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)

The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.
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PMID:Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment. 1167 48

Osteoclasts, cells that resorb bone, die once fully differentiated. Several factors including interleukin-1 (IL-1) have been shown to regulate the survival of mature osteoclasts. However, information on the mechanism underlying the regulation of osteoclast survival has been limited. In this study, we investigated the mechanism for the IL-1-stimulated survival of osteoclasts. Treatment of purified osteoclasts with IL-1alpha led to activation of the serine-threonine kinases Akt and ERK. Blocking the activation of Akt with LY294002, a specific inhibitor of the Akt up-stream molecule PI 3-kinase, or an with adenoviral vector for a dominant-negative form of Akt prevented the stimulation of osteoclast survival by IL-1alpha. PD98059, a specific inhibitor of the ERK-activating kinase MEK1, also abolished the effects of IL-1alpha on ERK activation and osteoclast survival. IL-1alpha reduced the apoptosis of osteoclasts by reducing caspase 3 activity. The IL-1alpha-mediated suppression of apoptosis was abolished by the PI 3-kinase/Akt or MEK1/ERK pathway inhibitor. These findings implicate the PI 3-kinase/Akt and ERK signaling pathways in the promotion of osteoclast survival by IL-1alpha.
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PMID:IL-1alpha stimulation of osteoclast survival through the PI 3-kinase/Akt and ERK pathways. 1175 48

The expression of the choline acetyltransferase (ChAT) enzyme that synthesizes the neurotransmitter acetylcholine (ACh) is upregulated by ciliary neurotrophic factor (CNTF). We studied the involvement of the mitogen-activated protein kinase (MAPK) pathway in regulating ChAT expression in a murine septal cell line. Surprisingly, we found that PD98059 and U0126, two structurally distinct inhibitors of MAPK kinase (MEK1), increased both basal and CNTF-induced ACh production. Transient transfections with ChAT promoter-luciferase reporter construct demonstrated synergy between PD98059 and CNTF at the transcriptional level. Moreover, in cotransfection studies, overexpression of constitutively activated MEK1 completely abrogated the CNTF-mediated induction of the reporter. Blocking MEK1 did not significantly alter CNTF-induced Tyr705 phosphorylation of the principal mediator of the CNTF pathway, the transcription factor Stat3. However, PD98059 inhibited Ser727 phosphorylation of Stat3, demonstrating that the latter is MEK1-dependent. Taken together, these results indicate that activation of the MEK1/MAPK pathway inhibits the CNTF-mediated stimulation of ChAT expression, possibly as a part of a feedback mechanism.
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PMID:Mitogen-activated protein kinase kinase negatively modulates ciliary neurotrophic factor-activated choline acetyltransferase gene expression. 1184 86

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.
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PMID:Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells. 1188 67

Glutathione S-transferase pi (GSTpi) protects cells from death by altering intracellular oxidative stress. In order to understand the mechanism of GSTpi protection, a cell death model induced by serum depletion as the stress was established. Cotransfection of apoptosis signal-regulating kinase 1 (ASK1) and GSTpi cDNA was performed to elucidate the impact of GSTpi on ASK1 activity, as well as on its downstream signals, MKK7 and JNK, and to elucidate the potential protection of GSTpi on 293 cell death induced by serum depletion. The dominant negative mutant of JNK was used to explore if the blocking of the JNK pathway led to cell death inhibition. It was found that GSTpi had a dose-dependent inhibitory effect on activation induced by serum depletion, and also on inhibition both on MKK7 and JNK. Intracellular expression of GSTpi significantly inhibited serum depletion-induced cell death. Blocking the JNK pathway by transfection of a dominant negative form of JNK (JNK (APF)) brought about significant inhibition of cell death induced by serum depletion with an inhibiting rate as high as 15%. All the results suggest that the mechanism of GSTpi protection on serum depletion-induced cell death works through an ASK1-MKK7-JNK pathway.
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PMID:Glutathione S-transferase pi Protects Serum Depletion-induced Cell Death by Inhibiting ASK1-MKK7-JNK Pathway in the 293 Cells. 1205 Aug 8

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.
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PMID:The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways. 1256 21

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70(S6K), in beta-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70(S6K) phosphorylation activation is undefined. Increased glucose metabolism increases [Ca(2+)](i) and [cAMP], and it was investigated if these secondary signals were linked to glucose-induced Erk-1/2 and p70(S6K) activation in pancreatic beta-cells. Blocking Ca(2+) influx with verapamil, or inhibiting protein kinase A (PKA) with H89, prevented glucose-induced Erk-1/2 phosphorylation. Increasing cAMP levels by GLP-1 potentiated glucose-induced Erk-1/2 phosphorylation via PKA activation. Elevation of [Ca(2+)](i) by glyburide potentiated Erk-1/2 phosphorylation, which was also inhibited by H89, suggesting increased [Ca(2+)](i) preceded PKA for glucose-induced Erk-1/2 activation. Adenoviral-mediated expression of dominant negative Ras in INS-1 cells decreased IGF-1-induced Erk-1/2 phosphorylation but had no effect on that by glucose. Collectively, our study indicates that a glucose-induced rise in [Ca(2+)](i) leads to cAMP-induced activation of PKA that acts downstream of Ras and upstream of the MAP/Erk kinase, MEK, to mediate Erk-1/2 phosphorylation via phosphorylation activation of Raf-1. In contrast, glucose-induced p70(S6K) activation, in the same beta-cells, was mediated by a distinct signaling pathway independent of Ca(2+)/cAMP, most likely via mTOR-kinase acting as an "ATP-sensor."
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PMID:Differential activation mechanisms of Erk-1/2 and p70(S6K) by glucose in pancreatic beta-cells. 1266 69

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.
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PMID:Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival. 1268 17

The effect of cadmium (Cd), a significant environmental contaminant, on the expression of glucose-6-phosphate dehydrogenase (G6PDH), has been investigated. G6PDH is the key rate-limiting enzyme in the pentose pathway and the expression of its gene has been shown to be redox-sensitive. We show that incubation of primary rat hepatocytes with Cd induces oxidative stress in a time- and concentration-dependent manner as measured by increases in the cytotoxic parameters, lactate dehydrogenase (LDH) and lipid peroxidation (LPO). Significant increases in LDH leakage and LPO can be measured after 12 and 24 h, respectively, in the presence of 4 microM cadmium chloride. However, prior to significant increases in cytotoxic parameters, and within only 6 h of Cd treatment, significant decreases in reduced glutathione and increases in the expression of G6PDH as measured by mRNA levels and enzyme activity are observed. The signal protein MAP kinase (MAPK) is also induced by Cd within 6 h. Blocking the Cd induction of MAPK using the antioxidant N-acetyl cysteine (10 mM) or Trolox (0.5 mM) or the MEK specific inhibitor PD098059 (20 microM) also blocks the Cd induction of G6PDH suggesting that MAPK is a signal protein involved in the redox regulation of this gene.
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PMID:Mediation of cadmium-induced oxidative damage and glucose-6-phosphate dehydrogenase expression through glutathione depletion. 1271 38

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.
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PMID:Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly. 1278 34


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