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)

Tumour progression to the metastatic phenotype is mainly dependent on tumour cell invasiveness. Cell migration is a crucial step in this process. Here we investigate the effect of hepatocyte growth factor (HGF) on the induction of in vitro invasiveness of poorly aggressive Caco-2 colonic cancer epithelial cells. Invasion assays through a Matrigel barrier were performed. Proteases were assessed by zymography, reverse transcription-polymerase chain reaction and immunoblotting. Caco-2 cells were found to express HGF receptor but not HGF and to secrete several proteases, namely matrix metalloproteinase-1 (MMP-1), MMP-2, possibly MMP-9 and urokinase plasminogen activator (uPA). Exogenous HGF promoted invasiveness of Caco-2 cells through an artificial basement membrane matrix and enhanced their production of proteases. In addition, analyses of media at the end of invasion assays indicated that anti-HGF antibody inhibited protease production in parallel with cell invasion. The involvement of proteases in the HGF-induced invasion process was further investigated using either a synthetic general MMP inhibitor or neutralizing antibodies against MMPs or uPA. All components significantly inhibited HGF-promoted cell invasion. Moreover, specific inhibitors of PKCalpha/beta1 and PI3 kinase also decreased both HGF-promoted cell invasion and protease expression in invasion assay media. Thus, our findings provide evidence that the process of HGF-activated invasiveness of Caco-2 cells involves PI3 kinase and PKC and results from close association of two events, stimulation of cell motile activity and concomitant overproduction of proteases, which permits cell migration through a degraded extracellular matrix.
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PMID:Hepatocyte growth factor induces colonic cancer cell invasiveness via enhanced motility and protease overproduction. Evidence for PI3 kinase and PKC involvement. 1140 46

TGF-beta strongly promotes local tumor progression in advanced epithelial tumors, though the underlying mechanisms are poorly understood. In the present study, we demonstrate the potential of TGF-beta to increase the invasiveness of the pancreatic cancer cell lines PANC-1 and IMIM-PC1. TGF-beta-induced tumor cell invasion occurred in a time-dependent manner, started after 12 hr and continued to increase even 48 hr after a single application of the growth factor. Blocking of secreted TGF-beta1 by application of neutralizing antibodies 24 hr after TGF-beta treatment completely prevented the sustained effects of TGF-beta on tumor cell invasion. Together with our previous observation that TGF-beta1 up-regulates its own expression in both cell lines, our data suggest that TGF-beta1 acts in an autocrine manner to maintain tumor cell invasion. As measured by Northern blot hybridization and zymography, TGF-beta treatment of PANC-1 and IMIM-PC1 cells resulted in strong up-regulation of expression and activity of both matrix metalloproteinase-2 (MMP-2) and the urokinase plasminogen activator (uPA) system. Treatment with MMP inhibitors or inhibitors of the uPA system caused significant reduction of TGF-beta-induced invasiveness in both cell lines. In contrast, expression and activity of MMP-2 and uPA as well as tumor cell invasiveness remained unaffected in cell lines with defects of the TGF-beta type II receptor (MiaPaca2) or the Smad4 gene (IMIM-PC2 and CAPAN-1). In these cell lines, TGF-beta also failed to auto-induce its own expression. In conclusion, our results suggest that TGF-beta1 is a strong promotor of pancreatic cancer progression. TGF-beta thereby acts in an autocrine manner to induce tumor cell invasion, which is mediated by MMP-2 and the uPA system.
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PMID:TGF-beta-induced invasiveness of pancreatic cancer cells is mediated by matrix metalloproteinase-2 and the urokinase plasminogen activator system. 1141 Aug 67

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.
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PMID:Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles. 1145 Jul 4

Plasminogen activator (PA) inhibitor-1 (PAI-1) has been recognized as a surrogate marker of endothelial dysfunction in diseases associated with impaired angiogenesis, including atherosclerosis, diabetic vasculopathy, and nephropathy. To establish the necessary and sufficient components of the PA system [PAI-1, urokinase-type PA (uPA), or tissue-type PA (tPA), and plasminogen (Plg)] for angiogenesis, we examined angiogenic competence of vascular explant cultures obtained from mice deficient in PAI-1, tPA, uPA, and Plg. To gain insight into the requirement for different matrix-degrading systems during endothelial cell migration across plasmin-degradable basement membranes compared with profibrotic areas containing plasmin-nondegradable collagen, we contrasted vascular sprouting in collagen with Matrigel lattices. PAI-1(-/-) vessels showed an increased capillary sprouting in both collagen and Matrigel. Deficiency of uPA significantly reduced the rate of sprouting, whereas tPA(-/-) vessels showed a profound inhibition of capillary sprouting. The Plg(-/-) vessels failed to sprout, a defect that was restored not only by exogenous Plg, but also by the addition of PAs; a nonproteolytic effect of tPA was observed in Matrigel. Zymography revealed no differences in the activity of metalloproteinase (MMP)-2 and -9 in wild-type and PAI-1(-/-) vessels, but demonstrated reduced MMP-9 activity in all angiogenesis-deficient vessels. In summary, 1) PAI-1 by itself is a modest inhibitor of endothelial sprouting, 2) tPA and Plg are indispensable for angiogenesis in this model, 3) Plg is not the only substrate for PAs, and 4) the activity of MMP-9 is undetectable in explant cultures from tPA and Plg knockout mice.
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PMID:Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis. 1155 72

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.
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PMID:Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer. 1183 93

Prostate cancers ability to invade and grow in bone marrow stroma is thought to be due in part to degradative enzymes. The formation of prostate skeletal metastases have been reproduced in vitro by growing co-cultures of prostatic epithelial cells in bone marrow stroma. Expression of urokinase plasminogen activator, matrix metalloproteinase 1 and 7 by prostatic epithelial cells were identified using immunocytochemistry. Also, in vivo tissue sections from human prostatic bone marrow metastases were stained. To establish the role of these enzymes on colony formation, inhibitory antibodies directed against urokinase plasminogen activator, matrix metalloproteinase 1 and matrix metalloproteinase 7 were added into primary prostatic epithelial cells and bone marrow stroma co-cultures. All prostatic epithelial cell cultures stained positively for matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator. Generally prostatic epithelial cells derived from malignant tissues showed increased staining in comparison to epithelia derived from non-malignant tissue. In agreement with in vitro co-cultures, the in vivo tissue sections of prostate bone marrow metastases showed positive staining for all three enzymes. Inhibition studies demonstrated that blocking matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator function reduced the median epithelial colony area significantly in bone marrow stroma co-cultures in vitro. Using a human ex-vivo model we have shown that matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator play an important role in the establishment of prostatic epithelial cells within bone marrow.
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PMID:Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies. 1195 62

Bisphosphonates (clodronate, alendronate, pamidronate and zoledronate) at therapeutically attainable non-cytotoxic concentrations inhibited MMP-3, -12, -13 and -20 as well as MMP-1, -2, -8 and -9, but not urokinase-type plasminogen activator (uPA), a serine proteinase and a pro-MMP activator. Dose-dependent inhibition was shown by three independent MMP assays. The inhibition was reduced in the presence of an increased concentration of Ca(2+) when compared to physiologic Ca(2+) concentration. Alendronate inhibited the in vitro invasion (Matrigel) of human HT1080 fibrosarcoma and C8161 melanoma cells, and the random migration of these malignant and endothelial cell lines capable of expressing MMPs and uPA. The concentration of alendronate required to inhibit 50% of the activity (IC(50)=40-70 microM) of MMPs corresponded to the IC(50) of down-regulation of in vitro invasion and migration. The ability of bisphosphonates to down-regulate the in vitro invasion and random migration was comparable or slightly better in relation to the selective gelatinase inhibitor CTTHWGFTLC peptide. Alendronate but not CTTHWGFTLC peptide promoted the adhesion of HT1080 fibrosarcoma and C8161 melanoma cell lines on fibronectin. Bisphosphonates are broad-spectrum MMP inhibitors and this inhibition involves cation chelation. Bisphosphonates further exert antimetastatic, anti-invasive and cell adhesion-promoting properties, which may prevent metastases not only into hard tissues but also to soft tissues.
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PMID:Bisphosphonates inhibit stromelysin-1 (MMP-3), matrix metalloelastase (MMP-12), collagenase-3 (MMP-13) and enamelysin (MMP-20), but not urokinase-type plasminogen activator, and diminish invasion and migration of human malignant and endothelial cell lines. 1198 68

Proteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86 paralal Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89 paralal Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.
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PMID:Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor. 1219 4

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

The effect of CNTF and BDNF on a proteolytic complement instrumental to invasion and on differentiation was studied in two murine neuroblastoma clones, N1 and N7. At the membrane level, gelatinase MMP-2--mainly the activated form--was restrained by CNTF and BDNF to a residual 34% with both factors; membrane-type 1 MMP was down-regulated to 50% (10 h) and 34% (24 h) with both factors; and urokinase-type plasminogen activator was restrained mainly by BDNF to 70%. In the medium, the two gelatinases MMP-2 and MMP-9 were mainly in zymogen form: only MMP-2 was restrained in N1 cells, while only MMP-9 was restrained in N7 cells by both factors, single or in combination. These effects were paralleled by the induction of neurite outgrowth, which was more stimulated in the less differentiated clone. These dose-dependent and transient effects make CNTF and BDNF ideal candidates for constraining the potentially invasive behavior of nervous system tumors.
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PMID:Modulation of proteolytic potential and differentiation by CNTF and BDNF in two mouse neuroblastoma clones: relation to invasion. 1255 77


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