Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Plasminogen activators (tPA and uPA) are serine proteases that convert the circulating zymogen plasminogen to active plasmin and mediate fibrin degradation. These multifunctional proteins trigger various biological events such as extracellular matrix degradation, cell adhesion, migration, and proliferation, through not yet fully characterized mechanisms. We report that, in smooth muscle cells and ECV-304 carcinoma cells, tPA and ATF (the N-terminal catalytically inactive fragment of tPA) elicited DNA synthesis that requires activation of the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P), signaling pathway and was blocked by D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and N-N'-dimethyl sphingosine (DMS), two classical inhibitors of sphingosine-1-phosphate biosynthesis. Binding of tPA to its receptor uPAR triggered the coordinated activation of two key enzymes of the Spm/Cer/S1P pathway, the neutral sphingomyelinase and the sphingosine kinase-1 that was mediated by a common pertussis toxin (PTX)-sensitive mechanism. The tPA-induced sphingosine kinase-1 activation was mediated by Src, since it was inhibited by herbimycin A and in SrcK- cells (overexpressing a dominant negative kinase defective form of Src) and by ERK1/2 (early phase peaking at 15 min). Sphingosine kinase-1 activation was followed by a second phase of ERK1/2 phosphorylation (peaking at 120 min) and subsequent DNA synthesis, which were inhibited by D-MAPP and DMS, by anti-EGD-1 antibodies and in SrcK- cells (in which the mitogenic signaling was rescued by sphingosine-1-phosphate). Altogether, these data underline a pivotal role for the Spm/Cer/S1P pathway in the tPA-induced mitogenic signaling.
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PMID:The sphingomyelin/ceramide pathway is involved in ERK1/2 phosphorylation, cell proliferation, and uPAR overexpression induced by tissue-type plasminogen activator. 1523 24

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.
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PMID:Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase. 1532 92

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.
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PMID:p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. 1537 54

Only limited comparable data are available on the clot lysis power of the clinically used plasminogen activators (PA). Here the PA were used at different clinically relevant concentrations, and the lysis of the microclots was determined. A microclot lysis assay was used to study thrombolysis by urokinase, tissue-PA (t-PA), streptokinase, plasminogen-streptokinase activator complex (PSAC), reteplase, or tenecteplase. The clot turbidity served as a tool to determine clot mass: 100 microL fresh microclots were incubated with 25 microL PA in 6% bovine serum albumin (BSA)-phosphate-buffered saline (PBS) and 100 microL BSA-PBS or pooled normal human plasma; that is, the PA were in the liquid supernatant of a plasma clot and were not entrapped in the clot, an assay system comparable to normal physiology. The turbidity was determined after 0 to 5 hours (37 degrees C) by a microtiter plate reader. The lysable clot turbidity (clot mass) was expressed in percent of 100% lysable clot control. The clot lysis activity is 100% minus the clot mass in percent. The effective doses at 50% (ED(50)) of lysis of fresh clots after 4 hours (37 degrees C) with 6% BSA or pooled normal human plasma in the clot-supernatant were urokinase 128 or 180 IU/mL; t-PA 0.3 or 0.2 microg/mL; streptokinase 215 or 1371 IU/mL; PSAC 60 or 91 U/mL; reteplase 664 or 996 U/mL; tenecteplase 0.2 or 0.2 microg/mL. The presence of a plasma thrombus with plasma supernatant increases the activity of t-PA approximately 20-fold and that of tenecteplase approximately 400-fold after 4 hours (37 degrees C), when compared to urokinase; in contrast, the lytic activity induced by reteplase decreases; i.e., the plasmin generated by reteplase is hampered on its lytic action against a thrombus. When comparing the clot lysability of microclots of 29 different donors, the only correlation (r > 0.6) was that between u-PA and t-PA. The lysability of individual clots by PA can be measured with the present routine-suited technique. It is suggested that different thrombolytic agents or concentrations thereof would have a different clinical outcome in different individuals.
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PMID:In vitro simulation of therapeutic thrombolysis with microtiter plate clot-lysis assay. 1644 31

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.
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PMID:Optimization of pro-urokinase secretion from recombinant Saccharomyces cerevisiae. 1860 Jun 87

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.
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PMID:[Effects of biogenic phosphates on protease-induced protein cleavage and functioning of plasminogen activators]. 1867 89

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.
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PMID:Cross-talk between LPA1 and epidermal growth factor receptors mediates up-regulation of sphingosine kinase 1 to promote gastric cancer cell motility and invasion. 1870 80

Glioblastoma multiforme is an invasive primary brain tumor, which evades the current standard treatments. The invasion of glioblastoma cells into healthy brain tissue partly depends on the proteolytic and nonproteolytic activities of the plasminogen activator system proteins, including the urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and a receptor for uPA (uPAR). Here we show that sphingosine-1-phosphate (S1P) and the inflammatory mediator interleukin-1 (IL-1) increase the mRNA and protein expression of PAI-1 and uPAR and enhance the invasion of U373 glioblastoma cells. Although IL-1 enhanced the expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, down-regulation of SphK1 had no effect on the IL-1-induced uPAR or PAI-1 mRNA expression, suggesting that these actions of IL-1 are independent of S1P production. Indeed, the S1P-induced mRNA expression of uPAR and PAI-1 was blocked by the S1P(2) receptor antagonist JTE013 and by the down-regulation of S1P(2) using siRNA. Accordingly, the inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and Rho-kinase, two downstream signaling cascades activated by S1P(2), blocked the activation of PAI-1 and uPAR mRNA expression by S1P. More importantly, the attachment of glioblastoma cells was inhibited by the addition of exogenous PAI-1 or siRNA to uPAR, whereas the invasion of glioblastoma cells induced by S1P or IL-1 correlated with their ability to enhance the expression of PAI-1 and uPAR. Collectively, these results indicate that S1P and IL-1 activate distinct pathways leading to the mRNA and protein expression of PAI-1 and uPAR, which are important for glioblastoma invasiveness.
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PMID:Sphingosine-1-phosphate and interleukin-1 independently regulate plasminogen activator inhibitor-1 and urokinase-type plasminogen activator receptor expression in glioblastoma cells: implications for invasiveness. 1881 34

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.
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PMID:Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor. 1903 7

Glioblastoma multiforme (GBM) is an aggressively invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid sphingosine-1-phosphate (S1P) stimulates in vitro invasiveness of GBM cells and that high expression levels of the enzyme that forms S1P, sphingosine kinase-1 (SphK1), correlate with shorter survival time of GBM patients. We also recently showed that S1P induces expression of CCN1 (also known as Cyr61), a matricellular protein known to correlate with poor patient prognosis, in GBM cells. In this study, we further explored the role of CCN1 as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, in S1P-induced invasion using a spheroid invasion assay. We also investigated the roles of various S1P receptors in stimulating invasiveness through these pathways. S1P induced expression of uPA and its receptor, uPAR, in GBM cells. Whereas S1P(1), S1P(2), and S1P(3) receptors all contribute, at least partially, S1P(1) overexpression led to the most dramatic induction of the uPA system and of spheroid invasion, even in the absence of added S1P. Furthermore, neutralizing antibodies directed against uPA or CCN1 significantly decreased both basal and S1P-stimulated GBM cell invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment S1P receptor-mediated enhancement of uPA activity or invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell invasion, whereas S1P receptor signaling enhances invasion, partially through uPA and CCN1.
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PMID:Sphingosine-1-phosphate regulates glioblastoma cell invasiveness through the urokinase plasminogen activator system and CCN1/Cyr61. 1914 34


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