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)

Previously, we demonstrated that the anti-inflammatory drug chloroquine (CQ) inhibited LPS-induced TNF-alpha transcription. To define further the mechanism of CQ, we studied the effect of this drug on mitogen-activated protein kinase signaling pathways involved in regulation of TNF production. CQ interfered with phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and the ERK-activating kinases mitogen-activating protein/ERK kinase (MEK)1/2. Both CQ and PD98059, a MEK1 inhibitor, reduced luciferase reporter activity driven by human TNF promoter sequences. However, CQ appeared to mediate these effects by deactivating Raf, the upstream activator of MEK. These findings were supported by functional data demonstrating that CQ and PD98059 interfered with TNF expression in several human and murine cell types while neither inhibitor blocked TNF production in murine RAW264.7 macrophages, a cell line that does not require MEK-ERK signaling for TNF production. Finally, we evaluated whether CQ could sensitize HeLa cells to undergo anti-Fas-mediated apoptosis, an effect observed when ERK activation is interrupted in this cell line. CQ rendered HeLa cells sensitive to anti-Fas treatment in a manner similar to PD98059. Taken together, these data argue that therapeutic concentrations of CQ interfere with ERK activation by a novel mechanism, an effect that could be responsible, at least in part, for the potent anti-inflammatory effects of this drug.
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PMID:Inhibition of mitogen-activated protein kinase signaling by chloroquine. 1199 88

Heme oxygenase-1 (HO-1) is an enzyme that is highly inducible by various cellular stressors, especially oxidant injury. Our laboratory and others have demonstrated that induction of HO-1 exerts an antiinflammatory effect both in vitro and in vivo. We hypothesized that carbon monoxide (CO), a major catalytic byproduct of heme catalysis by HO-1, may mediate this antiinflammatory effect by modulating signal transduction pathways, in particular the mitogen-activated protein (MAP) kinase pathway. Confluent primary cultures of rat pulmonary artery endothelial cells (RPAEC) were treated with tumor necrosis factor-alpha (TNF-alpha; 50 ng/ml), and whole-cell extracts were assayed for phosphorylated ERK1/2, JNK1/2, and p38 MAP kinases. These three major MAP kinase pathways were activated by TNF-alpha in a time-dependent manner. RPAEC treated with TNF-alpha in the presence of a low concentration of CO (1%) exhibited marked attenuation of the phosphorylation of ERK1/2 MAP kinase when compared with cells treated with TNF-alpha alone. A similar effect was seen on the upstream MEK 1/2 kinase. Interestingly, CO (1%) accentuated TNF-alpha-induced phosphorylated p38 MAP kinase. These effects of exogenous CO on the ERK1/2 and p38 systems could be replicated by overexpression of HO-1 in RPAEC, using an adenoviral vector. As these MAP kinases are implicated in the regulation of various inflammatory molecules and adhesion molecules, our data provide a potential mechanism by which HO-1, acting via CO, may modulate the inflammatory response by differential activation of the MAP kinase pathway.
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PMID:Differential modulation by exogenous carbon monoxide of TNF-alpha stimulated mitogen-activated protein kinases in rat pulmonary artery endothelial cells. 1200 75

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.
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PMID:Caspase inhibitors induce a switch from apoptotic to proinflammatory signaling in CD95-stimulated T lymphocytes. 1220 31

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-kappaB pathways downstream of TNF-alpha in the context of alpha(2) type I collagen gene (COL1A2) expression. In JNK1-/--JNK2-/- (JNK-/-) fibroblasts, TNF-alpha inhibited basal COL1A2 expression but had no effect on TGF-beta-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-kappaB essential modulator-/- (NEMO-/-) fibroblasts, lack of NF-kappaB activation did not influence the antagonism exerted by TNF-alpha against TGF-beta but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-alpha, critical kinases upstream of the JNK and NF-kappaB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-alpha to inhibit the expression of a given gene, COL1A2, depending on its activation state.
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PMID:Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha. 1239 55

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.
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PMID:Lipopolysaccharide down-regulates the leukotriene C4 synthase gene in the monocyte-like cell line, THP-1. 1257 84

Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through binding to receptor type 1 (TNFR1) molecules expressed on the cell surface. The majority of TNFR1 molecules are localized to the Golgi apparatus. Soluble forms of TNFR1 (as well as of TNFR2) can be shed from the EC surface and inhibit TNF actions. The relationships among cell surface, Golgi-associated, and shed forms of TNFR1 are unclear. Here we report that histamine causes transient loss of surface TNFR1, TNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi in cultured human EC. The Golgi pool of TNFR1 serves both to replenish cell surface receptors and as a source of shed receptor. Histamine-induced shedding is blocked by TNF-alpha protease inhibitor, an inhibitor of TNF-alpha-converting enzyme, and through the H1 receptor via a MEK-1/p42 and p44 mitogen-activated protein kinase pathway. Cultured EC with histamine-induced surface receptor loss become transiently refractory to TNF. Histamine injection into human skin engrafted on immunodeficient mice similarly caused shedding of TNFR1 and diminished TNF-mediated induction of endothelial adhesion molecules. These results both clarify relationships among TNFR1 populations and reveal a novel anti-inflammatory activity of histamine.
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PMID:Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating TNF receptor shedding from the cell surface and Golgi storage pool. 1264 54

Salmonella has developed ways to modulate host cellular response in order to survive. Although the steps required for such modulation have been incompletely characterized, there is increasing evidence for a role for SptP, a type III secretion protein. In part, the actions of SptP are thought to be mediated through its reported inhibition of the extracellular-regulated kinase (ERK) MAP kinase pathway. In the present studies, a series of transfections were performed in which various constitutively activated components of the MAP kinase pathway were co-transfected with SptP in order to determine the mechanism by which SptP inhibits this MAP kinase activation. SptP was found to inhibit the activation of ERK stimulated by both a constitutively active form of Ras and a partially activated form of Raf-1 containing a phospho-mimetic mutation (Raf Y340D). In contrast, the activation of ERK by constitutively active forms of MAP kinase kinase (MEK) was not inhibited, suggesting that the actions of SptP were mediated by Raf-1. In order to determine how SptP might interfere with activation of Raf, we utilized a membrane-localized form of Raf. Constitutive membrane-localization of Raf (RafCAAX), resulting in partial activation, did not prevent inhibition by SptP. However, introduction of an additional, partially activating (Y340D) phospho-mimetic mutation, to RafCAAX, dramatically reduced the ability of SptP to inhibit Raf action. Comparison of SptP mutants, lacking either GTPase-activating protein (GAP) activity or tyrosine phosphatase activity, further suggested that SptP inhibits both the membrane localization and subsequent phosphorylation required for activation of Raf. Both tyrosine phosphatase activity and GAP activity were responsible for SptP inhibition of Raf(Y340D)-induced ERK activation, but only GAP activity was responsible for inhibition of the membrane localized forms of Raf-1. To assess the biological significance of SptP, we examined tumour necrosis factor (TNF)-alpha induction following Salmonella infection. SptP gene deletion enhanced the capacity of Salmonella to induce TNF-alpha secretion following infection of J774A.1 macrophage cells.
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PMID:SptP, a Salmonella typhimurium type III-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation. 1267 84

Monocytes from patients with sickle cell disease (SCD) are in an activated state. However, the mechanism of activation of monocytes in SCD is not known. Our studies showed that placenta growth factor (PlGF) activated monocytes and increased mRNA levels of cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) and chemokines (monocyte chemotactic protein-1 [MCP-1], IL-8, and macrophage inflammatory protein-1beta [MIP-1beta]) in both normal monocytes and in the THP-1 monocytic cell line. This increase in mRNA expression of cytochemokines was also reflected in monocytes derived from subjects with SCD. We studied the PlGF-mediated downstream cellular signaling events that caused increased transcription of inflammatory cytochemokines and chemotaxis of THP-1 monocytes. PlGF-mediated cytochemokine mRNA and protein expression was inhibited by PD98059 and wortmannin, inhibitors of mitogen-activated protein kinase kinase (MAPK/MEK) kinase and phosphatidylinositol-3 (PI3) kinase, respectively, but not by SB203580, a p38 kinase inhibitor. PlGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), which was completely inhibited by wortmannin, indicating that activation of PI3 kinase preceded MEK activation. PlGF also induced transient phosphorylation of AKT. MEK and PI3 kinase inhibitors and antibody to Flt-1 abrogated PlGF-induced chemotaxis of THP-1 monocytes. Overexpression of a dominant-negative AKT or a dominant-negative PI3 kinase p85 subunit in THP-1 monocytes attenuated the PlGF-mediated phosphorylation of ERK-1/2, cytochemokine secretion, and chemotaxis. Taken together, these data show that activation of monocytes by PlGF occurs via activation of Flt-1, which results in activation of PI3 kinase/AKT and ERK-1/2 pathways. Therefore, we propose that increased levels of PlGF in circulation play an important role in the inflammation observed in SCD via its effects on monocytes.
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PMID:Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. 1268 30

Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-kappa B activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-kappa B p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-alpha and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.
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PMID:Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A. 1270 21

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.
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PMID:Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line. 1273 6


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