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 purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.
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PMID:Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2. 893 Jan 66

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces HL-60 cells to differentiate along the monocyte/macrophage pathway and stimulates expression of the extracellular adhesion protein osteopontin (OPN). In this study, the mechanism of TPA-mediated OPN mRNA expression and its relationship to differentiation were investigated. The induction of OPN mRNA by TPA was dose dependently inhibited by staurosporine (0.4-10.0 nM) and chelerythrine (0.1-5.0 microM), indicating that OPN expression requires PKC activation. Furthermore, the mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD 098059 (1.0-10.0 microM), inhibited the effect of TPA in a dose-dependent fashion. Cycloheximide (10 microg/ml) ablated the induction of OPN mRNA by TPA. To determine if OPN mRNA expression was associated with a particular differentiational pathway, HL-60 cells were treated with RA, 9-cis-RA, calcitriol, or sodium butyrate. None of these agents stimulated OPN mRNA. Treatment with TPA subsequent to a 120-h pretreatment with retinoic acid (RA), 9-cis-RA, or calcitriol resulted in a potentiation of the induction of OPN mRNA. These results support a role for protein kinase C (PKC) in promoting OPN expression because each of these agents increased PKC levels. An hOPN promoter/reporter construct was responsive to TPA, indicating that this effect is at the level of transcription. Thus, TPA-stimulated transcription of the OPN gene apparently occurs via a PKC/MAPK-dependent mechanism that is independent of that associated with differentiation and is not dependent on the maturational state of these cells.
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PMID:Stimulation of osteopontin mRNA expression in HL-60 cells is independent of differentiation. 922 25

Retinoic acid (RA) has profound effects on epidermal homeostasis; however, the molecular mechanisms by which retinoids regulate keratinocyte cell proliferation and differentiation are not well understood. Here we report that mRNA expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family of growth factors, is induced by RA in human keratinocytes and skin, and is overexpressed in the context of epidermal hyperplasia in vivo. Treatment of normal adult human keratinocytes with micromolar concentrations of RA significantly induced the expression of HB-EGF. The response was efficiently blocked by specific inhibitors of ErbB tyrosine kinase activity, MAP kinase kinase (MEK), or p38 stress-activated protein kinase. RA also enhanced the induction of HB-EGF mRNA in human skin organ culture, an ex vivo model system displaying many similarities to wound healing in vivo. HB-EGF transcripts were markedly increased in human skin by topical treatment with RA under conditions known to provoke epidermal hyperplasia. HB-EGF transcripts were also markedly overexpressed in the hyperplastic epidermis of psoriatic lesions, relative to normal skin. These results support the hypothesis that the effects of RA on epidermal hyperplasia are mediated at least in part by HB-EGF, and suggest that signal transduction mechanisms other than or in addition to nuclear RA receptors contribute to this effect.
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PMID:Retinoid regulation of heparin-binding EGF-like growth factor gene expression in human keratinocytes and skin. 985 42

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF- or TNF-induced superoxide (O2-) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF- or TNF-induced O2- release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.
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PMID:Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. 986 79

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.
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PMID:The Ras/Erk pathway induces primitive endoderm but prevents parietal endoderm differentiation of F9 embryonal carcinoma cells. 988 May 24

HL-60 cells treated by PMA develop the monocyte adherent phenotype and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCbeta gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCbeta gene expression or transfected with PKCbeta cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCbeta. In conclusion, we propose that the pathway PKCbeta-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.
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PMID:Activation of plasminogen activator inhibitor-1 synthesis by phorbol esters in human promyelocyte HL-60--roles of PCKbeta and MAPK p42. 1010 71

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCgamma, PI-3 kinase, or src-like kinase.
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PMID:Transformation-defective polyoma middle T antigen mutants defective in PLCgamma, PI-3, or src kinase activation enhance ERK2 activation and promote retinoic acid-induced, cell differentiation like wild-type middle T. 1022 45

The type 3 iodothyronine deiodinase (D3) metabolizes thyroid hormones to inactive metabolites in many tissues, including the brain. In the present studies, we have examined the mechanisms by which T3 (T3), retinoic acid, 12-O-tetradecanoyl phorbol 13-acetate (TPA), and basic fibroblast growth factor (bFGF) induce D3 expression in primary cultures of neonatal rat astrocytes. In untreated cells, D3 messenger RNA (mRNA) was essentially undetectable by Northern analysis and RT-PCR. However, all four agents induced expression of a 2.4-kb D3 transcript as well as D3 activity. Induction of D3 by TPA and bFGF was more rapid than that by T3 and retinoic acid, and T3 potentiated the stimulatory effects of TPA and bFGF. D3 induction by TPA was blocked by GF 109203X, an inhibitor of protein kinase C. In addition, the effects of TPA and bFGF were partially prevented by PD 98059, a specific inhibitor of MEK and the Erk signaling cascade. These studies demonstrate that multiple growth factors and hormones regulate D3 activity in cultured astrocytes by inducing D3 mRNA expression. In addition, the stimulatory effects of TPA and bFGF on D3 mRNA and activity appear to be mediated at least in part by activation of the MEK/Erk signaling cascade.
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PMID:Regulation of type 3 iodothyronine deiodinase expression in cultured rat astrocytes: role of the Erk cascade. 1034 85

The addition of all-trans retinoic acid (ATRA) in combination with basic fibroblast growth factor (bFGF) to human fibroblasts results in a synergistic induction of tissue inhibitor of metalloproteinases-1 (TIMP-1) protein production. The synergistic stimulation of TIMP-1 protein by ATRA and bFGF increased across 72 h. An incubation of 10 min to 12 h with bFGF alone followed by ATRA gave a similar synergistic induction of TIMP-1 protein to that seen with both agents together. Treatment of cells with ATRA first followed by bFGF was ineffective. Expression of RARbeta mRNA was induced by ATRA alone, but not further induced by ATRA and bFGF; expression of RARgamma mRNA was induced by both ATRA or bFGF alone, and further induced by both reagents together; expression of RXRgamma was repressed by ATRA alone, but not by ATRA in combination with bFGF. Steady-state levels of TIMP-1 mRNA were induced 14 to 40-fold above control by ATRA and bFGF. Treatment with ATRA and bFGF did not alter the stability of TIMP-1 mRNA. The induction of TIMP-1 mRNA by ATRA and bFGF was greatly diminished by cycloheximide and therefore required new protein synthesis. The tyrosine kinase inhibitor genistein caused a dose-dependent inhibition of TIMP-1 protein induction by ATRA and bFGF. A MEK1 inhibitor (PD98059) inhibited both basal and induced levels of TIMP-1. At high concentrations, p38 MAP kinase inhibitors further enhanced the synergistic stimulation of TIMP-1 protein by ATRA and bFGF, but at these concentrations, p42/44 MAP kinase was strongly activated. These data begin to elucidate the mechanisms by which TIMP-1 gene expression can be upregulated.
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PMID:Mechanisms of induction of human tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression by all-trans retinoic acid in combination with basic fibroblast growth factor. 1086 18

The SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor participates in the repression of target gene expression by a variety of transcription factors, including the nuclear hormone receptors, promyelocytic leukemia zinc finger protein, and B-cell leukemia protein 6. The ability of SMRT to associate with these transcription factors and thereby to mediate repression is strongly inhibited by activation of tyrosine kinase signaling pathways, such as that represented by the epidermal growth factor receptor. We report here that SMRT function is potently inhibited by a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) cascade that operates downstream of this growth factor receptor. Intriguingly, the SMRT protein is a substrate for phosphorylation by protein kinases operating at multiple levels in this MAPKKK pathway, including the MAPKs, MAPK-extracellular signal-regulated kinase 1 (MEK-1), and MEK-1 kinase (MEKK-1). Phosphorylation of SMRT by MEKK-1 and, to a lesser extent, MEK-1 inhibits the ability of SMRT to physically tether to its transcription factor partners. Notably, activation of MEKK-1 or MEK-1 signaling in transfected cells also leads to a redistribution of the SMRT protein from a nuclear compartment to a more perinuclear or cytoplasmic compartment. We suggest that SMRT-mediated repression is regulated by the MAPKKK cascade and that changes both in the affinity of SMRT for its transcription factors and in the subcellular distribution of SMRT contribute to the loss of SMRT function that is observed in response to kinase signal transduction.
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PMID:The SMRT corepressor is regulated by a MEK-1 kinase pathway: inhibition of corepressor function is associated with SMRT phosphorylation and nuclear export. 1093 35


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