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)

Mitogen-activated protein kinases (MAPKs) have been shown to play an important role in transducing extracellular signals into cellular responses. The classic MAPK pathway is commonly activated by growth factors and has been shown to play a crucial role in cell proliferation. Transforming growth factor-beta (TGF-beta)-activating kinase-1 (TAK1) is a novel MAPK kinase kinase that is reported to stimulate the MKK6-p38K pathway. To elucidate the functional roles of the TAK1 pathway, we transfected its constitutive active form (TAKdN) and negative form (TAKK63W) to LLC-PK1 cells. TAKdN stimulated MKK6 phosphorylation and p38K activity and inhibited the percentages of the S and G2/M phases. TAKK63W, the constitutive negative form, reduced TGF-beta-stimulated MKK6 phosphorylation and p38K activity and increased the percentages of the S and G2/M phases. The cyclin D1 protein level is reduced by the TAK1 pathway. We also examined the effects of the TAK1 pathway on cyclin D1 promoter-luciferase assay. The overexpression of TAKdN or p38K inhibited cyclin D1 promoter activity. In contrast, overexpression of the active form of MKK1, the classic MAPK-activator, MKK1 increased cyclin D1 promoter activity and protein level, as well as the percentages of S and G2/M phases.
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PMID:Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated protein kinase cascade. 1050 69

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.
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PMID:Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization. 1060 20

Several reports have indicated that protein kinase C (PKC) is an important regulator of proliferation in thyroid cells. Unlike TSH, the mitogenic effects of phorbol esters are accompanied by de-differentiation. The role of individual PKC isoforms in thyroid cell proliferation and differentiation has not been examined. Recent studies have implicated the atypical PKCzeta, a phorbol ester-unresponsive isozyme, in cell proliferation, death, and survival. We overexpressed PKCzeta in Wistar rat thyroid (WRT) cells and determined that PKCzeta conferred TSH-independent DNA synthesis and cell proliferation. Cells overexpressing PKCzeta show higher levels of phosphorylated p42/p44 MAPK compared with vector-transfected cells. Experiments using a luciferase reporter for Elk-1 revealed that PKCzeta overexpressing cells exhibit higher basal Elk-1 transcriptional activity than vector-transfected control cells. Interestingly, stimulation of Elk-1 transcriptional activity by MEK1, a p42/p44 MAPK kinase, was significantly enhanced in cells overexpressing PKCzeta. Strikingly, TSH retained the ability to stimulate Tg expression in cells expressing PKCzeta. These results suggest that PKCzeta stimulates TSH-independent mitogenesis through a p42/p44 MAPK-dependent pathway. Unlike overexpression of Ras or phorbol ester treatment, PKC overexpression does not impair thyroglobulin (Tg) expression.
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PMID:Atypical protein kinase C-zeta stimulates thyrotropin-independent proliferation in rat thyroid cells. 1061 33

The role of signal transducer and activator of transcription (STAT) signaling pathways in the interleukin-6 (IL-6)-induced morphological differentiation of PC12-E2 cells was assessed using wild type and dominant negative mutants of Stat1 and Stat3, containing Tyr --> Phe (YF), Ser --> Ala (SA), and the double mutations (DM), respectively. FS3-YF or FS3-DM markedly inhibited the IL-6-induced response, but overexpression of FS3-SA caused only a modest inhibition. Expression of all Stat3 mutants had no effect on NGF-induced neurite outgrowth. Overexpression of wild type Stat1 protein inhibited IL-6 activated DNA binding complexes containing Stat3 homodimers, which may explain the partial negative effect of Stat1 on IL-6-induced neurite outgrowth. Specificity of these STAT constructs was confirmed using luciferase reporter gene assays, which showed that IL-6-activated transcription was blocked by expression of FS3-YF and FS3-DM and that FS1 enhanced the interferon gamma-activated transcription. Thus, in PC12-E2 cells, Stat3 homodimers are preferentially activated by IL-6, indicating a role for Stat3 in the regulation of cellular differentiation. Furthermore, IL-6 induced robust neurite outgrowth in PC12-E2 cells expressing dominant negative forms of RAS or SHC or in cells pretreated with the mitogen-activated protein kinase mitogen-activated protein kinase kinase inhibitor, PD98059. Thus, activation of the Stat3 signaling pathway, but not RAS/ERK dependent pathways, is essential for differentiation of PC12-E2 cells by IL-6.
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PMID:Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12-E2 cells. 1063 20

Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin-induced PACAP mRNA expression. Down-regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.
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PMID:Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells. 1064

A decline in oxygen concentration perturbs endothelial function, which promotes local thrombosis. In this study, we determined whether hypoxia in the range of that observed in pathophysiological hypoxic states stimulates plasminogen activator inhibitor-1 (PAI-1) production in bovine aortic endothelial cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (P<0.05 versus normoxic control, n=4) at 12 hours after hypoxic stimulation. Northern blot analysis showed the progressive time-dependent increase in the steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7.5-fold increase (P<0.05 versus control, n=4) at 12 hours. Deferoxamine, which has been known to bind heme protein and to reproduce the hypoxic response, induced PAI-1 production at both the mRNA and protein levels. The half-life of PAI-1 mRNA, as determined by a standard decay assay, was not affected by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainly at the transcriptional level. Transient transfection assays of the human PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive region lies between -414 and -107 relative to the transcription start site, where no putative hypoxia response element is found. The hypoxia-mediated increase in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors genistein (50 micromol/L) and herbimycin A (1 micromol/L), whereas PD98059 (50 micromol/L, MEK1 inhibitor), SB203580 (10 micromol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 micromol/L, protein kinase C inhibitor) had no effect on the induction of PAI-1 expression by hypoxia and deferoxamine. Genistein but not daidzein blocked the production of hypoxia- and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stimulates PAI-1 gene transcription and protein production through a signaling pathway involving genistein-sensitive tyrosine kinases in vascular endothelial cells.
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PMID:Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells. 1076 87

In PC12 cells stably expressing alpha(1A)-adrenergic receptors (ARs), norepinephrine (NE) activates several mitogen-activated protein kinase pathways and causes differentiation (). Using retroviral luciferase reporters, we found that NE also activated both signal transducers and activators of transcription (Stat) and gamma-interferon-activated sequence-mediated transcriptional responses, with maximal effects similar to those caused by interleukin-6 (IL-6). UTP and epidermal growth factor had no effect, whereas nerve growth factor caused a small Stat activation. Responses to NE were blocked by prazosin and depended on receptor density. Responses to NE were not blocked by inhibitors of mitogen-activated protein kinase kinase (PD98059), protein kinase C (GFX203290), Src (PP2), Jak2 (AG490), or the calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The p38 mitogen-activated protein kinase inhibitors SB202190 and SB203580 blocked Stat activation by NE, the epidermal growth factor receptor inhibitor AG1478 caused a small inhibition, but the phosphoinositide 3 kinase inhibitor LY294002 potentiated both responses. Gel shifts confirmed formation of nuclear factors binding to both Stat and gamma-interferon-activated sequence consensus sequences in response to NE and IL-6. Immunoprecipitation experiments showed that IL-6 increased tyrosine phosphorylation of Stat1 and Stat3 in PC12 cells, whereas NE caused a sustained increase in tyrosine phosphorylation of Stat1. These results suggest that alpha(1A)-AR stimulation causes Stat-mediated transcriptional responses in PC12 cells that are not downstream of known second messenger or tyrosine kinase pathways.
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PMID:Activation of signal transducers and activators of transcription by alpha(1A)-adrenergic receptor stimulation in PC12 cells. 1077 80

Intraarticular injection of dexamethasone (DEX) accelerates cartilage degradation due to the suppression of chondrocyte proliferation and extracellular matrix formation. The present study first demonstrated the interaction between DEX and TGF beta, a potent growth factor for cultured rat articular chondrocytes (CRAC), and then investigated the molecular mechanism by which DEX counteracts TGF beta-induced chondrocyte proliferation and differentiation through the regulation of AP-1 activity. DEX reduced serum-deprived and TGF beta-stimulated cell growth and [(3)H]-thymidine incorporation of CRAC. DEX also inhibited the expression of (alpha)1 type II collagen with concomitant suppression of the promoter activity. Transfection studies using a reporter vector with AP-1 responsive elements showed that DEX reduced TGF beta-activated but not basal luciferase activities. Activation of 3TP-luc, another AP-1 responsive element containing reporter was also blocked by DEX. GAL4-Elk1 studies revealed that DEX suppressed TGF beta-induced ERK activation which led to c-fos gene expression followed by increase in AP-1 complex formation, whereas the Smad pathway was not involved in DEX-dependent negative regulation of AP-1 in a reporter assay that requires FAST1-Smad2 for the activation. DEX also eliminated TGF beta-induced c-fos mRNA expression and ERK activation in Northern analysis and in vitro kinase assay, respectively. Further, DNA synthesis and transactivation of type II collagen by TGF beta were inhibited by PD98059, an inhibitor of MEK. Our results indicate that DEX suppressed TGF beta-induced chondrocyte proliferation and type II collagen expression, probably through selective inhibition of ERK integrated AP-1 activation.
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PMID:Dexamethasone inhibition of TGF beta-induced cell growth and type II collagen mRNA expression through ERK-integrated AP-1 activity in cultured rat articular chondrocytes. 1096 45

We previously reported tumor necrosis factor-alpha (TNF) modulates transcriptional and post-transcriptional down-regulation of macrophage scavenger receptor (MSR) (Hsu, H. Y., Nicholson, A. C., and Hajjar, D. P. (1996) J. Biol. Chem. 271, 7767-7773); however, TNF-mediated signaling mechanisms are unknown. Here, we demonstrate that ligation of TNF receptor stimulates activity of p21-activated protein kinase (PAK) and mitogen-activated protein kinases (MAPK) as follows: ERK, JNK, and p38 in murine macrophage J774A.1 cells. Upon activation of protein kinases (PK), TNF rapidly increases MSR message and protein; later it markedly reduces MSR expression. Studies using PK inhibitors and dominant negative constructs demonstrate phosphatidylinositol 3-kinase/Rac1/PAK/JNK and phosphatidylinositol 3-kinase/Rac1/PAK/p38 pathways contribute to important roles in the late stage of TNF down-regulation of MSR expression and taking up of OxLDL. Alternatively, the PKC/MEK1/ERK pathway in the early stage plays a significant role in up-regulation of the MSR gene. By using anti-TNF-R1 agonist antibody, we further confirm TNF-R1-mediated MAPK in regulation of MSR. Furthermore, in MSR gene promoter-driven luciferase reporter assays with TNF, PKC activator increases, but antioxidant N-acetylcysteine, PK inhibitors, and dominant negative constructs decrease luciferase activity in MSR gene promoter-transfected cells. Our current results show the first evidence of crucial roles for TNF-mediated MAPK pathways in the transcriptional regulation of MSR gene and increase MSR expression; in contrast, with TNF longer treatment the pathways down-regulate MSR and foam cell formation probably via post-transcriptional process.
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PMID:Tumor necrosis factor-alpha -mediated protein kinases in regulation of scavenger receptor and foam cell formation on macrophage. 1096 71

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression is strongly activated by a variety of extracellular stimuli, acting through the Raf/MEK/MAP kinase pathway. To study the elements that respond to this pathway, we have isolated and sequenced a fragment of the rat HB-EGF gene promoter. By transfection of a series of promoter/reporter constructs into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, gave a fold-stimulation of activity no greater than that seen for the parental pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced little or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB-EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5'-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two species approximately 260 bp upstream of the transcriptional start site. PCR analysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in the HB-EGF gene.
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PMID:Characterisation of the rat heparin-binding epidermal growth factor-like growth factor gene promoter. 1100 14


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