Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors. J Biol Chem 265: 9904-8 (original) (raw)
Journal of Biological …, 1986
An approximately 75% pure form of a human M,-54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose. The inhibitor inhibited human urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator, but not plasmin. Rabbit antibodies against this plasminogen activator inhibitor also reacted with a plasminogen activator inhibitor with identical electrophoretic mobility in extracts of human blood platelets, indicating that the HT-1080-inhibitor is of the same type as the inhibitor of blood platelets. As revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin-agarose zymography, incubation of HT-1080inhibitor with the active form of human u-PA led to the formation of an equimolar sodium dodecyl sulfateresistant complex between them; in contrast, no complex formation was observed between the inhibitor and the proenzyme form of human u-PA (pro-u-PA). Likewise, using a column of anti-inhibitor antibodies coupled to Sepharose for removal of excess inhibitor and activator-inhibitor complexes, the potential enzymatic activity of pro-u-PA was found to be unaffected by incubation with inhibitor under conditions in which more than 95% of the active u-PA had formed complex with inhibitor. Plasminogen activators comprise a group of serine proteases, which by limited proteolysis convert the abundant extracellular proenzyme plasminogen to the active protease plasmin. Plasmin has a relatively broad trypsin-like specificity. Release of plasminogen activators from cells may thus initiate localized extracellular proteolysis. TWO types of plasminogen activators have been recognized in mammals, the urokinase-type (u-PA1) and the tissue-type (t-PA), with M I of-50,000 and-70,000, respectively. They are products of different genes (1-3). Several functions have been proposed
The urokinase plasminogen activator and its receptor
Thrombosis and Haemostasis, 2005
SummaryThe 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.
The urokinase plasminogen activator system as a novel target for tumour therapy
2000
Substantial data have been collected for numerous types of solid cancer, including cancer of the breast, the gastrointestinal and urological tract, the lung, and the brain, demonstrating a strong clinical value of the plasminogen activation system in predicting disease recurrence and survival in cancer patients. Elevated levels of certain members of the plasminogen activation system, the serine protease uPA (urokinase-type plasminogen activator), its receptor (uPA-R; CD87), and inhibitor (PAI-1), in tumour tissue or blood emphasize their fundamental role in tumour invasion and metastasis and provide the rationale for novel therapeutic strategies. uPA, besides its proteolytic action toward the extracellular matrix, in concert with uPA-R, PAI-1, and integrins contributes to tumour cell proliferation, adhesion, and migration. Several technical methods of affecting tumour growth and metastasis by targeting the uPA-system in cancer patients at the gene and protein level have been explored: (1) antisense oligodeoxynucleotides to uPA, uPA-R, or PAI-1; (2) antisense oligonucleotides to signal transduction pathway components such as Rel (NF-κ B), affecting uPA but not PAI-1 synthesis; (3) viral vectors delivering genes for components of the plasminogen activation system; (4) soluble, recombinant uPA-R as a scavenger for uPA; (5) monoclonal antibodies directed to uPA or uPA-R blocking uPA/uPA-R interaction; (6) enzymatically inactive uPA to compete for active uPA binding to uPA-R; (7) linear and cyclic uPA-derived peptides to block uPA/uPA-R interaction; (8) toxins, coupled to uPA or fractions thereof to kill tumour cells; (9) naturally occurring inhibitors to uPA and its derivatives for inhibition of uPA proteolytic activity; and (10) synthetic inhibitors to uPA to inhibit uPA proteolytic activity. There is substantial hope that substances designed to affect or turn off the plasminogen activation system will eventually be administered to cancer patients thereby opening a new vista for tumour biology-based, individualized cancer therapy.
Journal of Histochemistry & Cytochemistry, 1997
The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 ϫ 10 5 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One g of FITC-uPA efficiently stains 2 ϫ 10 5 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells. (J Histochem Cytochem 45:
Functional Analysis of the Cellular Receptor for Urokinase in Plasminogen Activation
Journal of Biological Chemistry
1 The abbreviations used are: DFP, diisopropyl fluorophosphate; uPA, urokinase-type plasminogen activator, both the activated two-chain protease and as a generic term; pro-uPA, single-chain form of uPA; uPAR, uPA receptor; s-uPAR, soluble recombinant uPAR residues 1-277; PAI-1, plasminogen activator inhibitor type-1; 6-AHA, 6-aminohexanoic acid; pNA, para-nitroaniline; AMC, 7-amido-4-methylcoumarin.
A Cellular Receptor for Urokinase-Type Plasminogen Activator
XIth International Congress on Thrombosis and Haemostasis, 1987
Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.The available information on the cellular binding of u-PA can be summarized as follows:1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional ...
Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor
Proceedings of the National Academy of Sciences, 1989
Urokin plasminogen activator (uPA) interacts with a surface receptor and with specifi inhibitors, such as plasminogen activator inhibitor type 1 (PAM-). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). We have now investigated whether PAMcan bind and inhibit receptor-bound uPA. Binding of 125I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-l complexes, but not by PAMalone. Preformed '2SI-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAMalso binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptorbinding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition ofPAM-. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-i. The possibility that in adherent cells a different localization ofPAI-i and uPA leads to protection of uPA from PAMis to be considered.
European Journal of Biochemistry, 1997
The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasniinogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-~-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Clul]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S22.51, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPAluPAR-(1-2Sl)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDSPAGE, when incubated with plasminogen and Spl in the presence of 100 M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(I-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(I-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.
Fibrinolysis and Proteolysis, 1997
Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in numerous physiological and pathological processes, e.g. tissue remodelling, embryogenesis, fibrinolysis, and tumour spread, uPA protease binds to a specific high-affinity receptor (uPAR; CD87) on normal and tumour cells. This binding is mediated by the growth factor domain of uPA and thus independent of its proteolytic activity. An ELISA-type, solid-phase microtitre plate assay is presented, designed for the quantitation of such uPA molecules capable of binding to the receptor uPAR. This solid-phase uPA-ligand binding assay makes use of the specific, high-affinity interaction of uPA with uPAR. This assay format is different from the common uPA-ELISA which measures uPA antigen but not uPAR-reactivity. Recombinant soluble uPAR (CHO-uPAR), attached to the well of a microtitre plate, serves as the capture molecule for uPA. uPA-containing samples are added to allow binding of uPA to immobilized uPAR. Receptor-bound uPA is then detected by reaction of uPA with biotinylated monoclonal antibody no. 377 (American Diagnostica, Greenwich, CT, USA) directed to the kringle domain of uPA followed by avidin-peroxidase. The solid-phase uPA-ligand binding assay detects various forms of uPA: pro-uPA, HMW-uPA, ATF, GFD but does not react with the low molecular weight form of uPA (LMW-uPA) lacking the uPAR-reactive domain, uPA molecules in which the uPAR-binding domain has been impaired by proteolysis and uPA/uPAR complexes are also excluded from detection. The high sensitivity of the solid-phase uPAligand binding assay (lower limit 2 pM = 0.1 ng uPA/ml) allowed to measure reactive uPA (and fractions thereof) in the supernatants of cultured ovarian cancer cells, in extracts of ovarian and breast cancer tissues, in placenta tissue extracts and in malignant ascites. The solid-phase uPA-ligand binding assay was also used to screen the receptor binding reactivity of recombinant human uPA-polypeptides synthesized by yeast cells and that of synthetic uPA-peptides. The uPAR-blocking capability of uPA-peptides uPA14-32, uPA14-32/H29A, and uPA14-32/N32A, determined by the solidphase uPA-ligand binding assay, was confirmed by flow cytofluorometric analysis employing fluorescent pro-uPA (FITCpro-uPA) and the uPAR-rich promyeloid cell line U937.
Blood, 1996
Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule totS cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affmity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of SCUPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor-bound tcuPA. Binding of PAL1 to suPARIscuPA complexes is totally ROKINASE-TYPE plasminogen activator (uPA) has
Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor
Febs Letters, 2000
The expression of the receptor for the urokinase-type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up-regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral. ß
Journal of Biological Chemistry, 1998
The multipotent drug suramin, which is currently being studied as an anticancer agent, was found to inhibit the interaction between the urokinase-type plasminogen activator (u-PA) and its cellular receptor. 60% inhibition of binding was obtained with a suramin concentration between 30 and 60 pg/ml when using U937 cells and a ligand concentration of 0.3 nM. This concentration of the drug is well below the serum levels found in suramin-treated patients. Inhibition of binding was also demonstrated at the molecular level, using chemical cross-linking or an enzyme-linked immunosorbent assay-type technique based on the ligand interaction. The inhibition was not caused by a mere polyanion effect since polysulfates such as heparin, heparan sulfate, and pentosan polysulfate were noninhibitory or showed only a very weak inhibition. However, polysulfonated compounds with structures resembling suramin (Le. trypan blue and Evans blue) did prove inhibitory. The inhibition found with suramin showed a concentration dependence consistent with a mixed competitive and noncompetitive mechanism. The off-rate of prebound ligand was accelerated by the drug. It is speculated that the present effect may contribute to the anti-invasive properties of suramin by destroying the cellular potential for localized plasminogen activation and proteolytic matrix degradation. The urokinase-type plasminogen activator (u-PA)' converts the abundant proenzyme, plasminogen, into active plasmin. Plasmin degrades a number of extracellular proteins, and plasminogen activation mediated by u-PA appears to be a central event in certain processes involved in degradation of the extracellular matrix. These processes are a prerequisite for tissue remodeling which occurs under various normal as well as pathological conditions, including the invasion and metastasis of cancer cells; for reviews, see Refs. 1-4. A cellular u-PA receptor (u-PAR) has been identified (5) and characterized on the molecular (6-10) and functional (5, 11-19) level. The binding of u-PA or its proenzyme to this
Biochemistry, 1991
The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 "C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 pg/mL, 10500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP-and dansyl-EGRck-treated Sc-uPA (0.45-2.25 pM), performed in the presence of these inhibitors and Trasylol (10 pM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 1251-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 1251-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors. The present findings indicate that, under conditions stringently excluding Tc-uPA contamination, neither recombinant human Sc-uPA expressed in Chinese hamster ovary cells nor Sc-uPA secreted by fetal kidney cells or a transformed line of kidney cells shows measurable amidolytic activity above the detection limit of 0.001 7% or plasminogen activator activity above the detection limit of 0.01% of Tc-uPA activity. These studies suggest that the intrinsic activities ascribed to Sc-uPA or its plasmin-resistant mutants arise from small amounts of Tc-uPA, possibly generated from Sc-uPA by the action of traces of contaminating proteases that are not susceptible to inactivation by usual inhibitors of trypsin-like serine proteases. U r o k i n a s e 4 ype plasminogen activator (uPA)' is synthesized and secreted by cells as a 50-kDa single-chain glycoprotein that is variously referred to as single-chain urokinase (Sc-uPA) or prourokinase [review by Lijnen et al. (1987a) and references cited therein]. Sc-uPA is converted to two-chain urokinase (Tc-uPA) following cleavage of the Lys'S8-Ile'S9 peptide bond by plasmin (Wun et al., 1982; Nielsen et al., 1982), plasma kallikrein, or some other serine proteases (Ichinose et al., 1986).
1992
266,109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe3+-Sepharose chromatography. Both forms exhibit indistinguishable K , and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis. Urokinase plasminogen activator (uPA)' regulates plasmin-* This work was supported by Progetto Finalizzato Biotecnologia e Biostrumentazione and Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica del Consiglio Nazionale delle Ricerche (to M. P. S.), the Danish Biotechnology Program, the Danish Medical Research Council, and the Commission of the European Communities
Emerging Therapeutic Targets, 1999
In the promotion of cancer progression, a classical role had previously been ascribed to the plasminogen activation system on the basis of urokinase plasminogen activator (uPA) proteolytic activity and plasminogen activation triggering a focalised pericellular activation cascade involving matrix metalloproteinases (MMPs). As a result, many pharmaceutical companies have undertaken the development of synthetic uPA inhibitors. However, during the last few years, data have accumulated that uPA, as well as urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are likely to play an essential role in tumour progression through non-proteolysis-related activities. Such activities endow them with new and likely key functions in tumour progressionassociated events, such as cellular adhesion, migration, invasion and angiogenesis. Since these activities essentially depend upon proteinprotein interactions, they represent new therapeutic targets.
Frontiers in oncology, 2018
The plasminogen activator (PA) system is an extracellular proteolytic enzyme system associated with various physiological and pathophysiological processes. A large body of evidence support that among the various components of the PA system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 and-2 (PAI-1 and PAI-2) play a major role in tumor progression and metastasis. The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby, cause tumor cell migration from the primary site of origin to a distant secondary organ. The components of the PA system show altered expression patterns in several common malignancies, which have identified them as ideal diagnostic, prognostic, and therapeutic targets to reduce cancer-associated morbidity and mortality. This review summarizes the various components of the PA system and focuses on the role of uPA-uPAR in different biological processes especially in the context of malignancy. We also discuss the current state of knowledge of uPA-uPAR-targeted diagnostic and therapeutic strategies for various malignancies.