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)

When cultured in type I collagen gels, two kidney-derived cell lines, Madin-Darby canine kidney (MDCK) cells and murine inner medullary collecting duct (mIMCD3) cells, from branching tubular structures in the presence of Swiss 3T3 conditioned medium, in which hepatocyte growth factor (HGF) is the major branching tubule inducing factor. However, upon incubation with transforming growth factor-beta (TGF-beta) in the presence of 3T3 conditioned medium, MDCK tubulogenesis and branching was markedly inhibited. In contrast, mIMCD3 cells, which are much less susceptible to growth and tubulogenesis inhibition by TGF-beta, formed long straight tubulelike structures in presence of TGF-beta, suggesting a dissociation between tubulogenesis and branching morphogenesis. Interestingly, those long tubules that did branch often superficially resembled the early branching ureteric bud in embryonic kidneys. Quantitation of branching events revealed a selective branch-inhibiting effect of TGF-beta on mIMCD3 cells at concentrations between 0.02 and 2 ng/ml. There was no qualitative or quantitative difference among TGF-beta 1, -beta 2, and -beta 3 on inhibition of branching events, suggesting existence of potentially redundant mechanisms for modulating branching morphogenesis. Concentrations of TGF-beta that resulted in long nonbranching tubules also altered the profile of extracellular matrix-degrading proteases and their inhibitors expressed by developing tubules. Ratios of urokinase type plasminogen activator (u-PA) to plasminogen activator inhibitor (PAI-l) and matrix metalloprotease (MMP)-1 to tissue inhibitor of metalloprotease (TIMP)-1 were both markedly decreased. In addition, apart from a direct effect on epithelial cell branching morphogenesis, TGF-beta downregulated the expression of HGF mRNA in Swiss 3T3 cells. Thus TGF-beta exerts at least three distinct effects relevant to tubulogenesis and branching morphogenesis inhibition of branching morphogenesis alone (mIMCD3 cells), inhibition of both tubulogenesis and branching morphogenesis (MDCK cells), and inhibition of the expression of growth factor which induce tubulogenesis and branching morphogenesis (3T3 cells). In the context of epithelial tissue development, which requires tightly regulated branching tubulogenesis of epithelial cells, the data suggest a model where branching patterns are regulated by a precise temporal and spatial balance between branching morphogens such as HGF and inhibitory morphogens such as members of the TGF-beta superfamily [e.g., TGF-beta isoforms, certain bone morphogenetic proteins].
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PMID:Transforming growth factor-beta selectively inhibits branching morphogenesis but not tubulogenesis. 903 60

Fetal bovine aortic endothelial GM 7373 cells were transfected with a viral expression vector harboring the human urokinase-type plasminogen activator (uPA) gene. The stable transfectant clone uPA-R5 overexpressed and secreted human uPA as shown by Northern blot analysis, immunoprecipitation of metabolically labeled proteins, plasmin chromogenic assays, and SDS-PAGE zymography of cell extracts and conditioned media. The uPA-R5 cells were analyzed for their invasive capacity in vitro in the Matrigel chemoinvasion assay in the presence of serine- or metalloprotease inhibitors. uPA overexpression enhanced the invasive capacity of GM 7373 cells through a mechanism which differs from that mediated by metalloproteases. Endothelial cell uPA transfectants may represent an useful experimental model to investigate the role of uPA in angiogenesis and angioproliferative diseases.
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PMID:Urokinase-type plasminogen activator overexpression enhances the invasive capacity of endothelial cells. 921 3

Much attention has recently focused upon hepatocyte growth factor (HGF) as a potential regulator of epithelial branching morphogenesis. However, since neither the HGF nor c-met "knockout" mice show abnormal kidney branching morphogenesis, we sought to analyze the relative importance of HGF in in vitro branching morphogenesis compared with other factors secreted by the embryonic kidney. Exploiting an assay that employs kidney epithelial cells (murine inner medullary collecting duct, mIMCD3) seeded in collagen cocultured with the embryonic kidney, we found that a tyrosine kinase inhibitor that is highly specific for the epidermal growth factor (EGF) receptor (EGFR), tyrphostin AG1478, inhibited mIMCD3 cell process formation (an early step in branching tubulogenesis) by 40%, whereas high concentrations of neutralizing anti-HGF antibodies had a lesser effect (20% inhibition), suggesting that EGFR ligands account for a larger fraction of branching morphogens secreted by the embryonic kidney than HGF. In addition, when an embryonic epithelial cell line derived from c-met (-/-) mice was cocultured with the embryonic kidney, these c-met (-/-) cells underwent process formation. EGFR ligands but not HGF were able to induce branching tubulogenesis in these cells. All EGFR ligands tested, including EGF, transforming growth factor-alpha, heparin-binding EGF, betacellulin, and amphiregulin, induced mIMCD3 cell tubulogenesis. EGFR ligands caused upregulation of urokinase, urokinase receptor, and matrix metalloprotease-1, and tubulogenesis could be inhibited by the metalloprotease inhibitor 1,10-phenanthroline. Our results support the notion that multiple parallel and potentially redundant growth factor-dependent pathways regulate branching tubulogenesis.
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PMID:EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney. 932 21

A number of tightly regulated proteolytic enzyme systems, including the plasminogen activation cascade and matrix metalloproteases, play integral roles in the remodelling of extracellular matrices during pregnancy and parturition. This study assessed these labour-associated changes in protease activity in human gestational tissues. Amnion, choriodecidua and placenta collected from women before (at caesarean section, not in labour), during (at caesarean section, in labour) and after (spontaneous-onset labour, normal vaginal delivery) labour were examined on gelatin-substrate SDS-PAGE zymography. All tissues displayed major 55 kDa plasminogen-dependent activity that was abolished by the serine protease inhibitors (10 mmol phenylmethyl-sulphonylfluoride l-1, 100 mmol epsilon aminocaproic acid l-1, 1 mmol Glu-Gly-Arg chloromethylketone l-1). The enzymic activity was identified as urokinase plasminogen activator on the basis of its co-migration with reference standard and western blot analysis, and did not vary with labour status. An additional protease with an apparent molecular mass of approximately 90 kDa was detected in all tissues. Densitometric measurement of these tissues showed a significant (P < 0.05) increase in this enzyme activity with labour onset. Heavy metal chelators (1 mmol 1.10 phenanthroline l-1 and 10 mmol EDTA l-1) selectively blocked the 90 kDa activity, consistent with the proposal that it is a metalloprotease. Co-migration with reference standard and western blot analysis confirmed the identity of this protease as the matrix metalloprotease 9 (MMP-9). Immunoreactive MMP-9 protein was also significantly (P < 0.05) increased during and after labour compared with before labour in all tissues examined. It is proposed that the upregulated expression of MMP-9 is involved in fetal membrane rupture and placental separation during and after labour onset, respectively. In conclusion, the regulated repertoire of protease activities expressed by human gestational tissues implies an important role for matrix-degrading enzymes during human parturition.
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PMID:Differential expression of proteases in human gestational tissues before, during and after spontaneous-onset labour at term. 1050 55

Tumor progression and metastasis may result in part from the selection of cell clones competent for survival, invasion and growth at secondary sites and characterized by loss of growth inhibitory responses, acquisition of increased adhesiveness and enhanced motility and protease expression. Transforming growth factor-beta1 (TGF-beta1) is produced by osteoblasts (OB) in a latent form and is activated by proteases in a cell-dependent manner. We show here that OB conditioned medium (OB CM) modulates Matrigel invasion of a bone metastatic prostate cancer cell line (PC3) and that this effect is blocked by antibody against TGF-beta1 and by uPA/plasmin inhibitors, suggesting that TGF-beta1 can modulate OB-mediated cell recruitment and that PC3 cells can activate TGF-beta1. TGF-beta1 induces uPA and PAI-1 secretion and promotes binding of uPA at the external plasma membrane with increased membrane-associated plasmin activity. Matrix metalloprotease-9 (MMP-9) is induced both in the medium and in the membrane associated form. Moreover, the balance between proteolytic activity and inhibition is crucial in the metastatic event. Indeed, the increment of PAI-1 could have an important regulatory role on the extracellular proteolysis and might explain the decrease of net PA and gelatinolytic activities measured in the medium. In addition, PAI-1 plays a regulative role localizing matrix degradation in some specific sites, such as areas of cell-to-cell or cell-to-ECM contacts. In conclusion, TGF-beta1 enhances PC3 Matrigel invasion by a uPA/plasmin-dependent mechanism, also involving the MMP-9, and thus may play a central role in malignant prostate tumor progression as a result of stimulating bone matrix invasion.
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PMID:Osteoblast-derived TGF-beta1 modulates matrix degrading protease expression and activity in prostate cancer cells. 1065 34

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.
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PMID:A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. 1066 46

A novel protease with fibrinolytic activity, designated as SW-1, was isolated and purified from the fermentation broth of Streptomyces sp. strain Y405, a soil isolate. The purification procedure involved ammonium sulphate fractionation, decolorization on 290 resin, gel filtration on Sephadex G75, anion-exchange chromatography on DEAE Sephadex A25, and affinity chromatography on Lysine Sepharose 4B. About 4.2 mg purified enzyme was obtained from a liter of fermentation broth and the recovery yield was 12.0%. The purified enzyme showed the specific activity of 2952.3 urokinase units per milligram, which was increased by 230.6 fold over the fermentation broth. The purity determined by HPLC was 83.5%. SW-1 is a single chain polypeptide with a predicted molecular weight of 30 kDa in SDS-PAGE and an isoelectric point of 8.5. The N-terminal sequence of SW-1 is R/N/F-P/D-G-M-T-M-T-A-I-A-N-Q-N-T-Q-I-N. There may be nonhomogeneity in the first and second amino acid residue of its N-terminal sequence. The analysis of amino acid composition showed that SW-1 consisted of 262 amino acids. The fibrinolytic activity of SW-1 was entirely inhibited by 10 mmol/L PMSF, 1 mmol/L EDTA and 1 mol/L lysine, respectively, suggesting that SW-1 is a serine protease and metalloprotease, and that the lysine binding site might play a role in the activity. The fibrinolytic activity of SW-1 is stable between 4-37 degrees C and pH 4.0-9.0, and the optimum pH is 8.0. On plasminogen-free fibrin plates, SW-1 showed the same fibrinolytic activity as the mixture of SW-1 with plasminogen, indicating that SW-1 is a fibrinolytic enzyme which affects fibrin directly, but not a plasminogen activator which affects fibrin by activating plasminogen.
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PMID:Purification and characterization of a novel fibrinolytic enzyme from Streptomyces spp. 1071 27

Components of the plasminogen activator (PA) and matrix metalloprotease (MMP) cascade have been characterized in multiple sclerosis lesions by immunohistochemistry, enzyme-linked immunosorbent assay and enzyme activity assays in order to establish a functional role for the enzyme sequence in lesion development. Highly significant quantitative increases in urokinase PA (uPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 were detected in acute multiple sclerosis lesions (P < 0.0001) and in uPAR in normal-appearing white matter (P < 0.0001) compared with control tissue. All three proteins were immunolocalized to mononuclear cells in perivascular cuffs and to macrophages in the lesion parenchyma. MMP-9 and the tissue inhibitor of metalloprotease-1 also increased during lesion development but the enzyme was present largely in the inactive pro-form. In contrast to uPA, the concentration and activity of tissue PA (tPA), the most abundant plasminogen activator in normal control brain, were reduced in multiple sclerosis specimens. In acute lesions tPA co-localized with fibrin(ogen) on large diameter axons also stained with SMI-32, an immunohistochemical marker of axonal damage. The uPA-uPAR complex, concentrated on inflammatory cells in the perivascular zone of the evolving lesion, may facilitate cellular infiltration into the CNS which is amplified by MMP- mediated degradation of blood vessel matrix. tPA localization on injured axons may be a marker of axonal damage or represent a protective mechanism aimed at removal of fibrin deposits and restoration of axonal function.
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PMID:Plasminogen activators in multiple sclerosis lesions: implications for the inflammatory response and axonal damage. 1157 Dec 16

To elucidate potential mechanisms of enhanced type 2 matrix metalloprotease levels and activity within the thickened aged rat aorta, the present study measured its mRNA and protein levels and those of its membrane bound activator, MT1-MMP, its endogenous tissue inhibitor, TIMP-2, tissue type, and urokinase plasminogen activators and their receptors, and an inhibitor of plasminogen activation in aortae from Fisher 344X Brown Norway rats, 2 to 30 months of age. Semiquantitative immunohistochemistry, in situ hybridization, and in situ zymography of aortae detected a marked age-associated increase in gelatinolytic activity of type 2 metalloprotease within the thickened intima, internal elastic lamina, and elastic fibers in the inner part of the thickened tunica media, whereas the intimal tissue inhibitor of metalloprotease-2 mRNA and protein levels were not age related. Both activators of plasminogen and their receptors increased approximately 2-fold within the intima between 2 to 30 months. Similar, but not identical, age-associated changes in factors that regulate protease activity within the aortic media were also observed. We conclude that discordant regulation of factors that determine the activation status of type 2 matrix metalloprotease, coupled with an increase in the expression of its zymogen, occur with aging, which lead to an increase in the amount of activated protease. These factors are candidate mechanisms for age-associated vascular remodeling, a potent risk factor for vascular diseases with advancing age.
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PMID:Altered regulation of matrix metalloproteinase-2 in aortic remodeling during aging. 1196 41

Invasive squamous cell carcinoma (SCC) cells degrade extracellular matrix (ECM) via an extracellular protease cascade that includes urokinase-type plasminogen activator (uPA), plasmin, and the metalloprotease (MMP) family of collagenases. In this study, treatment of oral SCC cells with epidermal growth factor (EGF) stimulated the cells to invade Matrigel (constructive basement membrane (BM) protein). EGF-induced cell invasion was inhibited by antibodies to uPA and by synthetic uPA inhibitors. EGF also induced increased expression of uPA and uPA receptor (uPAR) proteins and mRNA, as well as transcription factor activator protein-1 (AP-1)-DNA binding. These EGF-induced changes were inhibited by treatment with dexamethasone (DEX). DEX treatment also stimulated the production of plasminogen activator inhibitor type 1. Moreover, transfection of SCC cells with AP-1 decoy oligodeoxynucleotides (ODNs) resulted in the suppression of EGF-induced uPA and uPAR expression and Matrigel invasion. These results suggest that oral SCC cells invade Matrigel mainly through the uPA-plasmin cascade, which is mediated by the transcription factor AP-1. The uPA-uPAR interaction is essential for augmenting proteolytic activity and uPAR-mediated signaling, which ultimately induce motility and invasion. Since DEX inhibits the expression of both uPA and uPAR, it may be a useful treatment for oral SCC.
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PMID:Inhibition of epidermal growth factor-induced invasion by dexamethasone and AP-1 decoy in human squamous cell carcinoma cell lines. 1238 86


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