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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclooxygenase-2 (COX-2) and ERK-MAPK mitogenic signaling pathways are important in human hepatocellular carcinoma. We investigated the effect of COX-2 inhibition on ERK-MAPK signaling and the effect of combining MEK (MAPK kinase) and COX-2 inhibitors in human hepatocellular carcinoma in vitro. COX and ERK expression were determined by immunoblot in HepG2 and Hep3B cells. COX-2 and MEK activity were determined by prostaglandin E(2) assay and phosphospecific immunoblot, respectively. Cell growth was determined by cell proliferation and cell counts. Apoptosis was determined by DNA fragmentation enzyme-linked immunosorbent assay and flow cytometry. Cell cycle was determined by flow cytometry. HepG2 and Hep3B cells do not express COX-1 or COX-2. Correspondingly, basal and agonist (arachidonic acid, lipopolysaccharide)-stimulated COX-2 activity is undetectable. Treatment of HepG2 and Hep3B cells with NS398 resulted in an increase in ERK1/2 phosphorylation (MEK activity) in a concentration-dependent fashion (NS398, 1 to 100 micromol/L). Treatment with the COX-2 inhibitor NS398 in the presence of U0126 (MEK inhibitor) effectively suppressed ERK1/2 phosphorylation as determined by phosphospecific ERK1/2 immunoblot. Total ERK1/2 and COX-2 were unchanged with NS398 and U0126 treatments. In HepG2 cells, NS398 (1 to 100 micromol/L) decreased apoptosis as determined by DNA fragmentation enzyme-linked immunosorbent assay. Relative apoptosis was increased with U0126 alone or in combination with NS398 (9 to 10 times the control value), eliminating the anti-apoptotic effect of NS398. In Hep3B cells, apoptosis was unchanged with NS398 (1 to 50 micromol/L) or U0126 (1 to 10 micromol/L) alone. The combination of NS398 and U0126 in Hep3B cells resulted in a synergistic increase in apoptosis (10 times the control value). Relative apoptosis in both cell lines strongly correlated with changes in the expression of the antiapoptotic protein Bcl-xL. Cellular growth was assessed by colorimetric proliferation assay and cell counts. HepG2 and Hep3B cells had concentration-dependent inhibition of cell growth with NS398 or U0126 treatment alone. The combination of NS398 and U0126 resulted in complementary inhibitory effects on growth. Growth inhibitory effects in HepG2 and Hep3B cells with combination treatment appear to be, in part, secondary to the induction of G(0)/G(1) and G(2)/M cell cycle arrest, respectively, as determined by flow cytometry. Despite differential signaling in HepG2 and Hep3B cells, the sum effect of combining the COX-2 inhibitor NS398 and the MEK inhibitor U0126 results in enhanced antitumor actions. This novel combination may be useful for in vivo studies of hepatocellular carcinoma.
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PMID:Novel combination of cyclooxygenase-2 and MEK inhibitors in human hepatocellular carcinoma provides a synergistic increase in apoptosis. 1467 12

Growth factors upregulate cyclooxygenase-2 (COX-2) expression and extracellular signal-regulated kinase (ERK) activity, yet little is known regarding the interaction between COX-2 and ERK in terms of mitogenic signal transduction pathways in gastric epithelial cells. Therefore, we examined the role of COX-2 in EGF-induced proliferation of gastric epithelial RGM1 cells. EGF treatment significantly induced ERK activity (peaked at 30 min) and significantly increased COX-2 protein (peaked at 6 hr), production of prostaglandin E2 (PGE2), and cell proliferation. MEK inhibitor (PD98059) decreased ERK activity and cell proliferation induced by EGF. The selective COX-2 inhibitor (NS-398) significantly reduced EGF-induced cell proliferation. Exogenous PGE2 partly reversed the NS-398-induced inhibitory action on cell proliferation, clearly indicating the importance of PGE2 in mitogenic pathway. The induction of COX-2 protein by EGF was completely blocked by preincubation with MEK inhibitor. These results suggest that the ERK-COX-2 pathway is critical for EGF-induced proliferation of gastric epithelial cells.
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PMID:COX-2 is essential for EGF induction of cell proliferation in gastric RGM1 cells. 1471 10

Celecoxib, the first US FDA-approved selective cyclooxygenase-2 (COX-2) inhibitor initially developed for the treatment of adult rheumatoid arthritis and osteoarthritis, was reported to reduce the polyp burden in patients with familial adenomatous polyposis. This specific COX-2 inhibitor also protects against experimentally induced carcinogenesis, but molecular mechanisms underlying its chemopreventive activities remain largely unresolved. In the present work, we found that celecoxib inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of COX-2 in female ICR mouse skin when applied topically 30 min prior to TPA as determined by both immunoblot and immunohistochemical analyses. In another study, celecoxib attenuated the DNA binding activity of activator protein 1 (AP-1) through suppression of c-Jun and c-Fos expression in TPA-treated mouse skin. In addition, celecoxib inhibited both the catalytic activity and phosphorylation of p38 mitogen-activated protein (MAP) kinase. In the same animal model, TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2 and p38 MAP kinase, which are upstream of AP-1 in mouse skin. In order to clarify the roles of p38 and ERK in TPA-induced AP-1 activation, we utilized the pharmacologic inhibitors of these enzymes. The p38 inhibitor SB203580 blocked TPA-mediated AP-1 activation, while the MEK1/2 inhibitor U0126 was not inhibitory despite suppression of c-Fos expression in mouse skin. Furthermore, SB203580 markedly inhibited COX-2 expression induced by TPA. Taken together, these findings suggest that celecoxib down-regulates COX-2 by blocking activation of p38 MAP kinase and AP-1, which may represent molecular mechanisms underlying antitumor promoting effects of this drug on mouse skin tumorigenesis.
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PMID:Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin. 1472 83

Both UVB (280-320 nm) and UVA (320-400 nm) radiation lead to an enhanced expression of cyclooxygenase-2 (COX-2) in epidermal cells in various in-vitro and in-vivo models. It is demonstrated here that the expression of COX-2 is induced in artificial human epidermis exposed to simulated solar light (>290 nm). Employing filters eliminating specified regions from the simulated solar spectrum, the UVB and UVA-2 (320-350 nm) regions are shown to fully account for induction of COX-2 mRNA and protein as well as the enhanced production of prostaglandin E(2) after irradiation. At the protein level, approximately 70% of the total induction by solar light is due to light in the UVA-2 region. UVA-1 (350-400 nm), visible light and IR radiation are practically ineffective. COX-2 induction by simulated solar light is attenuated in the presence of inhibitors of p38(MAPK) or of c-Jun-N-terminal kinases (JNK), whereas COX-2 induction by UVA is blocked only by inhibition of JNK. UV-induced COX-2 expression is not affected by inhibition of the MEK 1,2/ERK 1,2 pathways.
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PMID:Contribution of UVB and UVA to UV-dependent stimulation of cyclooxygenase-2 expression in artificial epidermis. 1499 41

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.
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PMID:Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages. 1500 72

Based on evidence that arsenic modulates proinflammatory events that are involved in skin carcinogenecity, we hypothesized that in normal human epidermal keratinocytes (NHEK) arsenic increases expression of the procarcinogenic enzyme cyclooxygenase-2 (COX-2) and that this occurs via specific mitogen and stress signaling pathways. To test this hypothesis, NHEK were exposed to sodium arsenite, and COX-2 expression, prostaglandin E2 (PGE(2)) secretion, mitogen-activated protein kinase (MAPK) phosphorylation, and DNA synthesis were quantified. Inhibitors of p42/44 and p38 MAPKs were used to evaluate the contribution of mitogen and stress signaling to the modulation of COX-2. Our results demonstrate that arsenite (0.005-5 microM) elevates COX-2 expression, PGE(2) secretion (2.5-5 microM), and DNA synthesis (1-5 microM). Arsenite stimulated p42/44 but not p38 MAPK phosphorylation (2.5 microM), responses different than those produced by epidermal growth factor. Inhibition of mitogen-activated protein kinase kinase (MAPKK) and p38 MAPK using PD98059 (20 microM) and SB202190 (5 microM), respectively, attenuated the elevation of COX-2 protein induced by arsenite, whereas physiological concentrations of three COX-2 inhibitors (e.g., NS-398, piroxicam, and aspirin) reduced arsenite-stimulated DNA synthesis. These data indicate that arsenite elevates COX-2 in NHEK at the transcriptional and translational levels as well as increases PGE(2) secretion. Compounds that inhibit COX-2 expression and activity may be useful in the scientific study, prevention, and treatment of arsenic skin carcinogenesis and deserve further investigation.
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PMID:Micromolar concentrations of sodium arsenite induce cyclooxygenase-2 expression and stimulate p42/44 mitogen-activated protein kinase phosphorylation in normal human epidermal keratinocytes. 1505 98

High density lipoproteins (HDL) induce prostacyclin (PGI(2)) release in vascular smooth muscle cells (VSMC) by up-regulation of cyclooxygenase-2 (Cox-2). Our goal was to analyse the mechanisms underlying this effect, and its potential modulation by HMG-CoA reductase inhibition in human VSMC. The contribution of mitogen-activated protein kinase (MAPK) signalling pathways was assessed by Western blot analysis and using specific inhibitors [PD098059 for p42/44 MAPK kinase (MEK); SB203580 for p38 MAPK or L-JNKI1 for c-Jun N-terminal kinase-1 (JNK-1)]. HDL-induced PGI(2) release was inhibited by rofecoxib (a specific Cox-2 inhibitor, 5 microM). HDL induced the early activation of p42 MAPK, p38 MAPK and JNK-1. p42/44 MAPK was the major pathway involved in both Cox-2 up-regulation and PGI(2) synthesis; p38 MAPK was also involved in both processes while JNK inhibition only affected PGI(2) synthesis. Pertussis toxin (an inhibitor of Galphai/Galphao proteins) prevented MAPK activation and inhibited both Cox-2 up-regulation and PGI(2) release. Genistein (a tyrosine kinase inhibitor) inhibited PGI(2) release without affecting MAPK activation or Cox-2 up-regulation. Simvastatin (0.1-1 microM) increased HDL-induced PGI(2) release ( approximately 45% at 1 microM) but did not significantly modify early MAPK activation or Cox-2 expression. Simvastatin alone did not significantly affect PGI(2) release. Our results suggest that mechanisms associated with G protein-coupled receptor activation, trigger Cox-2 up-regulation and PGI(2) release via multiple MAPK signalling pathways in VSMC. The mechanism is independent of tyrosine kinase receptors, although cytosolic tyrosine kinases could activate Cox-2 post-translationally. The potential contribution of HDL to vascular homeostasis, via increases in PGI(2) synthesis, could be enhanced by HMG-CoA reductase inhibitors.
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PMID:Simvastatin potenciates PGI(2) release induced by HDL in human VSMC: effect on Cox-2 up-regulation and MAPK signalling pathways activated by HDL. 1513 60

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.
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PMID:Raf and RhoA cooperate to transform intestinal epithelial cells and induce growth resistance to transforming growth factor beta. 1514 Sep 45

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high-level prostaglandin production during inflammation and carcinogenesis. In this study, the transcriptional regulation of COX-2 expression induced by epidermal growth factor (EGF) in human epidermoid carcinoma A431 cells was studied. EGF treatment induced the expression of COX-2 mRNA, protein, promoter and enzyme activity in a time-dependent manner. EGF-induced COX-2 promoter activity was inhibited by overexpression of the dominant-negative forms of Ras and ERK2. Induction of COX-2 and c-Jun by EGF was completely suppressed by MEK inhibitor combined with JNK inhibitor. Analysis of the COX-2 promoter binding proteins by gel mobility shift assay and DNA affinity precipitation assay revealed that c-Jun and p300 binding to CRE/E-box site were responsible for the EGF-induced COX-2 gene transcription. Overexpression of p300 significantly enhanced COX-2 promoter activity in cells overexpressed of c-Jun or treated with EGF. EGF- and c-Jun-induced transcription of COX-2 promoter was repressed by cotransfection of E1A in a dose-dependent manner. All together, these results indicated that the EGF-induced expression of COX-2 in A431 cells was mediated through the Ras-ERK/JNK signaling pathway, and subsequent induction of c-Jun following MAPK activation, in cooperation with coactivator p300, was required for the EGF response.
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PMID:Essential role of c-Jun induction and coactivator p300 in epidermal growth factor-induced gene expression of cyclooxygenase-2 in human epidermoid carcinoma A431 cells. 1523 18

We monitored cyclooxygenase-2 (COX-2) expression in the insulin-like growth factor-II (IGF-II) treated human keratinocytes and explored the IGF-II signaling pathways with respect to the expression of COX-2. IGF-II induced COX-2 mRNA and protein levels, and the up-regulation of COX-2 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src and PI3-kinase. The inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) 1 also reduced the increased expression of COX-2 by IGF-II, but the inhibition of p38 did not. To further examine the roles of these mitogen-activated protein kinases (MAPKs) in IGF-II-induced COX-2 expression, we performed COX-2 promoter analysis using dominant negative plasmids of MEK1 (DN-MEK1), p38 (DN-p38) and JNK1 (DN-JNK1). Although IGF-II increased COX-2 promoter activity approximately 2.5-fold, this increase was blocked by cotransfection with DN-MEK1 or DN-JNK1. However, DN-p38 did not block the IGF-II-induced COX-2 promoter activity. In addition, inhibition of ERK or JNK1 reduced the increase of IGF-II-induced prostaglandin E(2) synthesis or cell proliferation. These results suggest that IGF-II induces COX-2 expression through the tyrosine kinase-Src-ERK and tyrosine kinase-PI3-kinase pathways, but not via p38 MAPK pathway, and that the basal JNK activity is required for the upregulation of COX-2 by IGF-II, as well.
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PMID:IGF-II-mediated COX-2 gene expression in human keratinocytes through extracellular signal-regulated kinase pathway. 1530 95


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