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)

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones.
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PMID:Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin. 1256 52

Release of transcellular tension upon disruption of actin stress fibers with cytochalasin D (CD) and associated changes in cell morphology are reflected in the rapid transcription of "deformation-responsive" genes. For certain genes (e.g., urokinase plasminogen activator and its type-1 inhibitor PAI-1), de novo mRNA synthesis appears to require cell shape-dependent activation of the MAP kinases ERK1/2. ERK activation in response to microfilament disruption was inhibited completely by the broad-spectrum tyrosine kinase inhibitor genistein and the relatively src-kinase selective compound PP1. Such inhibitor sensitivity profiles suggested that src-family members, likely pp60(c-src), were important upstream elements in deformation-related ERK activation. pp60(c-src) kinase activity was elevated fourfold within 15 min after CD addition to quiescent R22 smooth muscle cells and declined quickly thereafter. CD-induced increases in the phosphorylation levels of both pp60(c-src) and IgG heavy chain (a substrate target in the coupled immunoprecipitation/in vitro pp60(c-src) kinase assay) were ablated completely by pretreatment with the src-type kinase inhibitor PP1. Prior PP1 exposure similarly repressed CD-stimulated PAI-1 transcript accumulation. Consistent with the pharmacologic findings, transfection of a dominant-negative pp60(c-src) expression construct (DN-Src) effectively suppressed (in a concentration-dependent manner) CD-induced PAI-1 synthesis in R22 cells. To more specifically address the potential involvement of src kinases in CD-initiated ERK mobilization, R22 cells were transiently co-transfected with DN-Src and Myc-tagged ERK2 expression constructs, serum-deprived then stimulated with CD. The effect of DN-Src expression on endogenous ERK1/2 activation and nuclear translocation was assessed in separate experiments. The phosphorylation levels of both exogenous (Myc-ERK2) and endogenous ERK1/2 targets was significantly reduced by DN-Src; nuclear accumulation of pERK1/2 was completely inhibited. These data indicate that pp60(c-src) is a critical upstream activator of the ERK cascade leading to PAI-1 transcription in response to cellular deformation stimuli.
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PMID:Pp60c-src mediates ERK activation/nuclear localization and PAI-1 gene expression in response to cellular deformation. 1270 50

We have investigated the role of a classical isoform of protein kinase C (PKCgamma) in promoting immortalized mammary cell tumorigenesis in vivo and the contribution of proteases and adhesion molecules to this process. We hypothesized that overexpression of PKCgamma in immortalized mammary epithelial cells may initiate, by activating the mitogenic ERK pathway, early changes in proteases, adhesion molecules, and markers of an epithelium-to-mesenchyme transition that may contribute to in vivo tumorigenesis. Here we show that compared to vector-transfected cells, immortalized murine mammary epithelial cells (NMuMG) overexpressing PKCgamma have stronger activation of (approximately 5-fold) ERK1/2 MAPKs, which results in a similar increase in cyclin D1. In addition, PKCgamma-expressing cells showed increased levels of vimentin, fibronectin (FN), beta1-integrins, enhanced adhesion to fibronectin, and its organization into fibrils. Concomitantly, PKCgamma induced a dramatic down-regulation of E-cadherin protein levels and its localization to cell-cell junctions. NMuMG cells expressing PKCgamma became resistant to death by anoikis and formed colonies in soft agar. This effect was dependent on ERK activation, because Mek1/2 inhibition with PD98059 abrogated anchorage-independent growth. Most importantly, unlike control NMuMG cells, PKCgamma-transfected cells inoculated s.c. into nude mice displayed tumorigenic and invasive capacity and were able to spontaneously metastasize. This behavior correlated with increased production of uPA and MMPs-9/-2 induced by PKCgamma. These results suggest that PKCgamma overexpression in immortalized mammary epithelial cells may generate, through an increase in ERK, signaling changes in the expression of genes associated with an epithelium-to-mesenchyme transition that may be sufficient to favor tumor growth in vivo.
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PMID:Immortalized mammary epithelial cells overexpressing protein kinase C gamma acquire a malignant phenotype and become tumorigenic in vivo. 1293 3

The Ets1 proto-oncoprotein is a member of the Ets family of transcription factors that share a unique DNA binding domain, the Ets domain. The DNA binding activity of Ets1 is controlled by kinases and transcription factors. Some transcription factors, such as AML-1, regulate Ets1 by targeting its autoinhibitory module. Others, such as Pax-5, alter Ets1 DNA binding properties. Ets1 harbors two phosphorylation sites, threonine-38 and an array of serines within the exon VII domain. Phosphorylation of threonine-38 by ERK1/2 activates Ets1, whereas phosphorylation of the exon VII domain by CaMKII or MLCK inhibits Ets1 DNA binding activity. Ets1 is expressed by numerous cell types. In haemotopoietic cells, it contributes to the regulation of cellular differentiation. In a variety of other cells, including endothelial cells, vascular smooth muscle cells and epithelial cancer cells, Ets1 promotes invasive behavior. Regulation of MMP1, MMP3, MMP9 and uPA as well as of VEGF and VEGF receptor gene expression has been ascribed to Ets1. In tumors, Ets1 expression is indicative of poorer prognosis.
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PMID:The biology of the Ets1 proto-oncogene. 1297 29

We have recently reported that tyrosine kinase, p56(lck) regulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of IkappaBalpha following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56(lck) and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56(lck) in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells.
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PMID:Tyrosine kinase, p56lck-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways. 1469 20

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.
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PMID:Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. 1470 50

A Kunitz-type protease inhibitor, bikunin, downregulates expression of uPA and its receptor uPAR at the mRNA and protein levels in several types of tumor cells. Our recent work showed that, using a cDNA microarray analysis, pregnancy-associated plasma protein-A (PAPP-A) is a candidate bikunin target gene. To clarify how reduced levels of PAPP-A may confer repressed invasiveness, we transfected human ovarian cancer cell line HRA with antisense (AS)-PAPP-A cDNA and compared the properties of the transfected cells to those of parental HRA cells. Here, we show that regulation of uPA mRNA and protein by IGF-I depends on the PI3K and MAPK signaling pathways and phosphorylation of Akt and ERK1/2 is required for IGF-I-mediated cell invasion; that IGFBP-4 protease in HRA cells is identified as PAPP-A; that reduced PAPP-A expression is associated with the upregulation of IGFBP-4 expression; that higher intact IGFBP-4 levels were associated with low invasive potential and growth rate in AS-PAPP-A cells in response to IGF-I; that IGF-I stimulates Akt and ERK1/2 activation of both the control and antisense cells, but the relative potency and efficacy of IGF-I were lower in the antisense cells compared to the control; and that genetic downregulation of PAPP-A reduces the proliferation, invasion and metastasis of HRA cells. In conclusion, our data identify a novel role for PAPP-A as a bikunin target gene. IGF-I-induced IGFBP-4 proteolysis by PAPP-A may enhance cell growth and invasion through IGF-I-dependent Akt and ERK1/2 activation and subsequently upregulation of uPA.
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PMID:Genetic downregulation of pregnancy-associated plasma protein-A (PAPP-A) by bikunin reduces IGF-I-dependent Akt and ERK1/2 activation and subsequently reduces ovarian cancer cell growth, invasion and metastasis. 1496 70

We have previously reported in a series of papers that a Kunitz-type protease inhibitor, bikunin, suppresses up-regulation of urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and cancer cell invasion in vitro and peritoneal disseminated metastasis in vivo. In the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted, extracellular uPA, signal transduction involved in the expression of uPA and invasion in HRA human ovarian cancer cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk inhibitor (BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans; 2) that neither KTI nor BBI effectively inhibits enzymatic activity of uPA; 3) that uPA upregulation observed in HRA cells was inhibited by preincubation of the cells with KTI with an IC50 of approximately 2 microM, whereas BBI failed to repress uPA upregulation, as measured by enzyme-linked immunosorbent assay; 4) that cell invasiveness was inhibited by treatment of the cells with KTI with an IC50 of approximately 3 microM, whereas BBI failed to suppress cell invasion, as measured by an in vitro invasion assay; 5) KTI suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated by a binding protein(s) other than a bikunin binding protein and/or its receptor; and 6) that transforming growth factor-beta 1 (TGF-beta1)-mediated activation of ERK1/2 was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI, but not BBI, could inhibit cell invasiveness at least through suppression of uPA signaling cascade, although the mechanisms of KTI may be different from those of bikunin.
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PMID:A soybean Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation. 1516 33

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

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.
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PMID:Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation. 1524 85


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