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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)

We undertook a study to determine if the serine-threonine kinase-encoding v-mos oncogene regulated the expression of the urokinase-type plasminogen activator. An expression vector encoding v-mos, but not a kinase-inactive mutant, stimulated urokinase promoter activity in CAT assays employing a squamous cell carcinoma cell line. The induction of urokinase promoter activity by v-mos was mediated, in part, via an increased AP-1 activity since (a) mutation of 2 AP-1 binding sites (at -1967 and -1885), or the co-expression of a transactivation domain-lacking c-jun mutant reduced the induction of the urokinase promoter by v-mos and (b) expression of v-mos increased the activity of a CAT reporter driven by three AP-1 tandem repeats. The stimulation of the urokinase promoter by v-mos was partially countered by co-expression of an ERK1/ERK2-inactivating phosphatase. Western blotting and zymographic analysis indicated that v-mos-transformed NIH3T3 cells (MSV NIH-3T3) secreted more urokinase compared with NIH3T3 cells and this was associated with a higher level of activated ERK1 and ERK2. Expression of a catalytically-inactive MAPKK mutant reduced the activity of a urokinase promoter-driven CAT reporter in the MSV NIH-3T3 cells. In conclusion, the data herein indicate that urokinase expression is regulated by v-mos through a MAPKK-dependent signaling pathway.
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PMID:Regulation of urokinase-type plasminogen activator expression by the v-mos oncogene. 854 21

Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that the uPA gene is transcriptionally induced by FGF-2 as well as by 12-O-tetradecanoylphorbol-13 -acetate involving a PEA3/AP1 element located 2.4 kb upstream of the transcription initiation site; neither induction requires ongoing protein synthesis. Unlike 12-O-tetradecanoylphorbol-13-acetate induction, FGF-2 induction was not impaired by protein kinase C down-regulation. Analyses of various signaling molecules by Western blotting, extracellular signal-regulated kinase (ERK) activity assays, and transient transfection assays (cotransfection of a uPA-reporter gene construct with expression vectors for wild-type or dominant negative type of these molecules or for ERK-specific protein phosphatase MKP-1) showed that a Ras/Raf-1/MEK/ERK-2/JunD pathway is induced by FGF-2 and 12-O-tetradecanoylphorbol-13-acetate, leading to the activation of the uPA gene.
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PMID:Elucidation of a signaling pathway induced by FGF-2 leading to uPA gene expression in NIH 3T3 fibroblasts. 854 15

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.
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PMID:Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling. 971 May 99

The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA) and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged with uPA.PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the catabolism of [35S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t(1)/(2) was 8.2 h. uPA.PAI-1 complex accelerated uPAR catabolism (t(1)/(2) to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t(1)/(2) of 7.8 h) and absence (t(1)/(2) of 16.9 h) of uPA.PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 +/- 0.1-fold and by 4.4 +/- 0.3-fold when 1.0 nM uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures. These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular motility.
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PMID:The very low density lipoprotein receptor regulates urokinase receptor catabolism and breast cancer cell motility in vitro. 1006 6

HL-60 cells treated by PMA develop the monocyte adherent phenotype and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCbeta gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCbeta gene expression or transfected with PKCbeta cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCbeta. In conclusion, we propose that the pathway PKCbeta-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.
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PMID:Activation of plasminogen activator inhibitor-1 synthesis by phorbol esters in human promyelocyte HL-60--roles of PCKbeta and MAPK p42. 1010 71

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.
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PMID:The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network. 1066 86

Transformed PDV keratinocytes respond to TGF-beta(1) by stimulating cell motility and invasiveness concomitantly to enhancement of the urokinase-type plasminogen activator (uPA) expression/secretion. Depletion of extracellular signal-regulated kinase (ERK1, 2) proteins by treatment of PDV cells with antisense oligonucleotides reduced basal uPA production and abolished stimulation of uPA secreted levels and cell motility by TGF-beta(1). PD098059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK), decreased TGF-beta(1)-induced uPA mRNA expression, secreted activity in a dose-dependent manner, and abrogated TGF-beta(1)-stimulated cell motility and invasiveness. PDV-derived dominant-negative RasN17 cell transfectants secreted similar amounts of uPA and exhibited similar invasive abilities as the parental cells or control clones, but were unable to respond to TGF-beta(1) for stimulation of uPA-secreted levels and invasiveness. These results suggest that a Ras/MAPK transduction pathway is involved in the invasive response of transformed keratinocytes to TGF-beta(1).
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PMID:Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-beta(1) in transformed keratinocytes. 1087 38

Smad4 functions as a transcription factor in TGF-beta signalling. We have investigated the role of Smad4 in the TGF-beta1 cell responses of transformed PDV keratinocytes, which contain a Ras oncogene, and of non-tumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negative Smad4 construct. Smad4 mediates TGF-beta1-induced up-regulation of p21Cip1 and growth arrest in MCA3D cells. However, in PDV keratinocytes, Smad4 is only partially involved in TGF-beta1-induced growth inhibition, and does not mediate enhancement of p21Cip1 levels by the growth factor. TGF-beta1 activates Ras/Erk signalling activity in both cell lines. PD098059, a specific inhibitor of MEK, disminishes TGF-beta1-induced p21Cip1 levels in PDV but not in MCA3D cells, suggesting an involvement of Erk in up-regulation of p21Cip1 by TGF-beta1 in PDV cells. PDV dominant-negative Smad4 cell transfectants, but not MCA3D transfectants, showed constitutive hyperactivation of the Ras/Erk signalling pathway, increased secretion of urokinase, higher motility properties, and a change to a fibroblastoid cell morphology associated in vivo with the transition from a well differentiated to a poorly differentiated tumour phenotype. Infection of MCA3D control and dominant negative Smad4 cell transfectants with retroviruses carrying a Ras oncogene led to enhanced p21Cip1 and urokinase secreted levels, independently of TGF-beta1 stimulation, that were reduced by PD098059. These results suggest that Smad4 acts inhibiting Ras-dependent Erk signalling activity in Ras-transformed keratinocytes. Loss of Smad4 function in these cells results in hyperactivation of Erk signalling and progression to undifferentiated carcinomas. Oncogene (2000) 19, 4134 - 4145
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PMID:Blockade of Smad4 in transformed keratinocytes containing a Ras oncogene leads to hyperactivation of the Ras-dependent Erk signalling pathway associated with progression to undifferentiated carcinomas. 1096 74

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.
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PMID:Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C- and MEK/ERK/c-Jun-dependent signaling pathways. 1105 91

The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.
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PMID:Sequential activation of ERK and repression of JNK by scatter factor/hepatocyte growth factor in madin-darby canine kidney epithelial cells. 1107 4


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