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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)

The 5' flanking regions of the mouse and pig urokinase plasminogen activator (uPA) genes were sequenced and sequence homology interrupted by repeat elements was found to extend to -4.6kb in pig and -6.6kb in mouse. A transient transfection procedure was devised for the murine macrophage cell line RAW264. Pig uPA promoter-CAT constructs were more active than mouse constructs in this assay. This contrast may involve sequence differences within 100 bp of the transcription start site. The selective deletion of distal regions of the promoter (greater than 2.6 kb upstream), and of a conserved element, 5'-AGGAGGAAATGAGG-TCA-3' around -2 kb greatly reduced the activity of reporter constructs in RAW264 cells. Electrophoretic mobility shift assays using the latter sequence identified a single nuclear protein complex. This element has been referred to as PEA3/AP1-like, but the complex did not comigrate with either AP1 or known proteins that bind polypurines (including the macrophage-specific factor PU-1) and was not competed by AP1 or polypurine oligonucleotides. uPA promoters contain multiple AP1 and AP2-like DNA sequences, which were recognised by nuclear proteins expressed constitutively in RAW264 cells. They also contain multiple binding sites for NF kappa B but activated NF kappa B was not expressed in RAW264 cells. The conserved, transcribed 5' non-coding sequences were also required for maximal gene expression. Hence, the uPA promoter contains multiple weak cis-acting elements distributed over 7.0 kb 5' to the translation start site.
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PMID:Constitutive expression of the urokinase plasminogen activator gene in murine RAW264 macrophages involves distal and 5' non-coding sequences that are conserved between mouse and pig. 176 14

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.
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PMID:Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter. 812 5

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

Expression of polyomavirus middle-T antigen (middle-T) is involved in the formation of various tumors in vivo, e.g. hemangiomas and mammary gland tumors. Several genes have been shown to be activated in middle-T-expressing cells, but the underlying mechanisms have only been partially elucidated. Among the genes regulated by middle-T, the urokinase-type plasminogen activator (uPA) gene seems to be of primary importance for the development of the transformed phenotype. We have found that the uPA gene is highly expressed in eEnd2 cells derived from a hemangioma expressing middle-T. NIH3T3 cells show negligible levels of uPA mRNA but its expression was highly induced by infecting with a middle-T-expressing retrovirus. Middle-T did not affect uPA mRNA stability. Transient cotransfection experiments using a uPA-receptor gene construct and a middle-T expression vector showed that high uPA mRNA levels are due to increased uPA promoter activity. Analyses of various signaling molecules by transient cotransfection assays and in vitro kinase assays established that a signaling pathway involving c-Src, SOS, Ras, Raf-1 and ERK is activated by middle-T in NIH3T3 cells, resulting in the activation of the uPA gene promoter via PEA3/AP1 elements. In contrast, in eEND2 cells uPA gene induction is only partially dependent on this pathway, suggesting the involvement of additional signaling molecules in endothelial cells.
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PMID:Urokinase-type plasminogen activator gene regulation by polyomavirus middle-T antigen. 857 Jan 90

We have previously shown in NIH 3T3 fibroblasts that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or fibroblast growth factor-2 (FGF-2) activates the Ras/Erk signaling pathway in NIH 3T3 fibroblasts, leading to the induction of the urokinase-type plasminogen activator (uPA) gene. In this study, we characterize cis-acting elements involved in this induction. DNase I hypersensitive (HS) site analysis of the uPA promoter showed that two regions were enhanced after TPA and FGF-2 treatment. One was located 2.4kb upstream of the transcription start site (-2.4kb), where a known PEA3/AP1 (AGGAAATGAGGTCAT) element is located. The other was located in a previously undefined far upstream region. Sequencing of this region revealed a similar AP1/PEA3 (GTGATTCACTTCCT) element at -6.9 kb corresponding to the HS site. Deletion analysis of the uPA promoter in transient transfection assays showed that both PEA3/AP1 elements are required for full inducibility, suggesting a synergism between the two elements. When the two sites were inserted together upstream of a minimal promoter derived from the thymidine kinase gene, expression of the reporter gene was more strongly induced by TPA and FGF-2 than with either of the two elements alone. Alone, the -6.9 element was more potent than the -2.4 element. The involvement of AP1 as well as Ets transcription factors was confirmed by examining different promoter constructs containing deletions in either the AP-1 or the PEA3 element, and by using an expression plasmid for dominant negative Ets-2. Electromobility shift analyses using specific antibodies showed that c-Jun and, JunD bind to both elements with or without induction. In addition, ATF-2 binds to the -2.4-kb element even without induction and c-Fos to the -6.9-kb element only after induction. Accordingly, overexpression of c-Fos caused induction from the -6.9-kb element, but reduced induction from the -2.4-kb element. The involvement of the Ets-2 transcription factor was shown by using expression plasmids for wild-type and dominant negative Ets-2.
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PMID:Cooperation of two PEA3/AP1 sites in uPA gene induction by TPA and FGF-2. 940 85

The broad spectrum protease urokinase-type plasminogen activator (uPA) has been implicated in muscle regeneration in vivo as well as in myogenic proliferation and differentiation in vitro. These processes are known to be modulated by basic fibroblast growth factor (FGF-2) and serum. We therefore investigated the mechanism(s) underlying the regulation of uPA expression by these two stimuli in proliferating and differentiating myoblasts. The expression of uPA mRNA and the activity of the uPA gene product were induced by FGF-2 and serum in proliferating myoblasts. uPA induction occurred at the level of transcription and required the uPA-PEA3/AP1 enhancer element, since deletion of this site in the full promoter abrogated induction by FGF-2 and serum. Using L6E9 skeletal myoblasts, devoid of endogenous FGF receptors, which have been engineered to express either FGF receptor-1 (FGFR1) or FGF receptor-4 (FGFR4), we have demonstrated that both receptors, known to be expressed in skeletal muscle cell precursors, were able to mediate uPA induction by FGF-2, whereas serum stimulation was FGF receptor-independent. The induction of uPA by FGF-2 and serum in FGFR1- and in FGFR4-expressing myoblasts required the mitogen-activated protein kinase pathway, since treatment of cells with a specific inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase-2 kinase, PD98059, blocked uPA promoter induction. Although FGF-2 and serum induced uPA in proliferating myoblasts, their actions on cell-cell contact-induced differentiating myoblasts differed dramatically. FGF-2, but not serum, repressed uPA expression in differentiation-committed myoblasts, and these effects were also shown to occur at the level of uPA transcription. Altogether, these results indicate a dual regulation of the uPA gene by FGF-2 and serum, which ensures uPA expression throughout the whole myogenic process in different myoblastic lineages. The effects of FGF-2 and serum on uPA expression may contribute to the proteolytic activity required during myoblast migration and fusion, as well as in muscle regeneration.
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PMID:Differential regulation of urokinase-type plasminogen activator expression by basic fibroblast growth factor and serum in myogenesis. Requirement of a common mitogen-activated protein kinase pathway. 944 43

The activation status of the ras pathway was studied in eight ovarian tumor cell lines. Three biochemical parameters indicative of ras activation were tested: (a) the ratio of the ras-GTP:ras-GDP complex; (b) the activity of mitogen-activated protein kinases p42/p44; and (c) ets-2 phosphorylation at position threonine 72, a mitogen-activated protein kinase phosphorylation site in vivo. Four of the ovarian tumor cell lines had an activated ras pathway by these three parameters, whereas only one of these contained a mutated ras gene. In addition, ras/ets-2 responsive genes such as the urokinase plasminogen activator (uPA) were activated in these four cell lines. Transient transfection assays indicated that the compound ets-AP1 oncogene responsive enhancer present in the uPA gene was the target of ras signaling in ovarian tumor cells and that the combination of activated ras and ets-2 could superactivate the uPA enhancer element. Coexpression of the dominant-negative ras-Asn17 cDNA gene abrogated activity of this uPA element in ovarian tumor cells. These data indicate that ets-2 is a nuclear target of ras action in ovarian tumor cell lines and that ras signaling pathways may be activated in ovarian cancer by mechanisms independent of direct genetic damage to ras genes.
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PMID:Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines. 960 74

Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter.
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PMID:Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor. 1072 33

The monofunctional alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is a widespread environmental carcinogen that causes DNA lesions, leading to cell death. However, MNNG can also trigger a cell-protective response by inducing the expression of DNA repair/transcription-related genes. We demonstrate that the urokinase-type plasminogen activator (uPA) gene product, a broad spectrum extracellular protease to which no DNA repair function has been assigned, is transcriptionally induced by MNNG in C2C12 and NIH3T3 cells. This induction required an AP1-enhancer element located at -2.4 kilobase (kb), because it was abrogated by deletion of this site. MNNG was found to induce the activation of JNK/SAPK and p38 mitogen-activated protein kinases (MAPKs). Accordingly, we attempted to assess the contribution of each of these MNNG-inducible MAPKs to uPA gene induction by this alkylating agent. Coexpression of dominant negative versions of kinases of the JNK pathway, such as catalytically inactive forms of MEKK1, MKK7, and JNKK, and of cytoplasmic JNK-inhibitor JIP-1, as well as treatment of cells with curcumin (which blocks JNK activation by MNNG), inhibited MNNG-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 nor SB203580, which specifically inhibit p38 MAP kinase activation, abrogated the MNNG-induced effect. Taken together, our results show that the JNK signaling pathway links external MNNG stimulation and AP1-dependent uPA gene expression, providing the first functional dissection of a transcription-coupled signal transduction pathway for MNNG. (Blood. 2000;96:1415-1424)
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PMID:The cJun N-terminal kinase (JNK) signaling pathway mediates induction of urokinase-type plasminogen activator (uPA) by the alkylating agent MNNG. 1094 86

The inducible urokinase enhancer contains three essential elements: a combined PEA3/AP1 and a downstream AP1 site, separated by a 74-bp DNA region called COM (cooperation mediator), that is required for the synergism between the three sites. The 5' half of COM (uCOM) forms four retarded complexes with HeLa or HepG2 nuclear proteins (UEF1-4). We now demonstrate that the UEF4 complex is the transcription factor Oct-1. Because of functional redundancy of the UEF sites, single mutations in UEF4 have no phenotype; we have changed UEF4 from a low to a high affinity binding site for Oct-1. In vitro, this mutation increases the DNA binding of Oct-1 and disturbs the binding of the Prep-Pbx complexes to the nearby UEF3 site. In vivo, this mutation reduces the basal transcriptional activity of the urokinase enhancer, while not affecting its phorbol ester inducibility. This is in keeping with the effect of the deletions of the COM region, which result in an increase in the basal level and, as a consequence, in the loss of 4beta-phorbol 12-myristate 13-acetate inducibility. Oct-1 therefore is not involved in the inducibility of the urokinase enhancer but only in determining its basal activity level.
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PMID:Oct-1 specifically binds the UEF4 site of the human AP1-regulated urokinase enhancer. 1095 Dec 1


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