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)

The urokinase-urokinase receptor system plays a dominant role in the degradation and invasion of extracellular matrix (ECM) by tumor cells. In this system, urokinase bound to its cell receptor converts plasminogen to plasmin, a broad-spectrum serine protease that participates in the degradation and invasion of connective tissues by tumor cells. In this study, we evaluated whether these activities of plasmin are inhibited by a newly characterized human 32 kDa recombinant serine protease inhibitor called trypsin/tissue factor pathway inhibitor-2 (rTFPI-2). We found that rTFPI-2 dose-dependently inhibited fluid-phase plasmin as well as plasmin generated on the ECM and/or the cell surface of HT-1080 fibrosarcoma cells. The degradation of radiolabeled matrix as well as Matrigel invasion by these tumor cells is also inhibited by rTFPI-2 in a dose-dependent fashion. We have reported that rTFPI-2 is identical to 33 kDa extracellular matrix-associated serine protease inhibitor (33 kDa MSPI), whereas the 31 and 27 kDa MSPIs are under-glycosylated forms of the 33 kDa MSPI. We therefore evaluated the ability of MSPIs from the ECM of dermal fibroblasts to inhibit plasmin and found that the plasmin activity was effectively blocked by the MSPIs. We have also evaluated whether the HT-1080 cells synthesize and secrete the MSPIs and found that the synthesis and secretion of the MSPIs was undetectable in these cells. Collectively, our results suggest that rTFPI-2/33 kDa MSPI inhibits plasmin on the tumor cell and ECM surfaces as well as the degradation and invasion of matrix by HT-1080 fibrosarcoma cells.
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PMID:HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. 961 Jul 35

Cancer cells are known to shed extracellular membrane vesicles both in vitro and in vivo. To analyse their possible involvement in the metastatic behaviour of tumours, we measured the Matrigel invasion capability and amounts of vesicles shed by four human tumour cell lines (8701-BC, MCF-7, MDA-MB-231 and HT-1080), and by MCF-10A, an immortalised human breast cell line. The proteolytic activity content of vesicles was analysed by gelatin and casein zymographies. While MCF-10A cells do not release a measurable amount of vesicles, all tumour lines analysed, when cultured in presence of serum, shed vesicles rich in MMP-9. Other vesicle-associated proteinases include MMP-2 and uPA. Amounts and proteolytic activities of shed vesicles correlate with the in vitro invasiveness of cells. Since vesicles appear to promote the proteolytic cascade required for the localised degradation of the extracellular matrix, their shedding from cancer cells might represent an important feature of tumour progression.
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PMID:The amount and proteolytic content of vesicles shed by human cancer cell lines correlates with their in vitro invasiveness. 985 20

Tumor invasion into the extracellular matrix (ECM) and basement membrane (BM) is a crucial step of tumor metastasis. In order to investigate the possible therapeutic procedure for the tumor invasion, we investigated the anti-invasive activities of several synthetic serine protease inhibitors. FOY-305, a serine protease inhibitor, showed no cytotoxic activity against human HT-1080 fibrosarcoma cells at concentrations ranging from 0.1 to 100 micrograms/ml, while its analogs ONO-3403 and FO-349 showed slight cytotoxic activities at the concentration of 100 micrograms/ml. These compounds inhibited the activity of urokinase-type plasminogen activator (u-PA) which is one of serine proteases and considered to be associated with tumor invasion and metastasis in fibrin zymography. FOY-305 more potently inhibited the invasion of HT-1080 cells into the reconstituted BM Matrigel, as well inhibited u-PA activity, compared with ONO-3403 and FO-349. These results suggest that the anti-invasive activity of these compounds is consistent with their anti-fibrinolytic activities. In addition, the combined treatment of FOY-305 with FC-336 processing anti-invasive and anti-MMP properties resulted in marked enhancement of anti-invasive activity. In conclusion, FOY-305 inhibited the invasion of tumor cells through interference with the u-PA activity of tumor cells, and this inhibitory activity was augmented by the combination with a MMP inhibitor.
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PMID:Anti-invasive activity of synthetic serine protease inhibitors and its combined effect with a matrix metalloproteinase inhibitor. 989 76

Tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a 32- to 33-kDa Kunitz-type serine protease inhibitor, inhibits plasmin and trypsin. Because plasmin and trypsin are involved in the activation of promatrix metalloproteases proMMP-1 and proMMP-3, we investigated the role of TFPI-2/MSPI in the activation of these proenzymes. Both plasmin and trypsin activated proMMP-1 by converting the 53-kDa proenzyme to the partially active 43-kDa polypeptide; this activity was inhibited by TFPI-2/MSPI. Similarly, TFPI-2/MSPI inhibited the conversion of 66-kDa proMMP-3 to the activated 45- and 30-kDa polypeptides by plasmin and trypsin. Because plasmin is involved in the physiological activation of proMMP-3, we tested whether TFPI-2/MSPI inhibits the activation of proMMP-3 by HT-1080 fibrosarcoma cells and urokinase-charged HeLa cells. We found that the inhibitor inhibited proMMP-3 activation by HT-1080 cells and urokinase-charged HeLa cells. Collectively, our results suggest that TFPI-2/MSPI indirectly regulates MMP-1- and MMP-3-catalyzed matrix proteolysis by regulating the activation of proMMP-1 and proMMP-3.
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PMID:Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor. 1008 61

Vitamin D and its derivatives (deltanoids) are potent regulators of cell proliferation and differentiation. Targeted production of proteolytic enzymes like serine proteases and metalloproteinases is an important part of the invasive process of cancer cells. Treatment with 1 alpha25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] decreases the invasive properties of breast carcinoma cells. Here we have analyzed the effects of 1alpha,25(OH)2D3 and its synthetic analogues on the secretion and cell surface association of the components of the plasminogen activator (PA) system and on the secretion of certain matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231 breast carcinoma cells. Deltanoids were able to decrease the secretion of urokinase PA and tissue-type PA activity in a dose-dependent manner and to increase PA inhibitor 1 secretion, leading to reduced total PA activity. CB1093 was the most potent analogue, effective at concentrations several logarithms lower than 1alpha,25(OH)2D3. Transient transfection of different urokinase PA promoter reporter constructs to HT-1080 fibrosarcoma indicator cells indicated that vitamin D-responsive sequences were located between nucleotides -2350 and -1870 in the 5' region of the promoter. Treatment of MDA-MB-231 cells with 1alpha,25(OH)2D3 or other deltanoids also resulted in decreased MMP-9 levels in association with increased tissue inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or proteolytic processing were not appreciably affected by deltanoids. Vitamin D and its analogues caused a decrease in Matrigel invasion assays of MDA-MB-231 cells. Cancer cell invasion is associated with coordinated secretion of proteolytic enzymes and their inhibitors. Vitamin D and its derivatives can evidently influence invasive processes by two means: (a) decreasing the expression and activity of cell invasion-associated serine proteases and metalloproteinases; and (b) inducing their inhibitors.
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PMID:1alpha,25-dihydroxyvitamin D3 and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells. 1077 39

The effects of heat treatment on the viability and fibrinolytic potential of four cultured human carcinoma cell lines, fibrosarcoma cells (HT-1080), lung adenocarcinoma cells with highly metastatic potential (HAL-8), melanoma cells (Bowes) and osteosarcoma cells (NY), determined by measuring their levels of urokinase-type plasminogen activator (u-PA) and its specific receptor (u-PAR), were investigated by comparing them with those of human umbilical vein endothelial cells (HUVECs). HUVECs incubated at 43 degrees C for 120 min exhibited no decrease in viability but exhibited an increase in both u-PA and u-PAR. HT-1080 and HAL-8 showed a moderately high heat-resistance (viability, 60-90%) that correlated with the reduction of u-PAR but not u-PA. On the other hand, Bowes and NY cells, with poor heat-resistance (viability, 20-50%), exhibited stronger cell-associated u-PA activity when they survived at 43 degrees C for 120 min. Since the u-PA/u-PAR system is directly involved in the invasiveness and metastatic potential of carcinoma cells, hyperthermia would alter the biological activity of these carcinoma cells.
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PMID:Effect of hyperthermia on the viability and the fibrinolytic potential of human cancer cell lines. 1080 68

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.
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PMID:The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity. 1100 77

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.
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PMID:Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. 1105 58

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.
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PMID:Isolation and characterization of cell lines with reduced urokinase binding. 1120 35

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that inhibits urokinase. Constitutive and regulated PAI-2 gene expression involves post-transcriptional events, and an AU-rich mRNA instability motif within the 3'-untranslated region of PAI-2 mRNA is required for this process (Maurer, F., Tierney, M., and Medcalf, R. L. (1999) Nucleic Acids Res. 27, 1664-1673). Here we show that instability determinants are present within various exons of the PAI-2 coding region, most notably within exon 4. Deletion of exon 4 from the full-length PAI-2 cDNA results in a doubling in the half-life of PAI-2 mRNA, whereas a 28-nucleotide region within exon 4 contains binding sites for cytoplasmic proteins. Inducible stabilization of PAI-2 mRNA in HT-1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. Hence, PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3'-untranslated region and that cytoplasmic mRNA binding factors may influence steady state and inducible PAI-2 mRNA expression. Finally a 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist.
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PMID:Plasminogen activator inhibitor type 2 contains mRNA instability elements within exon 4 of the coding region. Sequence homology to coding region instability determinants in other mRNAs. 1127 13


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