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)

We previously described that the major promoter (M) of human choline acetyltransferase (ChAT) gene is activated by three inhibitors of histone deacetylase, butyrate, trichostatin and trapoxin, in transfected CHP126 neuroepithelioma cells. We now show that trapoxin and butyrate triggered a rapid and transient phosphorylation of ERK1/2 kinases, that was suppressed by PD98059, a highly specific inhibitor of MAP kinase kinase MEK1. The stimulation of ChAT promoter activity by trapoxin or butyrate did not require ongoing protein synthesis, and was suppressed by PD98059. The overexpression of dominant negative mutants of H-ras or ERK2 proteins depressed ChAT promoter activation by trapoxin in transient transfection assays. Conversely, the overexpression of constitutively active mutants of H-ras or MEK1 proteins had little or no effect on ChAT promoter activity, but strongly synergized with trapoxin. These data thus suggest that the activation of the MEK/ERK kinase cascade plays a necessary, but not sufficient, role in the regulation of ChAT promoter by inhibitors of histone deacetylase.
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PMID:Activation of the MAP kinase cascade by histone deacetylase inhibitors is required for the stimulation of choline acetyltransferase gene promoter. 960 89

We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.
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PMID:Cooperation between phosphorylation and acetylation processes in transcriptional control. 1020 71

Conditioned medium from stimulated microglia and from the monocyte/macrophage cell line (RAW 264.7; MC-CM) promotes the differentiation of cholinergic neurons from undifferentiated progenitors in the septal nuclei and adjacent basal forebrain (BF). We have studied the regulation of this process by measuring the activity of choline acetyltransferase (ChAT) in cultured BF taken from embryonic day 16 rat brain. Inhibition of either xanthine oxidase with allopurinol or nitric oxide synthase with N(G)-monomethyl-l-arginine produces a small but significant improvement in the efficacy of MC-CM while inclusion of pyrrolidine dithiocarbamate, a hydroxyl radical scavenger widely used as an antioxidant, lowers MC-CM-induced ChAT activity. Addition of nerve growth factor (NGF) but not brain-derived neurotrophic factor or glial-derived neurotrophic factor together with MC-CM has a synergistic effect on both ChAT activity and ChAT mRNA, raising ChAT activity as much as 29-fold and ChAT mRNA almost 15-fold. While MC-CM raised mRNA for trkA, the effect was not synergistic with NGF. mRNA for the common neurotrophin receptor (p75NTR) showed a modest synergistic increase. Blockade of the Ras/Raf/ERK [extracellular signal-regulated kinase, also known as mitogen-activated protein [(MAP) kinase] signal transduction pathway with either PD28059 (an inhibitor of MAP kinase/ERK kinase kinase or MEK) or N-acetyl-S-farnesyl-l-cysteine (an inhibitor of Ras farnesylation and, hence, activation) inhibited the action of MC-CM. Moreover, a subpopulation of cells responded rapidly to MC-CM with an increased appearance of phosphorylated ERK. Because NGF also utilizes this pathway, synergy may occur along this signal transduction pathway.
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PMID:Macrophage cell-conditioned medium promotes cholinergic differentiation of undifferentiated progenitors and synergizes with nerve growth factor action in the developing basal forebrain. 1068 94

The common neurotrophin receptor (p75NGFR) can signal in vitro through activation of the c-Jun N-terminal kinase (JNK) pathway and nuclear translocation of NFKappaB. Activation of JNK and its substrate c-Jun can lead to apoptosis. We investigated these activities in vivo by comparing immunoreactivity for phosphorylated(p) SEK-1 (or MKK4, which activates JNK), c-Jun (ser63, ser73) and nuclear translocation of NFKappaB-p50 in tissue sections through the forebrain of control and p75NGFR-deficient mice. During postnatal development, SEK1p-immunoreactivity was detectable in p75NGFR-positive cholinergic neurons and p75NGFR-negative neurons throughout the forebrain in control mice. During development, few cells contained c-Junp, although many neurons contained c-Jun. No obvious c-Jun immunostaining was present in the adult forebrain. At any age, NFKappaB-p50 immunoreactivity was seen in nuclei of most cells throughout the forebrain. Following fimbria fornix transection in adult mice, few basal forebrain neurons contained SEK1p while many axotomized choline acetyltransferase (ChAT)-positive neurons contained c-Junp and nuclear NFKappaB-p50. The immunostaining patterns of SEK1p, c-Junp and NFKB during development and following injury were largely similar in p75NGFR-deficient mice. During development, cells throughout the forebrain had TdT-mediated dUTP-biotin nick end labelling (TUNEL)-labelling (a potential marker for apoptosis), however, their presence was not predicted by number of neurons stained for SEK1p or c-Junp. These results suggest that the expected activation of the JNK pathway by p75NGFR, as well as the expected relationship between SEK1 and downstream activation of c-Jun do not occur in the mammalian forebrain. Also, these results suggest that this activation does not necessarily lead to cell death.
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PMID:SEK1/MKK4, c-Jun and NFKappaB are differentially activated in forebrain neurons during postnatal development and injury in both control and p75NGFR-deficient mice. 1088 28

We investigated the effects of antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) on the expression of choline acetyltransferase (ChAT) in cultured cholinergic precursors from the embryonic rat septal nuclei and basal forebrain. Carboxy-dichlorofluorescein fluorescence confirmed that 2-ME inhibited intracellular oxidation. Low micromolar concentrations of 2-ME produce as much as a 12-fold increase in ChAT; this is enhanced further by inclusion of nerve growth factor (NGF). NAC effects are biphasic: 0.15 mM produces profound increases in ChAT while 1.5 mM has no effect. Immature (E16) cultures respond with increases in ChAT while more highly differentiated cultures (E18) do not. Labeling of single precursors with a lacZ-expressing retrovirus reveals that the increase in ChAT is due primarily to an increased number and size of clones, not an increase in cholinergic neurons per clone, suggesting an effect on precursor survival. Inhibition of ras farnesylation inhibits both 2-ME and NAC induction of ChAT suggesting a ras-mediated pathway. Inclusion of the MEK inhibitor PD98059 does not affect low doses of NAC, but at doses of NAC that fail to increase ChAT activity, inhibition of the pathway actually raises ChAT. Immunocytochemical investigation of the cultures indicates that cells exposed to low doses of NAC develop healthy neuronal arbors in the apparent absence of glial support. At higher concentrations of NAC, neurons were found in association with astrocytes, making contact via elaborate varicose fibers. Treatment of the cultures with PD98059 to inhibit MEK returned cultures to a 'low-dose' phenotype. These data suggest that redox status of basal forebrain precursors affect both their survival and differentiative potential.
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PMID:Antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) affect the survival and differentiative potential of cholinergic precursors from the embryonic septal nuclei and basal forebrain: involvement of ras signaling. 1167 23

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

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single "cholinergic locus," can be differentially or synergistically regulated, depending on the activated signaling pathways.
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PMID:Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways. 1267 45

Postganglionic parasympathetic neurons in guinea-pig cardiac ganglia exhibit choline acetyltransferase (ChAT)-immunoreactivity, and a large fraction (60%) of the ChAT-positive cardiac neurons co-express somatostatin-immunoreactivity. This co-expression remained when the cardiac ganglia explants were maintained in culture for 72 h (40% somatostatin-immunoreactive). The guinea-pig cardiac ganglia neurons express the high affinity pituitary adenylate cyclase activating polypeptide (PACAP)-selective PAC1 receptor, and treatment of the ganglia explants with 20 nM PACAP27 for 72 h to evaluate PACAP regulation of somatostatin expression revealed a dramatic 85% decrease in the number of somatostatin-IR neurons (6% somatostatin-IR neurons) compared with untreated control explant preparations. The decrease in percentage of somatostatin-IR neurons by PACAP27 was time- and concentration-dependent, and selective for PACAP27; PACAP38 and vasoactive intestinal polypeptide were less effective. PACAP6-38, a PACAP antagonist, eliminated the PACAP27-induced change in somatostatin positive neurons. The PACAP-mediated decrease in somatostatin-IR neurons was eliminated in calcium-deficient solutions and by the addition of nifedipine, indicating a requirement for calcium influx through L-type calcium channels. The addition of either the calmodulin inhibitor N-(4-aminobutyl)-1-naphthalenesulfonamide or the MEK inhibitor PD98059, also eliminated the PACAP27-induced decrease in somatostatin-IR cells. The PACAP27-mediated effect on somatostatin expression was not affected by inhibitors of protein kinase A or phospholipase C, but was reduced by the adenylyl cyclase inhibitor SQ22356, suggesting cAMP involvement. Semiquantitative and quantitative reverse transcription PCR prosomatostatin transcript measurements showed that cardiac ganglia prosomatostatin mRNA levels were not diminished by chronic PACAP27 exposure despite the dramatic decrement in somatostatin-expressing neurons. Neuronal peptide-IR content represents a balance between production and secretion. These results suggested that one of the primary effects of PACAP exposure may be enhanced levels of neuropeptide release that exceeded production levels, resulting in somatostatin depletion and a decrement in the number of identifiable somatostatin-expressing cardiac neurons.
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PMID:Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia. 1520 51

Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.
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PMID:Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol-3'-kinase pathway. 1568 78

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.
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PMID:PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases. 1799 38


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