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

Human alveolar macrophages are known to synthesize urokinase (uPA) and a specific plasminogen activator inhibitor, PAI-2. In this study we have identified a uPA receptor expressed by these cells and defined the influence of PAI-2 on the interaction of uPA with its receptor. Alveolar macrophages from four normal volunteers were incubated with 55 kDa 125I-labeled uPA (0.24-8 nM) in the presence or absence of excess unlabeled uPA. Specific and saturable binding was demonstrable in all cases. Scatchard plots were linear; regression analysis revealed a mean Kd of 5.25 nM (range 3.2-6.7) and mean Bmax of 30.7 femtomoles/10(5) cells (range 21.5-34.5). The structure of the uPA receptor was defined by electroblotting membrane fractions of macrophages and sequentially exposing filters to uPA and uPA antibodies. Membranes from macrophages demonstrated binding of either uPA or a 15-kDa amino-terminal fragment of uPA to a 55- to 60-kDa glycosylated membrane protein. Binding of uPA to filters was blocked by a synthetic oligopeptide containing the known receptor binding region of native uPA. Preincubation of 125I-uPA with PAI-2 dramatically reduced the rate of association of uPA with macrophage uPA receptor. Conversely, receptor-bound uPA activity was less susceptible to inhibition by PAI-2 than soluble uPA activity. These data indicate that normal alveolar macrophages express uPA receptors. The receptor preferentially binds and protects free uPA over complexed enzyme, indicating that one function of the receptor is to allow the cells to express active uPA in an inhibitor-rich environment.
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PMID:Alveolar macrophage urokinase receptors localize enzyme activity to the cell surface. 217 57

Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the fibrinolytic system could be inhibited by alpha 1-antitrypsin reactive site mutants. Wild-type alpha 1-antitrypsin contains a Met residue at P1 (position 358), the central position of the reactive center. It did not inhibit plasma kallikrein, beta-factor XIIa, plasmin, tissue-type plasminogen activator (t-PA), or urokinase. In contrast, these serine proteases were inhibited by alpha 1-antitrypsin Arg358. For the inhibition of C1s, a double mutant having Arg358 and a Pro----Ala mutation at P2 (position 357) was required. This double modification was made because C1-inhibitor, the natural inhibitor of C1s, has Arg and Ala residues at positions P1 and P2. Plasminogen activator inhibitor 1, the natural inhibitor of t-PA, also has Arg and Ala residues at positions P1 and P2. In a purified system, alpha 1-antitrypsin Ala357-Arg358 was 150-fold less efficient against C1s than C1-inhibitor and 27,000-fold less efficient against t-PA than plasminogen activator inhibitor-1. In plasma, 2.3 microM alpha 1-antitrypsin Ala357-Arg358 reduced by 65% the formation of a complex between kallikrein and C1-inhibitor following activation of the intrinsic pathway of blood coagulation by kaolin. Furthermore, after supplementation by 2.0 microM alpha 1-antitrypsin Ala357-Arg358, zymographic analysis showed that the majority of the free t-PA of normal plasma formed a bimolecular complex with the double mutant. In contrast, 3.4 microM alpha 1-antitrypsin Ala357-Arg358 did not prevent the activation of the classical pathway of complement observed when normal serum is supplemented with anti-C1-inhibitor F(ab')2 fragment. These results demonstrate that alpha 1-antitrypsin Ala357-Arg358 has therapeutic potential for disorders with unregulated activation of the intrinsic pathway of blood coagulation and the fibrinolytic system; however, the double mutant is not an efficient inhibitor for the classical pathway of complement.
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PMID:Reactivity of alpha 1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement, and fibrinolytic systems. 219 58

Normal human bronchial epithelial (NHBE) cells respond to signals initiated by the binding of transforming growth factor-beta type 1 (TGF-beta 1) to its surface receptors by activating pathways that result in terminal squamous differentiation. By use of both normal and SV40 T-antigen-immortalized cells, it was found that treatment with TGF-beta 1 transiently increases mRNA levels for urokinase (uPA) and plasminogen activator inhibitor type 1 (PAI-1) approximately 5- and 50-fold, respectively, within 4 h. In NHBE cells, PAI-1 protein is increased by TGF-beta 1 in both extracellular matrix and medium. The net effect of TGF-beta 1 on plasminogen activator activity in the medium was a 50% reduction as measured by a caseinolytic assay. A T-antigen-immortalized bronchial epithelial cell line that does not undergo squamous differentiation in response to TGF-beta 1 but binds this growth factor did not respond to TGF-beta 1 by modulation of either uPA or PAI-1 expression. Comparison of human bronchial epithelial, pleural mesothelial, and lung fibroblastic cell strains indicated that the epithelial cells have a constitutively higher ratio of uPA to PAI-1 mRNA expression. These data suggest that modulation of pericellular proteolysis in bronchial epithelial cells in response to TGF-beta 1 represents a significant biological change in their pericellular environment. The induction of uPA and PAI-1 expression in human bronchial epithelial cells may be related to the ability of the cell to undergo squamous differentiation in response to TGF-beta 1. These observations identify specific changes in gene expression that may serve as markers for the differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:TGF-beta 1 modulation of urokinase and PAI-1 expression in human bronchial epithelial cells. 222 Oct 87

Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.
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PMID:Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro. 223 Feb 25

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.
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PMID:Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha. 223 28

Cells of the monocyte/macrophage lineage are known to produce urokinase type plasminogen activator (u-PA) and are active participants in the inflammatory response. Modulation of cellular u-PA production, for instance in response to LPS, may have an important impact on the evolution of inflammatory lesions. A definitive picture of how monocyte u-PA production and activity are regulated by LPS is lacking. We addressed this issue directly by measuring u-PA Ag and activity in mononuclear cell cultures. By using a competition ELISA to quantitate u-PA Ag, we found that LPS-stimulated mononuclear cells in culture increased u-PA production in a dose-dependent manner and that all the u-PA detected was attributable to the monocytes therein. Increasing amounts of u-PA were secreted into the medium, bound to the cell surface, and found intracellularly. Although the absolute amounts of u-PA varied from donor to donor, the increases seen with LPS stimulation were a consistent and statistically significant finding. Only the cell-surface-bound u-PA was fibrinolytically active, however, with this activity increasing upon LPS stimulation. All monocyte cell-surface-associated fibrinolytic activity was attributed to u-PA, as shown by plasminogen dependence, neutralization by antibodies to u-PA, and identification of fibrinolytically active molecules eluted from the cell surface. The surface bound u-PA was not inhibited by its physiologic inhibitors, PAI-1 or PAI-2, whereas free u-PA was. Hence LPS stimulation results in monocytes exhibiting increased cell-surface-associated u-PA Ag and fibrinolytic activity, in spite of concomitant high levels of plasminogen activator inhibitor type 2 production. This surface-bound enzymatic activity may influence the ability of monocytes to migrate in and interact with an inflammatory microenvironment.
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PMID:Lipopolysaccharide-induced modulation of human monocyte urokinase production and activity. 225 14

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.
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PMID:The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2. 230 45

Increasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125I-fibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI1 remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.
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PMID:Detection of both type 1 and type 2 plasminogen activator inhibitors in human monocytes. 233 62

Endothelial cells from both human retinal microvessels (HME) and fetal bovine aortic endothelium (FBAE) were grown in aggregate cultures alone, or with either retinal pigment epithelium (RPE) cells or fibroblasts. The levels of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in the conditioned media of the various aggregate types were measured. High PA levels were detected in the conditioned medium of pure endothelial cell aggregates (equal to 140% and 124% of urokinase control for HME and FBAE, respectively), and high PAI levels were associated with pure RPE aggregates (inhibiting 93% of the urokinase control). The conditioned medium of pure fibroblast aggregates had very low levels of either PA or PAI. When RPE cells were aggregated with FBAE or HME cells into mixed (heterogenous) aggregates, the PA measured in the conditioned medium was equal to 22% and 30% of the urokinase control, respectively. The PA level in the conditioned medium of mixed fibroblast-FBAE cell aggregates was higher, 104% of the control, and the difference was statistically significant (P less than 0.001). Co-incubation of pure RPE aggregates with pure FBAE aggregates or with pure HME aggregates resulted in PA activity in the conditioned medium that was equal to 110% and 96% of the control, respectively. The PA level found when pure FBAE cell aggregates were co-incubated with pure fibroblast aggregates was higher, 134% of the control, and the difference was statistically significant (P less than 0.001). Our results indicate that RPE cells can reduce endothelial cell PA, probably through both direct contact between the cells and PAI production. Fibroblasts did not have this influence on endothelial cell PA.
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PMID:Retinal pigment epithelium cells can influence endothelial cell plasminogen activators. 235 10

This laboratory recently reported that laminin degradation by cultured colon cancer was plasminogen dependent and reflected the presence of urokinase bound to cell surface receptors. (Schlecte, W.; Murano, G.; Boyd D. Cancer Res., 49:6064-6069; 1989). The present study was undertaken to determine the sensitivity of urokinase receptor directed proteolysis to the type I plasminogen activator inhibitor (PAI-1). Colon cancer cell types, that were highly effective in degrading laminin in vitro, elaborated into their conditioned medium an inhibitor which was indistinguishable from PAI-1 on the basis of its performance in reverse zymography, western blotting, and immunoprecipitation assays. A fraction of this PAI-1 was active, as evidenced by complex formation with the active site of radioactive urokinase. Laminin degradation by the colon cancer cells, however, did not appear to be affected by the endogenous inhibitor, since an antibody to the inhibitor, which blocked urokinase-PAI-1 interactions, had little effect on laminin turnover. Further, addition of exogenous PAI-1, activated by guanidine hydrochloride pretreatment, to the colon cancer cells did not perturb laminin degradation. Because laminin degradation by colonic cells was a function of receptor bound urokinase, presumably immobilized plasminogen activator escaped the neutralizing effect of the inhibitor. These data suggest either a shielding effect of the receptor on the plasminogen activator or a physical separation of activator and inhibitor. Either way, for cultured colon cancer at least, laminin degradation directed by urokinase receptor bound plasminogen activator appeared unaffected by the presence of this inhibitor.
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PMID:Insensitivity of laminin degradation directed by receptor bound urokinase to PAI-1 in cultured colon cancer. 239 Apr 19


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