Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.
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PMID:Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane. 751 99

Using immunohistochemistry and in situ hybridization, we have characterized the expression and localization of components of the plasminogen activator proteolytic cascade in an organotypic coculture system which consists of a "dermal" portion (human dermal fibroblasts throughout a collagen matrix) and a stratified, well-differentiated epidermal portion. Specifically, the following components were examined: the enzymes urokinase-type plasminogen activator and tissue-type plasminogen activator and their type 1 and type 2 inhibitors. Urokinase plasminogen activator mRNA and antigen were found predominantly in the least differentiated, basal keratinocytes; in some fields there was also faint deposition of antigen beneath the basal cells. The distribution of plasminogen activator inhibitor type 1 was similar to that of urokinase, except that inhibitor type 1 antigen deposition beneath the basal cells appeared more intense and uniform. In contrast to the results with urokinase plasminogen activator and inhibitor type 1, tissue plasminogen activator mRNA and antigen were localized focally in the suprabasal, i.e. more differentiated, keratinocytes. Plasminogen activator inhibitor type 2 mRNA and antigen were detected in most epidermal layers, but were more intense suprabasally and often spared the basal layer. These studies demonstrate that the same type of cell, i.e. the keratinocyte, can express different components of the plasminogen activator cascade depending on its state of differentiation. The change in expression of plasminogen activator cascade components with keratinocyte differentiation suggests distinct epidermal functions for these components, related to cell-matrix interaction and epidermal differentiation.
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PMID:Differential expression of plasminogen activators and their inhibitors in an organotypic skin coculture system. 827 Jun 42

Urokinase plasminogen activator (uPA) and its receptor are key components of a cell surface proteolytic cascade used by tumor cells and capillary endothelial cells for basement membrane invasion, a process required for metastasis and angiogenesis. We have cloned, expressed, and purified the epidermal growth factor-like domain of murine uPA alone and fused it to the Fc portion of human IgG as high-affinity murine urokinase receptor antagonists. These molecules are potent inhibitors of murine urokinase binding to its receptor and inhibit angiogenesis in an in vitro model of capillary tube formation in fibrin gels. In vivo, basic fibroblast growth factor-induced neovascularization and B16 melanoma growth in syngeneic mice are also substantially suppressed by these molecules. Coupled with previous studies showing inhibition of metastasis, these findings suggest that urokinase receptor antagonists may be useful therapeutically as inhibitors of tumor progression.
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PMID:Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. 862 23

We compared macrophage density, assessed by enumeration of peritumoral mononuclear cell immunoreactivity for HAM 56, to clinicopathologic features and to immunostaining for two "invasion-associated" proteases (Cathepsin D and Urokinase plasminogen activator) in 80 breast carcinomas. Diffuse (2+) infiltrates of HAM 56- positive mononuclear cells were present in 27 cases (34%) and 43 (54%) exhibited focal (1+) infiltrates. Presence of 2+ macrophage infiltrates correlated significantly with poor differentiation. None of the seven well-differentiated cases exhibited 2+ infiltrates, whereas 9/43 (21%) moderately differentiated and 18/30 (60%) poorly differentiated tumors were diffusely infiltrated (p = .001). Wide-spread macrophage infiltrates were also more frequent in cases with advanced stage (23% of node negative vs 40% of node positive cases, p = NS). Forty-four percent of the cases with diffuse macrophage infiltrates were cathepsin D positive (i.e. in host derived cells) vs only 18% with focal macrophage infiltrates (p = .002). A similar relationship was observed between staining for HAM 56 and urokinase-type plasminogen activator (p = .02). Disease recurrences (50 months median follow-up) were more frequent in patients with 2+ (17/27, 63%) as opposed to 0+ (1/10, 10%) macrophage infiltrates (p = .01). We conclude that the density of stromal macrophage infiltrates is associated with clinical aggressiveness in breast carcinomas. Further, this relationship may reflect contribution of host derived macrophages to invasion and metastasis through elaboration of proteases which putatively mediate degradation and remodeling of extracellular matrix.
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PMID:Clinicopathologic analysis of macrophage infiltrates in breast carcinoma. 882 15

Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen activator inhibitor type-1 (PAI-1) is often associated with the extracellular matrix. The spatial localization of these three molecules could account for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical force transmission across the cell surface to the cytoskeleton. Here we investigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matrix ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-1 at increasing concentrations. The number of adherent cells and their projected area were quantitated after 3 hours of plating. PAI-1 promoted cell adhesion and spreading in a dose dependent manner. Addition of antibodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dose dependent way. The PAI-1 mediated cell adhesion required the presence of urokinase at the cell surface. Removal of the glycosylphosphatidylinositol (GPI)-linked proteins abolished cell adhesion on PAI-1 dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against alpha v beta3 integrin completely inhibited cell adhesion on PAI-1, suggesting that alpha v beta3 might be the transmembrane molecule that physically connects the complex of PAI-1, urokinase, and urokinase receptor to the cytoskeleton. Visualization of spread cells stained for filamentous actin with confocal microscopy showed a dose-dependent increase of filopodia on PAI-1 coated dishes and cytoskeletal reorganization, suggesting a migratory profile. These data indicate that PAI-1 plays a direct role in dynamic cell adhesion particularly at the leading edge, where increased levels of urokinase plasminogen activator (uPA) and its receptor (uPAR) are localized in migrating cells. Immobilized PAI-1 could therefore serve to bridge the cell surface with the extracellular matrix via the formation of a multimolecular complex that includes alpha v beta3 integrins in myogenic cells.
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PMID:Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading. 917 5

Changes in plasminogen activator are associated with the reproductive tissue remodelling that occurs during growth. Given the trophic effects of relaxin on the pig uterus and cervix, the present study was designed to examine the impact of relaxin on urokinase and tissue-type plasminogen activator (uPA and tPA) protein and activity in the uterus and cervix of prepubertal pigs. After relaxin administration in vivo to induce growth of the immature uterus and cervix, plasminogen activator activity was measured in uterine flushes and uterine and cervical tissue using a chromogenic substrate assay. Immunoreactive uPA and tPA protein in uterine flushes and uterine and cervical tissue was detected by western blotting. Urokinase plasminogen activator activity was significantly higher (P < 0.05) in uterine flushes from relaxin-treated animals than in controls. However, there was no change in uterine flush tPA activity or protein in response to in vivo relaxin treatment. There was no evidence for acid-labile inhibitors of plasminogen activator in uterine flushes of any of the animals. Cell-associated uterine tissue uPA and tPA activity, as well as protein, were similar in relaxin-treated and control prepubertal pigs. In the cervix, cell-associated tPA activity decreased significantly (P < 0.05) in relaxin-treated animals, while cervical uPA activity was unchanged. These results support the view that at least one means by which relaxin promotes pig uterine growth is by increasing uterine secretion of uPA. In addition, these studies suggest that relaxin administration in vivo to prepubertal gilts has tissue-specific effects with respect to plasminogen activator.
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PMID:Regulation of urokinase- and tissue-type plasminogen activator by relaxin in the uterus and cervix of the prepubertal gilt. 987 63

1. The present study compares plasma urokinase plasminogen activator (uPA) peptide levels, plasma plasminogen inhibitor (PAI-1) activity and urokinase receptors (uPAR) on peripheral blood monocytes of patients with stable coronary artery disease (SCAD) and healthy volunteers. 2. Urokinase plasminogen activator levels were analysed by ELISA and PAI-1 activity was determined by a plasmin generation method using the chromogenic substrate S2390. Relative uPAR numbers and the adhesion molecules CD11b/CD18 on peripheral blood monocytes were estimated using specific antibodies and flow cytometry. 3. Patients with SCAD were found to have higher plasma uPA peptide levels than age-matched healthy subjects (10.40 +/- 0.99 vs 8.25 +/- 0.53 pmol/L, respectively; P < 0.05). 4. Plasma PAI-1 activity was also higher in patients with SCAD than in healthy subjects (13.6 +/- 2.5 vs 5.2 +/- 1.0 IU/mL, respectively; P < 0.05). 5. Relative uPAR and CD11b/CD18 adhesion molecules were similar on peripheral blood monocytes of patients with SCAD and in healthy subjects. 6. The data indicate a pattern of expression/activity of uPA and PAI-1 in patients with SCAD suggestive of an impaired fibrinolytic ability.
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PMID:Urokinase plasminogen activator system in humans with stable coronary artery disease. 1022 48

Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His(204) to Gln) (r-uPA(H/Q)), urokinase with mutation of His(204) to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC(50)) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC(50) of about 6 nm, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r-uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.
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PMID:The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis. 1074 81

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-beta1 (TGF-beta1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-beta1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-beta1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-beta1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-beta1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-beta1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-beta1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-beta1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-beta1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.
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PMID:Transforming growth factor beta-1 stimulates invasivity of hepatic stellate cells by engagement of the cell-associated fibrinolytic system. 1176 74

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT-PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia.
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PMID:Expression pattern of the urokinase-plasminogen activator system in rat DS-sarcoma: role of oxygenation status and tumour size. 1195 98


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