uPA Proteolytic Activity Research Articles

Transcriptional Induction of SERPINE1 and Fibrinolysis Inhibition as Predominant Effects of Glucocorticoids on the Cancer Coagulome.

How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.

Prevention of Bleeding-Induced Vascular Abnormalities in the Hemophilic Mouse Joint By Increasing TAFI Levels or Inhibiting uPA Activity

Hemophilic arthropathy is characterized by vascular abnormalities in the joint soft tissue that may cause rebleeding and progressive joint destruction. Defective thrombin generation in hemophilia leads to impaired activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI). Activated TAFI (TAFIa) is a carboxypeptidase that cleaves C-terminal Lysine from fibrin or cell plasminogen receptors, thereby diminishing fibrinolysis or pericellular plasmin generation. In addition, TAFIa inactivates pro-inflammatory (i.e. C3a, C5a, bradykinin) and anti-angiogenic (i.e. VEGF-A, SDF-1α) substrates. We have recently shown that TAFI-gene deficiency causes vascular abnormalities in the mouse joints that phenocopy those in hemophilic arthropathy, either after joint bleeding or spontaneously with aging. Based on these mechanistic insights, we evaluated treatment with TAFI for its ability to mitigate bleeding-induced vascular abnormalities and associated bleeding susceptibility in the FVIII KO mouse joint. Knee joint bleeding in FVIII KO mice was induced by a subpatellar puncture injury that was standardized to cause severe bleeding as defined by a hematocrit (Hct) drop from 47 ± 2 % at baseline (BL) to 29 ± 11 % two days after injury. The vascular abnormalities formed in the hemophilic mouse joint 2 weeks after injury capture those in patients with hemophilic arthropathy: i.e. increased vascular permeability and abnormal vessel enlargement. Hydrodynamic gene delivery was used in mice for continuous treatment with human wild type TAFI (hTAFI-WT). To compensate for lower TAFI activation in hemophilia, two TAFI mutants were tested with extended enzymatic stability: hTAFI-CYQ (8-fold increased) and hTAFI-CIIYQ (180-fold increased). The TAFI constructs had comparable antigen expression profiles (reaching a similar level as in human plasma at day 2 and gradually returning to BL by week 2). Treatment efficacy to reduce vascular permeability, however, differed according to TAFIa's enzymatic stability in FVIII KO mice 2 weeks after injury. Without treatment, Evans blue-BSA extravasation in the joints was increased (1.8-fold compared to the contralateral joint; p= 0.0001 vs BL). hTAFI-WT overexpression initiated 2 days after injury had no effect, whereas hTAFI-CYQ and hTAFI-CIIYQ overexpression reduced the vascular permeability to 1.5-fold (n= 10; p= 0.10 vs untreated) and 1.3-fold (n= 8; p= 0.0032 vs untreated), respectively, indicating that hTAFI-CIIYQ was most effective. Histology at week 2 confirmed that hTAFI-CIIYQ reduced the number of excessively enlarged CD31+ vessels (with ≥ 20 µm diameter) by 3-fold (n= 6; p= 0.0011 vs untreated). To determine whether the protective effect of TAFI overexpression on bleeding-induced vascular abnormalities was associated with inhibition of the plasminogen activation system, continuous administration of tranexamic acid (TXA; 50 mg/ml in drinking water) or antibody-mediated blockage of uPA's proteolytic activity (mU1; 60 mg/kg; 2x weekly) was initiated in FVIII KO mice 2 days after injury. Histology at week 2 showed mU1, but not TXA, reduced vessel enlargement to the same extent as hTAFI-CIIYQ. Finally, the joint bleeding model was adopted to appreciate functional consequences of hypervascularity to changes in bleeding susceptibility. Aged TAFI KO mice (8 months) were used for their spontaneously (3-fold) increased number of enlarged vessels (≥ 40 µm diameter; p= 0.0076) in the joint that correlates with Power Doppler (PD) signal. A mild injury was optimized to cause minimal bleeding and showed that PD signals in joints of aged TAFI KO mice correlated negatively with the Hct 2 days after mild injury with injection of a FVIII inhibitor (r= -0.53, p= 0.024; n= 18), supporting hypervascularity as a major contributor to hemophilic joint bleeding. Furthermore, the same mild injury caused minimal bleeding in naive FVIII KO mice with low BL vascularity (median Hct 46 % vs 48 %; p= 0.81 vs BL; n= 7), but excessive bleeding in FVIII KO mice injured 2 weeks previously with vascular abnormalities (median Hct 38 %; p= 0.021 vs BL; n= 7). hTAFI-CIIYQ effectively reduced bleeding severity upon re-injury (median Hct 47 %; p> 0.99 vs BL; n=5). Our data provide an experimental link between vascular abnormalities and bleeding susceptibility in hemophilic joints and conclude that the vascular abnormalities were alleviated by increasing TAFI levels or inhibiting uPA activity. Disclosures von Drygalski: University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Mosnier:The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Effect of urokinase type plasminogen activator on in vitro bovine oocyte maturation

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on the in vitro maturation (IVM) of bovine oocytes. Cumulus-oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or were in vitro fertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls (P > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group (P < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine.

199 EFFECT OF UROKINASE TYPE PLASMINOGEN ACTIVATOR DURING IN VITRO MATURATION OF BOVINE OOCYTES AND EARLY EMBRYO PRODUCTION

The study of molecules involved in the oocyte maturation and early embryo development is crucial to improve the conditions of in vitro embryo production. The plasminogen activation system is involved in the initial steps of reproduction and urokinase type plasminogen activator (uPA) is expressed in the granulosa cells (GC) of bovine cumulus-oocyte complexes (COC). The aim of this study was to analyse the effect of uPA in bovine oocyte in vitro maturation (IVM). We have analysed whether the addition of uPA or the inhibition of its proteolytic activity affects IVM. We have evaluated (1) nuclear and cytoplasmic maturation, (2) developmental competence, and (3) oocyte and GC gene expression. Immature COC were obtained by aspiration of ovarian follicles of slaughtered heifers. Selected COC were in vitro-matured in 4 groups: control, 10 nM uPA, dimethyl sulfoxide control, and 100 μg mL–1 amiloride, a specific inhibitor of uPA proteolytic activity (4 replicates). After 24 h of IVM, oocytes of each treatment were either fixed and stained with Hoëscht (to evaluate nuclear maturation) or LCA-FITC to analyse the cortical granules distribution as a marker of cytoplasmic maturation (n = 10/group per treatment per replicate). In addition, pools of 10 oocytes and their separated GC were snap-frozen to analyse by qRT-PCR their profile expression of genes related with apoptosis (BAX, BCL2, TP53, SHC1), cell junctions (GJA1, TJP1), cell cycle (CCNB1), oxidative stress (SOD2, GPX1), oocyte quality (BMP15, GDF9), and serpin proteases inhibitors (SRP1, SRP5), normalised respect 2 housekeeping genes (H2AFZ, ACTB). The remaining COC were fertilised (Day 0) and in vitro cultured to evaluate developmental competence in terms of cleavage rate (Day 2) and blastocyst yield (Days 7–9). All data were analysed by one-way ANOVA. In the presence of amiloride, a significant reduction in the oocyte maturation was observed at both levels; 83.33% of oocytes remained in vesicle stage, and 75.0% showed a cortical granules distribution of type I. The rest of the groups (62.67%, 62.65%, and 60.29%) reached metaphase II (MII), and 51.66%, 32.9%, and 25.44% showed granule distribution of type III. For embryo development, the amiloride group had a cleavage rate and blastocyst yield significantly lower compared with controls (23.23% v. 80.85% and 83.83%; 4.45% v. 25.21% and 25.21%, respectively), whereas uPA treatment had no effect (84.28% and 24.16%). In presence of amiloride, the transcript levels of TJP1, GJA1, and CCNB1 were up-regulated, whereas SOD2, SRP1, and SRP5 were down-regulated in GC compared with all other groups (P &lt; 0.05). No differences were observed in oocytes gene expression between treatments. In conclusion, although the addition of exogenous uPA does not alter oocyte maturation, the specific inhibition of the proteolytic activity of uPA by amiloride reduced IVM of bovine oocytes and altered the expression of genes related to cell junctions, cell cycle, oxidative stress, and serpins of GC, indicating that proteolytic activity of uPA is critical for oocyte IVM in bovines.

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo.

The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface

The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail.We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/β1 integrin and FPR1/β1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/β1 integrin co-localization. By contrast, a low FPR1/β1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations.The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells.Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum.Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking.We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and β1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

BackgroundPreviously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.MethodsRetroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.ResultsWe have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.ConclusionsPresented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.

Clinical Applications of the Urokinase Receptor (uPAR) for Cancer Patients

Since decades the urokinase plasminogen activator (uPA) system has been associated with the invasion of malignant cells. The receptor of urokinase (uPAR) is one of the key players in this proteolytic cascade, because it focuses uPA's proteolytic activity to the cell surface and in addition functions as a signaling receptor. uPAR is highly expressed in virtually all human cancers, suggesting possible clinical applications as diagnostic marker, predictive tool of survival or clinical response, and as a target for therapy and imaging. This review summarizes the possibilities of uPAR in clinical applications for cancer patients.

Abstract 516: Differential in vivo effects of uPA overexpression: role of endogenous tumor PAI-1 expression

Abstract We have previously reported that the protease activity of uPA delays tumor progression in a syngeneic model of breast cancer, and that the inhibitory effects are due to the protease activity of uPA (Merchan et. al, JNCI, 2006: 7; 98(11)). We also reported that changes in the uPA/PAI-1 balance favoring the proteolytic activity of uPA are associated with delayed tumor growth and reduction in lung metastases in vivo (Jing, et al, AACR 2009 meeting, #1124). The aim of this study was to extend the above findings in other in vivo syngeneic cancer models and to characterize the changes in gene expression mediated by uPA overexpression. We generated stably uPA overexpressing murine renal cancer (RENCA) and murine colon cancer (MC-38) cell lines using lentiviral vectors carrying m-uPA and selecting high uPA expressing clones for in vivo studies. uPA protein expression and in vitro growth was similar between the uPA overexpressing and control cancer cells. Similar with our findings in the 4T1 model, mice harboring RENCA tumors overexpressing uPA had reduced tumor growth than controls. Conversely, mice carrying uPA-overexpressing MC-38 tumors had significantly enhanced tumor growth compared to controls. To understand the causes for the opposite in vivo effects of uPA overexpressing RENCA and MC-38 tumors, we first compared uPA's proteolytic activity vs. protein concentration between both cell lines. To our surprise, the uPA proteolytic activity of (uPA overexpressing) MC-38 conditioned medium (CM) was significantly lower (&amp;gt; 15-fold) than that of stable RENCA cell lines even though uPA protein levels were comparable. To assess if the differences in uPA proteolytic activity between MC38 and RENCA transfectants was due to differential endogenous PAI-1 expression, we measured PAI-1 levels in MC38 and RENCA transfectants CM (ELISA). We found that PAI-1 expression in MC 38 was significantly higher (30-fold) than RENCA cells. The above data strongly suggest that in MC-38 tumors, uPA overexpression promoted tumor growth in part due to high endogenous PAI-1 levels. On the other hand, in the RENCA model, uPA overexpression (and low PAI-1 expression) was associated with significantly enhanced proteolytic activity and delayed tumor growth. Gene expression arrays of early murine mammary tumors overexpressing active uPA, showed significant tumor down regulation of several members of the MMP family, which may explain the in vivo antitumor effects. The above data suggest that the pro-or anti tumorigenic/metastatic effects of uPA overexpression depend on its proteolytic activity, and indicate that the status of endogenous tumor PAI-1 may regulate uPA's effects on tumor progression and metastases. The above data may help reconcile contradictory experimental and clinical findings on the role of proteases and protease inhibitors on tumor progression. 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 516.

Multiple activities of a multifaceted receptor: roles of cleaved and soluble uPAR

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell-surface receptor involved in many physiological and pathological events that include cell migration and tissue invasion. uPAR traditional role was considered the focusing of uPA proteolytic activity on the cell surface; however, different uPAR activities have been demonstrated in the last years. In fact, cell surface uPAR functionally interacts with integrins, fMLP-receptors (fMLP-Rs) and growth factor receptors, thus regulating cell adhesion, migration and proliferation. uPAR also exists in a soluble form (suPAR) that has been detected in human body fluids. Both cell surface and suPAR can be proteolytically cleaved, thus generating truncated forms lacking the N-terminal domain and exposing the specific sequence able to interact with the fMLP-Rs. The cleaved form of suPAR binds and activates the fMLP-Rs and regulates the activity of MCP-1, RANTES and SDF1 receptors. Here, we review the role that shedding and cleavage could play in regulating uPAR structural/functional interaction with other cell-surface receptors and in uPAR-mediated biological and pathological processes.

Inhibition of Plasminogen Activators Attenuates the Death of Differentiated Retinal Ganglion Cells and Stabilizes Their Neurite Network In Vitro

Although previous studies have indicated that elevated levels of the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA) associate with the death of retinal ganglion cells (RGCs), it was unclear whether these proteases directly cause cell death. With the use of a transformed and undifferentiated retinal ganglion cell line, RGC-5, which does not express tPA, and by treating this cell line with staurosporine, which induces not only the differentiation of RGC-5 cells but also the expression of uPA and tPA in other neuronal cells, the authors sought to determine whether these proteases regulate the differentiation of RGC-5 cells and whether elevated levels of these proteases directly cause the death of RGC-5 cells. Transformed RGC-5 cells were cultured in serum-free medium and were treated with 0.5 muM to 2.0 muM staurosporine to induce their differentiation. Neurite outgrowth was assessed by phase-contrast microscopy and calcein AM staining and quantified with imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by LIVE/DEAD viability assay kit. Compared with untreated RGC-5 cells, cells treated with staurosporine differentiated as early as 1 to 6 hours. However, proteolytic activities of neither tPA nor uPA were observed within this time frame. Differentiated RGC-5 cells expressed detectable levels of uPA proteolytic activity starting at 24 hours and tPA proteolytic activity only at 48 hours. RGC-5 cells synthesized and secreted uPA and tPA into the conditioned medium, depending on staurosporine concentration and treatment time. At lower concentrations of staurosporine, differentiated RGC-5 cells had longer neurites and expressed lower levels of tPA and uPA. At higher concentrations of staurosporine, differentiated RGC-5 cells expressed higher levels of tPA and uPA, had smaller neurites, and most of them died. In contrast, when RGC-5 cells were treated with staurosporine along with inhibitors specific to tPA and uPA, proteolytic activities of both PAs were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived, showed increased neurite outgrowth, and established their neurite network in vitro. Results presented in this study indicate that RGC-5 cells do not require tPA and tPA for their differentiation. In fact, differentiated RGC-5 cells synthesize elevated levels of tPA and uPA, and elevated levels of these proteases acting in an autocrine-fashion in turn lead to the death of RGC-5 cells.

The urokinase plasminogen activator and its receptor

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis). The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.

Anti-angiogenic Activity of the Recombinant Kringle Domain of Urokinase and Its Specific Entry into Endothelial Cells

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.

Small molecule inhibitors of urokinase-type plasminogen activator

The urokinase-type plasminogen activator (uPA) protein is a multifunctional protein involved in a myriad of biological activities including extracellular matrix degradation and cell invasion. Active uPA is a 411 amino acid protein consisting of 3 domains, each of which confers a particular biological function to the overall protein. The amino terminal domain or growth factor domain (GFD), comprised of amino acid residues 1 – 48, is involved in uPA interaction with its cell surface receptor, urokinase-type plasminogen activator receptor (UPAR). The interaction of uPA with UPAR promotes, in part, cell adhesion, migration and invasion. A second domain is the kringle domain, comprising amino acid residues 49 – 135. Initially thought to bind heparin, the kringle domain has more recently been shown to possess antiangiogenic activity. A third domain comprising amino acid residues 159 – 411, the serine protease domain, is involved in the proteolytic activation of plasminogen to plasmin. The production of plasmin by uPA begins a cascade of events manifested by extracellular matrix degradation. The recent patent literature describes small molecule compounds, which inhibit the interaction of uPA with UPAR, inhibit the proteolytic activity of the uPA serine protease domain and inhibit the interaction of uPA with its natural inhibitor, plasminogen activator inhibitor-1 (PAI-1). Small peptides encompassing residues 19 – 31 of the GFD have been developed which exhibit potent inhibition of the uPA–UPAR interaction and show efficacy in tumour-bearing animal models. Small molecules have been disclosed by Corvas, which are reported to be inhibitors of PAI-1. Finally, two approaches toward the development of inhibitors of the uPA serine protease domain have been described in the recent patent literature. The first approach describes non-covalent peptidederived inhibitors discovered by phage display techniques, which bind in the substrate-binding groove of the uPA active site. An alternative approach describes non-covalent small molecule inhibitors, which bind in the enzyme active site in a slightly different binding mode than the peptide-derived inhibitors. These small molecule non-peptide analogues inhibit the uPA proteolytic activity quite effectively and are reported to possess excellent enzyme selectivity and highly improved oral activity. The clinical utility of small molecule uPA enzyme inhibitor analogues awaits the results of a preliminary clinical evaluation of compounds described by Wilex.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) expression and activity during early embryo development in the cow.

Several extracellular matrix (ECM)-degrading proteinases are hypothesised to play important roles during early mammalian development. In particular, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are expressed in peri-implantation mouse, sheep, and pig embryos and are implicated in the implantation process. These proteinases are not expressed in early (pre-blastocyst) mouse, sheep or pig embryos. The aim of this study was to establish the gene expression and proteolytic activity of uPA and MMP-9 in in vitro-produced (IVP) cow embryos. Using RT-PCR, mRNA transcripts for uPA and MMP-9 were detected during the first 7 days of development. To investigate the activity of these proteinases, conditioned media from various stages of development (days 2, 3, 4, 5 and 7) were assayed for uPA activity by chromogenic assay and MMP-9 activity by gelatin zymography. Both uPA and MMP-9 activities were detected in the media samples indicating the production and secretion of these proteinases. This pattern of proteinase expression is novel in comparison to the mouse where uPA and MMP-9 are only expressed from the blastocyst stage onwards. The results of this study suggest that these ECM proteinases have a role prior to implantation in the cow, in contrast to that exhibited by mouse, sheep and pig embryos.

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.

Urokinase activates calcium-dependent potassium channels in U937 cells via calcium release from intracellular stores.

The urokinase receptor (uPAR) is highly expressed in the human promyelocytic cell line U937 and contributes to transmembrane signalling. However, the signalling mechanisms are poorly understood. We used the patch-clamp technique to demonstrate that urokinase (uPA) binds to uPAR and thereby stimulates Ca(2+)-activated K+ channels in U937 cells. uPA transiently increased K+ currents within 30 s. The K+ currents were pertussis toxin-sensitive and were also observed in Ca(2+)-free solution. However, when cells were dialysed with EGTA, uPA did not affect K+ currents. The intracellular Ca2+ response to uPA was independent of extracellular Ca2+, was pertussis toxin-sensitive, and was blocked by both thapsigargin and the phospholipase C inhibitor U-73122. The uPA-induced increase in intracellular Ca2+ was independent of uPA proteolytic activity. Furthermore, uPA initiated a rapid formation of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]. The amino-terminal uPA fragment and uPA inactivated with diisopropyl fluorophosphate or with inhibitory antibody, elicited the same Ca2+ signal. On the other hand, Ca2+ signalling required the intact uPAR because the effects were abrogated by PtdIns-specific phospholipase C, which removes the uPAR from the cell surface. The prevention of glycosyl phosphatidylinositol moiety synthesis and interference with uPAR anchoring to the cell surface using mannosamine also abolished Ca2+ signals. Taken together, our findings indicate that uPA binds to uPAR and stimulates the production of Ins(1,4,5)P3 via a G-protein- and phospholipase C-dependent mechanism. Ins(1,4,5)P3 in turn liberates Ca2+ from intracellular stores, which leads to the stimulation of Ca(2+)-activated K+ channels.

Dissemination Risk Index Based on Plasminogen Activator System Components in Primary Breast Cancer

To study interactions between disease-free survival (DFS) and four components of the plasminogen activator system: urokinase-type plasminogen activator (uPA), its two inhibitors (PAI-1 and PAI-2), and its membrane receptor uPAR. We conducted a retrospective study of 499 primary breast cancer patients (median follow-up, 6 years). uPA, PAI-1, and PAI-2 were determined on cytosols and uPAR on solubilized pellets, using enzyme-linked immunoadsorbent assay kits (American Diagnostica, Greenwich, CT). Classical univariate and multivariate statistical methods were used together with multiple correspondence analysis to graphically examine interactions between the variables and outcome. By univariate analysis, higher uPA and PAI-1 values were significantly related to shorter DFS (P =.002; P <.00002). PAI-2 was not significantly related to DFS, although patients with high and very low PAI-2 values had a longer DFS. Multiple correspondence analysis showed the parallel impact of uPA and PAI-1 on outcome, and the clearly different behavior of PAI-2 compared with PAI-1. The prognostic contribution of uPAR seemed weak by both methods. A dissemination risk index [uPA x PAI-1/(PAI-2 + 1)], taking into account the modulation of uPA proteolytic activity by the ratio of its two inhibitors, was then tested. Dissemination risk index was selected as an independent variable in the Cox model in the overall population (P <.000001) and in node-positive patients (P <.00001). It was the only variable selected in node-negative patients (P =. 003). A dissemination risk index determined on primary tumor and taking into account the different effects of PAI-1 and PAI-2 on uPA can be of major help in clinical management of breast cancer, particularly in node-negative patients.

Chemotactic Effect of Urokinase Plasminogen Activator: a Major Role for Mechanisms Independent of Its Proteolytic or Growth Factor Domains

Urokinase type plasminogen activator (uPA) converts plasminogen to plasmin and is highly chemotactic for many cell types. We examined, using recombinant wild type and mutated forms of uPA, the extent to which its proteolytic properties, its growth-like domain (GFD) and/or interactions with the specific receptor (uPAR) contribute to the chemotactic activity towards vascular smooth muscle cells (SMC). Recombinant wild type uPA (r-uPA) stimulated cell migration nearly 5,8-fold, inactive r-uPA, with a mutation in the catalitic domain (r-uPA(H/Q)), 3-fold, uPA without growth factor like domain (r-uPA(GFD)), 2.6-fold, and a form containing both mutations (r-uPA(H/Q, GFD), 3.3-fold. All recombinant forms of uPA, wild type and those with mutations were equally and highly effective (IC5∼20 nM) in displacing 125I-r-uPA bound to SMC. These results indicate that additional mechanisms, not dependent on uPA's proteolytic activity or the binding ability of its GFD to uPAR, are the major contributors to its chemotactic action on SMC

The expression of an Ets1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells.

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.