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)

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

Endothelial cells form a multifunctional cell lining that covers all of the inner surface of blood vessels and regulates several important physiological and pathological reactions. These include inflammation/immune reaction, blood vessel tonus, hemostasis/thrombosis, angiogenesis and so on. Thus, abnormalities of endothelial function may play crucial roles in the development of angitis syndrome, thrombosis/embolism, bleeding disseminated intravascular coagulation (DIC), and neovascularization in some pathological states including tumor growth and diabetic retinopathy. Research on endothelial cells now forms a new frontier termed 'Endotheliology'. Recent advances of the functional and structural aspects of endothelial cells are reviewed here mainly from the viewpoint of endothelial regulation of coagulation and the fibrinolytic system. First we show that the natural endothelial membrane protein thrombomodulin is localized not only on apical endothelial surface but also in caveolae. Since it has been reported that such factors involved in coagulation/fibrinolysis as tissue factor, tissue factor pathway inhibitor (TFPI), thrombin receptor and urokinase receptor are also localized in the caveolae, this membrane structure may act as a special component to regulate coagulation/fibrinolysis on the endothelial membrane surface. Next we demonstrate the signaling pathway of the thrombin receptor. Thrombin cleaves the N-terminus of the receptor as a substrate, exposing a new N-terminus. This newly exposed N-terminus acts as a ligand and activates platelets, endothelial cells and vascular smooth-muscle cells. We have identified that the signal from the thrombin receptor activates NF-kappaB through the activation of protein C kinase, tyrosine kinase and MAP kinase, and results in proliferation of the cells. We have also shown that the receptor is over-expressed on platelets from diabetes patients.
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PMID:Biology of endothelium. 981 71

We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR-ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system.
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PMID:An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line. 1072 21

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase-like plasminogen activator (u-PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u-PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u-PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u-PAR in this proliferative phenomenon. Our results show that in vitro u-PA stimulates proliferation of SaOS-2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS-2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u-PAR, and that culturing SaOS-2 cells in the presence of u-PA represents a stimuli for u-PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate-derived u-PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u-PA receptor.
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PMID:ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line. 1150 Sep 57

u-PA contributes to CaP progression, especially in the metastatic androgen-insensitive state. In vitro, u-PA is expressed by androgen-insensitive, but not androgen-sensitive, CaP cell lines. We hypothesized that in androgen-sensitive CaP an activated ARE represses u-PA expression but in androgen-insensitive CaP this repression is lost and u-PA is upregulated through MAP kinase signaling pathways. To determine whether binding of the DHT-AR complex to AREs in the u-PA promoter region represses u-PA transcription in androgen-sensitive CaP, we studied 2 PC3 androgen-insensitive human CaP cell lines stably transfected with AR [PC3(AR)(2) and PC3(AR)(13)] and 1 mock-transfected cell line [PC3(M)]. In the presence of the synthetic androgen mibolerone, both PC3(AR)(2) and PC3(AR)(13), but not PC3(M), cells showed decreased u-PA expression as assayed by Western and Northern blotting. The AR inhibitor flutamide abrogated mibolerone's effect. Androgen regulation of a second gene, PSA, was also demonstrated in the PC3(AR)(2) cell line. To explore the pathway stimulating u-PA expression in CaP, we performed transient transfections in PC3(AR)(2) cells using u-PA promoter-regulated CAT reporter constructs. Compared to full-length u-PA promoter-CAT constructs, either deletion or mutation of the 5' AP-1 or PEA3 site reduced CAT expression. The location of androgen responsiveness in the u-PA promoter was not identified through the combination of promoter search and transient transfection assays, indicating that a more complicated mechanism is involved in the AR-mediated downmodulation of u-PA expression.
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PMID:Regulation of u-PA gene expression in human prostate cancer. 1174 19

Bikunin (bik, also known as 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) in order to evaluate the effect on expression of urokinase-type plasminogen activator (uPA). Preincubation of the cells with bik reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. We next asked whether the mechanism of inhibition of uPA expression by bik is due to interference with MAP kinase, since PMA could also activate a signaling pathway involving MEK/ERK/c-Jun-dependent uPA expression. When cells were preincubated with bik, we could detect suppression of phosphorylation of these proteins, demonstrating that bik markedly suppresses the cell motility possibly through negative regulation of 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. To clarify the role of bik on tumor metastasis, HRA cells were transfected with an expression vector harboring a cDNA encoding for human bik. Transfection of HRA with the bik cDNA resulted in five variants stably expressing functional bik and significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells. Animals with bik* transfectants induced reduced peritoneal dissemination and long term survival. These results suggest that transfection with the bik gene induces the suppression of tumor cell invasion and peritoneal dissemination, and can prolong survival. This pre-clinical animal model offers the possibility to explore gene therapy as a new treatment modality for ovarian cancer.
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PMID:Suppression of urokinase expression and tumor metastasis by bikunin overexpression [mini-review]. 1177 42

The tumor suppressor PTEN possesses lipid and protein phosphatase activities. It has been well established that the lipid phosphatase activity is essential for its tumor-suppressive function via the phosphoinositide 3-kinase (PI3K) and Akt pathways. The precise role of the protein phosphatase activity is still unclear. In the current study, we demonstrate that overexpression of wild-type PTEN in the MCF-7 breast cancer line results in phosphatase activity-dependent decreases in the phosphorylation of ETS-2, which is a transcription factor whose DNA-binding ability is controlled by phosphorylation. Exposure of MCF-7 cells to insulin, insulin-like growth factor 1 (IGF-1) or epidermal growth factor (EGF) can lead to the phosphorylation of ETS-2, Akt and ERK1/2. The MEK inhibitor PD590089 abrogates insulin-stimulated phosphorylation of ETS-2. In contrast, the PI3K inhibitor LY492002 has no effect on insulin-stimulated phosphorylation of ETS-2, despite the fact that it diminishes insulin-stimulated phosphorylation of Akt. Interestingly, overexpression of PTEN in MCF-7 leads to blockade of insulin-stimulated, but not EGF-stimulated, phosphorylation of ERK, accompanied by dramatic decreases in ETS-2 phosphorylation. We further show that the relationship of PTEN and ETS-2 has functional significance by demonstrating that PTEN abrogates activation of the uPA Ras-responsive enhancer, a target of ETS-2 action, in a phosphatase-dependent manner, irrespective of the presence or absence of insulin. Our observations, therefore, suggest that PTEN blocks insulin-stimulated ETS-2 phosphorylation through inhibition of the ERK members of the MAP kinase family independently of PI3K, and that the PTEN effect on the phosphorylation status of ETS-2 may be mediated through PTEN's protein phosphatase activity.
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PMID:PTEN blocks insulin-mediated ETS-2 phosphorylation through MAP kinase, independently of the phosphoinositide 3-kinase pathway. 1209 11

Changes in cellular morphology induced as a consequence of direct perturbation of cytoskeletal structure with network-specific targeting agents (i.e. microfilament- or microtubule-disrupting drugs) results in the stimulated expression of a specific subset of genes. Transcription of c-fos, collagenase, transforming growth factor-beta, actin, urokinase plasminogen activator and its type-1 inhibitor (PAI-1) appears to be particularly responsive to shape-activated signaling pathways. Cytochalasin D (CD) or colchicine treatment of contact-inhibited and serum-deprived vascular smooth muscle (R22) cells was used, therefore, as a model system to evaluate morphology-associated controls on PAI-1 gene regulation in the absence of added growth factors. PAI-1 transcript levels in quiescent R22 cells increased rapidly and in a CD-concentration-dependent fashion, with kinetics of expression paralleling the morphological changes. Colchicine concentrations that effectively disrupted microtubule structure and reduced the cellular 'footprint' area (to approximately that of CD treatment) also stimulated PAI-1 synthesis. Shape-related increases in PAI-1 mRNA synthesis were ablated by prior exposure to actinomycin D. Unlike the mechanism of induction in growth-factor-stimulated cells, CD- and colchicine-induced PAI-1 expression required on-going protein synthesis (i.e. it was a secondary response). Although PAI-1 is a TGF-beta-regulated gene and TGF-beta expression is also shape dependent, an autocrine TGF-beta loop was not a factor in CD-initiated PAI-1 transcription. Since CD exposure resulted in actin microfilament disruption and subsequent morphological changes, with uncertain effects on interactions between signaling intermediates or 'scaffold' structures, a pharmacological approach was selected to probe the pathways involved. Signaling events leading to PAI-1 induction were compared with colchicine-treated cells. CD- as well as colchicine-stimulated PAI-1 expression was effectively and dose dependently attenuated by the MEK inhibitor PD98059 (in the 10 to 25 microM concentration range), consistent with the known MAP kinase dependency of PAI-1 synthesis in growth-factor-stimulated cells. Reduced PAI-1 mRNA levels upon exposure to genistein prior to CD addition correlated with inhibition of ERK1/2 activity, implicating a tyrosine kinase in shape-dependent MEK activation. Src-family kinases, moreover, appeared to be specific upstream elements in the CD- and colchicine-dependent pathways of PAI-1 transcription since both agents effectively activated pp60(c-src) kinase activity in quiescent R22 cells. The restrictive (src-family) kinase inhibitor PP1 completely inhibited induced, as well as basal, ERK activity in a coupled immunoprecipitation myelin-basic-protein-phosphorylation assay and ablated shape-initiated PAI-1 mRNA expression. These data suggest that PP1-sensitive tyrosine kinases are upstream intermediates in cell-shape-associated signaling pathways resulting in ERK1/2 activation and subsequent PAI-1 transcription. In contrast to the rapid and transient kinetics of ERK activity typical of serum-stimulated cells, the ERK1/2 response to CD and colchicine is both delayed and relatively sustained. Collectively, these data support a model in which MEK is a focal point for the convergence of shape-initiated signaling events leading to induced PAI-1 transcription.
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PMID:MEK/ERK pathway mediates cell-shape-dependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks. 1211 65

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI-1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.
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PMID:CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells. 1240 8

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous study defined a functional role of CD40 activation in MM cell homing and migration. In this study, we examine signaling events mediating CD40-induced MM cell migration. We show that cross-linking CD40, using either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb), induces phosphatidylinositol 3-kinase (PI3K) activity and activates its downstream effector AKT in MM.1S cells. CD40 activation also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of extracellular signal-regulated mitogen-activated protein kinase (ERK), but not c-jun amino-terminal kinase (JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as adenoviruses expressing dominant-negative and constitutively expressed AKT, we demonstrate that PI3K and AKT activities are required for CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK phosphorylation only partially (10%-15%) prevents migration, suggesting only a minor role in regulation of CD40-mediated MM migration. We further demonstrate that CD40 induces nuclear factor (NF)-kappa B activation as a downstream target of PI3K/AKT signaling, and that inhibition of NF-kappa B signaling using specific inhibitors PS1145 and SN50 completely abrogates CD40-induced MM migration. Finally, we demonstrate that urokinase plasminogen activator (uPA), an NF-kappa B target gene, is induced by CD40; and conversely, that uPA induction via CD40 is blocked by PI3K and NF-kappa B inhibitors. Our data therefore indicate that CD40-induced MM cell migration is primarily mediated via activation of PI3K/AKT/NF-kappa B signaling, and further suggest that novel therapies targeting this pathway may inhibit MM cell migration associated with progressive MM.
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PMID:CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling. 1243 78


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