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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Binding of the urokinase-type plasminogen activator (uPA) to its receptor activates diverse cell signaling pathways. How these signals are integrated so that cell physiology is altered remains unclear. In this study, we demonstrated that migration of MCF-7 breast cancer cells and HT-1080 fibrosarcoma cells on serum-coated surfaces is stimulated by agents that activate ERK, including uPA, epidermal growth factor, and constitutively active MEK1. The promigratory activity of these agents was entirely blocked not only by the MEK-specific antagonist PD098059, but also by antagonists of the Rho-Rho kinase pathway, including Y-27632 and dominant-negative RhoA (RhoA-N19). uPA did not significantly increase the level of GTP-bound RhoA, suggesting that the constitutive activity of the Rho-Rho kinase pathway may be sufficient to support ERK-stimulated cell migration. Paradoxically, Y-27632 and RhoA-N19 increased ERK phosphorylation in MCF-7 cells, providing further evidence that ERK activation alone does not promote cell migration when Rho kinase is antagonized. When MCF-7 cell migration was stimulated by ERK-independent processes such as expression of the beta(3) integrin subunit or changing the substratum to type I collagen, Y-27632 and RhoA-N19 failed to inhibit the response. This study supports a model in which the Ras-ERK and Rho-Rho kinase pathways cooperate to promote cell migration. Neutralizing either pathway is sufficient to block the response to agents that stimulate cell migration by activating ERK.
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PMID:Cooperativity between the Ras-ERK and Rho-Rho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration. 1180 8

Activation of focal adhesion kinase (FAK), overexpressed in several human cancers, induces survival, proliferation and motility of cells in culture, but its functional importance in human tumor growth in vivo has not been elucidated. I explored the role of FAK in regulating tumorigenicity of human carcinoma cells, HEp3. These cells overexpress urokinase receptor (uPAR) which, by activating alpha5beta1 integrin, initiates an intracellular signal through FAK and Src leading to ERK activation and tumorigenicity in vivo. Down regulation of uPAR in these cells led to an approximately 3-5-fold reduction in FAK phosphorylation and association with Src and dormancy in vivo. Both FAK phosphorylation and ability to grow in vivo were restored by re-expression of uPAR. The FAK signaling pathway in T-HEp3 cells, measured by FAK phosphorylation, GTP-loaded Ras and ERK activation, was inhibited by transient or stable transfection of FAK related non-kinase (FRNK), known to have a dominant negative function, but not by a FRNK mutant version (S1034-FRNK). Most importantly, while vector- and mutant-S1034-FRNK transfected cells inoculated onto chicken embryo CAMs formed progressively growing tumors, the HA-FRNK-expressing T-HEp3 cells did not proliferate in vivo and remained dormant for at least 6 weeks. Both cell types had similar rate of apoptosis in vivo and the p38(SAPK) or PI3K-Akt signaling pathways were unaffected by FRNK. FRNK induced dormancy could be reverted by expression of an active-R4F-Mek1 mutant. These results show that active FAK is an important mediator of uPAR-regulated tumorigenicity of HEp3 cells and that interruption of FAK mitogenic signaling either through down-regulation of uPAR or by expression of FRNK can force human carcinoma cells into dormancy.
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PMID:Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo. 1197 Nov 86

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

In order to identify metastasis-associated and promoting genes of pancreatic carcinoma we investigated the transcriptional profile of rat pancreatic carcinoma cell lines BSp73-AS (non-metastatic) and BSp73-ASML (highly metastatic) with Affymetrix GeneChip Array technology. We analyzed the expression profile of 7000 genes. Two hundred and ten genes (3%) were up-regulated and 247 genes (3.5%) were down-regulated in the metastatic cell line based on a fold change of expression of at least 3 and a change factor quality of > or = 2. In order to classify the de-regulated genes we defined the following categories: proteases and protease-related genes, cytokines, receptor tyrosine kinases, other transmembrane proteins/receptors, transcription, cell cycle/apoptosis, signaling, adhesion/extracellular matrix, metabolism, detoxification, protein modification, trafficking, immune response and other genes. We identified de-regulated AP1, FRA-1 and c-myc-mediated transcription in cell line BSp73-ASML. Up-regulation of transmembrane tyrosine kinase receptors c-met, IGFR1, IGFR2 and EGFR family-related ligands such as HB-EGF, TGFa, amphiregulin and neuregulin as well as c-met ligand HGF point to a possible role of this system in metastasis. We identified 56 non-tyrosine kinase transmembrane receptors as new target candidates for inhibition of metastasis, four of them representing already validated targets. In addition, we identified MMP9, uPA, uPAR, cyclin D1 and S100A4 (mts1) as possible contributors of the metastatic phenotype.
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PMID:Identification of rat pancreatic carcinoma genes associated with lymphogenous metastasis. 1217 79

To explore the hypothesis that aging not only increases breast cancer incidence but also alters breast cancer biology, we correlated patient age and diagnosis with tumor histology, stage and biomarkers independently determined from two different tumor archives: an American collection of approximately 800 paraffin-embedded and immunohistochemically analyzed primary breast cancers, and an European collection of approximately 3000 cryobanked primary breast cancers analyzed by ligand-binding and enzyme immunoassay (EIA). The prognostic biomarkers chosen for comparison represented surrogate measures of tumor: (i). proliferation, growth and genetic instability (mitotic and apoptotic indices, Ki-67/MIB-1-positivity, nuclear grade, p53-positivity), (ii). endocrine-dependence (estrogen receptor (ER), progesterone receptors (PR), pS2, Bcl2), (iii). growth factor receptor-dependence (ErbB2, EGFR/ErbB1), and (iv). angiogenic, invasive and proteolytic potential (uPA, PAI-1, Cathepsin D, VEGF). No biomarker reflecting tumor angiogenic, invasive or proteolytic potential showed a significant correlation with patient age at diagnosis. In contrast, significant inverse correlations (|r|>0.1; P< or =0.05) were observed for all measures of tumor growth and genetic instability as well as growth factor receptor overexpression (ErbB2 or EGFR positivity). Only one marker of endocrine-dependence, ER expression, showed a significant positive correlation with patient age at diagnosis. In summary, these findings support the hypothesis that breast cancer biology is significantly affected by patient age. In particular, breast tumors arising in older patients have slower growth rates, are more likely to be ER-positive, and are less likely to be p53-positive, EGFR-positive or ErbB2-positive.
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PMID:Age-associated biomarker profiles of human breast cancer. 1220 28

The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88-92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88-92 (P88-92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88-92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88-92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88-92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions.
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PMID:The cleavage of the urokinase receptor regulates its multiple functions. 1229 5

Activation of phosphoinositide 3-kinase (PI3-kinase) is involved in many cellular responses. FGF-2 is one of the potent inducers of urokinase-type plasminogen activator (uPA) production in endothelial cells. However, little is known about the molecular mechanisms underlying FGF-2-mediated uPA production. Here we examined the signal transduction pathways involved in the regulation of uPA production by FGF-2-treatment. FGF-2 potently upregulated uPA production in murine brain capillary endothelial cells (IBE cells), as well as porcine aortic endothelial (PAE) cells and L6 myoblasts ectopically expressing FGFR1. PI3-kinase inhibitors, wortmannin and LY294002, both enhanced FGF-2-dependent uPA production by these cells. Stable expression of activated mutant p110alpha catalytic subunit of PI3-kinase into IBE cells decreased FGF-2-mediated uPA production, suggesting that PI3-kinase exhibited the negative regulatory effect on uPA production. No increase in FGF-2-induced PI3-kinase activity was observed in proteins immunoprecipitated by anti-phosphotyrosine antibody. Although stable expression of deleted mutant p85alpha regulatory subunit, which lacks association with p110 catalytic subunit, in IBE cells showed no dominant negative effect, transient expression of dominant negative Ras inhibited FGF-2-mediated PI3-kinase activation. These results suggest that only activated Ras contributed the FGF-2-mediated PI3-kinase activation. In cells stably expressing mutant p85alpha subunit, FGF-2 efficiently induced uPA production. Taken together, activation of PI3-kinase by FGF-2 is Ras-dependent and results in down-regulation of uPA production.
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PMID:Negative regulation of urokinase-type plasminogen activator production through FGF-2-mediated activation of phosphoinositide 3-kinase. 1237 Aug 24

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.
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PMID:A Kunitz-type protease inhibitor, bikunin, inhibits ovarian cancer cell invasion by blocking the calcium-dependent transforming growth factor-beta 1 signaling cascade. 1249 70


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