR and PAI-1 expression, and subsequent phenotypic modifications in terms of uPA/uPAR-dependent invasion and proliferation. uPA, uPAR and PAI-1 levels were studied by ELISA, RT-PCR (uPAR) and zymography (uPA) in synoviocytes from four RA patients and four healthy controls. Chemoinvasion was assessed by the Boyden chamber invasion assay, using Matrigel as the invasion substrate. Proliferation was evaluated by cell counting. Both invasion and proliferation were measured upon treatment with deflazacort 5 muM with or without parallel stimulation with uPA 500 ng/ml or in the presence of monoclonal anti-uPA and anti-uPAR antibodies. Invasion and proliferation of RA synoviocytes require a proper functional balance of the fibrinolytic system. Both deflazacort and monoclonal antibodies against uPA and uPAR reduced expression and activity of the system, thus inhibiting invasion and proliferation. In RA synoviocytes, deflazacort induced higher PAI-1 and lower uPA and uPAR levels, as well as a decrease in uPA enzymatic activity. The levels of uPAR mRNA were concomitantly reduced, as was uPA-induced chemoinvasion. All these effects were also shown in controls, though to a lesser extent. Deflazacort might control RA synovial proliferation and invasion by differential modulation of single members of the fibrinolytic system.

Involvement of urokinase‐type plasminogen activator in sphingosylphosphorylcholine‐induced angiogenesis

Sphingosylphosphorylcholine (SPC) has been shown to accelerate wound healing. As angiogenesis is fundamental to proper wound healing, we examined the effect of SPC on angiogenesis using a well-established rat aortic ring assay. SPC significantly stimulated the sprouting of endothelial cells from rat aortic ring. Recognizing its potential effect on angiogenesis, we further investigated the action of SPC using human umbilical vein endothelial cells (HUVECs) cultured in vitro. SPC significantly accelerated the closure of in vitro wound. In addition, SPC markedly enhanced the chemotactic migration and capillary-like tube formation. Subsequently, we examined whether SPC affected the production of urokinase-type plasminogen activator (uPA), an important regulator of angiogenesis, and found that SPC stimulated the expression of uPA at both the transcriptional and translational levels. Consistent with these results, SPC increased the activity of cell-surface-associated plasminogen activator. Pretreatment with antiuPA antibody significantly diminished both the chemotactic migration and capillary-like tube formation, indicating the potential importance of uPA in SPC-induced angiogenesis. Together, these results suggest that SPC may affect angiogenesis in the wound-healing process via regulation of uPA production.

Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3.

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

Isolation and characterization of cell lines with reduced urokinase binding.

Six cell lines have been generated from the human fibrosarcoma HT-1080 by mutagenesis. They were selected on the basis of reduced urokinase (uPA) binding on replicate polyester filters. Single cell clones were then isolated by limited dilution cloning. All cloned cells showed less uPA binding on filters, and as cell monolayers. These cell lines were able to bind only 10 to 65% as much uPA as the wild-type HT-1080 cells. Surface-bound uPA proteolytic activity and surface activation of plasminogen from these cells were also reduced relative to the wild-type. uPA could activate MAP kinases in the wild-type and two of the cell lines with the least uPA-binding, but the amount of the activated forms of the signalling molecules were reduced. Immunoblotting using two different anti-uPA receptor antibodies showed two cross-reacting protein species of approximately 53 kDa and approximately 38 kDa. The proportion of the lower Mr band to the higher Mr band was found to be reduced in all the cell lines relative to the wild-type. Chemical cross-linking with single-chain urokinase (scuPA) showed only one high-molecular-weight adduct, with Mr approximately 90 kDa, in all the cell lines tested. Similarly, cross-linking with the amino terminal fragment of uPA yielded a single approximately 70 kDa adduct. These would indicate that only the approximately 53 kDa band was responsible for cross-linking reactions. Equilibrium binding experiments showed that only one set of high-affinity binding sites for the wild-type cells. However, the binding of scuPA to two of these cell lines was best fitted to a two-site model, one of which was similar to the high-affinity binding sites of the wild-type, although the number of sites was reduced, while the other was of much lower affinity but was large in number. These results are discussed in relation to changes in the structure of ligand binding machinery in these cells, which affect other cellular functions.

Complexes between urokinase-type plasminogen activator and its receptor in blood as determined by enzyme-linked immunosorbent assay.

Complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) were assessed in plasma and serum from 39 breast cancer patients and from 20 healthy individuals, applying a recently developed enzyme-linked immunosorbent assay (ELISA) for the analysis of these complexes in tumor tissue extracts. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies for catching and a mouse anti-uPAR monoclonal antibody (MAb) for detection. The specificity of the assessment of uPA:uPAR complexes was verified by simultaneous analysis of the individual blood samples in corresponding non-sense ELISA formats, in which either the anti-uPA catching antibody or the anti-uPAR detecting antibody was substituted with an irrelevant antibody. Assessment of native uPA:uPAR complexes was ascertained by demonstrating the absence of any de novo formation of uPA:uPAR complexes in plasma and serum during the sample incubation step in the ELISA, as verified by the use of a peptide antagonist for uPAR. Plasma and serum samples contained almost identical levels of uPA:uPAR complexes. The levels of uPA:uPAR complexes were found to be significantly lower in serum from breast cancer patients compared to the serum of healthy donors, while the levels of (total) uPAR in plasma from breast cancer patients were significantly higher than in plasma from the healthy controls. In addition, the free, uncomplexed uPAR levels, estimated by subtraction of uPA:uPAR complex levels from (total) uPAR levels, were significantly elevated in plasma as well as in serum from breast cancer patients compared to healthy individuals. The uPA:uPAR complex levels were highly comparable to the uPA levels analyzed in the same plasma and serum samples, indicating that most if not all of the uPA present in these samples is complexed with uPAR.